COMPOSITIONS FOR DETECTING BIOLOGICAL AGENTS

The present invention is directed to the detection of biological agents.

Currently the detection of biological agents in communities is performed in the laboratory using samples collected from sampling sites that are analysed by traditional cell culture methods, next generation sequencing (i.e., sequencing that allows large number of DNA sequences to be analysed in parallel) or biotyping mass spectrometry. The sensitivity and specificity of next generation sequencing for biological agent identification and detection means that it is the gold standard method to identify biological agents in both bodily fluids and environmental settings. Next generation sequencing is available in many clinical settings. However, its cost, complexity, relatively slow analysis speed and the requirement for highly specialized equipment remain barriers for its wide-spread, continual use to detect biological agents in various environmental settings.

A number of additional approaches have been developed to monitor the cleanliness of workspaces in the food industry. For example, the most commonly used is visual inspection and adenosine triphosphate (ATP) assays. ATP tests can typically detect 10 ⁴ CFU/mL of bacteria, but they do not detect viruses or bacterial spores. These tests also suffer from contamination issues as some foods such as ultra-high treatment (UHT) milk have a high ATP content, but a low microbiological load. Other biochemical tests that detect protein, reducing sugars, and nicotinamide suffer from similar deficiencies.

A further major drawback of these existing detection systems is that they are not able to deliver immediate results for the user. The 2020-22 COVID-19 pandemic has highlighted the challenges in managing a biological agent’s transmission, especially when there is a delay in its detection or because it is undetectable to the unaided eye. For example, by the time a lab report comes back a few days later after checking a surface for the presence of COVID-19 in a hospital, it will have swept through the building and community, resulting in the loss of many lives. A new technology is required to effectively manage biological agent identification and eradication. For the public to feel safe, there is a need for a technology that rapidly detects multiple biological agents, to then be able react promptly to manage the threat.

Lateral flow detection systems have been applied to a diverse set of biological agents and typically comprise three major components: 1) A chemical detector; 2) A detection signal amplifier; 3) A signal off-on switch. Given the requirement of such systems to detect a single biological agent, bespoke antibodies, aptamers or nucleotide sequences are typically used as the detection systems because of their high affinity and specificity against the target biological agents. Nanomaterials, particularly gold nanoparticles, are commonly used as the detection amplifiers as their large surface area provides a high amplification capacity. Particle aggregation, redox reactions and fluorescent quenching are the most common off-on switches for signal detection.

Prior technologies have focused on using a specific capture substrate that detects a specific biological agent. For example, Bui et al, Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay. Nano Lett. 2015; 15(9) focuses on detection of three pathogens using three specific detection substrates, i.e., “a one capture substrate to one pathogen approach”.

None of the technologies described to-date aim to rapidly detect multiple biological agents, either alone or in the context of biological agent communities found in environments inhabited by humans who therefore may be at risk. A threat of a biological agent should be removed before a biological agent spreads, to mitigate any risk. Current technologies, even if used in different formats, are not able to meet the need for rapid and broad detection of a wide variety of biological agents and their eradication. Instead, the delay in detection of a biological agent using current technologies may result in the biological agent spreading and potentially causing disease.

The present invention overcomes one or more of the above-mentioned problems.

The present inventors have surprisingly found that a composition of the invention (e.g. for detecting at least two genetically different biological agents on a surface or in a sample, the composition comprising: a capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different; and a nanomaterial associated with the capture substrate (preferably wherein the association between the nanomaterial and capture substrate is reversible)) finds particular use in the detection of biological agents in industrial and/or care settings (e.g. in food preparation premises and/or hospitals). In more detail, the compositions of the invention may comprise discrete populations of nanomaterials that are coated with one or more different capture substrates mixed in specific ratios to control the specificity of the interactions with the biological agents. Advantageously, in some instances the present invention may allow for the rapid and/or simple determination of whether or not a surface has been sufficiently cleaned. In such instances, the specific identity of a biological agent may not be of particular interest. Instead, for such applications, a composition, by detecting multiple genetically different biological agents, may be particularly sensitive to any biological agents present on the surface which may be indicative of insufficient cleaning.

Conventional technologies have focused on using one specific capture substrate to detect one specific biological agent. Signal interference, which may affect the detectability of a signal, may arise when an increasing number of capture substrates, that each bind to a different biological component of a different biological agent, are used. Advantageously, the invention may provide a composition that is capable of detecting at least two genetically different biological agents (such as bacteria, fungi, viruses, parasites and protozoa) on a surface or in a sample without significant interference. Moreover, where in preferred embodiments the composition produces an optically-detectable signal, this may avoid the use of specialist laboratory equipment, allowing the composition to be used in a multitude of environments. Furthermore, by using the composition to detect at least two genetically different biological agents, it may be determined whether a surface or sample exceeds a predetermined safety level and thus, needs cleaning to prevent pathogenic disease (for example, in the food industry and/or in a clinical setting). The composition of the invention may further comprise a detergent, allowing for the lysis of a biological agent (and thus, the killing and/or inactivation of the biological agent) whilst the biological agent is being detected.

In one aspect, the invention provides a composition for detecting a biological agent on a surface or in a sample, the composition comprising: a capture substrate for binding to a component of a biological agent; and a nanomaterial. The nanomaterial may be associated with the capture substrate (preferably wherein the association between the nanomaterial and capture substrate is reversible). Also provided are methods of using said composition to detect a biological agent on a surface or in a sample.

In one aspect the present invention provides a composition for detecting at least two genetically different biological agents on a surface or in a sample, the composition comprising:

(a) a capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different; and (b) a nanomaterial associated with the capture substrate (preferably wherein the association between the nanomaterial and capture substrate is reversible).

In one aspect the present invention provides a composition for detecting at least two genetically different biological agents on a surface or in a sample, the composition comprising:

(a) a capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different; and

(b) a nanomaterial.

A capture substrate (e.g. a single capture substrate molecule) that independently binds to a component of a first biological agent and a component of a second (or further) biological agent may be a capture substrate that is incapable of binding to a component of a first and second (or further) biological agent simultaneously. For example, such a capture substrate when bound to a component of a first biological agent may be incapable of additionally binding to a component of a second (or further) biological agent, or when bound to a component of a second (or further) biological agent may be incapable of additionally binding to a component of a first biological agent. Capture substrates that independently bind to a component of a first biological agent and a component of a second (or further) biological agent may comprise a single binding site for both the first and second (or further) biological agents. Once that binding site is bound to a component of a first biological agent it may be occupied and unable to be bound by a second (or further) biological agent.

Alternatively, a capture substrate (e.g. a single capture substrate molecule) may bind to both a component of a first biological agent and a component of a second (or further) biological agent simultaneously. Such capture substrates may comprise multiple binding sites such that when a single binding site is bound to a component of a first biological agent, a further binding site is free to bind a component of a second (or further) biological agent.

The binding sites may be the same or different (preferably the same).

Preferably the component of a first and second (or further) biological agent bound by a capture substrate of the invention is the same component (e.g. both components are nucleic acids, sugars, or proteins). In one aspect the present invention provides a composition for detecting at least two genetically different biological agents on a surface or in a sample, the composition comprising:

(a) a nanomaterial;

(b) a first capture substrate for binding to a component of a first biological agent; and

(c) a second capture substrate for binding to a component of a second biological agent; wherein the first and second biological agents are genetically different and optionally wherein the composition is capable of producing an optically-detectable signal when at least one of the capture substrates binds to the component of the biological agent.

At least one of the capture substrates may be capable of binding to at least two genetically different biological agents. Thus, the first capture substrate may be capable of binding to at least two genetically different biological agents. The second capture substrate may be capable of binding to at least two genetically different biological agents. The first and second capture substrates may be capable of binding to at least two genetically different biological agents.

In one aspect the present invention provides a composition for detecting at least two genetically different biological agents on a surface or in a sample, the composition comprising:

(a) a nanomaterial;

(b) a first capture substrate for binding to a component of a first biological agent; and

(c) a second capture substrate for binding to a component of a second biological agent; wherein the first and second biological agents are genetically different; optionally: wherein the composition is capable of producing an optically-detectable signal that is indicative of binding of at least one of the capture substrates to the component of the biological agent; and wherein at least one of the capture substrates is capable of binding to at least two genetically different biological agents.

The term “nanomaterial” as used herein refers to a material of nanoscale size. A nanomaterial may have at least one dimension of less than or equal to 1000 nm in size. The size of a nanomaterial may be determined by laser light scattering, by atomic force microscopy or by other suitable techniques. The size of a nanomaterial may be an average size of a nanomaterial, e.g. an average diameter of a nanomaterial where a nanomaterial is spherical (e.g. substantially spherical). In one embodiment, the term “average particle size” refers to average agglomerate particle size dso as measured by laser diffraction.

In one embodiment, the term “nanomaterial” refers to a material of a size of less than 1000 nm. For example, the nanomaterial may be less than or equal to 900, 800, 700, 600, 500, 400, 300 or 200 nm. In one embodiment, a nanomaterial has a size of less than or equal to 200, 180, 160, 140 or 120 nm, preferably less than or equal to 110 nm, more preferably less than or equal to 105 nm (e.g. less than or equal to 100 nm). In one embodiment, the nanomaterial has a size of less than or equal to 100, 90, 80, 70, 60, 50, 40 or 30 nm, preferably less than or equal to 20 nm, more preferably less than or equal to 17 nm (e.g. less than or equal to 15 nm).

In one embodiment, the term “nanomaterial” refers to a material of a size of at least 0.5, 1 , 1.5, 5, 10, 30, 50, 70, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 nm. In one embodiment, a nanomaterial has a size of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nm, preferably at least 13 nm, more preferably at least 14 nm (e.g. at least 15 nm). In one embodiment, a nanomaterial has a size of at least 10, 20 or 30 nm, preferably at least 40 nm, more preferably at least 45 nm (e.g. at least 50 nm).

In one embodiment, the term “nanomaterial” refers to a material of a size of 0.5-1000, 5-900, 10-800, 50-700, 100-600, 200-500 or 300-400 nm. In other embodiments the term “nanomaterial” refers to a material of a size of 10-20 nm, 11-19 nm, 12-18 nm, preferably 13- 17 nm, more preferably 14-16 nm (e.g. 15 nm). In one embodiment, a nanomaterial has a size of 10-100, 20-90 or 30-80 nm, preferably 40-70 nm, more preferably 45-65 nm (e.g. 50 nm).

In one embodiment, the nanomaterial is a gold nanoparticle. In one embodiment, the nanomaterial is a gold nanoparticle that has a size of less than or equal to 100, 90, 80, 70, 60, 50, 40 or 30 nm, preferably less than or equal to 20 nm, more preferably less than or equal to 17 nm (e.g. less than or equal to 15 nm).

In one embodiment, a nanomaterial is a gold nanoparticle that has a size of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nm, preferably at least 13 nm, more preferably at least 14 nm (e.g. at least 15 nm). In one embodiment, a nanomaterial is a gold nanoparticle that has a size of 10-25, 15-20, 10-20, 11-19, 12-18 nm, preferably 13-17 nm, more preferably 14-16 nm (e.g. 15 nm).

In one embodiment, the nanomaterial is a metal-organic framework (MOF). In one embodiment, a nanomaterial is a metal-organic framework (MOF) that has a size of less than or equal to 200, 180, 160, 140 or 120 nm, preferably less than or equal to 110 nm, more preferably less than or equal to 105 nm (e.g. less than or equal to 100 nm).

In one embodiment, a nanomaterial is a MOF that has a size of at least 10, 20 or 30 nm, preferably at least 40 nm, more preferably at least 45 nm (e.g. at least 50 nm).

In one embodiment, a nanomaterial is a MOF that has a size of 10-100, 20-90 or 30-80 nm, preferably 40-70 nm, more preferably 45-65 nm (e.g. 50 nm).

Advantageously, due to the large surface area (e.g. less than or equal to 1000 nm) of a nanomaterial, this allows, in one embodiment, for the nanomaterial to be associated with one or more capture substrates. The association of one or more capture substrates may allow for an increase in detection capability of a detectable signal (preferably an optically-detectable signal).

Thus, the nanomaterial may be associated with a capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different. The nanomaterial may be associated with a first capture substrate for binding to a component of a first biological agent. The nanomaterial may be associated with a second (or further) capture substrate for binding to a component of a second (or further) biological agent.

The nanomaterial may be associated with a plurality of capture substrates for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different. The nanomaterial may be associated with a capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different and a further capture substrate for binding to a further biological agent. The nanomaterial may be associated with a first capture substrate for binding to a component of a first biological agent and, optionally independently, a component of a second biological agent, wherein the first and second biological agents are genetically different, and a second capture substrate for binding to a component of a third biological agent, and optionally independently, a component of a fourth biological agent, wherein the third and fourth biological agents are genetically different.

The nanomaterial may be associated with a plurality of first capture substrates for binding to a component of a first biological agent. The nanomaterial may be associated with a plurality of second (or further) capture substrates for binding to a component of a second (or further) biological agent. The nanomaterial may be associated with a first capture substrate for binding to a component of a first biological agent and a second (or further) capture substrate for binding to a component of a second (or further) biological agent.

The use of a nanomaterial in a composition of the invention may amplify a detectable signal and increase the sensitivity of detection. Thus, unlike current detection means (and associated methods), in some embodiments, the compositions and methods of the invention do not require the use of a molecular technique to amplify the detectable signal and increase the sensitivity of detection. Advantageously, by omitting these molecular techniques, this allows for more rapid detection of a biological agent. Said molecular technique may be polymerase chain reaction (PCR), reverse transcriptase (RT-PCR), DNA sequencing, and/or a DNA microarray. Preferably, the invention does not comprise the use of one or more of said techniques, more preferably does not comprise the use of PCR.

A nanomaterial may be available in different formats without limitation, e.g., as a solid (e.g. a metal such as silver, gold, iron, titanium), as a non-metal, as a lipid-based solid, as a polymer, as a suspension of nanoparticles, or combinations thereof. A nanomaterial may also be metal, dielectric, and/or semiconductive and may also be prepared as a hybrid structure (e.g., a core-shell nanoparticle). Where a nanomaterial comprises a semiconducting material, such a nanomaterial may be a quantum dot.

The nanomaterial may comprise an inorganic nanomaterial, an organic nanomaterial, or a combination thereof.

A nanomaterial may have any suitable configuration. A configuration may be selected from: a nanoparticle, a nanocomposite, a nanorod, a nanoshell, a nanocube, a nanocluster, a two- dimensional (2-D) or three-dimensional (3-D) nanoparticle array, a nanobipyramid, a colloidal nanoparticle, a nanowire, a core-shell nanoparticle, a nanosphere, a nanoprism, a nanotriangle, a nanograting, a nanodot, a nanocylinder, a nanogap, a nanodome, a nanopillar, a nanoantenna, a nanotube, a nanosheet, a fullerene, a nanocrystal, or a quantum dot, preferably a nanoparticle. A nanomaterial may be linear, triangular, square, tetrahedral, hexagonal or octagonal in shape. The shape may be selected to result in different properties, e.g. different surface area properties.

The term “inorganic nanomaterial” as used herein may be a nanomaterial that does not comprise carbon, such as a nanomaterial that comprises a metal or non-metal element. In one embodiment, an inorganic nanomaterial does not comprise carbon. The inorganic nanomaterial may be selected from: gold, nanosilica, a magnetic nanomaterial, silver, titanium, alumina, zirconia, palladium, platinum, copper or hydroxyapatite, preferably the inorganic nanomaterial is gold, more preferably the inorganic nanomaterial is a gold nanoparticle.

A magnetic nanomaterial may be a paramagnetic or a superparamagnetic nanomaterial. Said paramagnetic or superparamagnetic nanomaterial may be manufactured with a paramagnetic or superparamagnetic nanoparticle core, respectively.

A nanomaterial may be a metal nanomaterial comprising gold, nanosilica, a magnetic nanomaterial, silver, titanium, alumina, zirconia, palladium, platinum, copper or hydroxyapatite. A metal nanomaterial may be a MOF.

The term “organic nanomaterial” as used herein may refer to a nanomaterial that comprises carbon. An organic nanomaterial may comprise a lipid and/or a carbohydrate. A lipid-based nanomaterial may comprise one or more lipids. A carbohydrate-based nanomaterial may comprise one or more carbohydrates. A carbohydrate-based nanomaterial may comprise an oligosaccharide, a polysaccharide (such as a peptidoglycan, a lipopolysaccharide, and/or an exopolysaccharide) or a monosaccharide, preferably a polysaccharide. Preferably, the carbohydrate-based nanomaterial comprises chitosan. An organic nanomaterial may be selected from the group consisting of: a micelle, a dendrimer or a liposome.

The term “chitosan” as used herein may refer to a linear polysaccharide comprising at least two repeating copolymers. A copolymer may comprise a unit of 2-acetamido-D-glucose (preferably N-acetyl-2-amino-2-D-glucopyranose) and a unit of 2-amino-D-glucose (preferably 2-amino-2-deoxy-d-glucopyranose) linked by a glycosidic bond (preferably a - (1 — 4)-glycosidic bond). The repeating copolymers may each be linked by a glycosidic bond (preferably a p-(1— >4)-glycosidic bond).

The nanomaterial may comprise a combination of inorganic and organic nanomaterials. Preferably, when the nanomaterial comprises a combination of inorganic and organic nanomaterials the nanomaterial is a MOF.

The term “MOF” as used herein may refer to a structure that is produced by a coordination interaction between one or more metal ion(s)/cluster(s) and one or more organic ligand(s). As a MOF may have an adjustable micropore structure, defined geometry, a large specific surface area, and/or (preferably and) one or more exposed active site(s), these properties are advantageous for the association (e.g. adsorption) of one or more capture substrates to the MOF and/or in the preparation of artificial nanozymes. Moreover, a MOF may be cost- efficiently manufactured on a large scale and may quench the fluorescence of a fluorescent label.

A nanomaterial may comprise graphene or graphene oxide (GO). A graphene or GO-based nanomaterial may be effective at associating with a capture substrate such as one or more nucleic acid(s), protein(s) and/or peptide(s). An advantage of a graphene or GO-based nanomaterial may be that such a nanomaterial may quench the fluorescence of a fluorescent label.

A biological agent may be a bacterium, a fungus, a virus, a parasite, or a protozoan.

A biological agent may be a bacterium from the family Acetobacteraceae, Moraxellaceae, Actinomycetaceae, Rhizobiaceae, Ehrlichiaceae, Xanthobacteraceae, Pseudomonadaceae, Bacillaceae, Paenibacillaceae, Bacteroidaceae, Bartonellaceae, Alcaligenaceae, Borreliaceae, Brucellaceae, Burkholderiaceae, Enterobacteriaceae, Campylobacteraceae, Chlamydiaceae, Clostridiaceae, Corynebacteriaceae, Coxiellaceae, Ehrlichiaceae, Neisseriaceae, Enterococcaceae, Fusobacteriaceae, Bifidobacteriaceae, Pasteurellaceae, Helicobacteraceae, Lactobacillaceae, Streptococcaceae, Legionellaceae, Leptospiraceae, Listeriaceae, Methylobacteriaceae, Methanobacteriaceae, Microbacteriaceae, Micrococcaceae, Moraxellaceae, Mycobacteriaceae, Mycoplasmataceae, Neisseriaceae, Pasteurellaceae, Porphyromonadaceae, Prevotellaceae, Pseudomonadaceae, Rhizobiaceae, Rickettsiaceae, Yersiniaceae, Spirillaceae, Staphylococcaceae, Xanthomonadaceae or Streptococcaceae, preferably Enterobacteriaceae. A biological agent may be a bacterium from the genus Acetobacter, Acinetobacter, Actinomyces, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Eikenella, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leptospira, Listeria, Methanobacterium, Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Porphyromonas, Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shigella, Spirillum, Staphylococcus, Stenotrophomonas or Streptococcus, preferably Escherichia.

A biological agent may be a bacterium from the species Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacian, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetiid, Ehrlichia chaffeensis, Ehrlichia ewingii, Eikenella corrodens, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus maloratus, Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Leptospira interrogans Leptospira noguchii, Listeria monocytogenes, Methanobacterium extroquens, Micro bacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma Mexican, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Porphyromonas gingivalis, Prevotella melaninogenica, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Spirillum volutans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis or Streptococcus sobrinus, preferably Escherichia coii.

A fungus may be a mould.

A fungus may be a yeast.

A biological agent may be a fungus from the family Pleosporaceae, Trichocomaceae, Arthrodermataceae, Orbiliaceae or Saccharomycetaceae, preferably Arthrodermataceae.

A biological agent may be a fungus from the genus Helminthosporium, Aspergillus, Penicillium, Trichophyton, Dactylella or Candida, Saccharomyces, preferably Trichophyton.

A biological agent may be a fungus from the species Helminthosporium solani, Helminthosporium gramineum, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Penicillium chrysogenum, Penicillium citrinum, Penicillium janthinellum, Penicillium marneffei, Penicillium purpurogenum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Dactylella acrochaeta, Dactylella beijingensis, Dactylella Candida, Dactylella dasguptae, Dactylella formosana, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae or Saccharomyces cerevisiae, preferably Trichophyton rubrum. A biological agent may be a virus from the family Adenoviridae, Coronaviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papoillomaviridae, Rhabdoviridae or Togaviridae, preferably Coronaviridae.

A biological agent may be a virus from the genus Atadenovirus, Mastadenovirus, Aviadenovirus, Betacoronavirus, Enterovirus, Lymphocryptovirus, Simplexvirus, Cytomegalovirus, Rhadinovirus, Varicellovirus, Orthohepadnavirus, Hepacivirus, Lenti virus, Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, Deltainfluenzavirus, Morbillivirus, Orthorubulavirus, Respirovirus, Orthopneumovirus, Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, Nupapillomavirus, Lyssavirus or Rubivirus, preferably Betacoronavirus.

A biological agent may be a virus from the species adenovirus (A to G), Severe acute respiratory syndrome coronavirus 2, coxsackievirus, hepatitis A virus, poliovirus, Epstein- Barr virus, herpes simplex (type 1), herpes simplex (type 2), human cytomegalovirus, human herpesvirus (type 8), varicella-zoster virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, influenza virus (A to D), measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, papillomavirus, rabies virus or Rubella virus, preferably Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

In one embodiment, the virus may be a bacteriophage.

A bacteriophage may be from the family Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Cystoviridae or Inoviridae, preferably Inoviridae.

A bacteriophage may be from the genus Tequatrovirus, Muvirus, Punavirus, Peduovirus, Lambdavirus, Tequintavirus, Hendrixvirus, Teseptimavirus, Gammalipothrixvirus, Icerudivirus, Cystovirus or Inovirus, preferably Inovirus.

A bacteriophage may be from the species Escherichia virus T4, Escherichia virus Mu, Escherichia virus P1, Escherichia virus P2, Escherichia virus Lambda, Escherichia virus T5, Escherichia virus HK97, Escherichia virus T7, Acidianus filamentous virus 1, Icerudivirus SIRV1, Pseudomonas virus phi6 or Escherichia virus M13, preferably Escherichia virus M13. A biological agent may be a protozoan from the family Plasmodiidae, Trypanosomatidae or Babesiidae.

A biological agent may be a protozoan from the genus Plasmodium, Leishmania, Trypanosoma or Babesia.

A biological agent may be a protozoan from the species Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, Leishmania tropica, Trypanosoma cruzi, Trypanosoma brucei, Babesia microti or Babesia duncani.

A biological agent may be a parasite from the family Onchocercidae, Dipylidiidae or Hymenolepididae.

A biological agent may be a parasite from the genus Wuchereria, Brugia, Dirofilaria, Mansonella, Onchocerca, Loa, Dipylidium or Hymenolepis.

A biological agent may be a parasite from the species Wuchereria bancrofti, Brugia malayi, Dirofilaria tenuis, Mansonella perstans, Mansonella streptocerca, Mansonella ozzardi, Onchocerca volvulus, Loa, Dipylidium caninum, Hymenolepis diminuta or Hymenolepis nana.

In one embodiment, a biological agent is not a proteinaceous biological agent (e.g. a prion).

Genetically different biological agents preferably comprise different genetic information. Preferably said difference is a difference in the genome of a biological agent when compared to the genome of a second biological agent. Thus, where there are two biological agents with the same genome but one of the two biological agents comprises a further genetic element (e.g. a plasmid or vector), in some embodiments, said biological agents are not considered to be different.

Genetically different biological agents may be genetically different by virtue of a difference in genetic material of at least 10, 25, 50, 100, 150, 200, 250 or 300, 500, 1000, 2500, 5000, or 10,000 nucleotides. For example, genetically different biological agents may be genetically different by virtue of a difference in genetic material of less than or equal to 50,000, 25,000, 10,000, 5000, 1000, 800, 600, 400 or 200 nucleotides.

Genetically different biological agents may be genetically different by virtue of a difference in genetic material of 10-50,000, 10-25,000, 10-1000, 50-900, 100-800, 200-700 or 300-600 nucleotides.

A genetic difference may result in at least two biological agents being classified as biological agents from different domains, different kingdoms, different phyla, different classes, different orders, different genera, different species or different strains.

The term “at least two” as used herein may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. For example, the term “at least two” when used in the context of biological agents (e.g. genetically different biological agents) described herein may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 biological agents.

The at least two genetically different biological agents may comprise:

(a) a first family of bacterium and a second family of bacterium genetically different to the first family of bacterium;

(b) a first family of fungus and a second family of fungus genetically different to the first family of fungus;

(c) a first family of virus and a second family of virus genetically different to the first family of virus;

(d) a first family of parasite and a second family of parasite genetically different to the first family of parasite; or

(e) a first family of protozoan and a second family of protozoan genetically different to the first family of protozoan.

The at least two genetically different biological agents may comprise:

(a) a first genus of bacterium and a second genus of bacterium genetically different to the first genus of bacterium;

(b) a first genus of fungus and a second genus of fungus genetically different to the first genus of fungus;

(c) a first genus of virus and a second genus of virus genetically different to the first genus of virus; (d) a first genus of parasite and a second genus of parasite genetically different to the first genus of parasite; or

(e) a first genus of protozoan and a second genus of protozoan genetically different to the first genus of protozoan.

The at least two genetically different biological agents may comprise:

(a) a first species of bacterium and a second species of bacterium genetically different to the first species of bacterium;

(b) a first species of fungus and a second species of fungus genetically different to the first species of fungus;

(c) a first species of virus and a second species of virus genetically different to the first species of virus;

(d) a first species of parasite and a second species of parasite genetically different to the first species of parasite; or

(e) a first species of protozoan and a second species of protozoan genetically different to the first species of protozoan.

The at least two genetically different biological agents may comprise:

(a) a bacterium and a virus;

(b) a bacterium and a fungus;

(c) a fungus and a virus;

(d) a bacterium and a parasite;

(e) a bacterium and a protozoan;

(f) a fungus and a parasite;

(g) a fungus and a protozoan;

(h) a virus and a parasite;

(i) a virus and a protozoan; or

(j) a parasite and a protozoan.

The at least two genetically different biological agents may comprise a first strain of a species of bacterium, fungus, virus, parasite or protozoan and a second strain of the same species of bacterium, fungus, virus, parasite or protozoan. The term “strain” as used herein may refer to a genetic variant or subtype of a bacterium, fungus, virus, parasite or protozoan. A first strain of a species of bacterium, fungus, virus, parasite or protozoan may differ from at least a second strain of the same species of bacterium, fungus, virus, parasite or protozoan by a genetic difference of at least 1 , 2, 3, 4 or 5 nucleotides.

A first strain of a species of bacterium, fungus, virus, parasite or protozoan may differ from at least a second strain of the same species of bacterium, fungus, virus, parasite or protozoan by a genetic difference of less than or equal to 50, 40, 30, 20 or 10 nucleotides.

A first strain of a species of bacterium, fungus, virus, parasite or protozoan may differ from at least a second strain of the same species of bacterium, fungus, virus, parasite or protozoan by a genetic difference of 1-50, 5-45, 10-40, 15-35 or 20-30 nucleotides.

A biological agent herein may be pathogenic and/or non-pathogenic. The pathogenic biological agent may cause disease in a subject. Preferably, the pathogenic biological agent may cause disease in a human, animal and/or in a plant, more preferably in a human. A non- pathogenic biological agent may not cause disease in a human, animal and/or in a plant, more preferably in a human.

A “subject” as used herein may be a mammal, such as a human or other mammal. Preferably “subject” means a human subject.

The composition may be capable of detecting at least two of: Escherichia coli (E. coli), Trichophyton Rubrum (T. rubrum), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The composition may be capable of detecting: Escherichia coli (E. coli), Trichophyton Rubrum (T. rubrum), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The composition may be capable of detecting at least three, at least four, at least five, or at least six genetically different biological agents on a surface or in a sample, preferably at least three genetically different biological agents on a surface or in a sample.

In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 capture substrates may be associated with a single nanomaterial (e.g. a single nanoparticle). For example 1-500, 10-250, 50-150, or 100-200 capture substrates may be associated with a single nanomaterial. Thus, a composition of the invention may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 capture substrates. For example, a composition of the invention may comprise 1-500, 10-250, 50-150 or 100-200 capture substrates.

In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different capture substrates may be associated with a single nanomaterial (e.g. a single nanoparticle). For example 1-500, 10-250, 50-150, or 100-200 different capture substrates may be associated with a single nanomaterial. Thus, a composition of the invention may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 different capture substrates. For example, a composition of the invention may comprise 1-500, IQ- 250, 50-150 or 100-200 different capture substrates.

A capture substrate may comprise a nucleic acid (such as an oligonucleotide, a cDNA or an RNA, a primer, or a probe), a lectin, an aptamer, a polypeptide (e.g. an antibody or a peptide), a small molecule, a fatty acid, or a combination thereof. The detection of a component that is found in multiple (genetically different) bacteria, fungi, viruses, parasites or protozoa is advantageous as this allows a single capture substrate to bind (e.g. via hybridisation) to numerous biological agents.

A capture substrate may comprise a binding site for binding to an intracellular component of a biological agent, an extracellular component of a biological agent, a cell surface component of a biological agent, or a combination thereof. A capture substrate may comprise a binding site for binding to an extracellular component of a biological agent. A capture substrate may comprise a binding site for binding to a cell surface component of a biological agent. Preferably, a capture substrate comprises a binding site for binding to an intracellular component of a biological agent, for example, a substrate for binding to a nucleic acid of a biological agent.

The term “hybridisation” as used herein may refer to a process in which a first nucleic acid joins with a second nucleic acid that has a nucleic acid complementary to the first nucleic acid. The hybridisation conditions may be based on the melting temperature (Tm) of the complex of a first and second nucleic acid (see Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). The melting temperature may affect the stringency. Maximum stringency may occur at about 5°C below the Tm; high stringency may occur at about 5°C to 10°C below the Tm; intermediate stringency may occur at about 10°C to 20°C below the Tm; and low stringency may occur at about 20°C to 25°C below the Tm. The skilled person will appreciate that whilst a maximum stringency hybridisation may be used to identify or detect nucleic acids that are identical to one another, an intermediate (or low) stringency hybridisation may be used to identify or detect nucleic acids that are similar or related.

The nucleic acid may be an oligonucleotide. An oligonucleotide may comprise less than or equal to 50, 45, 40, 35 or 30 bases, preferably less than or equal to 25 bases, more preferably may be less than or equal to 22 bases in length (e.g. 20 bases in length). In other words, the oligonucleotide may comprise less than or equal to 22 bases (e.g. 20 bases). An oligonucleotide may comprise at least 5 or 10 bases, preferably at least 15 bases, more preferably at least 18 bases in length (e.g. 20 bases in length). In other words, an oligonucleotide may comprise at least 18 bases (e.g. 20 bases). An oligonucleotide may comprise 5-50, 5-35, 10-30, or 12-16 bases, preferably at least 15-25 bases, more preferably may be at least 18-22 bases in length (e.g. 20 bases in length). In other words, an oligonucleotide may comprise at least 18-22 bases (e.g. 20 bases).

An oligonucleotide may comprise less than or equal to 50, 45, 40, 35 or 30 nucleotides, preferably less than or equal to 25 nucleotides, more preferably may be less than or equal to 22 nucleotides in length (e.g. 20 nucleotides in length). In other words, the oligonucleotide may comprise less than or equal to 22 nucleotides (e.g. 20 nucleotides). An oligonucleotide may comprise at least 5 or 10 nucleotides, preferably at least 15 nucleotides, more preferably at least 18 nucleotides in length (e.g. 20 nucleotides in length). In other words, an oligonucleotide may comprise at least 18 nucleotides (e.g. 20 nucleotides). An oligonucleotide may comprise 5-50, 5-35, 10-30, or 12-16 nucleotides, preferably at least 15- 25 nucleotides, more preferably may be at least 18-22 nucleotides in length (e.g. 20 nucleotides in length). In other words, an oligonucleotide may comprise at least 18-22 nucleotides (e.g. 20 nucleotides).

Thus, in one embodiment, the capture substrate is a nucleic acid, preferably an oligonucleotide. The nucleic acid (e.g. oligonucleotide) is preferably single-stranded.

A nucleic acid may be a primer or a probe.

A nucleic acid capture substrate for binding to a component of a biological agent may bind (e.g. via hybridisation) a region of a nucleic acid (e.g. DNA or RNA) found in biological agents (e.g. a plurality of biological agents) that are genetically different. The detection of such a nucleic acid sequence is advantageous as detection may be effected regardless of genetic variation (e.g. substantial genetic variation) of (or between) said biological agents.

In one embodiment, a capture substrate of the invention does not comprise (or consist of) a nucleic acid isolated from a subject, preferably a human subject. In one embodiment, a capture substrate of the invention comprises (or consists of) a nucleic acid isolated from a subject, such as a biological agent as described herein.

A capture substrate may bind (e.g. via hybridisation) a ribosomal RNA of a biological agent, preferably a 16S rRNA of a biological agent. 16S rRNA is highly conserved between different species of bacteria and archaea. Accordingly, a capture substrate which binds 16S rRNA of a biological agent may also bind 16S rRNA of a genetically different biological agent. The capture substrate may comprise (or consist of) a nucleotide sequence complementary to a 16S rRNA of a biological agent. The capture substrate may comprise (or consist of) a nucleotide sequence of a portion of a 16S rRNA of a biological agent.

A capture substrate may comprise (or consist of) a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1. For example, a capture substrate may comprise (or consist of) a nucleic acid sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1. Preferably, a capture substrate may comprise (or consist of) SEQ ID NO: 1.

A capture substrate may comprise (or consist of) a fragment of nucleic acid corresponding to a nucleic acid sequence found in a biological agent (e.g. a genetically similar or genetically identical biological agent). The biological agent may be a bacterium, preferably E. coli. The biological agent may be one to which a capture substrate is targeted. Said fragment may be at least 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% identical to a nucleic acid sequence found in a biological agent. Said fragment may be obtainable by any method known in the art, for example, by de novo nucleic acid synthesis. However, it is preferred that said fragment of nucleic acid is obtainable by fragmenting nucleic acid obtainable from a biological agent. Fragmented nucleic acid from a biological agent may be obtainable by subjecting nucleic acid from (e.g. isolated from) a biological agent to fragmentation. Preferably, said fragment of nucleic acid may be obtainable by subjecting nucleic acid from a biological agent to nuclease (e.g. DNase) treatment. Said fragment of nucleic acid may be spliced nucleic acid (e.g. spliced DNA). The fragment of nucleic acid may be single-stranded or double-stranded nucleic acid, preferably single-stranded. Said single-stranded fragment of nucleic acid corresponding to a nucleic acid sequence found in a biological agent may be capable of binding to its complementary nucleotide sequence obtainable from the biological agent (e.g. a genetically similar or genetically identical biological agent). Said single-stranded fragment of nucleic acid corresponding to a nucleic acid sequence found in a biological agent may be capable of binding to its complementary nucleotide sequence obtainable from a genetically different biological agent. Thus, a capture substrate comprising (or consisting of) a fragment of nucleic acid corresponding to a nucleic acid sequence found in a biological agent may be able to bind to complementary nucleotide sequences obtainable from a plurality of biological agents (e.g. a plurality of genetically different biological agents). The nucleic acid is preferably DNA. A capture substrate may comprise a plurality of fragments of nucleic acid, each corresponding to a different nucleic acid sequence found in a biological agent (e.g. a genetically similar or identical biological agent). A composition of the invention may comprise a plurality of capture substrates, each comprising a fragment of nucleic acid corresponding to a nucleic acid sequence found in a biological agent. Each of said fragments may be different.

A nuclease may be a deoxyribonuclease (DNase) and/or a ribonuclease (RNase). A DNase may be DNase I or DNase II. A RNase may be an endoribonuclease or an exoribonuclease. An endoribonuclease may be RNase A, RNase H, RNase III, RNase L, RNase P, RNase E or RNase G. An exoribonuclease may be RNase PH, RNase R, RNase D or RNase T.

A fragment of nucleic acid may comprise less than or equal to 50, 45, 40, 35 or 30 nucleotides, preferably less than or equal to 25 nucleotides, more preferably less than or equal to 22 nucleotides (e.g. 20 nucleotides). A fragment of nucleic acid may comprise at least 5 or 10 nucleotides, preferably at least 15 nucleotides, more preferably at least 18 nucleotides (e.g. 20 nucleotides). A fragment of nucleic acid may comprise 5-50, 5-35, 10- 30, or 12-16 nucleotides, preferably at least 15-25 nucleotides, more preferably at least 18- 22 nucleotides (e.g. 20 nucleotides).

A capture substrate may bind (e.g. via hybridisation) a viral nucleic acid sequence. Preferably, a capture substrate may bind (e.g. via hybridisation) a viral M13 sequence.

A capture substrate may comprise (or consist of) a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 2. For example, a capture substrate may comprise (or consist of) a nucleic acid sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 2. Preferably, a capture substrate may comprise (or consist of) SEQ ID NO: 2.

A capture substrate may comprise (or consist of) a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3. For example, a capture substrate may comprise (or consist of) a nucleic acid sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 3. Preferably, a capture substrate may comprise (or consist of) SEQ ID NO: 3.

A capture substrate may be an aptamer. An “aptamer” as used herein may refer to an oligonucleotide or peptide (e.g. an oligonucleotide or peptide as defined herein), preferably an oligonucleotide.

In one embodiment, the capture substrate is a lectin. The term “lectin” as used herein may refer to a carbohydrate-binding protein that has binding specificity to a sugar group.

A lectin may be a lectin of the genus Agaricus, Allium, Amaranthus, Arachis, Artocarpus, Astragalus, Bauhinia, Cicer, Canavalia, Crotalaria, Datura, Dolichos, Erythrina, Euonymus, Galanthus, Glycine, Griffonia, Hippeastrum, Iris, Lens, Lotus, Lycopersicon, Maackia, Madura, Momordica, Morus, Musa, Narcissus, Phaseolus, Phytolacca, Pisum, Polygonatum, Psophocarpus, Sambucus, Solanum, Sophora, Styphnolobium, Triticum, Tulip, Ulex, Urtica, Vida, Vigna, Viscum or Wisteria, preferably Canavalia.

A lectin may be a lectin of the plant species Agaricus bisporus, Allium sativum, Amaranthus caudatus, Arachis hypogaea, Artocarpus integrifolia, Astragalus membranaceus, Bauhinia purpurea, Cicer arietinum, Canavalia ensiformis, Crotalaria juncea, Datura stramonium, Dolichos biflorus, Erythrina cristagalli, Euonymus europaeus, Galanthus nivalis, Glycine max, Griffonia simplicifolia, Lens culinaris, Lotus tetragonolobus, Lycopersicon esculentum, Maackia amurensis, Madura pomifera, Momordica charantia, Morus alba, Morus rubra, Musa paradisiaca, Narcissus pseudonarcissus, Phaseolus acutifolius, Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisum sativum, Polygonatum cyrtonema, Polygonatum odoratum, Psophocarpus tetragonolobus, Sambucus nigra, Solanum tuberosum, Sophora flavescens, Styphnolobium japonicum, Triticum vulgaris, Ulex europaeus, Urtica dioica, Vida ervilia, Vicia faba or Vicia villosa, preferably Canavalia ensiformis. Preferably, the lectin is Concanavalin A (Con A).

Examples of lectins may be found at https://www.glycomatrix.com/collections/affypure- lectins.

In one embodiment, the capture substrate is an antibody. The term “antibody” as used herein may refer to a whole antibody or an antigen-binding fragment thereof (for example an antigen-binding fragment may be Fv, Fab, Fab', F(ab')2, Fc, or a single chain Fv fragments (scFv), or a fragment of an immunoglobulin that specifically binds to an antigen expressed by a biological agent. An antibody or an antigen binding fragment may comprise a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a bispecific antibody, a mini body, or a linear antibody, or a fragment thereof.

Where a capture substrate is an antibody, the antibody may have a binding specificity to its antigen with a Kd of less than or equal to 70, 60, 40, 30, 20, 10 or 5nM, preferably less than or equal to 4nM.

In one embodiment a polypeptide may comprise a chain of at least 51 or more amino acids, with each amino acid being linked by a peptide bond. A polypeptide may comprise less than or equal to 1500, 1000, 750, 500, 200, 180, 160 or 140 amino acids. A polypeptide may comprise at least 51, 60, 70 or 80 amino acids. A polypeptide may comprise 51-1500, 51- 750, 51-200, 70-180, 80-160 or 100-140 amino acids. Where a polypeptide comprises less than or equal to 50 amino acids, said polypeptide may be referred to as a peptide. A peptide may comprise a chain of 2-50 amino acids, with each amino acid being linked by a peptide bond. A peptide may comprise less than or equal to 40 amino acids, 30 amino acids or 20 amino acids. A peptide may comprise at least 2 amino acids, 5 amino acids, 10 amino acids or 20 amino acids. A peptide may comprise 2-50 amino acids, 5-45 amino acids, 10-40 amino acids or 15-35 amino acids.

The term “small molecule” as used herein may refer to a molecule with a molecular weight of less than or equal to 1000 Daltons. A small molecule may comprise a ribonucleotide, a deoxyribonucleotide, a sugar (such as glucose), an amino acid, cholesterol, a lipid, a glycoside, an alkaloid, a hormone, a metabolite, a drug or a phenolic compound. A capture substrate may comprise a fatty acid. A fatty acid may be saturated or unsaturated. A saturated or unsaturated fatty acid may be branched or unbranched. A capture substrate comprising a fatty acid may be a lipid, e.g. a phospholipid, diglyceride, or triglyceride, or a functional equivalent thereof.

A capture substrate may have a binding specificity to its respective target (component of a biological agent) with a Kd of less than or equal to 70, 60, 40, 30, 20, 10 or 5nM, preferably less than or equal to 4nM.

The binding specificity may be determined by any suitable assay such as surface plasmon resonance (SPR) (e.g. via a Biacore system) or enzyme linked immunosorbent assay (ELISA).

The capture substrate may bind (e.g. via hybridisation) the same component (e.g. 16S rRNA) of a first biological agent and, independently, the same component (e.g. 16S rRNA) of a second (genetically different) biological agent.

The term “component” as used herein may refer to a nucleic acid, a carbohydrate, a polypeptide (e.g. a peptide), a fatty acid, or a small molecule (e.g. a metabolite) of a biological agent. A component may be a soluble component or a membrane-bound component. A component may be an intracellular component, an extracellular component, or a cell-surface (e.g. membrane-bound) component. Thus, a component of a biological agent may comprise (or consist of) a nucleic acid, a carbohydrate, a polypeptide (e.g. a peptide), a fatty acid, or a small molecule (e.g. a metabolite) of a biological agent. A component of a biological agent may comprise a fragment of a nucleic acid, carbohydrate, polypeptide (e.g. a peptide), fatty acid, or small molecule (e.g. a metabolite) of a biological agent.

A component that is a nucleic acid of a biological agent may comprise DNA or RNA. Said nucleic acid may be complementary to a nucleic acid capture substrate. A component may comprise a nucleic acid that encodes for 16S rRNA or may be the 16S rRNA itself. A component may comprise a nucleic acid comprising a sequence corresponding to that of bacteriophage M13. A component may comprise a nucleic acid that is single stranded. A component may comprise a nucleic acid that is the single stranded DNA of bacteriophage M13. A component that is a carbohydrate of a biological agent may comprise an oligosaccharide, a polysaccharide (such as a peptidoglycan, a lipopolysaccharide, and/or an exopolysaccharide) or a monosaccharide. A polysaccharide may comprise a glycan or a chitin. A monosaccharide may comprise fucose, mannose or a sialic acid. Where a capture substrate is a lectin, said capture substrate may have binding affinity to a component of a biological agent that is a carbohydrate.

A component that is a polypeptide (e.g. a peptide) of a biological agent may comprise a structural protein (such as tubulin, a spike protein, a nucleocapsid protein, a membrane protein or an envelope protein), an enzyme (such as a sortase, an integrase, a reverse transcriptase, a viral neuraminidase, an endoglucanase, a cellobiohydrolase or a p- glucosidase), a transport protein (such as a Sec protein, a Tat protein, an ATP-binding cassette or a protein of the major facilitator superfamily), a regulatory protein (such as RecA, Rev or a protein from the family Velvet) or a hormone (such as SCB1). Where a capture substrate is a polypeptide (e.g. an antibody or a peptide), a fatty acid, or a small molecule, said capture substrate may have binding affinity to a component of a biological agent that is a polypeptide (e.g. a peptide).

A component that is a small molecule of a biological agent may comprise a metabolite. A component that is a small molecule of a biological agent may comprise a ribonucleotide, a deoxyribonucleotide, a sugar (such as glucose), an amino acid, cholesterol, a lipid, a glycoside, an alkaloid, a hormone, a metabolite, a drug or a phenolic compound. Where a capture substrate is a nucleic acid, a lectin, a polypeptide (e.g. an antibody or a peptide), a fatty acid, or a small molecule, said capture substrate may have binding affinity to a component of a biological agent that is a small molecule.

A component of a biological agent may comprise a fatty acid.

A component may comprise a combination of a carbohydrate and a protein, such as a glycoprotein.

In some embodiments, a component of a biological agent may be associated with (and not isolated from) the biological agent prior to binding by a capture substrate of the invention. For example, in some embodiments, a component of a biological agent (e.g. protein) on the surface of a biological agent may be associated with an outer membrane of a biological agent and not isolated or separated from the biological agent using standard molecular biology techniques (such as by protein precipitation) prior to binding. For example, in some embodiments a nucleic acid is not isolated or separated (such as by DNA precipitation) from other components of a biological agent prior to binding.

In some embodiments, a component of a biological agent may be isolated from the biological agent prior to binding by a capture substrate of the invention. For example, in some embodiments, a component of a biological agent (e.g. protein or nucleic acid) may not be associated with the biological agent or may have been isolated or separated from the biological agent using standard molecular biology techniques (such as protein precipitation) prior to binding. For example, in some embodiments, a nucleic acid is isolated or separated (such as by DNA precipitation) from other components of a biological agent prior to binding.

Where the composition comprises a first capture substrate and second capture substrate, the ratio of the first and second capture substrates in the composition may be 1000:1, 800:1, 600:1, 400:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:100, 1:200, 1:400, 1:600, 1:800 or 1:1000.

The ratio of the first and second capture substrates in the composition may be 100:1-5:1; 50:1-10:1; 40:1-20:1, 40:1-30:1, 1:5-1:100, 1:10-1:50 or 1:20-1:40.

The ratio of a nanomaterial to a capture substrate may be less than or equal to 5000:1, 4000:1, 3000:1, 2000:1, 1000:1, 500:1, 400:1, 200:1, 100:1, 50:1, 10:1, 1:10, 1:50, 1:100, 1:200, 1:400, 1:500, 1:1000, 1:2000, 1:3000, 1:4000 or 1:5000. The ratio of a nanomaterial to a capture substrate may be 5000:1-10:1, 4000:1-100:1, 3000:1-1000:1, 1:10-1:5000, 1:100- 1:4000 or 1:1000-1:3000, preferably, 1:10-1:1000, e.g. 1:50-1:150, such as 1:60 to 1:120.

The ratio of a gold nanoparticle to a capture substrate may be less than or equal to 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30,1:20 or 1:10. The ratio of a gold nanoparticle to a capture substrate may be 150:1-10:1, 130:1-20:1, 120:1- 30:1, 110:1-40:1, 100:1-50:1, 90:1-60:1, 80:1-70:1, 1:10-1:150, 1:20-1:130, 1:30-1:120, 1:40- 1:110, 1:50-1:100, 1:60-1:90 or 1:70-1:80, preferably 1:60-1:120.

The ratio of a MOF to a capture substrate may be less than or equal to 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 80:1, 60:1, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:80 or 1:60. The ratio of a MOF to a capture substrate may be 150:1-40:1; 140:1-50:1, 130:1-60:1, 120:1- 70:1 , 1 :40-1:150, 1:50-1:140, 1:60-1 :130 or 1:70-1 :120, preferably 1 :50-1:140, more preferably 1 :60-1:130.

A capture substrate may be associated with a nanomaterial. The term “associated” as used herein may refer to the direct or indirect interaction of a capture substrate with a nanomaterial. A direct or indirect interaction of a capture substrate with a nanomaterial may be through the surface of the nanomaterial or the core of the nanomaterial. A direct interaction of a capture substrate with a nanomaterial may involve an interaction without an additional molecule (such as a linker) to link the capture substrate to the nanomaterial. An indirect interaction of a capture substrate with a nanomaterial may involve an additional molecule (such as a linker) that links the capture substrate to the nanomaterial. Preferably, a capture substrate directly interacts with a nanomaterial. The direct or indirect association of a capture substrate with a nanomaterial may be covalent or non-covalent. For example, a capture substrate may be covalently bonded at one end of the capture substrate to the surface of the nanoparticle.

A plurality of capture substrates may be associated with a nanomaterial. For example, a plurality of different capture substrates may be associated with the same nanomaterial (e.g. same nanoparticle). Advantageously, this may allow improved detection of biological agents.

In one embodiment, the association of the capture substrate with a nanomaterial may be through adsorption of the capture substrate to the nanomaterial. The term “adsorption” as used herein may refer to the physical process of a nanomaterial interacting with a capture substrate without the formation of a covalent bond. Preferably, a capture substrate directly interacts with a nanomaterial through adsorption.

Adsorption may be carried out by mixing and incubating a sample of a capture substrate and a nanomaterial at an acidic pH at room temperature. Following incubation at an acidic pH, the sample of the capture substrate and the nanomaterial may be incubated at a neutral pH at room temperature. A buffer may be used to change the pH of the sample (such as a liquid (e.g. solution)) in which the capture substrate and the nanomaterial are present, by changing the pH to acidic and the pH to neutral and vice versa. The sample of the capture substrate and the nanomaterial may be centrifuged to remove any supernatant and the resultant pellet comprising the capture substrate and the nanomaterial may be washed and centrifuged again. The wash may be repeated several times. The sample of adsorbed capture substrate and nanomaterial may be resuspended in a liquid (e.g. a buffer solution) for further use. The extent of capture substrate adsorption may be determined using fluorescent quantification. A Malvern Nanosizer may be used to assess the extent of capture substrate adsorption on a nanomaterial. In one embodiment, the capture substrate may be a nucleic acid (preferably an oligonucleotide) and the nanomaterial may be a nanoparticle (preferably a gold nanoparticle).

Adsorption may be carried out by using a capture substrate at a final concentration of less than or equal to 1000, 800, 600, 400, 200 or 100 pM. Adsorption may be carried out by using a capture substrate at a final concentration of at least 1 , 10, 50, 100, 200 or 500 pM. Adsorption may be carried out by using a capture substrate at a final concentration of 1- 1000, 10-900, 100-800 or 200-700 pM.

Adsorption may be carried out by using a nucleic acid capture substrate (preferably an oligonucleotide) at a final concentration of less than or equal to 100, 90, 80, 70, 60 or 50 pM. Adsorption may be carried out by using a nucleic acid capture substrate (preferably an oligonucleotide) at a final concentration of at least 1 , 5, 10 or 20 pM. Adsorption may be carried out by using a nucleic acid capture substrate (preferably an oligonucleotide) at a final concentration of 1-100, 10-70 or 20-60 pM.

Adsorption may be carried out by using a nanomaterial at a final concentration of less than or equal to 1000, 800, 600 or 400 nM. Adsorption may be carried out by using a nanomaterial at a final concentration of at least 10, 50, 100, 200 or 500 nM. Adsorption may be carried out by using a nanomaterial at a final concentration of 10-1000, 100-900, 200-700 or 300-600 nM.

Adsorption may be carried out by using a gold nanoparticle at a final concentration of less than or equal to 100, 80, 60, 40 or 20 nM, preferably 11 nM, more preferably 10.5 nM (e.g. 10 nM). Adsorption may be carried out by using a gold nanoparticle at a final concentration of at least 1, 2, 3, 4 or 5 nM, preferably 9 nM, more preferably 9.5 nM (e.g. 10 nM). Adsorption may be carried out by using a gold nanoparticle at a final concentration of 1-100, 2-80, 3-60, 4-40 or 5-20 nM, preferably 9-11 nM, more preferably 9.5-11.5 nM (e.g 10 nM).

In one embodiment, adsorption may be carried out by incubating the capture substrate and the nanomaterial at an acidic pH for less than or equal to 10, 9, 8, 7, 6 or 5 minutes, preferably 4 minutes, more preferably 3 minutes and 30 seconds (e.g. 3 minutes). In one embodiment, adsorption may be carried out by incubating the capture substrate and the nanomaterial at an acidic pH for at least 10, 20, 30, 40, 50 or 60 seconds, preferably 2 minutes, more preferably 2 minutes and 30 seconds (e.g. 3 minutes). In one embodiment, adsorption may be carried out by incubating the capture substrate and the nanomaterial at an acidic pH for 10 seconds to 10 minutes, 20 seconds to 8 minutes, 30 seconds to 6 minutes, 1 to 5 minutes, preferably 2 to 4 minutes, more preferably 2 minutes and 30 seconds to 3 minutes and 30 seconds (e.g. 3 minutes).

In one embodiment, the capture substrate and the nanomaterial may be incubated at a neutral pH for less than or equal to 20, 18, 16, 14 minutes, preferably 12 minutes, more preferably 11 minutes and 30 seconds (e.g. 10 minutes). In one embodiment, the capture substrate and the nanomaterial may be incubated at a neutral pH for at least 30 seconds, 1 , 2, or 3 minutes, preferably 4 minutes, more preferably 4 minutes and 30 seconds (e.g. 5 minutes). In one embodiment, the capture substrate and the nanomaterial may be incubated at a neutral pH for 30 seconds to 20 minutes, 1 to 18 minutes, 2 seconds to 16 minutes, 3 minutes to 14 minutes, preferably 4-12 minutes, more preferably 4 minutes and 30 seconds to 11 minutes and 30 seconds (e.g. 5-10 minutes).

A capture substrate may adsorb to a nanomaterial by changing the pH of a liquid (e.g. solution) in which the capture substrate and nanomaterial may be present. A buffer may be used to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present. A buffer may be a citrate buffer, a hydrochloric acid buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, a oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, a sulfonic acid buffer and/or a trimethylamine salt. Preferably, a citrate and hydrochloric acid buffer may be used to adjust the pH to acidic. Preferably, a sulfonic acid buffer (e.g. a HEPES buffer) may be used to adjust the pH to neutral. The term “neutral” as used herein in reference to pH may refer to a pH of 7.

A buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of less than or equal to 1000, 800, 600 or 400 mM. A buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of at least 10, 50, 100, 200 or 500 mM. A buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of 10-1000, 100-900, 200-700 or 300-600 mM.

A citrate and hydrochloric acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of less than or equal to 500, 100, 50 or 20 mM, preferably 11 mM, more preferably 10.5 mM (e.g. 10 mM). A citrate and hydrochloric acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of at least 1, 3, 5, or 7 mM, preferably 9 mM, more preferably 9.5 mM (e.g. 10 mM). A citrate and hydrochloric acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of 1-100, 2-80, 3-70, 4-60, 5-50, 6-40 7-30 or 8-20 mM, preferably 9-11 mM, more preferably 9.5-10.5 mM (e.g. 10 mM).

A sulfonic acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of less than or equal to 100, 90, 80, 70, 60 or 50 mM, preferably 35 mM, more preferably 32 mM (e.g. 30 mM). A sulfonic acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of at least 1, 5, 10 or 20 mM, preferably 25 mM, more preferably 28 mM (e.g. 30 mM). A sulfonic acid buffer may be used (to adjust the pH of the liquid (e.g. solution) in which the capture substrate and nanomaterial may be present) at a final concentration of 1-100, 10-70 or 20-60 mM, preferably 25-35 mM, more preferably 28-32 mM (e.g 30 mM).

A capture substrate may adsorb to a nanomaterial at an acidic pH. The term “acidic pH” as used herein may refer to a pH of 0-6.9. A capture substrate may adsorb to a nanomaterial at a pH of less than or equal to 6.9, 6, 5, 4, 3, 2 or 1. A capture substrate may adsorb to a nanomaterial at a pH of 0-6.9, 1-6, 2-5 or 3-4.

A centrifugation step may be carried out at 1000-20,000, 2000-18,000, 3000-17,000 rpm, preferably 4000-16,000 rpm.

A wash step may be repeated 1-2, 3-4, 4-5 or 6-7 times, preferably 4-5 times.

Preferably, the association of the capture substrate with the nanomaterial may be reversible. The term “reversible” when used in this context may mean that a capture substrate associated with a nanomaterial is capable of dissociating from said nanomaterial and a capture substrate that is not associated with a nanomaterial is capable of associating with said nanomaterial. Whether or not the capture substrate associates with the nanomaterial may be based on whether or not the capture substrate is bound to a component of a biological agent. A capture substrate may be associated with (preferably conjugated to) a nanomaterial. The term “conjugated to” as used herein may refer to a reaction between at least two functional groups. A conjugation reaction may result in the formation of a covalent bond.

A capture substrate may be associated with (preferably conjugated to) the nanomaterial via a chemical moiety. For example, a chemical moiety may be a functional group.

A nanomaterial may comprise a functional group.

A capture substrate may comprise a functional group.

Said functional group may promote or may be suitable for promoting association between a nanomaterial and a capture substrate.

A capture substrate may be associated with (preferably conjugated to) the nanomaterial via a functional group. The term “functional group” as used herein may include a thiol group (-R- SH), a sulfhydryl group (-SH), an amino group (-NH2), a hydroxyl group (-OH), a carboxyl group (-COOH), an azido group (-N3), a guanidyl group (-NH2-C(NH)-NH2), and/or a carbohydrate. Such functional groups may conjugate directly or indirectly to a capture substrate via, for example, an amino, sulfhydryl, or a phosphate group. Such functional groups may conjugate directly or indirectly to a nanomaterial via, for example, an amino, sulfhydryl, or a phosphate group. A nanomaterial (e.g. nanoparticle) may be thiolated. A nanomaterial may be a thiolated gold nanomaterial, a thiolated chitosan nanomaterial, or a thiolated lipid-based nanomaterial. A nanoparticle may be a thiolated gold nanoparticle, a thiolated chitosan nanoparticle, or a thiolated lipid-based nanoparticle. Thioglycolic acid may be used to introduce a sulfhydryl (SH) group in the nanomaterial, thereby facilitating adsorption of a capture substrate.

Thiolation may result in the nanomaterial and/or capture substrate having a thiol or sulfhydryl group.

A capture substrate may be thiolated. For example, the capture substrate may be a thiolated nucleic acid (such as a thiolated cDNA, a thiolated RNA, a thiolated primer, a thiolated probe, or a thiolated oligonucleotide), a thiolated lectin, a thiolated aptamer, a thiolated polypeptide (e.g. a thiolated antibody or a thiolated peptide), a thiolated small molecule, a thiolated fatty acid, or a combination thereof. Preferably, the capture substrate is a thiolated oligonucleotide. Thus, a capture substrate may comprise a nucleic acid described herein that has been thiolated, e.g. at the 3’ or 5’ end, preferably at the 5’ end. A nucleotide (preferably terminal nucleotide, i.e. the most 5’ or 3’ nucleotide) of the capture substrate may be modified, e.g. modified from a nucleotide described in a SEQ ID NO described herein. The modification may be to introduce a thiol group or sulfhydryl group. For example, a capture substrate may comprise a nucleic acid having at least 70% sequence identity to SEQ ID NO: 1, 2, or 3, such as a nucleic acid sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1, 2, or 3 or preferably comprising or consisting of SEQ ID NO: 1, 2, or 3 but wherein a nucleotide thereof has been modified (preferably the 5' terminal nucleotide has been modified, preferably to introduce a thiol group or sulfhydryl group). Preferably, a capture substrate may comprise a nucleic acid having at least 70% sequence identity to SEQ ID NO: 1 , such as a nucleic acid sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1 or preferably comprising or consisting of SEQ ID NO: 1 but wherein a nucleotide thereof has been modified (preferably the 5' terminal nucleotide has been modified, preferably to introduce a thiol group or sulfhydryl group).

A capture substrate may be associated indirectly with a nanomaterial, preferably a capture substrate may be conjugated indirectly to a nanomaterial. A linker may be used to conjugate the capture substrate indirectly to the nanomaterial. A linker may comprise any type of molecule. For example, a linker may be an aliphatic chain comprising at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more carbon atoms), and may be substituted with one or more functional groups including a sulfoxide, sulfone, sulfonamide, ketone, ether, ester, amide, alcohol, amine, urea, thiourea, and/or disulfide.

A linker may include a disulfide at the free end that conjugates to the nanomaterial surface. A disulfide may comprise a C2-C10 disulfide, which is an aliphatic chain that terminates in a disulfide that includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. A longer aliphatic chain may be used that terminates in a disulfide of more than 10 carbon atoms. A linker may comprise either one or more sulfhydryl group(s) (SH) or one or more disulfide group(s) (S-S) or a multitude of sulfur atoms. In particular embodiments, a linker is not required to add a thiol modification.

The term “functionalisation” as used herein may refer to modifying an object (e.g. a nanomaterial) to impart a new property that the object did not have prior to functionalisation. A nanomaterial may be functionalised by associating a capture substrate with the nanomaterial.

A component of a biological agent (preferably at least two genetically different biological agents) on a surface or in a sample may be free from a nanomaterial (i.e. not associated with a nanomaterial).

The composition may comprise at least a further capture substrate that binds to a component of a genetically different biological agent. The further capture substrate may be a second capture substrate.

The composition may comprise:

(a) a first nanomaterial associated with the first capture substrate and a second nanomaterial associated with at least a further (e.g. second) capture substrate; or

(b) the nanomaterial associated with the first and the at least further (e.g. second) capture substrates.

A first nanomaterial and a second nanomaterial may be of the same nanomaterial type (e.g. where the first and second nanomaterials are first and second gold nanoparticles) or may be different nanomaterial types (e.g. where the first nanomaterial is a MOF and the second nanomaterial is a gold nanoparticle).

The composition may comprise:

(a) a first nanomaterial associated with the first capture substrate and a second capture substrate; and

(b) a second nanomaterial associated with at least a third capture substrate.

The composition may comprise a first nanomaterial, and at least a second nanomaterial that is different to the first nanomaterial. For example, a first nanomaterial may be a nanoparticle and at least a second nanomaterial may be a MOF.

The composition may comprise a first nanomaterial associated with the first capture substrate and a second nanomaterial that is different to the first nanomaterial and is associated with at least a further (e.g. second) capture substrate.

The composition may comprise: (a) a first nanomaterial associated with the first capture substrate and a second capture substrate; and

(b) a second nanomaterial that is different to the first nanomaterial and is associated with at least a third capture substrate.

The ratio of a first capture substrate and an at least further (e.g. second) capture substrate in a composition may be adjusted to accommodate the detection of one of the at least two genetically different biological agents that may be particularly prevalent in any given environment. In such an instance, the ratio of the first capture substrate and the at least further capture substrate may be adjusted in the composition to increase or decrease the detection sensitivity to be able to bind to (e.g. via hybridisation) the desired biological agent(s).

For example, the ratio of capture substrates in a composition may be adjusted to increase the amount of a capture substrate for binding to a component of a biological agent, such as a foodborne biological agent (e.g. E. coli) when being used in an environment susceptible to, or at high risk of, said foodborne biological agent (e.g. in a kitchen). The ratio of capture substrates in a composition may be adjusted to increase the amount of a capture substrate for binding to component of a biological agent, such as a pathogenic biological agent (e.g. SARS-CoV-2 or T. rubrum) when being used in an environment susceptible to, or at high risk of, said pathogenic biological agent (e.g. in a hospital).

The composition is preferably capable of producing a detectable signal (preferably optically- detectable signal) when the capture substrate is bound to the component a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent).

The term “detectable signal” as used herein may refer to a signal that is capable of being perceived or identified by a subject (e.g. a user that has applied the composition to a sample or surface). The perception or identification of a detectable signal by the subject may be with (or may require) a suitable tool (e.g. a microscope). The skilled person will appreciate that whilst a detectable signal may be produced, it may not necessarily be perceived or identified by a subject unless there is input from an external source. For example, the production of fluorescence emission from a fluorescent label may be capable of being perceived by a subject once an external LED light source is used. The term “optical ly-detectable signal” as used herein may refer to a visual signal that is perceived or identified by a subject. The skilled person will appreciate that said visual signal may be perceived or identified either by a subject’s unaided eye or by a subject’s aided eye (e.g. via the aid of an external source such as an LED light source, a microscope, etc.).

The skilled person will appreciate that in some embodiments where there is a very low amount of a biological agent on a surface or in a sample, the capture substrate may still bind to the biological agent but no signal may be detectable, as it is below a detectable limit. Then, a signal may be detectable only when an amount of a biological agent present on a surface or in a sample exceeds a threshold corresponding to the detectable limit of any detectable signal. Moreover, the skilled person will appreciate that the amount of a biological agent on a surface or in a sample may vary between environments in which the composition of the invention is used.

A signal may be detectable when at least 1 , 10, 50, 100, 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ cells are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal. A signal may be detectable when less than or equal to 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ cells are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal. A signal may be detectable when 1-10 ⁶ , 10-10 ⁶ , 10 ² -10 ⁵ or 10 ³ -10 ⁴ cells are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal.

A signal may be detectable when at least 1 , 10, 50, 100, 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ viral particles are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal. A signal may be detectable when less than or equal to 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ viral particles are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal. A signal may be detectable when 1-10 ⁶ , 10-10 ⁶ , 10 ² -10 ⁵ or 10 ³ -10 ⁴ viral particles are present on a surface or in a sample, thereby exceeding the threshold corresponding to the detectable limit of any detectable signal.

A signal may be detectable upon a threshold of an amount of capture substrate-biological agent binding being met. A signal may be detectable upon at least 20, 30, 40, 50 or 60% of capture substrates in a composition being bound to their corresponding component(s) of the biological agent(s). A signal may be detectable upon less than or equal to 100, 90, 80, 70 or 60% of capture substrates in a composition being bound to their corresponding component(s) of the biological agent(s). A signal may be detectable upon 10-100, 20-90, 30- 80 or 40-70% of capture substrates in a composition being bound to their corresponding component(s) of the biological agent(s).

A capture substrate may dissociate from a nanomaterial upon binding (e.g. when bound) to a component of a biological agent. The dissociation may result in the production of a detectable signal and/or a detectable difference. This may occur due to a capture substrate having a stronger affinity to a component of a biological agent than to the nanomaterial.

A capture substrate may comprise a label detectable upon binding of the capture substrate to the component of a biological agent. A capture substrate may comprise a label detectable upon binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and/or a component of the second (or further) biological agent). Optionally, the detectable label is not detectable when the capture substrate is not bound to the component of a biological agent (e.g. a component of the first biological agent and/or a component of the second (or further) biological agent). Optionally, the detectable label is always detectable but produces a detectable difference, e.g. a different signal when the capture substrate is bound to the component of a biological agent (e.g. a component of the first biological agent and/or the component of the second biological agent).

A label that is detectable upon binding of the capture substrate may be a fluorescent probe, an organic dye, a fluorescent stain or a fluorescent dye, such as a fluorescent chromophore, preferably a fluorescent dye. Accordingly, a capture substrate may comprise a fluorescent probe, an organic dye, a fluorescent strain or a fluorescent dye, such as a fluorescent chromophore. A fluorescent probe may be a deoxyuridine triphosphate (dllTP) probe conjugated to a fluorophore. An organic dye may be N-719 (a Ruthenium-based dye), methylene blue, ellipticine, Victoria Pure Blue BO (VPBBO, a triarylmethane dye), ethidium bromide or fluorescein (preferably Fluorescein 9). A fluorescent stain may be rose Bengal, propidium iodide, crystal violet, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258, Hoechst 33342, Hoechst 34580, YOYO-1 , a carbocyanine dimer with green fluorescence similar to FITC (CAS Number: 143413-85-8 (e.g. DiYO-1 ™), CAS Number: 143413-84-7 (e.g. DiTO™-1), Quinolinium, 1-T-[1 ,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4- [(3-methyl-2(3H)-benzothiazolylidene)methyl]]-, tetraiodide (CAS Number: 143413-84-7 (e.g. TOTO™-1)) or a nucleic acid gel stain (CAS Number: 172827-25-7 (e.g. SYBR™ Green II), CAS Number: 163795-75-3 (e.g. SYBR™ Green I), C60H72I2N8O5 (PubChem CID: 117700725 (e.g. Biotium GelRed™)), CseHso^NsOs (PubChem CID: 11857532 (e.g. Biotium GelGreen®)), preferably C60H72I2N8O5 (PubChem CID: 117700725 (e.g. Biotium GelRed™), more preferably CAS Number: 172827-25-7 (e.g. SYBR™ Green II). A fluorescent dye may be fluorescein, fluorescein isothiocyanate (FITC), cyanine, acridine orange, rhodamine, 7- AAD (7-Aminoactinomycin D) or boron dipyrromethene (BODIPY). In one embodiment, a fluorescent dye may be an organic dye.

In some embodiments, the label may not be detectable when the capture substrate is not bound to the component of a biological agent (e.g. the first biological agent). For example, the label may be fluorescent (i.e. detectable) but the fluorescence is quenched by the nanomaterial (i.e. non-detectable). In some embodiments, upon binding of the capture substrate to the component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent), the fluorescent label is no longer quenched by the nanomaterial and is detectable.

The label may be detectable (e.g. always detectable when intact) but produces a detectable difference, e.g. a different signal. Where the label may be detectable (e.g. always detectable when intact), fluorescence quenching may produce a detectable difference. For example, the label may be fluorescent (i.e. detectable) and upon binding of the capture substrate to the component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent) this may quench the fluorescence of the label. Such a detectable difference may be detected by an external light source such as an LED or a fluorescent microscope.

The term “detectable difference” as used herein may refer to a change in the state of the composition (or a component thereof) or a detectable signal as defined herein. Said detectable difference may be capable of being perceived or identified by a subject (e.g. a user that has applied the composition to a sample or surface, e.g. contacted a sample or surface with the composition). The perception or identification of said difference by the subject may be with (or may require) a suitable tool (e.g. a microscope). In one embodiment, a detectable difference may be a change in any detectable signal described herein (e.g. an appearance of said detectable signal).

The detectable difference may be a change in an optical property of a nanomaterial. For example, upon binding of the capture substrate to the component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent), the nanomaterial(s) may aggregate, thereby producing a detectable difference (e.g. a change in the optical property of the nanomaterial, such change may be detected by a change in the colour of the composition [e.g. from colourless to coloured]) which is capable of being perceived or identified by a subject. The detectable difference may be a change in the fluorescent state of a fluorescent label. For example, a fluorescent signal may be quenched and upon binding of the capture substrate to the component of a biological agent (e.g. a component of a first biological agent and/or a component of the second biological agent), the fluorescent label may no longer be quenched and may thus fluoresce, thereby producing a detectable difference.

The composition may comprise a plurality of capture substrates comprising different labels. For example, a first capture substrate may comprise a first label as described herein and a second (or further) capture substrate may comprise a second (or further) label as described herein. The composition may comprise a plurality of capture substrates, each capture substrate comprising a different (e.g. distinct) label. Advantageously, this may allow more accurate identification of biological agents.

The composition may comprise a plurality of the nanomaterials. Said plurality of nanomaterials may be reversibly associable with one another.

The composition may comprise a plurality of the nanomaterials associated with at least one capture substrate, and wherein the nanomaterials are reversibly associable with one another.

The term “plurality” when used in the context of a nanomaterial described herein may mean at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the nanomaterials. A plurality of nanomaterials may comprise 2-100, 10-90, 20-80, 30-70 or 40-60 nanomaterials, preferably 30-70 nanomaterials.

The term “reversibly associable with one another” when used in the context of a nanomaterial described herein may mean that the nanomaterials associate or disperse from one another as a response to a change in the composition (e.g. the binding of a capture substrate to a biological agent). The nanomaterials may simultaneously or non- simultaneously associate or dissociate from one another. A change in the composition may be a capture substrate binding to a component of a biological agent (e.g. a component of a first biological agent and, optionally independently, a component of a second biological agent) as described herein. A detectable signal may be produced by aggregation of the nanomaterial, dispersion of the nanomaterial, a catalytic reaction (e.g. catalysis of a reaction by a nanomaterial (e.g. a MOF)), an enzymatic reaction, a colorimetric change of a photonic crystal, fluorescence emission, a redox reaction, fluorescence quenching, Forster resonance energy transfer (FRET), or a combination thereof, preferably aggregation of the nanomaterial, more preferably fluorescence emission. Preferably, the detectable signal is optically-detected using an external source. An external source may include a light source such as an LED.

The detectable signal may comprise a colorimetric change, a fluorescence emission, a quenched fluorescence emission, or a combination thereof. A colorimetric change may be optically-detectable. A fluorescence emission or a quenched fluorescence emission may be optically-detectable via an external source or component. An external source or component may include a light source such as an LED.

A nanomaterial may comprise an optical property, wherein the optical property is surface plasmon resonance (SPR). The term “SPR” as used herein may refer to the oscillation of electrons at the interface between negative and positive permittivity material when interacting with incident light. A nanomaterial may comprise the optical property of localised surface plasmon resonance (LSPR). The term “LSPR” as used herein may refer to the production of one or more electron oscillations when light interacts with a surface or structure which has dimensions lower than its wavelength, and, where the light is localised within the subwavelengths to produce a localised electromagnetic field.

Therefore, a detectable signal may be produced based on a detectable difference (e.g. a change in an optical property) of the nanomaterial. Preferably, where a nanomaterial comprises the optical property of LSPR, the nanomaterial is a gold or silver nanoparticle, more preferably a gold nanoparticle. Such a detectable signal may be a detectable change. A change in the optical property of the nanomaterial may produce the detectable change. For example, where a nanomaterial comprises the optical property of SPR or LSPR, a change in the SPR or LSPR of the nanomaterial may produce a detectable change. Such a detectable change may be a colorimetric change. Such a detectable change may be advantageous as an additional reagent such as an organic dye or a fluorescent dye are not required for the signal to be detectable (preferably optically-detectable). The term “colorimetric change” as used herein may refer to a change in colour (for example, red to purple) of the composition comprising the nanomaterial.

Where a nanomaterial comprises the optical property of SPR or LSPR, aggregation of the nanomaterial or dispersion of the nanomaterial may produce a change in the SPR or the LSPR which is detectable via a colorimetric change.

A detectable signal may be produced by aggregation of the nanomaterial.

The term “aggregation” as used herein may refer to the process of two or more nanomaterials associating with one another to form a nanomaterial aggregate. A nanomaterial aggregate in the composition may comprise two or more nanomaterials within less than or equal to 1 , 0.9, 0.8, 0.7 or 0.6 nm distance from one another.

The term “dispersion” as used herein may refer to the process of two or more nanomaterials (preferably, a nanomaterial aggregate) in the composition dissociating from one another. A dispersion of nanomaterials in the composition may comprise two or more nanomaterials within at least 2, 3, 4, 5 or 6 nm distance from one another.

A detectable signal may be a colorimetric change. A colorimetric change may be produced when a dispersed nanomaterial aggregates. In one embodiment, a nanomaterial may be dispersed in the presence of a biological agent and a salt. The biological agent and salt may protect the nanomaterial and prevent the nanomaterial from aggregating. In the absence of a biological agent and a salt, the dispersed nanomaterial may aggregate, thereby producing a colorimetric change. A salt may comprise a cation such as sodium, potassium, magnesium and calcium and an anion such as lactate, chloride, propionate and gluconate or a combination thereof.

In one embodiment, the nanomaterial may be dispersed in the absence of a biological agent and a salt. In the presence of a biological agent and a salt, the dispersed nanomaterial may aggregate, thereby producing a colorimetric change.

The composition may comprise a plurality of the nanomaterials, and the nanomaterials may be associated with one another forming an aggregate (preferably in the absence of binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent)). When the capture substrate is bound to the component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent), the nanomaterials may disperse from one another, thereby producing a change in an optical property of the nanomaterial. Such a change in the optical property of the nanomaterial may produce a colorimetric change.

The composition may comprise a plurality of the nanomaterials, wherein the nanomaterials are dispersed in the composition (preferably in the absence of binding of the capture substrate to a component of a biological agent, e.g. a component of the first biological agent and/or a component of the second biological agent). When the capture substrate is bound to a component of a biological agent (e.g. a component of the first biological agent and/or a component of the second biological agent), the nanomaterials may associate with one another, thereby producing a change in the optical property of the nanomaterial. Such a change in the optical property of the nanomaterial may produce a colorimetric change.

A detectable signal may be fluorescence emission. The term “fluorescence emission” as used herein may refer to the emission of a wavelength of light by a fluorescent dye when said fluorescent dye is in an excited state. A physical (such as the absorption of light), mechanical (such as friction) or chemical mechanism may cause the fluorescent dye to reach an excited state. A fluorescence emission may be detected by an external light source such as an LED or a fluorescent microscope. A fluorescence emission may be detected when the capture substrate comprises a fluorescent label that is detectable upon binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and, optionally independently, a component of the second biological agent). Upon binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and, optionally independently, a component of the second biological agent), the capture substrate may dissociate (e.g., desorb) from the nanomaterial, and its label may fluoresce as the fluorescent signal is no longer quenched by the nanomaterial surface. Preferably, wherein a detectable signal comprises fluorescence emission, the capture substrate comprises a fluorescent label, more preferably, an oligonucleotide comprising a fluorescent label.

In one embodiment, fluorescence emission may be detected by the addition of a further fluorescent label, e.g. that is not associated with the initial capture substrate used to bind the component of the biological agent. Upon binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and, optionally independently, a component of the second biological agent), any unbound biological agent may be removed (for example through a wash step). A further fluorescent label may be added to the capture substrate-biological agent complex to produce a detectable signal. In some embodiments a further fluorescent dye may be conjugated to an antibody, said antibody having binding specificity to a component of a biological agent (e.g. a component of the first and/or second biological agent). In some embodiments a further fluorescent dye may be conjugated to an antibody, said antibody having binding specificity to the capture substrate-biological agent complex.

A detectable signal may be quenched fluorescence emission. The term “quenched fluorescence emission” or “fluorescence quenching” as used herein may refer to the decrease or absence in the emission of a wavelength of light which would normally be emitted by a fluorescent dye when in an excited state. A label may be fluorescent (and thus detectable) and upon binding of the capture substrate to a component of a biological agent (e.g. a component of the first biological agent and, optionally independently, a component of the second biological agent), the fluorescent of the label may be quenched. Quenching of fluorescence emission may be by the nanomaterial. A nanomaterial that may quench fluorescence emission may comprise a graphene-based nanomaterial or a MOF.

A detectable signal may be produced in the presence of a substrate. The substrate may refer to a molecule that undergoes a change in state due to its involvement in a catalytic (e.g. enzymatic) reaction. A substrate may be 3,3,5,5-tetramethylbenzidine (TMB) or glucose.

A catalytic and/or an enzymatic reaction may produce a colorimetric change as a detectable signal. A catalytic and/or an enzymatic reaction may be a redox reaction or a hydrolysis reaction. A redox reaction may comprise a peroxidase enzyme. For example, a redox reaction may comprise the oxidation of TMB via horseradish peroxidase which produces a colorimetric change that may be quantified. In one embodiment, a redox reaction may comprise the oxidation of glucose by glucose oxidase (Gox) to produce H2O2 products. In the presence of TMB, H2O2 may oxidise TMB to produce a colorimetric change. In some embodiments an antibody conjugated to a reporter enzyme (e.g. a peroxidase capable of catalysing production of a detectable product) with said antibody having binding specificity to a component of a biological agent (e.g. a component of the first and/or second biological agent) may be added to facilitate detection. In some embodiments an antibody conjugated to a reporter enzyme (e.g. a peroxidase capable of catalysing production of a detectable product) with said antibody having binding specificity to a capture substrate-biological agent complex may be added to facilitate detection.

In some embodiments, a capture substrate may comprise a reporter enzyme (e.g. a peroxidase reporter). The reporter enzyme may be conjugated thereto.

A nanomaterial may comprise inherent enzymatic properties and/or catalytic properties. For example, where the nanomaterial comprises a MOF, said MOF may have inherent catalytic properties. A MOF may replicate a reaction catalysed by a natural enzyme, e.g. lowering down the energy barrier for the chemical reaction to be favoured. The term “inherent enzymatic properties and/or catalytic properties” as used herein may refer to a nanomaterial that may directly catalyse a reaction, without the use of (or requirement for) an externally- sourced enzyme and/or catalyst. A nanomaterial may be a nanozyme and/or nanocatalyst, wherein the nanozyme and/or nanocatalyst comprises inherent enzymatic and/or catalytic activity to directly catalyse a reaction. The inherent enzymatic and/or catalytic activity of a nanomaterial may be advantageous as this may omit the requirement of externally-sourced enzymes and/or catalysts to catalyse a reaction and produce a detectable signal.

A redox reaction as used herein may refer to the transfer of an electron from a substance to another. A redox reaction may comprise a change in the oxidation state of a substrate. A redox reaction may be catalysed by an enzyme and/or catalyst. In other embodiments, a redox reaction may not be catalysed by an enzyme. An enzyme in a redox reaction may be an oxidoreductase (e.g. hydrogen peroxidase).

A reaction herein (e.g. catalysed by a nanomaterial comprising inherent enzymatic and/or catalytic properties or a reporter enzyme) may be determined according to Paia L, Sirec T, Spitz II. Modified Enzyme Substrates for the Detection of Bacteria: A Review. Molecules. 2020 Aug 13;25(16):3690. A reaction herein (e.g. catalysed by a nanomaterial comprising inherent enzymatic and/or catalytic properties or a reporter enzyme) may comprise the use of the corresponding substrate conjugated to a signalophor. Following the reaction, the signalophor may be released from the substrate, generating a specific and measurable signal. A signalophor may be chromogenic, fluorogenic, luminogenic, electrogenic or redox. A chromogenic and fluorogenic signalophor may comprise nitrophenol, 4- Methylumbelliferone (4-Mll), 7-Amino-4-methylcoumarin (7-AMC), 7-Hydroxycoumarin-3- carboxylate (EHC), resorufin, fluorescein, dihydroxynaphthalene or indoxyl. A luminogenic signalophor may comprise pro-luciferins-luciferase, luminol or dioxetane. An electrogenic signalophor may comprise indoxyl or p-Aminophenol. A redox signalophor may comprise formazan or resorufin. Fluorescein-based signalophors have high sensitivity and brightness, biocompatibility, and excitation (485 nm) and emission (514 nm) in the visible range of the light spectrum. Preferably, a fluorescein-based signalophor is Fluorescein 9.

A colorimetric change may be produced by a photonic crystal. The term “photonic crystal” as used herein may refer to a type of nanomaterial comprising a refractive index that changes periodically and a photonic band gap (PBG) as a property, which may make certain wavelengths in the PBG difficult to pass through. A photonic crystal may produce discoloured materials which are responsive to a change in the environment based on the colour change of a photonic crystal structure. A photonic crystal may transform the change in the environment into a colorimetric change.

FRET may produce a fluorescence emission or a quenched fluorescence emission as a detectable signal.

In one aspect of the present invention, there is provided a method for detecting the presence or absence of a biological agent on a surface or in a sample, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable signal (e.g. optical ly-detectable signal), thereby indicating the presence of the biological agent on the surface or in the sample, respectively; or

(c) not detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the absence of the biological agent on the surface or in the sample, respectively.

In one aspect of the present invention, there is provided a method for detecting the presence or absence of a biological agent on a surface or in a sample, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the presence of the biological agent on the surface or in the sample, respectively; or

(c) not detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the absence of the biological agent on the surface or in the sample, respectively. A biological agent may be detected following contact of a surface or sample with the composition. In one embodiment, the detection of a biological agent may be perceived or identified by a subject’s unaided eye by nanomaterial aggregation. For example, a solution comprising the nanomaterials may not change colour (e.g. the colour may remain red) in the absence of a biological agent but in the presence of a biological agent, the nanomaterials may aggregate and cause the solution to change colour (e.g. from red to purple), indicating the successful detection of the biological agent.

In one embodiment, where a capture substrate may be labelled with a fluorescent label, such as a fluorescent dye, an LED light source or a fluorescent microscope may be used to detect the fluorescence. For example, after contact of the composition with the surface or sample, an LED light source may be shone over the composition. In the presence of a biological agent, fluorescence emission may be produced as a detectable signal and may indicate the successful detection of the biological agent.

The composition or method may produce a detectable signal (preferably optically-detectable signal) that allows for the distinction between the type of biological agent being detected. The method may distinguish between the presence or absence of a bacterium, fungus, a virus, a parasite and/or a protozoan. For example, a first fluorescent marker may be used to identify a bacterium, a second fluorescent marker different to the first fluorescent marker may be used to identify a fungus and a third fluorescent marker, different to the first and second fluorescent markers may be used to identify a virus, or any combination thereof.

The term “presence of the biological agent” as used herein may refer to the existence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetically different biological agent(s) on the surface or sample. The term “presence of the biological agent” as used herein may refer to the quantity of the biological agent being at or above (preferably above) the threshold of detection.

The term “absence of the biological agent” as used herein may refer to the biological agent being absent from the surface or sample or the quantity of biological agent being less than the threshold of detection.

The skilled person will appreciate that the threshold of detection will be dependent on the specific nature of the composition, capture substrates, nanomaterial, and the nature of any detectable label employed. Said threshold may be determined empirically for any given composition. The skilled person will appreciate that the composition and methods of the invention may detect the total quantity of biological agents (bacteria, viruses, fungi, parasites and/or protozoa) present or absent on a surface or in a sample. The composition and methods of the invention may be tailored to detecting the total quantity of a particular biological agent(s) on a surface or in a sample.

In one aspect of the present invention, there is provided a method for determining whether or not the quantity of a biological agent on a surface or in a sample exceeds a pre-determined safety level, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable signal (e.g. optical ly-detectable signal), thereby indicating the quantity of the biological agent on the surface or in the sample, respectively, exceeds the pre-determined safety level; or

(c) not detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the quantity of the biological agent on the surface or in the sample, respectively, does not exceed the pre-determined safety level.

In one aspect of the present invention, there is provided a method for determining whether or not the quantity of a biological agent on a surface or in a sample exceeds a pre-determined safety level, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the quantity of the biological agent on the surface or in the sample, respectively, exceeds the pre-determined safety level; or

(c) not detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the quantity of the biological agent on the surface or in the sample, respectively, does not exceed the predetermined safety level.

In a method of the invention at least two genetically different biological agents are preferably detected (or not detected). The method of the invention may detect (or not detect) at least two genetically different biological agents on a surface or in a sample. In one aspect of the present invention, there is provided a method for detecting the presence or absence of at least two genetically different biological agents on a surface or in a sample, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the presence of at least two genetically different biological agents on the surface or in the sample, respectively; or

(c) not detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the absence of at least two genetically different biological agents on the surface or in the sample, respectively.

In one aspect of the present invention, there is provided a method for detecting the presence or absence of at least two genetically different biological agents on a surface or in a sample, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the presence of at least two genetically different biological agents on the surface or in the sample, respectively; or

(c) not detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the absence of at least two genetically different biological agents on the surface or in the sample, respectively.

In one aspect of the present invention, there is provided a method for determining whether or not the quantity of at least two genetically different biological agents on a surface or in a sample exceeds a pre-determined safety level, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the quantity of the at least two genetically different biological agents on the surface or in the sample, respectively, exceeds the pre-determined safety level; or

(c) not detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the quantity of the at least two genetically different biological agents on the surface or in the sample, respectively, does not exceed the predetermined safety level.

In one aspect of the present invention, there is provided a method for determining whether or not the quantity of at least two genetically different biological agents on a surface or in a sample exceeds a pre-determined safety level, the method comprising:

(a) contacting the surface or sample with the composition of the invention; and

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the quantity of the at least two genetically different biological agents on the surface or in the sample, respectively, exceeds the pre-determined safety level; or

(c) not detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the quantity of the at least two genetically different biological agents on the surface or in the sample, respectively, does not exceed the pre-determined safety level.

When a bacterial agent is determined to be present or when a quantity of a biological agent on a surface or in a sample, respectively, exceeds the pre-determined safety level, this may indicate that the surface is unclean and/or contaminated.

A wash step may be used to remove any capture substrate and its associated nanomaterial that is not bound to a component of a biological agent from the surface or the sample, prior to the detection of the biological agent. Advantageously, in some embodiments, the method of the invention may not require any wash step prior to detection of the biological agent (preferably at least two genetically different biological agents).

In some embodiments, the method may not require any separation step of a biological agent prior to its detection. In more detail, in some embodiments, the first biological agent and the second biological agent are not separated from each other before detection (e.g. the method may comprise the detection of the at least two genetically different biological agents on the same surface or in the same sample).

The term “surface” as used herein may refer to any natural (e.g. wood or stone) or nonnatural (e.g. metal or plastic) solid surface in an environment. The composition may be directly applied to such a surface for the detection of a biological agent (e.g. detection of at least two genetically different biological agents). The composition may be directly contacted with such a surface for the detection of a biological agent (e.g. detection of at least two genetically different biological agents). A suitable surface for directly applying the composition may comprise the surface of an object (e.g. a table, kitchen counter-top, ventilator or respirator) in an environment. The surface is preferably a food preparation surface or a medical surface (e.g. an operating table).

The term “sample” as used herein may refer to a sample that has been collected from an environment. The sample may be isolated. The sample may be collected from a surface (e.g. by swabbing). The sample may be a solid, a liquid or a gas. The sample may then be directly contacted with the composition. The sample may be collected from a surface and treated to lyse a biological agent present in said sample. Following treatment, the lysed sample may then be contacted with the composition. The sample may be collected from a surface (e.g. by swabbing) and applied to the composition without any prior lysis step.

Preferably, the sample is not collected from a human subject.

The term “contacting” as used herein may refer to the application of the composition to the surface or sample (such as by spraying the composition onto the surface or sample) or may refer to the indirect application of the composition to the surface or sample (such as by swabbing). Following contacting there may be an incubation period where the composition is allowed to interact with any biological agent that may be present on the surface or in the sample prior to a detection step.

An incubation period may be at least 30 seconds, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes. An incubation period may be less than or equal to 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 minutes. An incubation period may be 1-20, 2-19, 3-18, 4-17, 5-16, 6-15 or 7-14 minutes. Preferably, an incubation period may be 0 minutes (i.e. no incubation period may be required).

An incubation period may occur at at least 5, 10, 15 or 20 °C. An incubation period may occur at less than or equal to 40, 35, 30 or 25 °C. An incubation period may occur at 5-40, 10-35, or 15-30 °C. Preferably, an incubation period may occur at room temperature (e.g. at 25 °C).

The term “detecting” as used herein may refer to any process that enables a detectable signal to be identified. The term “detecting” as used herein may refer to any process that enables a detectable difference to be identified. For example, an LED light source may be used to detect fluorescence emission.

The term “quantity of the biological agent” as used herein may refer to the amount of a biological agent that is present on the surface or in the sample. The quantity of the biological agent may be less than 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ cells. The quantity of biological agent may be at least 10 , 50, 100 , 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ cells. The quantity of biological agent may be 10-10 ⁶ , 10 ² -10 ⁵ or 10 ³ -10 ⁴ cells. The number of cells may be per cm ² (solid), per ml (liquid) or per cubic meters (gas) dependent on whether the surface or sample is a solid, liquid or gas.

The quantity of the biological agent may be less than 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ viral particles. The quantity of biological agent may be at least 10, 50, 100 , 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ viral particles. The quantity of biological agent may be 10-10 ⁶ , 10 ² -10 ⁵ or 10 ³ -10 ⁴ viral particles. The number of viral particles may be per cm ² (solid), per ml (liquid) or per cubic meters (gas) dependent on whether the surface or sample is a solid, liquid or gas.

The term “pre-determined safety level” as used herein may refer to a quantity of a biological agent that is considered to be non-hazardous to a subject’s health. The skilled person will appreciate that the pre-determined safety level will vary dependent on the nature of the sample or surface to which the composition of the invention is to be contacted. For example, for a food preparation surface the pre-determined safety level may be much lower than the pre-determined safety level of a work desk. In some embodiments, a “pre-determined safety level” may be less than or equal to 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ cells. The pre-determined safety level may be a quantity of a biological agent of at least 10, 50, 100, 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ cells. The number of cells may be per cm ² (solid), per ml (liquid) or per cubic meters (gas) dependent on whether the surface or sample is a solid, liquid or gas.

In some embodiments, a “pre-determined safety level” may be less than or equal to 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² or 10 ¹ viral particles. The pre-determined safety level may be a quantity of a biological agent of at least 10, 50, 100, 500, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ viral particles. The number of viral particles may be per cm ² (solid), per ml (liquid) or per cubic meters (gas) dependent on whether the surface or sample is a solid, liquid or gas.

The composition may be in the form of a liquid. A liquid composition may comprise a carrier. A carrier may comprise saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and/or combinations thereof.

A carrier may comprise a buffer. A buffer may be a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, a oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, a sulfonic acid buffer and/or a trimethylamine salt, preferably a sulfonic acid buffer (e.g. a HEPES buffer).

A carrier comprising a buffer may be present at a final concentration of less than or equal to 1000, 800, 600 or 400 mM. A carrier comprising a buffer may be present at a final concentration of at least 10, 50, 100, 200 or 500 mM. A carrier comprising a buffer may be present at a final concentration of 10-1000, 100-900, 200-700 or 300-600 mM.

A carrier comprising a sulfonic acid buffer may be present at a final concentration of less than or equal to 100, 80, 60, 40 or 20 mM, preferably 10 mM, more preferably 7 mM (e.g. 5 mM). A carrier comprising a sulfonic acid buffer may be present at a final concentration of at least 1, 2, 3 or 4 mM, preferably 4.5 mM, more preferably 4.8 mM (e.g. 5 mM). A carrier comprising a sulfonic acid buffer may be present at a final concentration of 1-9, 2-8, 3-7 or 4- 6 mM, preferably 4.5-5.5 mM, more preferably 4.8-5.2 mM (e.g. 5 mM).

The composition may comprise a stabiliser. A stabiliser may provide advantageous properties to the composition including preventing adherence of the composition to its packaging wall. A stabiliser may be one or more of a polyhydric sugar alcohol; an amino acid (for example arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L- leucine, 2-phenylalanine, glutamic acid, and threonine); an organic sugar or a sugar alcohol (for example lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol); PEG; an amino acid polymer; a sulfur- containing reducing agent (for example urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate); a low molecular weight polypeptide (i.e., <10 residues); a protein (for example HSA, bovine serum albumin, gelatin or an immunoglobulin); a hydrophilic polymer (for example polyvinylpyrrolidone); a monosaccharide (for example xylose, mannose, fructose and glucose); a disaccharide (for example lactose, maltose and sucrose); a trisaccharide (for example raffinose), and a polysaccharide (for example dextran). The composition may comprise a disinfectant. For example, the composition may comprise any disinfectant known in the art to be suitable for slowing the growth, inactivating, and/or rendering inviable (e.g. killing) a biological agent as described herein. Advantageously, said disinfectant may allow for slowing the growth, inactivating, and/or rendering inviable (e.g. killing) a biological agent following detection. Suitable disinfectants may include a surfactant and/or an enzyme (e.g. a lytic enzyme).

The composition may comprise a surfactant. A surfactant may be anionic, cationic, amphoteric or non-ionic. The surfactant may have lysis activity. For example, the surfactant may be suitable for lysing at least one of the biological agents. Preferably, the surfactant is suitable for lysing the biological agents to be detected. A composition comprising a surfactant having lysis activity may be particularly relevant when directly applied to a surface, as this allows for the lysis of a biological agent (e.g. a biological agent to be detected or that has been detected) present on said surface. An anionic surfactant may comprise an anionic sodium lauryl sulfate, a docusate sodium, a cationic benzalkonium chloride, a cetylpyridinium chloride or a lecithin phosphatide. A non-ionic surfactant may comprise a polyoxyethylene sorbitan fatty acid ester (e.g. Polysorbate, Tween®), a polyoxyethylene 15 hydroxy stearate (e.g. Macrogol 15 hydroxy stearate, Solutol HS15®),a polyoxyethylene castor oil derivative (e.g. Cremophor® EL, ELP, RH 40), a polyoxyethylene stearates (e.g. Myrj®), a sorbitan fatty acid ester (e.g. Span®), a polyoxyethylene alkyl ether (e.g. Brij®), or a polyoxyethylene nonylphenol ether (e.g. Nonoxynol®).

The composition may comprise a lysis agent. As used herein, the term “lysis agent” refers to any agent which has the ability to lyse a biological agent. Thus, the lysis agent may comprise a surfactant as described herein or a detergent as described herein. A composition comprising a lysis agent may be particularly relevant when directly applied to a surface, as this allows for the lysis of a biological agent (e.g. a biological agent to be detected or that has been detected) present on said surface.

The composition may comprise an enzyme (e.g. a lytic enzyme and/or an enzyme that degrades DNA, RNA and/or a protein).

Lysis of a biological agent may be by any suitable lytic enzyme (such as a lysozyme or a lysin, protease K, bromelain, papain, clostridiopeptidase, trypsin, lyticase, collagenase, or lipase). A method of the invention may comprise a disinfecting step. Said disinfecting may be achieved by employing a disinfectant as described herein. The disinfectant may be part of or separate to the composition.

A method of the invention may comprise a step of contacting a surface or a sample with a disinfectant.

The composition may comprise an enzyme that degrades DNA, RNA and/or a protein. The composition may comprise an enzyme such as a deoxyribonuclease (DNase), a ribonuclease (RNase) and/or a protease. A DNase may be DNase I or DNase II. A RNase may be an endoribonuclease or an exoribonuclease. An endoribonuclease may be RNase A, RNase H, RNase III, RNase L, RNase P, RNase E or RNase G. An exoribonuclease may be RNase PH, RNase R, RNase D or RNase T. A protease may be an aspartic protease, a glutamic protease, a metalloprotease, a cysteine protease, a serine protease or a threonine protease. The composition may comprise a combination of different DNases, RNases and/or proteases. For example, the composition may comprise DNase I, DNase II, RNase A, RNase H, RNase III, RNase L, RNase P, RNase E, RNase G, RNase PH, RNase R, RNase D, RNase T, an aspartic protease, a glutamic protease, a metalloprotease, a cysteine protease, a serine protease, a threonine protease or a combination thereof. Such a composition may be particularly relevant when contacted with a surface or sample as this may allow for the degradation of DNA, RNA and/or proteins present on the surface or in the sample. Advantageously, this may allow for the degradation of DNA, RNA and/or protein components of biological agent(s) following their detection. This may be particularly advantageous where a capture substrate binds to said DNA, RNA, and/or protein component, as following detection, by degrading said DNA, RNA, and/or protein component, this may serve to end the production of the detectable signal and/or any further detectable signal and/or a detectable difference associated with a composition of the invention (or a component thereof). By ending the production of said detectable signal and/or detectable difference, this may serve as an indicator that a biological agent(s) is no longer present, is inactive, and/or is no longer viable (e.g. where a disinfectant has slowed the growth, inactivated or rendered the biological agent(s) inviable). For example, a detectable signal and/or detectable difference may comprise a colorimetric change, which may indicate the presence of a biological agent. By contacting a composition comprising an enzyme that degrades DNA, RNA and/or a protein to a surface or a sample, this may allow for the degradation of said DNA, RNA and/or protein of a biological agent, thereby terminating the production of the colorimetric change, and resulting in an absence of the colorimetric change. Advantageously, this may avoid the need to apply the composition again to detect whether the biological agent(s) are still present. This may be equally relevant to other detectable signals and/or detectable differences, such as fluorescence emission or quenched fluorescence. The skilled person understands that the enzyme should be carefully selected (e.g. to ensure that it does not degrade a nanomaterial or capture substrate present) and/or controlled (e.g. controllably activated/inactivated) such that it does not substantially affect a nanomaterial or capture substrate of the composition before detection of a biological agent. For example, where a capture substrate is a nucleic acid capture substrate, use of a DNase may be avoided. Thus, in one embodiment, the enzyme may be inactive during detection of a biological agent on a surface or in a sample and only active during the cleaning of the surface or the sample. In one embodiment the enzyme may not degrade a component of a biological agent when said component is a nucleic acid and/or a polypeptide. A suitable DNase may be one that specifically degrades double-stranded DNA and not single-stranded DNA. Such a DNase may be suitable for use with a composition of the invention wherein a capture substrate is a single-stranded nucleic acid. For example, when a capture substrate and a component of a biological agent are both a nucleic acid (e.g. a DNA), such a DNase may only degrade said nucleic acids when said capture substrate is bound to said component of a biological agent, thereby forming double-stranded DNA. When unbound, the capture substrate and the component of the biological agent may only be present as single-stranded DNA and therefore, not degraded by the DNase specific to double-stranded DNA. A DNase that degrades double-stranded DNA may be isolated from an arctic shrimp from the family Pandalidae. For example, a DNase that degrades double-stranded DNA may be isolated from an arctic shrimp from the species Pandalus borealis. A DNase that degrades doublestranded DNA may be one as taught in Nilsen et al PLoS ONE. 2010; 5(4): e10295, which is incorporated herein by reference. A DNase that degrades double-stranded DNA may be commercially available from ArcticZymes Technologies ASA, Norway, e.g. sold as dsDNase (Article No. 70600-201) or HL-dsDNase (Article No. 70800-201). An enzyme, preferably a DNase, may be inactivated by the use of dithiothreitol (DTT) and/or Ethylenediaminetetraacetic acid (EDTA). An enzyme, preferably a DNase, may be inactivated at a high temperature, for example at a temperature of 50-80, 52-78, 54-76, 56- 74, 58-72 or 60-70°C. An enzyme, preferably a DNase, may be inactivated in an alkaline pH, such as at a pH of 8-14, 9-13 or 10-12. The skilled person will appreciate that these methods of inactivating an enzyme may be used separately or in combination.

In one embodiment, the composition does not comprise an enzyme, e.g. does not comprise an enzyme that degrades DNA, RNA and/or a protein. A method of the invention may comprise a step of contacting a surface or a sample with an enzyme described herein.

A method of the invention may employ the use of a further composition that is different to the composition comprising the capture substrate and nanomaterial described herein. Said further composition may comprise a disinfectant as described herein. Said further composition preferably comprises an enzyme as described herein. Said further composition preferably does not comprise a capture substrate or a nanomaterial. A kit of the invention may comprise said further composition (e.g. in a separate container to the composition comprising a capture substrate and a nanomaterial).

A method of the invention may comprise a step of contacting a surface or a sample with a further composition. Said step is preferably carried out after having detected the presence a biological agent (e.g. via detecting a detectable signal and/or a detectable difference). The further composition preferably comprises an enzyme that degrades DNA, RNA and/or protein, preferably DNA, that is present on the surface or sample. The enzyme may be an enzyme that degrades a capture substrate described herein. The DNA, RNA or protein present on the surface or sample may be a capture substrate and/or a component of a biological agent, preferably, a DNA component from a biological agent. In one embodiment, the further composition comprising the enzyme may be contacted with the surface or sample, and may degrade DNA, RNA and/or protein, resulting in a detectable difference (e.g. a reduction or loss of a detectable signal) of the composition (or a component thereof). In a preferred embodiment, a further composition comprising a DNase may be contacted with the surface or sample and degradation of DNA (preferably a DNA component of a biological component) may result in a detectable difference (e.g. a reduction or loss of a detectable signal) of the composition (or a component thereof). A detectable difference may be a change in an optical property. A detectable difference (e.g. a different signal) may be a change in the fluorescent state of a fluorescent label (e.g. fluorescence quenching or fluorescence emission) and/or a colorimetric change, preferably a colorimetric change. The detectable difference may indicate that the surface or the sample has been disinfected.

In one aspect of the invention, there is provided a method for cleaning a surface or sample, the method comprising contacting the surface or sample with a composition of the invention. The method may further comprise detecting a detectable signal (e.g. optically-detectable signal) and/or detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the presence of the biological agent on the surface or sample, respectively.

The method may further comprise cleaning the surface or sample with a further composition (e.g. comprising a disinfectant and/or an enzyme, such as a DNase). The further step may take place when a detectable signal (e.g. optically-detectable signal) is detected, thereby indicating the presence of the biological agent on the surface or sample. Advantageously, a further cleaning step may improve cleaning of the surface or sample.

In one aspect of the invention, there is provided a method for cleaning a surface or sample, the method comprising:

(a) contacting the surface or sample with the composition as claimed;

(b) detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the presence of the biological agent on the surface or sample, respectively; and

(c) cleaning the surface or sample by contacting the surface or sample, respectively, with a further composition (e.g. comprising a disinfectant and/or an enzyme, such as a DNase).

In one aspect of the invention, there is provided a method for cleaning a surface or sample, the method comprising:

(a) contacting the surface or sample with a composition of the invention;

(b) detecting a detectable signal (e.g. optically-detectable signal), thereby indicating the presence of the biological agent on the surface or sample, respectively; and

(c) cleaning the surface or sample by contacting the surface or sample, respectively, with a further composition (e.g. comprising a disinfectant and/or an enzyme, such as a DNase).

In one aspect of the invention, there is provided a method for cleaning a surface or sample, the method comprising:

(a) contacting the surface or sample with the composition as claimed;

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the presence of the biological agent on the surface or sample, respectively; and (c) cleaning the surface or sample by contacting the surface or sample, respectively, with a further composition (e.g. comprising a disinfectant and/or an enzyme, such as a DNase).

In one aspect of the invention, there is provided a method for cleaning a surface or sample, the method comprising:

(a) contacting the surface or sample with a composition of the invention;

(b) detecting a detectable difference (e.g. of or in the composition or a component thereof, such as the nanomaterial), thereby indicating the presence of the biological agent on the surface or sample, respectively; and

(c) cleaning the surface or sample by contacting the surface or sample, respectively, with a further composition (e.g. comprising a disinfectant and/or an enzyme, such as a DNase).

The cleaning may result in a decrease (preferably a complete loss) of the detectable signal and/or a further detectable difference. This may indicate that the surface or sample has been sufficiently cleaned. Alternatively, when there is no decrease (or substantially no decrease) of the detectable signal and/or when there is no (or substantially no) further detectable difference, this may indicate that the surface has not been sufficiently cleaned. Thus, a method may further comprise a step of detecting a detectable difference. A method of the invention may further comprise a step of detecting a detectable signal.

A method of the invention preferably comprises a surface (e.g. rather than a sample).

A detectable difference (e.g. a different signal) in a method for cleaning a surface may be a change in the fluorescent state of a fluorescent label (e.g. fluorescence quenching or fluorescence emission) and/or a colorimetric change, preferably a colorimetric change.

The method may further comprise contacting the surface or the sample with a further composition (e.g. comprising an enzyme, preferably a DNase), when the biological agent(s) is/are present or wherein the quantity of the biological agent(s) on the surface exceeds the pre-determined safety level.

The method may further comprise cleaning and/or treating the surface when the biological agent(s) is/are present or wherein the quantity of the biological agent(s) on the surface exceeds the pre-determined safety level. The cleaning and/or treating of the surface may comprise cleaning and/or treating with a disinfectant and/or an enzyme, preferably a DNase. The cleaning and/or treating of the surface may comprise cleaning and/or treating with a further composition described herein (e.g. comprising a disinfectant and/or enzyme).

The detectable signal may be detected within 0-60 minutes, 10-50 minutes, 20-40 minutes or 25-35 minutes following contact of the composition with a surface or a sample. Preferably, the detectable signal may be detected less than 5 minutes (preferably within 1 minute) following contact of the composition with a surface or a sample (e.g. in real time).

The detectable difference may be detected within 0-60 minutes, 10-50 minutes, 20-40 minutes or 25-35 minutes following contact of the composition with a surface or a sample. Preferably, the detectable difference may be detected less than 5 minutes (preferably within 1 minute) following contact of the composition with a surface or a sample (e.g. in real time).

In one aspect the invention provides a kit for detecting at least two genetically different biological agents on a surface or in a sample, the kit comprising:

(a) the composition of the invention; and

(b) instructions for use of the same.

A kit may further comprise a component for producing the detectable (e.g. optically- detectable) signal, such as a source as described herein, e.g. an external light source. For example, an LED may be attached to the packaging of the composition, or a light filter may be attached to a smart phone.

A source may be employed to detect a detectable signal and/or detectable difference herein. Said source may be a light source, such as an LED, a laser, or a light (e.g. a flash of a smart phone or light filter attached to a smart phone). The source is preferably separate to a composition of the invention. The source may be present on a spray bottle (e.g. comprising the composition) and/or swab box. The spray bottle and/or swab box may be comprised in a kit of the invention.

The composition may be packaged. When formulated as a liquid, the packaging may include a spray or a swab (e.g. a spray and a swab). Alternatively, the composition may be available in a dry format and require re-constitution into a liquid, e.g. before use. The packaging may include a suitable diluent. The composition may be used in different environmental settings. In one embodiment, the composition may be used to detect the presence of at least two genetically different biological agents in a restaurant environment. For example, a swab may be taken from the work surface in a restaurant kitchen. The swab may be placed into a solution comprising one or more lytic enzymes to lyse any biological agents in the sample. The solution may be transferred into the composition of the invention and an LED light source may be shone over said composition. Alternatively, the composition comprising a surfactant with lysis activity may be sprayed directly onto the work surface in a restaurant kitchen. An LED light source may be shone over said composition.

In one embodiment, the composition of the invention may be used to detect the presence of at least two genetically different biological agents in a hospital environment. For example, a swab may be taken from a ventilator in a hospital. The swab may be placed into a solution comprising one or more lytic enzymes to lyse any biological agents in the sample. The solution may be transferred into the composition of the invention and an LED light source may be shone over said composition. Alternatively, the composition comprising a surfactant with lysis activity may be sprayed directly onto the ventilator. An LED light source may be shone over said composition.

Embodiments related to the various compositions of the invention are intended to be applied equally to the methods and the kits, and vice versa.

SEQUENCE HOMOLOGY

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align- M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides I amino acids divided by the total number of nucleotides I amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

ALIGNMENT SCORES FOR DETERMINING SEQUENCE IDENTITY

A 4

R-1 5

N -2 06

D-2-2 1 6

C 0-3 -3 -3 9

Q-1 1 0 0-3 5

E-1 0 02-42 5

G 0-2 0-1-3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3-1 -3 -3 -4 -34

L -1 -2 -3 -4 -1 -2 -3 -4-32 4

K-1 2 0-1 -3 1 1 -2-1 -3-2 5

M -1 -1 -2-3-1 0-2 -3 -2 1 2-1 5

F -2 -3 -3 -3 -2 -3 -3 -3-1 0 0-3 06

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0-1 0 0 0-1 -2-2 0-1 -2-1 4

T 0 -1 0-1-1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5

W-3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1-4-3-211

Y -2 -2 -2 -3 -2 -1 -2 -32 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0-3-3 -3 -1 -2 -2 -3-3 3 1 -2 1 -1 -2 -2 0-3-1 4

The percent identity is then calculated as:

Total number of identical matches x 100

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

CONSERVATIVE AMINO ACID SUBSTITUTIONS

Basic: arginine lysine histidine

Acidic: glutamic acid aspartic acid

Polar: glutamine asparagine

Hydrophobic: leucine isoleucine valine

Aromatic: phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991 ; Ellman et al., Methods Enzymol. 202:301, 1991 ; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a nanomaterial” includes a plurality of such candidate agents and reference to “the capture substrate” includes reference to one or more capture substrates and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the following Figures.

Figure 1 shows two types of nanomaterials; one nanomaterial associated with an oligonucleotide which binds to a nucleic acid from a biological agent (A) and a second nanomaterial associated with a lectin which binds to a cell-surface carbohydrate of a biological agent (B). An optically-detectable signal (such as fluorescence emission) may be produced when one of the capture substrates binds to the component of the biological agent.

Figure 2 shows adsorption of (A) 16S rRNA oligonucleotides and (B) spliced DNA from E. coli on gold nanoparticles. The control is a no nanoparticle negative control.

GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT TV

EXAMPLES

EXAMPLE 1 of onto the surface of a

Gold nanoparticles are sourced from a commercial supplier (Abeam Ltd) and their size is characterised using a Malvern Nanosizer. Discrete sets of short oligonucleotides (primers), supplied commercially (Integrated DNA Technologies), are adsorbed onto the surface of gold nanoparticles as follows.

1. A small volume (i.e. , 2 pL) of oligonucleotide stock solution (100 pM in 5 mM HEPES buffer, pH 7.4) is added to 200 pL of as-prepared AuNP solutions (10 nM) and mixed via a brief vortex mixing.

2. 4 pL of 500 mM pH 3 citrate HCI buffer (final 10 mM) is added to the AuNP solution (e.g., 1 pL of buffer/50 pL of AuNP solution). The solution is mixed via a brief vortex and incubated at room temperature for 3 min.

3. The pH of the AuNP solution is adjusted back to neutral by adding 12 pL of 500 mM HEPES buffer (pH 7.6, 3 pL of buffer/50 pL of AuNP solution). The sample is allowed to incubate for another 5-10 min at room temperature.

4. The DNA-AuNP mixture is centrifuged for 10 minutes at 15,000 rpm (ultracentrifuge usually 60k-200k, benchtop usually up to 15k, but top models), and the supernatant is removed. The pellet is washed four to five times with 5 mM HEPES buffer and centrifuged to remove any free DNA strands. The final DNA-AuNP conjugate is redispersed in 5 mM HEPES buffer for further use.

The extent of oligonucleotide adsorption onto the gold nanoparticles is determined using fluorescent quantification of the oligonucleotides (using SYBR Green II dye). The effects of the oligonucleotide adsorption on the gold nanoparticle size are determined using the Malvern Nanosizer.

The gold nanoparticles demonstrate a high fluorescence intensity and an increase in their size/surface area, indicating that oligonucleotides are successfully adsorbed to the surface of the gold nanoparticles. EXAMPLE 2

Testina the detection of Escherichia coli (E. coli} DNA (viral plasmid DNA) with a composition comprising gold nanoparticles adsorbed with oligonucleotides

Materials & Methods

Gram-negative E. coli bacteria are cultured to develop a concentrated broth. The bacteria are lysed and their DNA extracted and concentrated (using GenElute™ Bacterial Genomic DNA Kits, Sigma-Aldrich (NA2110)). Gold nanoparticles adsorbed with oligonucleotides are used to detect E. coli. Detection is visualised through aggregation, and subsequent colour change of the solution containing the oligonucleotide decorated gold nanoparticles. The limit of detection of the E. coli DNA is determined using up to 3 different types of oligonucleotide adsorbed nanomaterials.

Results

In the absence of E. coli DNA, a solution comprising the gold nanoparticles remains red in colour, indicating that the gold nanoparticles are dispersed in the solution. Upon the addition of extracted and concentrated E. coli DNA to the solution, the gold nanoparticles aggregate, causing the solution to change colour from red to purple and indicating the successful detection of E. coli.

EXAMPLE 3 the detection of and severe acute viral clone DNA with adsorbed with

Materials & Methods

The fungi T. rubrum are cultured to develop a concentrated broth. The fungi are lysed, and their DNA extracted and concentrated (using Qiagen DNeasy Plant mini Tissue Kit). Gold nanoparticles adsorbed with oligonucleotides are used to detect T. rubrum and SARS-CoV-2 clone DNA (commercially supplied). Detection is visualised through aggregation, and subsequent colour change of the solution. The limit of detection of the T. rubrum and SARS- CoV-2 is determined using up to 3 different types of oligonucleotide adsorbed nanomaterials.

Results

In the absence of T. rubrum and SARS-CoV-2 DNA, a solution comprising the gold nanoparticles remains red in colour, indicating that the gold nanoparticles are dispersed in the solution. Upon the addition of extracted and concentrated T. rubrum and SARS-CoV-2 DNA to the solution, the gold nanoparticles aggregate, causing the solution to change colour from red to purple and indicating the successful detection of T. rubrum and SARS-CoV-2. EXAMPLE 4 the detection of a mixture of adsorbed with

Materials & Methods

Gold nanoparticles adsorbed with oligonucleotides are used to detect mixtures comprising T. rubrum DNA, E. coli DNA and purchased viral clone SARS-CoV-2 DNA. Different mixtures of the species and different ratios of the gold nanoparticles adsorbed with oligonucleotides are tested.

Results

In the absence of E. coli, T. rubrum and SARS-CoV-2 DNA, a solution comprising the gold nanoparticles remains red in colour, indicating that the gold nanoparticles are dispersed in the solution. Upon the addition of extracted and concentrated E. coli, T. rubrum and SARS- CoV-2 DNA to the solution, the gold nanoparticles aggregate, causing the solution to change colour from red to purple and indicating the successful detection of E. coli, T. rubrum and SARS-CoV-2.

The mixtures of gold nanoparticles are more effective at detecting the biological agents, shown by a lower limit of detection, than the individual particles and thus physical mixing has enriched the detection system.

EXAMPLE 5 the detection of a mixture of biological agents with MOFs adsorbed with

Materials & Methods

MOFs adsorbed with oligonucleotides are used to detect mixtures comprising T. rubrum DNA, E. coli DNA and purchased viral clone SARS-CoV-2 DNA. Different mixtures of the species and different ratios of the MOFs adsorbed with oligonucleotides are tested. Oligonucleotides are labelled with a fluorescent label, such as a fluorescent dye.

Results

In the absence of E. coli, T. rubrum and SARS-CoV-2 DNA, a solution comprising the MOF shows fluorescence quenching (i.e. no detectable fluorescence). Upon the addition of extracted and concentrated E. coli, T. rubrum and SARS-CoV-2 DNA to the solution, oligonucleotides desorb from the MOF and bind to the corresponding biological agent DNA. A LED light source is shone over the solution, producing fluorescence emission as a detectable signal and indicating the successful detection of E. coli, T. rubrum and SARS- CoV-2.

EXAMPLE 6

Detection of E.coli in a restaurant environment

Materials & Methods

A composition of the invention comprising MOFs adsorbed with oligonucleotides is used to detect T. rubrum DNA, E. coli DNA and SARS-CoV-2 DNA on a surface or in a sample from a restaurant kitchen. The ratio of MOFs in the composition is altered to have a higher number of MOFs comprising oligonucleotides directed to detecting E. coli DNA. Oligonucleotides are labelled with a fluorescent label, such as a fluorescent dye.

A swab is taken from the work surface in a restaurant kitchen. The swab is placed into a solution comprising one or more lytic enzymes to lyse any biological agents in the sample. The solution is then transferred into the composition of the invention and an LED light source is shone over said composition.

Alternatively, the composition comprising a surfactant with lysis activity is sprayed directly onto the work surface in a restaurant kitchen. An LED light source is then shone over said composition.

Results

In the absence of E. coli, a composition comprising the MOF shows fluorescence quenching (i.e. no detectable fluorescence). In the presence of E. coli, and using a LED light source, fluorescence emission is produced as a detectable signal. This indicates the successful desorption of the oligonucleotides from the MOF, and, the successful lysis and detection of E. coli.

EXAMPLE 7

Detection of E.coli and SARS-CoV-2 DNA in a hospital

Materials & Methods

A composition of the invention comprising MOFs adsorbed with oligonucleotides is used to detect E. coli DNA and SARS-CoV-2 DNA on a surface. The ratio of MOFs in the composition is altered to have a higher number of MOFs comprising oligonucleotides directed to detecting E. coli and SARS-CoV-2 DNA. Oligonucleotides are labelled with a fluorescent label, such as a fluorescent dye. A swab is taken from a ventilator in a hospital. The swab is placed into a solution comprising one or more lytic enzymes to lyse any biological agents in the sample. The solution is then transferred into the composition of the invention and an LED light source is shone over said composition.

Alternatively, the composition comprising a surfactant with lysis activity is sprayed directly onto the ventilator. An LED light source is then shone over said composition.

Results

In the absence of E. coli and SARS-CoV-2, a composition comprising the MOF shows fluorescence quenching (i.e. no detectable fluorescence). In the presence of E. coli and SARS-CoV-2, and using a LED light source, fluorescence emission is produced as a detectable signal. This indicates the successful desorption of the oligonucleotides from the MOF, and, the successful detection of E. coli (and successful lysis) and SARS-CoV-2.

EXAMPLE 8

Detection of Klebsiella pneumoniae, Candida tropicalis and influenza virus DNA in a

Materials & Methods

A composition of the invention comprising MOFs adsorbed with oligonucleotides is used to detect Klebsiella pneumoniae, Candida tropicalis and influenza virus DNA on a surface. The ratio of MOFs in the composition is altered to have a higher number of MOFs comprising oligonucleotides directed to detecting Klebsiella pneumoniae, Candida tropicalis and influenza virus DNA. Oligonucleotides are labelled with a fluorescent label, such as a fluorescent dye.

A swab is taken from a ventilator in a hospital. The swab is placed into a solution comprising one or more lytic enzymes to lyse any biological agents in the sample. The solution is then transferred into the composition of the invention and an LED light source is shone over said composition.

Alternatively, the composition comprising a surfactant with lysis activity is sprayed directly onto the ventilator. An LED light source is then shone over said composition. Results

In the absence of Klebsiella pneumoniae, Candida tropicalis and influenza virus, a composition comprising the MOF shows fluorescence quenching (i.e. no detectable fluorescence). In the presence of Klebsiella pneumoniae, Candida tropicalis and influenza virus, and using a LED light source, fluorescence emission is produced as a detectable signal. This indicates the successful desorption of the oligonucleotides from the MOF and the successful detection of Klebsiella pneumoniae, Candida tropicalis and influenza virus.

EXAMPLE 9 of lectins onto the surface of a

Gold nanoparticles are sourced from a commercial supplier (Abeam Ltd) and their size is characterised using a Malvern Nanosizer. Discrete sets of lectins are supplied commercially (for example, Con A; Product Code: 22070010; glycoMatrix) and adsorbed onto the surface of gold nanoparticles as follows.

1. A small volume (i.e., 2 pL) of lectin stock solution (100 pM in 5 mM HEPES buffer, pH 7.4) is added to 200 pL of as-prepared AuNP solutions (10 nM) and mixed via a brief vortex mixing.

2. 4 pL of 500 mM pH 3 citrate HCI buffer (final 10 mM) is added to the AuNP solution (e.g., 1 pL of buffer/50 pL of AuNP solution). The solution is mixed via a brief vortex and incubated at room temperature for 3 min.

3. The pH of the AuNP solution is adjusted back to neutral by adding 12 pL of 500 mM HEPES buffer (pH 7.6, 3 pL of buffer/50 pL of AuNP solution). The sample is allowed to incubate for another 5-10 min at room temperature.

4. The lectin-AuNP mixture is centrifuged for 10 minutes at 15,000 rpm (ultracentrifuge usually 60k-200k, benchtop usually up to 15k, but top models), and the supernatant is removed. The pellet is washed four to five times with 5 mM HEPES buffer and centrifuged to remove any free lectin. The final lectin-AuNP conjugate is redispersed in 5 mM HEPES buffer for further use.

The extent of lectin adsorption onto the gold nanoparticles is determined using fluorescent quantification of the lectins (using SYBR Green II dye). The effects of the lectin adsorption on the gold nanoparticle size are determined using the Malvern Nanosizer. The gold nanoparticles demonstrate a high fluorescence intensity and an increase in their size/surface area, indicating that lectins are successfully adsorbed to the surface of the gold nanoparticles.

EXAMPLE 10

Detection of E.coli, Bacillus subtilis and SARS-CoV-2 in a restaurant environment

Materials & Methods

A composition of the invention comprising gold nanoparticles adsorbed with lectins is used to detect E. coli, Bacillus subtilis and SARS-CoV-2 on a surface or in a sample from a restaurant kitchen. Lectins are labelled with a fluorescent label, such as a fluorescent dye.

The composition is sprayed directly onto the work surface in a restaurant kitchen. An LED light source is then shone over said composition.

Results

In the absence of E. coli, Bacillus subtilis and SARS-CoV-2, a composition comprising the gold nanoparticles shows fluorescence quenching (i.e. no detectable fluorescence). In the presence of E. coli, Bacillus subtilis and SARS-CoV-2, and using a LED light source, fluorescence emission is produced as a detectable signal. This indicates the successful desorption of the lectins from the gold nanoparticles, and, the successful detection of E. coli, Bacillus subtilis and SARS-CoV-2.

EXAMPLE 11

Detection of E.coli, Bacillus subtilis and SARS-CoV-2 in a restaurant environment

Materials & Methods

A composition of the invention comprising MOFs adsorbed with lectins is used to detect E. coli, Bacillus subtilis and SARS-CoV-2 on a surface or in a sample from a restaurant kitchen. Lectins are labelled with a fluorescent label, such as a fluorescent dye.

The composition is sprayed directly onto the work surface in a restaurant kitchen. An LED light source is then shone over said composition.

Results

In the absence of E. coli, Bacillus subtilis and SARS-CoV-2, a composition comprising the MOFs shows fluorescence quenching (i.e. no detectable fluorescence). In the presence of E. coli, Bacillus subtilis and SARS-CoV-2, and using a LED light source, fluorescence emission is produced as a detectable signal. This indicates the successful desorption of the lectins from the MOFs, and, the successful detection of E. coli, Bacillus subtilis and SARS-CoV-2.

EXAMPLE 12

Gold nanoparticles are sourced from a commercial supplier (Abeam Ltd) and their size is characterised using a Malvern Nanosizer. Discrete sets of short oligonucleotides (primers), supplied commercially (Integrated DNA Technologies), are adsorbed onto the surface of gold nanoparticles as follows.

1. A small volume (i.e. , 2 pL) of oligonucleotide stock solution (100 pM in 5 mM HEPES buffer, pH 7.4) is added to 200 pL of as-prepared AuNP solutions (10 nM) and mixed via a brief vortex mixing.

2. 4 pL of 500 mM pH 3 citrate HCI buffer (final 10 mM) is added to the AuNP solution (e.g., 1 pL of buffer/50 pL of AuNP solution). The solution is mixed via a brief vortex and incubated at room temperature for 3 min.

3. The pH of the AuNP solution is adjusted back to neutral by adding 12 pL of 500 mM HEPES buffer (pH 7.6, 3 pL of buffer/50 pL of AuNP solution). The sample is allowed to incubate for another 5-10 min at room temperature.

4. The DNA-AuNP mixture is centrifuged for 10 minutes at 15,000 rpm, and the supernatant is removed. The pellet is washed four to five times with 5 mM HEPES buffer and centrifuged to remove any free DNA strands. The final DNA-AuNP conjugate is redispersed in 5 mM HEPES buffer for further use.

The extent of oligonucleotide adsorption onto the gold nanoparticles is determined using fluorescent quantification of the oligonucleotides loss from the adsorption solution (using SYBR Green II dye). The effects of the oligonucleotide adsorption on the gold nanoparticle size are determined using the Malvern Nanosizer.

The combination of the gold nanoparticles with the oligonucleotides results in a loss of oligonucleotides from solution, indicating that oligonucleotides are successfully adsorbed to the surface of the gold nanoparticles.

EXAMPLE 13 16S rRNA oligonucleotides (SEQ ID NO: 1) or oligonucleotides from spliced (fragmented) E. coli DNA were adsorbed onto the surface of gold nanoparticles as follows. Spliced (fragmented) E. coli DNA was prepared by a method comprising lysing E. coli and extracting DNA from the E. coli, concentrating the DNA and splicing the E. coli DNA using DNase I before concentrating the spliced (fragmented) E. coli DNA.

1. 8.6 pL of oligonucleotide stock solution (250ng) was added to 300 pL of commercial AuNP solution (Abeam Ltd). 25.4 uL of nuclease free water was added to the mixture, and the resulting AuNP-oligonucleotide solution was mixed via a 5 second vortex mixing.

2. 6 pL of 500 mM pH 3 citrate- HCI buffer (final 10 mM) was added to the AuNP- oligonucleotide solution (1 pL of buffer/50 pL of AuNP solution) and the resulting solution was mixed via a 5 second vortex mixing and incubated at room temperature for 3 min.

3. The pH of the AuNP-oligo solution was adjusted back to neutral by adding 150 pL of 500 mM HEPES buffer (pH 7.6, 20 pL of buffer/50 pL of AuNP solution). The pH was checked with litmus paper by dropping 1 uL of solution onto the paper.

4. The total volume was 490 uL.

5. The sample was incubated for another 10 min at room temperature.

6. The sample was centrifuged for 15 min at 14,000 rpm and the supernatant was removed.

7. 50 uL of H2O was added to 400 uL of the removed supernatant. The resulting supernatant solution was diluted in 550 uL of SYBR Green II solution.

8. The supernatant solution was mixed well by pipetting up and down 20 times.

9. The supernatant solution was incubated with SYBR Green II for 20 minutes to ensure homogeneity.

10. The supernatant solution was analysed in a Plate reader for unbound primers.

11. The pellet was washed two times with 1000ul of nuclease free water to remove the free RNA strands and centrifuged at 14,000 rpm for 15 minutes. The supernatant was removed.

12. The experiment was repeated 3 times (3 stocks) with 2 readings each in a plate reader (using 2 well plates).

SYBR Green II solution was prepared by diluting SYBR Green II (10,000X concentrate) in TBE buffer (89 mM Tris base, 89 mM Boric acid, 1 mM EDTA, pH 8) to provide 1X SYBR Green II in TBE buffer. As shown in Figure 2, 16S rRNA oligonucleotides and oligonucleotides from spliced E. coli DNA adsorbed onto the surface of gold nanoparticles.

EXAMPLE 14 detection bv 16S rRNA adsorbed to

The ability of gold nanoparticles coated with 16S rRNA to detect E. coli was assessed as follows.

1. 90 ul of washed gold nanoparticle conjugates was diluted with 210 ul of nuclease free water to a final volume of 300ul.

2. Using a Gilson pipette, 100 ul of gold nanoparticle solution was added to each of 3 wells of a transparent 96 well plate.

3. Spliced DNA from E. coli (600ng in 20 pl nuclease free water) was added to the 1 ^st well.

4. The 2 ^nd well was used as a negative control (20 pl nuclease free water).

5. Salt in 20 pl nuclease free water was added to the 3 ^rd well as a positive control to force gold nanoparticles to aggregate.

6. The plate reader was run at 24°C with absorbance wavelength of 682. The reading was taken.

The results showed increased absorbance with nanoparticles coated with 16S rRNA and with aggregated nanoparticles compared to negative control wells. This indicates that gold nanoparticles coated with 16S rRNA are suitable for detecting spliced DNA from E. coli.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.