Description

Title: _ Filter System

Cross-Reference to Related Applications

This application contains claims benefit to and priority of UK Patent Application No. UK 2009244. Ifiled on 17 June 2020.

Field of the Invention

[0001] The invention relates to system and method for detecting a neuraminidase using and indicating a presence of the marker using a filtering device of a silk-based material.

Background of the Invention

[0002] Testing for Covid and other viroids will be a major component of public safety in the future. This testing can be done, for example, by a process of capturing particles in or on a filter. Filter media in the filter is dependent on the properties and conditions of the incoming flow of air with the particles, and the flow of air through the internal microstructure of the filter, as well as the particle’s transport (aerosol) properties. For very small particles, such as viruses, the predominance of diffusion means that the adhesive nature of the filter dominates. Only advanced and specialized filters have this capacity.

[0003] It is known that silks are insulators and, as such, keep the charges generated during their melt-spinning process and/or inherent in the polarities of the amino acid composition of their proteins (Morley & Robert 2018). Silkworm silk cocoons already known to act as selective filters (Horrocks et al. 2013). It is also known that dipolar surfaces can equally attract positive and negatively charged particles and this property is used to great effect in the spider’s capture threads (Vollrath and Edmonds, 2013), and in the design of hybrid materials (Singh, Bollella et al. 2020, Singh, Dey et al. 2020).

[0004] As far as known, the corona virus as such is not airborne. Like most enveloped viruses, the corona virus is hydrated or in solution and transmitted via aerosol droplets. When the corona virus dehydrates, the lipid membrane of the corona virus collapses and the proteins denature and thus the corona virus is rendered inactive within a certain timetemperature ratio. [0005] The aerosolized droplets of the virus contain mucus and other biological materials, with direct influence on airborne particle size, composition, and infectivity (Gralton et al., 2011).

[0006] The net charge of the virus in solution (water, typically) depends on the pH. It is generally accepted that the isoelectric points (pl) of viruses (Michen and Graule 2010) are lower than 7, suggesting that most of the viruses would be net negatively charged at neutral pH. An estimate of the covid-19 virion pl (from sequence: http://www.rcsb.org/structure/6VXX) suggests a pl of 5.8, which is thus net negative. In addition, most pathogenic microbes and viruses express strong or moderate cell surface hydrophobicity.

[0007] The decay in static electricity of standard electrocharged polymer fibre under humid conditions (due to exhalation) is fast - the electrocharged layer decays in over a few hours.

Summary of the Invention

[0008] The average adult human re-cycles ca 10,000 litres of air per day which provides a ready-made airstream that could potentially be filtered of its viral load. An appropriate analysis of the viral load is able to act as an indicator for the coronavirus particles as well as other particles. Silks are able to provide a perfect material for both the filter and the diagnostics because of their specific properties including electrostatic charges.

[0009] A selective enzymatic assay (neuraminidase) for specific identification of the virus is taught in this document. The concentration and expected preservation of the virus particles on filter meshes in the filter of this disclosure will be able enhance the robustness of a more detailed genetic analysis (qPCR).

[0010] The facemask or air flow device disclosed in this document may have two separate filters - an inwards filter for the inhaled air and an outwards filter for the exhaled air. A standard filter of a high-quality mask may separate these two separate filters. In another aspect, there is a single filter in the facemask or the air flow device.

[0011] The device can also be a liquid flow device and may be a microfluidic device. In another aspect, the device can be a liquid device in which the liquid (i.e. fluid) in the device is able to be shaken or stirred. [0012] The filter disclosed in this document may also consist of one principal component with separate elements sandwiching a middle element and facing inwards as well as outwards.

[0013] This document teaches a filter system for detection of a marker in a fluid. The filter system comprises a filter membrane comprising at least a silk-based material, such as a silk fibre or silk filament, a silk membrane or a silk analogue. The silk-based material comprises a biosensor. The biosensor comprises at least one bioelement for detecting a presence of the marker in the fluid and at least one transducer for indicating the presence of the marker in the fluid. The marker of the filter system is, for example, a neuraminidase.

[0014] The marker is a microorganism comprising a recognisable molecule, wherein the molecule is a bacterial neuraminidase, and wherein the neuraminidase comprises at least one bacterial neuraminidase or a viral neuraminidase.

[0015] The biosensor of the filter system is impregnated in the silk-based material . The filter system is adapted to change colour in response to the presence of the presence of the marker in the fluid.

[0016] The silk-based material comprises a membrane or a fibre generated from a natural silk or from a regenerated or bio-synthetic silk considered to be a silk analogue.

[0017] The filter membrane of the filter system further comprises the silk-based material being interlaced, meshed, or woven with at least one other material or a different type of silk-based material.

[0018] A method for detecting a marker in a fluid using a silk based material is also disclosed. The method comprises the steps of providing a filter system in the fluid and recognizing in the marker in the fluid. The method further comprises reporting in the presence of the marker in the fluid.

[0019] A method for impregnating a silk-based material with a biosensor is also disclosed. The method comprises the synthesising of polydiacetylene (PDA) vesicles using a solvent injection method. The method further comprises impregnating in the silk based material with the synthesised polydiacetylene (PDA) vesicles. The method also comprises attachment of ligands to the impregnated silk-based material by incubating the synthesised polydiacetylene (PDA) vesicles in a buffer solution.

[0020] It will be appreciated that the fluid can a gas, such as but not limited to air, a liquid, or an aerosol. [0021] The method of this document can be used in a testing device. This can be a microfluidic device, a lateral flow device, a breathing device (such as a facemask), or a cleaning device (such as a vacuum cleaner).

Description of the Figures

[0022] Fig. 1 shows filtration mechanism.

[0023] Fig. 2 shows the filter efficiency versus particle diameter.

[0024] Figs. 3A-3C show facemasks with a filter.

[0025] Fig. 3D shows a pipe with a filter.

[0026] Fig. 4 shows the filter membrane with fibres.

[0027] Figs. 5A and 5B show micrographs of a silk with trapped particles.

[0028] Fig. 6 shows a biosensor element for detection of the neuraminidase.

[0029] Fig. 7A-7C shows selected transducing mechanisms.

[0030] Fig. 8 shows the photo-polymerisation of diacetylene lipid monomer.

[0031] Fig. 9 shows a Diacetylene monomers' nomenclature.

[0032] Fig. 10 shows diacetylene amphiphiles with a polar head group and diacetylene tail(s) form various self-assembled structures that can be photo-polymerised.

[0033] Fig. 11 shows an illustration of the concept for a virus-detecting fibre.

[0034] Fig. 12A-12D show a characterisation of neuraminidase from C. perfringens; effect of a) protein concentration (Fig. 12A), b) substrate concentration (4MU-NANA) (Fig. 12B), c) pH (Fig. 12C), and d) pH without enzyme (Fig. 12D).

[0035] Fig. 13 A shows how PCDA micelles are converted into PDA vesicles by UV (254 nm) induced photo-polymerisation.

[0036] Fig. 13B shows the colour shift of PDA (0.4 mg/mL) in water after the addition of 10 mM cyclodextrin.

[0037] Fig. 14 shows Silk (Eri) before and after impregnation with polydiacetylene (PDA) vesicles resulting in a deep blue colour of the fibre that remains after several washes.

[0038] Fig. 15A and 15B show silk containing PDA treated with various amount of a- cyclodextrin causing a colour shift of the fibre; the colours red and blue were measured using RBG-tool in ImageJ.

[0039] Fig. 16A and 16B show silks containing PDA with various inhibitors attached in different quantities. [0040] Fig. 17A and 17B show silks containing PDA with various inhibitors attached in different quantities.

[0041] Fig. 18A and 18B show silks containing PDA with Zanamivir attached.

[0042] Fig. 19 shows a flow chart further describing the method for filtering a fluid flow and detecting neuraminidase in the fluid flow using impregnated silk fibres.

[0043] Fig. 20 shows a flow chart describing the method for impregnating silk fibres with a biosensor.

Detailed Description of the Invention

[0044] Figs. 3A-3C show several examples of the use of a filter system 10 as set out in this document. The filter system 10 is used in a face mask 310 in the example shown in Figs. 3A-3C. In Fig. 3A a filter membrane 20 covers most of the face mask 310. In Fig. 3B, the filter membrane 20 is inserted into a pocket 320 on an inside of the face mask 310 and, in Fig. 3C, an insert is inserted into a box 330 on an inside of the face mask 310.

[0045] Fig. 3D shows a pipe 350 which could be attached to a blower or vacuum tube through which a fluid flow 15 such as an air flow is pumped or sucked. The filter membrane 20 is inserted into the pipe 350. This could be attached to an air flow device, such as but not limited to a fan, an air conditioning device, a hair dryer, or a vacuum cleaner.

[0046] Particle size and composition in aerosolized pathogen transmission.

[0047] Expiratory human activities such as breathing, coughing, sneezing, or laughing result in droplet 33 generation by the wind shear forces. Droplet 33 atomization from the respiratory tract arises from the passage of the air flow at a sufficiently high speed over the surface of a liquid; liquid is drawn out from the surface, pulled thin and broken into columns of droplets 33 (Hickey and Mansour 2019).

[0048] Each of these processes leads to droplets 33 comprising particles 35 of different size and originating from different areas of the upper respiratory tract. Differences in size result from a variation in air pressure and speed in different parts of the respiratory tract, in much the same way as explained above for the atomization process (Morawska, 2005; Morawska, 2008). The significance of each of these activities in the spread of infection depends on a number of factors, including: (1) number of droplets 33 produced by the activities, (2) size of the droplets, (3) content of infectious agents in the droplets and, (4) the frequency of its performance (Morawska, 2005; Morawska 2008). There is some understanding of the size of droplets 33 generated directly by humans indoors, during various human expiratory activities, and the region in the respiratory tract from which the droplets 33 originate from (Ai and Melikov 2018, Fatemeh and Hatam 2018). There is also some understanding of the content of the infectious agents in the droplets 33 from experiments on healthy individuals artificially “marked” with the agents (Burton, Grinshpun et al. 2007). However, there is much less knowledge of the content of real infecting agents expelled by infected individuals, which is of importance in understanding the actual spread of viral infections.

[0049] The critical diameter, also known as the cut-off diameter, is around 5-10pm (WHO 2007, Gralton, Tovey et al., 2011). Due to their importance, the determination of droplet 33 size and its distribution has been a focus of several studies (Chao, Wan et al. 2009, Lindsley, Reynolds et al. 2013, Zhang, Zhu et al. 2017). Many have agreed that the sizes of cough droplets 33 have at least two main ranges (Gralton, Tovey et al. 2011). The first range of these at least two main ranges is a group of fine droplets 33 typically from sub-micron to about 10pm. The second range of these at two main ranges is the range of coarse droplets 33 from several tens of microns to sub-millimetres. Generally, the droplet 33 size is governed by (i) relative humidity at evaporation, (ii) aggregation, and (iii) mucus properties (Gralton, Tovey et al. 2011). The first two aspects can be estimated, whereas the latter remains unclear. For example, an early study (Hersen, Moularat et al. 2008), followed by others, found that particles from infected individuals were larger than those from healthy individuals. Disease- induced changes, such as increases in mucus composition, quantity, and viscosity, have also been observed ((Vanthanouvong, Kozlova et al. 2006) which may suggest that the increase in size is directly related to increases in mucus viscosity. Differences in mucus composition at the mucus-air interface may be accountable for the inter-individual variability observed in studies.

[0050] Regardless of the complexities and limitations of sizing particles and the contention of size cut-offs, it remains that particles have been observed to occupy a size range between 0.05 and 500 mm. This size range indicates that particles do not exclusively disperse by airborne transmission or via droplet 33 transmission but rather avail of both methods simultaneously. This suggestion is further supported by the simultaneous detection of both large and small particles.

[0051] Filter sampling of airborne viruses. [0052] Several questions have been raised regarding the use of respirators against biological agents (Green 1965). The primary issue is whether or not particulate respirators can filter small particles such as fungal spores (2 to 5 pm), bacteria (0.3 to 10 pm), or viruses (0.02 to 0.3 pm) (MacIntyre, Wang et al. 2014, Offeddu, Yung et al. 2017, Garcia Godoy, Jones et al. 2020). Table 1 summarizes the sizes of selected viruses.

Table 1. Size and infectivity of selected viruses

Optimal relative Microorganism

Physical size (pm) humidity (rH) for

(common name or disease) maximum infectivity

Hepatitis virus (Hepatitis B) 0.042 - 0.047 30% < rH < 70 %

Adenovirus (respiratory 0.07 - 0.09 30% < rH < 70 % infections) 0.08 diameter 30% < rH < 70 %

Filoviruses (Ebola) 0.79 - 0.97 length

Buyaviridae (Hantavirus) 0.08 - 0.12 30% < rH < 70 %

Orthomyxoviridae (Influenza A, 0.08 - 0.12 < 30%

B, C) Coronaviridae (SARS-CoV, 30% < rH < 70 %

0.12 MERS-CoV, SARS-CoV-2) 0.14 - 0.26 diameter 30% < rH < 70 %

Variola Virus (Smallpox) 0.22 - 0.45 length

Mycobacterium tuberculosis (TB) < 1 to > 5 diameter 30% < rH < 70 %

Bacillus anthracis spore (Anthrax 1.0 - 1.5 diameter 30% < rH < 70 % infection)

[0053] Many particulate respirators use a non-woven fibrous filter media to capture particles

35. An example of this is shown in Figs. 4 and 5. Fig. 4 shows the fluid flow 15comprising the feed air 16 with charged particles 35 comprising the neuraminidase 40 which are trapped in the filter membrane 20. The neuraminidase 40 is, for example, a bacterial neuraminidase or a viral neuraminidase. The filter membrane 20 comprises a number of silk fibres 30 which range in size from less than 1 pm up to 100 pm in size crisscross and thus form a web of many layers which is mostly air due to the spaces between the silk fibres 30. It is these spaces between fibres that allow for breathability. The particles 35 are trapped or captured when flowing through the layers of the filter membrane 20. That capture can happen through four basic mechanisms (as shown in Fig 1 from CDC webpage at https://blogs.cdc.gov/niosh- science-blog/2009/10/14/n95): (i) interception, (ii) inertial impaction, (iii) diffusion, and, (iv) electrostatic attraction (Hinds, 1999). An example of the trapped particles 35 in the filter membrane 20 is shown in Fig. 5 A. Fig. 5B shows airborne particles 35 captured by and firmly adhering to Nephila spider Major Ampullate dragline thread.

[0054] Interestingly, all filters have their minimum efficiency at 0.3 pm (Hinds 1999). That is the reason why most efficiency tests are performed for particles 35 at 0.3 pm (Hinds 1999). [0055] The current gold standard is based on the melt-blowing method pioneered by scientists at the US Naval Research Laboratory in 1954 ((Wente 1954, Wente 1956). Melt- blown is made by extruding molten polypropylene polymers through small nozzles into a stream of heated air: the randomly deposited fibres then form a fabric sheet. The network of microfilaments and the fact that it is has an electrostatic charge make it highly effective at blocking particles 35. Fibres of the three polymers: nylon, polyethylene terephthalate), and polystyrene possess high air resistance values which show extreme fibre fineness. As a consequence of their small size, these three exhibit high filtration efficiencies with nylon fibre being most effective.

[0056] Fibres made by this technique possess measurable electric charges and these, in turn, are known to influence filtration performance. Effects of the charges and means of controlling them are yet unresolved, particularly on the more hydrophobic thermoplastics for which discharge rates are extremely low.

[0057] The use of electrostatic filtration media (Frederick 1974, Smith, East et al. 1988) based on layered polymer in filters (e.g., formed by electrospinning) is a cheaper alternative and is commonplace in particulate respirators. Electrets (the charged polymer layer) have a semi-permanent electric field (just as magnets have a permanent magnetic field) and the electrostatic charge on the electret fibre improves the filtration efficiency over that of purely mechanical filters. In particular, mixtures of both positively charged and negatively charged fibres form a good basis for an electrostatic filter (Smith, East et al. 1988).

[0058] A number of mechanisms have been proposed to explain this phenomenon and these are shown in Fig. 1. For instance, it is thought that neutralization of the charge on the silk fibre 30 by opposite charges of the captured aerosol particles 35 may be a factor. Alternatively, a layer of captured particles 35 may be shielding the charged fibres. In the case of liquid aerosols, there is a possibility that ionic conduction occurs through the liquid film on the silk fibre 30, resulting in discharge of the electret. Finally, there is also a possibility that, depending upon the nature of the silk fibre 30 and the aerosol, the aerosol modifies the electret silk fibre 30 itself due to chemical reaction or dissolution.

[0059] Noteworthy, work on large-scale air flow devices, such as industrial air purifiers, indicates that airborne virus capture, and inactivation are best achieved by electrostatic particle 35 collector devices (Kettleson, Ramaswami et al. 2009). Unfortunately, these industrial air purifiers are not portable nor cost-effective nor useful in the context of personalized filter function.

[0060] Preserving Infectivity for the analytic step.

[0061] Most air sampling technologies depend on the aerodynamic diameter of the airborne particles 35, the adhesion properties of airborne particles 35, Brownian motion, thermal gradients, and the inertia of the particles 35. Aerosolized particles 35 attach to any surface with which they come into contact. Adhesive forces such as van der Waals forces, electrostatic forces, and surface tension partly explain this adhesion (Hinds 1999).

[0062] In the above framework, two particular aspects of viral particles 35 capture need to be controlled: (i) the optimal relative humidity for maximal infectivity, and (ii) the filter induced virus structural damage (Ijaz, Karim et al. 1987). There is no absolute correlation between relative humidity and the preservation of viral infectivity in aerosols and that the impact of rH should be determined for each virus (see table 1). However, it appears that low rH tends to preserve the infectivity of enveloped viruses, while the stability of non-enveloped viruses is best preserved at high rH (Verreault, Moineau et al. 2008). As a consequence, the analytic method may be misleading.

[0063] Filters generally cause structural damage to the virus in addition to desiccation (Burton, Grinshpun et al. 2007). For laboratory setup, while gelatine filters can be handy for a functional sampling of viruses, environmental conditions can limit their use. Low humidity can cause the gelatine to dry out and break, while high humidity or water droplets 33 can cause them to dissolve. On the positive side, this property can be used to recover viruses or virus-laden particles 35 by dissolving the filters in water. Nevertheless, 0.3-um PTFE filters appear to be the best option for a long-term sampling of 10- to 900-nm diameter virus-laden particles 35 (Burton, Grinshpun et al. 2007).

[0064] Interestingly Samandoulgou et al. found that although electrostatic effects and hydrophobic interactions are competitive when objects bear the same charges, the hydrophobic nature of the capture surface (polystyrene, polyethylene, etc.) would favour maximal interaction with the viruses close to the isoelectric point (Samandoulgou, Fliss et al. 2015).

[0065] Collectively, the general trend is that pleated hydrophobic membrane filters have a superior filtration performance compared with electrostatic filters and that there is a difference between the filtration performance of electrostatic filters from different manufacturers. However, the necessity of choosing a breathing system filter with a high filtration performance remains controversial. (Wilkes, Benbough et al. 2000)

[0066] The biggest challenge for a breathing mask material is to accommodate the large volume of air of varying temperature and relative humidity (3M 2020). The inhaled streams are geography dependent, but the exhaled streams are at about 37° C and relative humidity above 70% (3M 2020). Besides, most of the masks have to balance inhalation/exhalation resistance with filter efficiency. As a result, only a few filtering face piece respirators are legally adequate.

[0067] It is, therefore, our aim to look to Nature as an inspiration for the fabrication of viral filters.

[0068] Lessons from Nature - Silks providing natural optimized dipolar filter materials.

[0069] Many biological systems use the attraction between oppositely charged objects. This charging facilitates, for example, the landing of pollen on the stigma during pollination by wind (Bowker and Crenshaw 2007), spiderlings ballooning (Morley and Robert, 2018), and, the geometrical layering of threads in the silkworm cocoons that allows respiration while filtering any pathogens (Horrocks, Vollrath et al. 2013).

[0070] More relevant to the present proposal are the spiders' capture thread mechanisms. Vollrath and Edmonds (2013) demonstrated how the viscid (wet) orb-web capture threads use the mobility of electrostatic charges to capture airborne particles 35. (Kronenberger and Vollrath 2015), as well as (Elettro, Neukirch et al. 2016)demonstrated how the dry cribellate silk capture threads are charged and use electrical charges. Joel et al. found that the cribellate silks attracts positive and negative charges and acts more like a dipole (Joel and Baumgartner, 2017). But even single, dry spider safety -line silk threads collect micron and sub-micron sized airborne particles 35 (Vollrath unpublished data, as shown in Fig. 5B), [0071] In the viscid capture system, the spiders simply avoid the glue-like threads. In the latter, however, neither a compensatory behaviour nor an attraction between spiders and their capture threads were observed. This suggested that spiders cannot employ a permanent larger charge during capture or dragline thread production ((Hawthorn and Opell 2003, Elettro, Neukirch et al. 2016, Joel and Baumgartner 2017 and Vollrath unpublished data).

[0072] A direct consequence of such dipolar surfaces is that they are not affected by processing and environment (humidity and temperature). There is instead a dependence on the silk chemical and structural composition.

[0073] Recently, Hegemann et al. produced some new plasma polymer surfaces with a dipolar nature. They demonstrated that nanoconfmed water could orient and cause long- range dipolar interactions with biomolecules (Hegemann, Hocquard et al. 2017). Earlier, Manciu et al. provided a theoretical framework for the polarization of water near dipolar Surfaces ((Manciu and Ruckenstein 2005). Unfortunately, plasma polymer surfaces are difficult to produce and expensive.

[0074] The selection and layering of a silk fibre can be used as a dipolar trap to concentrate the virus particles 35 for a cost-effective point of care detection. Surfaces with varying electrostatics can match the electrostatic nature and variation of the single viruses (Karlin and Brendel 1988, Hernando-Perez, Cartagena-Rivera et al. 2015, Maginnis 2018) as well as the airborne droplets 33 (Verreault, Moineau et al. 2008). There is strong evidence that the natural system of the spider’s web is excellent at capturing some airborne particles 35 (Hose, M. et al. 2002, Rutkowski, Rybak et al. 2018) and Vollrath (unpublished data) and apparently in some cases as good as commercial filters (Ling et al., 2016; Min et al., 2018; Vollrath unpublished data).

[0075] Introduction to the system 10 and method 100 for filtering of the fluid flow 15 and detection of the neuraminidase 40 in the fluid flow 15. The system and method for detection of the neuraminidase 40 in the fluid flow 15 is based on a generic biosensor 50 design comprising a bioelement 55 and a transducer 60, as shown in Fig. 6. In the biosensor 50, the bioelement 55 recognizes the neuraminidase 40 and the transducer 60 reports a presence of the neuraminidase 40 in the fluid flow 15, for example by a change in colour of the filter system 10. The fluid flow 15 is, for example, a flow of an aqueous solution. This aqueous solution can be a bodily fluid such as blood or urine. The fluid flow 15 can also be a flow of gas such as, for example, oxygen or air.

[0076] Relevant factors for the interaction between the virus and the bioelement 55 for a successful reaction are specific binding, efficient binding, strong binding, long-lasting binding (ideally irreversible), and robust binding (insensitive to environmental conditions). There are two main glycoproteins — hemagglutinin (HA) and neuraminidase (NA) on the surface of a virus, which are responsible for the invasion of the virus and the release of offspring virus, respectively (Maginnis 2018). Remarkably, over 10 amino acid residues of NA's active site are highly conserved. Hence, NA is an attractive target for anti-influenza research (Bhowmik, Nandi et al. 2020).

[0077] Bioelement.

[0078] The rationale of the present system 10 and method 100 stem from traditional antiviral drug development. Generally, antiviral drug therapies are designed to reduce the virus replication rate and thus the total virus load to ease symptoms caused by infection. The drug itself cannot destroy the virus to cure the disease, which requires the involvement of the humoral immune system. But the drug is highly effective and specific to its target. In the present case, the natural choice (i.e. highly conserved active site) of a surface target is neuraminidase (NA).

[0079] Transducing mechanism.

[0080] Transducing mechanisms are as crucial as bioelement 55. Typically, the reporting methods are optical, mass, thermal, and electrochemical (Vigneshvar, Sudhakumari et al. 2016). For simplicity, scalability, and robustness, we focused on a direct colourimetric and fluorescent reporter. In principle, the virus interaction with the bioelement 55 should be visually detectable, e.g., change of colour (Zhao, Wong et al. 2020). Figs. 7A-7C illustrate the three transducer mechanism considered. Figure 7A, Polydiacetylene (Qian and Stadler 2020), carbon dots (Zhang and Yu 2016, Ting, Dong et al. 2018) and molecular rotors (Kato, Kawaguchi et al. 2010). Figs. 7A-7C also lay out the two challenges to solve for successful fabrication and integration of the biosensor 50 with silk. The first challenge is the recognition with the bioelement 55. The second challenge is the immobilisation of the transducing element in the silk.

[0081] PDA - poly diacetylene. [0082] Figure 8 shows a photo-polymerisation of diacetylene lipid monomer Polydiacetylenes (PDA) which are conjugated polymers produced by UV (254nm) free radical polymerisation of lipid diacetylene monomers and that have a self-assembling property. Their microstructures were first reported by Yager et al. (Yager and Schoen 1984). For polymerisation, diacetylene monomers need to be well-aligned. This alignment is achieved by molecular self-assembly of the diacetylene monomers. The self-assembly process often generates interesting nanostructures, such as nano- and micro-tubes and micelles of lipidic diacetylenes. The lipid is further functionalised by the Ri and R2 groups (see also Fig. 11). Fig. 9 shows a Diacetylene monomers' nomenclature. The nomenclature is based on the number of carbons in each section, including the hydrophobic tail and hydrophilic head groups. Fig. 10 shows diacetylene amphiphiles with a polar head group and diacetylene tail(s) form various self-assembled structures that can be photo-polymerised. [0083] Final design.

[0084] Various approaches to fabricate a viral-detection system that is simple, cheap, and quick have been analysed. Fig. 11 shows an illustration of the concept for the virus- responsive silk fibre. The polydiacetylene polymer (PDA) embedded within the silk fibres changes colour upon stimulation caused by virus-ligand interactions.

[0085] Materials.

[0086] Spider silks collected directly from Nephila eduHs. Araneus diadematus and Uloborus plumipes (in our animal collections) and silkworm silks collected from Bombyx mori (Mulberry silkworm), Antheraea pernii (Tussar silkworm), Sarnia ricini (Eri silkworm), and Antheraea assamensis (Muga silkworm). Polystyrene beads commercially obtained from Sigma Aldrich. A range of dyes commercially obtained from Sigma Aldrich. Diacetylene monomers 10,12-pentacosadiynoic acid (PCD A), linker - 2,2'- (Ethylenedioxy)bis(ethylamine), neuraminidase 40, EDC, NHS, periodate, silk yarns. [0087] Methods.

[0088] Fibres electrostatics. The particle 35 adherence to a wide range of silks (with differing surface charges) as a function of material and conditions is analysed. An electrostatic induction mill is used for the measurement of e-charges. The analysis focuses on passing charged nano-size particles 35 by threads of silk and study their path and accumulation on the thread. The stability of the particles 35 on the silk is examined as an estimate for re-infection due to release from the silk. The filaments are packed into a non- woven sheet as well as woven into a very fine-meshed textile and their performance as filter medium examined.

[0089] Filter characterizations.

[0090] The surface morphology of the filters is characterized by electron microscopy. The chemistry is measured by Fourier transform infrared spectroscopy. The surface charges are characterized by potentiometric titration and calculation of the pK spectrum identifies the point of zero charges (PZC), which describes the pH value at which no surface charge is present on the material; and, the isoelectric point (IEP), which represents a pH value at which a molecule on the surface of the material has no net electrical charge. Note that the filter is for analytical purposes and part of a standard filter.

[0091] Model system.

[0092] Because of the infection risk, standard polystyrene beads are used. The polystyrene beads sizes vary from the standard 50 nm to a 500 nm. The polystyrene beads are functionalized with a dye molecule (e.g. Alexa or Uranine/Fluorescein salt) for fluorescence detection. The focus is on beads with a net negative charge. To mimic the complex environment of the airborne particles 35, the polystyrene beads are made in solutions of increasing complexity: from pure water to solution that mimic mucus (Anwarul Hasan et al., 2010). In addition, non-toxic and non-infectious pseudo typed SARS2-CoV-2 virus particles 35 are used for further in-depth analysis of the filter effectiveness and efficiency, using luciferase base assays.

[0093] Aerosol generation.

[0094] The polystyrene beads and other test particles 35 are nebulized, in a custom design nebulizer, inside a high-quality flow-cabinet. Guided by Zhang et al cough simulator (Zhang et al., 2017), with bimodal particle 35 sizes being nebulized. The nebulized particles 35 are directed to the filters at a selected temperature and relative humidity. Of interest are the relative humidity values of 30% (low), 50% (medium) and 80% (high). The fluorescent colloidal particles 35 in the fluid flow 15 are shot at the material at 30 liters/min (air speed for normal respiration) and 85 liters/min (air speed for increased respiration). For convenience, a Wetsys system (Setaram) is used to produce continuous air flows at controlled temperatures and humidities. A split flow system enables a reference stream and the filtered stream to be collected and compared. The number of particles 35 is measured using a laser light diffraction system. [0095] Detection of model nanoparticles.

[0096] Silk filters or parts after exposure are characterized as follows: morphology, light transmission attenuation, fluorescence microscopy and spectroscopy.

Examples

[0097] Description of the method 100 for impregnating silk fibres 30 with a biosensor 50 as shown in Fig. 20.

[0098] Neuraminidase assay.

[0099] Neuraminidase assay was created by adding 90 uL of pre-heated (37°C) 4MU- NANA substrate to 10 uL neuraminidase solution (step S300). The neuraminidase assay was incubated at 37°C for 30 minutes. The neuraminidase assay was quenched with 1.0 mL Glycine-NaOH buffer (0.2 M, pH 10.6) to stop the reaction and increase the fluorescence of the neuraminidase assay. The fluorescence emission was measured on a fluorimeter with excitation at 365 nm and emission at 445 nm. The neuraminidase assay refers a characterization of a purchased enzyme. It is, for example, determined, if the neuraminidase is active. It is further determined under which conditions the neuraminidase assay becomes active. This determining comprises, for example, the determining of the pH value of the neuraminidase or the temperature of the neuraminidase. The determining can be done using a standard procedure available from Sigma Aldrich (Neuraminidase assay kit, catalogue number MAK121).

[00100] Immobilisation of neuraminidase.

[00101] The neuraminidase enzyme was immobilised (step S310) on an amine functionalised polystyrene (PS) of 50 nm in diameter. A stock solution of PS beads was diluted to 3.125 mg/mL, where glutaraldehyde was added to 10 mM. The neuraminidase assay was incubated for 2 hours at room temperature. After that, the neuraminidase assay was added to a concentration of 1.55 mg/mL, and the sample was left to incubate for an additional 2 hours. The solution was dialysed against 12 kDa filter overnight to remove unreacted glutaraldehyde.

[00102] Synthesis of PDA vesicles.

[00103] Design of the assay comprises the synthesis of PDA in step S320. This synthesis was carried out according to (Tang, Weston et al. 2020) with modifications. 4 mg of PCDA was dissolved in 0.5 mL of absolute ethanol. Larger quantities of PCDA can also be added up to the maximum solubility of the PCDA in a suitable organic solvent such as, for example, ethanol. Ethanol can be used for reasons of low toxicity and cost effectiveness. To completely dissolve PCDA, a solution comprising the PCDA was sonicated for 15 minutes. The solution comprising the PCDA, also referred to as “PCDA solution”, was then added to 10 mL of distilled water at 85°C under rapid stirring (1000 rpm). After the addition of PCDA, the PCDA solution was maintained at 85°C under stirring for 30 minutes to allow the ethanol to evaporate (cover to avoid excess evaporation). A different temperature and/or stirring time may be used when relying on a different solvent than ethanol. The evaporated liquid was replaced with distilled water. The self-assembled PCDA vesicles in water were allowed to cool to room temperature thereby creating a PCDA vesicle solution. The PCDA vesicle solution was spread out on a glass petri dish and exposed to a preferred UV irradiation of 254 nm for a preferred duration of the UV irradiation of 20 minutes to obtain blue-phase PDA. The UV irradiation may be in a range from 254 nm to 400 nm. The duration of the UV irradiation may be in a range from 1 minute to 6 hours.

[00104] Impregnation of silk with PDA.

[00105] Three threads of Silk, more specifically Eri-silk, were rinsed with distilled water to rinse/wash out sericin from the Eri-silk. After removing the water, the Eri-silk threads were transferred to the PDA solution). The silk was incubated with PDA on a shaking board for 1 hour, allowing the Eri-silk fibres to absorb the PDA vesicles. After that, the coloured Eri-silk fibres were rinsed repeatedly with distilled water. The wet threads were kept in containers until further modification. This step is also referred to as impregnation of the Eri-silk fibres (step S330).

[00106] Oxidation of sialic acid.

[00107] The biolement 55 (sialic acid) was attached to the PDA micelles. This so- called “functionalisation” makes the biosensor system 55 analytical. Sialic acid was dissolved in 0.1 mL phosphate buffer (0.1 M, pH 7.4) to a final concentration of 50 mM. A periodate solution was prepared to contain 11 mM sodium periodate in phosphate buffer. A volume of 10 pL of the periodate solution was added to the sialic acid solution, and the sample was allowed to incubate at 4°C for 30 minutes.

[00108] Attachment of ligands to PDA. EDC and NHS were added to MES buffer (0.1 M, pH 5.5) to a final concentration of 100 mM each. 1 mL of the EDC/NHS solution was then added to 3.5 mg Eri-silk threads containing PDA (step S340). The Eri-silk threads were incubated (step S350) in the EDC/NHS solution for 30 minutes under shaking to activate the PDA carboxyl groups. After activation of the incubate Eri-silk threads, the incubated Eri- silk threads were rinsed with distilled water. The wet rinsed Eri-silk threads, also called “NHS-activated PDA-silk threads”, were then incubated with various ligands (Oseltamivir, Zanamivir, or sialic acid) as described below. The attachment of other mole molecules is, however, also possible. If other molecules are attached, the attachment chemistry must be tuned accordingly.

[00109] Oseltamivir.

[00110] The NHS-activated PDA-silk threads were incubated in a 1 mL solution containing 0.01, 0.1, or 5 mM of Oseltamivir in sodium phosphate buffer (0.1 M, pH 7.4). The crosslinking reaction was carried out under shaking overnight at preferably 4°C A temperature of up to 10°C might also be used. Finally, the NHS-activated PDA-silk threads containing Oseltamivir were washed thoroughly with distilled water and dried at room temperature.

[00111] Zanamivir.

[00112] The NHS-activated PDA-silk threads containing Oseltamivir were incubated in 1 mL solution containing 0.01, 0.1, or 5 mM of Zanamivir in sodium phosphate buffer (0.1 M, pH 7.4). The crosslinking reaction was carried out under shaking overnight at 4°C. Finally, the NHS-activated PDA-silk threads containing Oseltamivir and containing Zanamivir were washed thoroughly with distilled water and dried at room temperature.

[00113] Sialic Acid.

[00114] The NHS-activated PDA-silk threads containing Oseltamivir and/or containing Zanamivir were incubated in 1 mL solution containing 1 mM of 2,2'- (Ethylenedioxy)bis(ethylamine) in sodium phosphate buffer (0.1 M, pH 7.4). The ratio of the ligand to the PDA micelles should be less than 1 : 1 for optimal performance. The crosslinking reaction was carried out under shaking for 30 minutes at room temperature. After that, oxidised sialic acid was added. The reaction was allowed to proceed at least 10 hours and no more than 24 hours at 4°C. Finally, the NHS-activated PDA-silk threads containing Oseltamivir, Zanamivir, and sialic acid were washed thoroughly with distilled water and dried at room temperature.

[00115] Incubation of functionalized silk with neuraminidase. [00116] Single NHS-activated PDA-silk threads containing Oseltamivir, Zanamivir, and sialic acid were incubated with 100 pg/mL Neuraminidase for 30 minutes at 37 °C at either pH 7 or pH 5. Oseltamivir, Zanamivir, and sialic acid are also referred to as “ligands”. The colours red and blue were measured and analysed using RBG-tool in Image!

[00117] Further work is likely to reveal for example that virus from table 1 can be detected by targeting specific interactions with ligands.

[00118] Surface properties and interaction modelling can be assessed based on experimentally measured pK spectrum and morphology. The thus modelled surface is tested for its properties to capture and hold selected particles 35. The modelling is done using monte Carlo simulation, as found on the Faunus software (Stenqvist et al., 2013). This approach allows for continuous upgrading of the filter properties and thus improvements of the quality of its analytical properties.

[00119] Results.

[00120] Neuraminidase activity.

[00121] Fig. 12A-12D show a characterisation of neuraminidase from C. perfringens; effect of a) protein concentration (Fig. 12A), b) substrate concentration (4MU-NANA) (Fig. 12B), c) pH (Fig. 12C), and d) pH without enzyme (Fig. 12D). This characterisation allows to determine if the enzyme is still active in a defined range for the pH and the concentration of the enzyme.

[00122] Properties of PDA.

[00123] Fig. 13A-13B illustrate the preparation of the PDA micelles (Fig. 13A) and their response to stimuli (Fig. 13B). Note that PDA is sensitive to numerous stimuli (Reppy and Pindzola 2007).

[00124] Properties of silk impregnated with PDA.

[00125] Fig. 13 illustrates the PDA uptake/absorption by Eri-silk. Note that extensive washing did not yield any leakage (checked by UV-visible absorption of the wash solution). The Eri-silk absorbs the blue PDA particles. Before the absorption, the liquid containing the PDA is blue. The blue micelles are absorbed by the Eri-silk leaving behind a clear solution. [00126] Response of functional Eri-silk fibres to stimuli (a-cyclodextrin).

[00127] Fig. 15A and 15B illustrate the proof of principle of the stimuli-responsive PDA/silk system. The change of colour was triggered by increasing the concentration of a- cyclodextrin (top panel, Fig. 15 A). The scanned image (top panel, Fig. 15 A) is analysed for blue/red content on the lower panel (Fig. 15B). The plot suggests a step response with an activation concentration at approximately 0.75 to 1 mM of a-cyclodextrin. Additionally, the change of colour can also be observed by fluorescence spectroscopy or imaging. The reacted PDA/silk system fluoresces in the red to near-infrared region. [00128] Response of functional Eri-silk fibres to neuraminidase.

[00129] In this section, the complete sensor (modified PDA in silk) is tested against neuraminidase 40. Note that the ligand and inhibitor used are specific to the human viral neuraminidase. The neuraminidase 40 has a significantly lower specificity. Table 2 shows various conditions tested for producing silk-PDA crosslinked to ligand/inhibitor. Table 2: Various conditions tested for producing silk-PDA crosslinked to ligand/inhibitor.

[00130] Response at pH5.

[00131] Figs. 16A and 16B show the response to neuraminidase at pH5. Zanamivir modified PDA changed from blue to purple/red. More specifically, Fig. 16A and 16B show silks containing PDA with various inhibitors attached in different quantities; all samples were incubated with 100 pg/mL Neuraminidase for 30 minutes at 37 °C and pH 5; a) shows the measured silk threads, and b) shows the visual appearances of single wet silk threads from' Control' and' Zanamivir (Medium)' samples.; the colours red and blue were measured using RBG-tool in ImageJ; SA = Sialic acid (oxidised), OS = Oseltamivir, ZA = Zanamivir. [00132] Response at pH7.

[00133] Figs. 17A and 17B show the response to neuraminidase at pH7. None of the modified PDA showed a colour change. More specifically, Figs. 17A and 17B show silks containing PDA with various inhibitors attached in different quantities; all samples were incubated with 100 pg/mL Neuraminidase for 30 minutes at 37 °C and pH 7; the colours red and blue were measured using RBG-tool in ImageJ; SA = Sialic acid (oxidised), OS = Oseltamivir, ZA = Zanamivir

[00134] Response of functional silk fibres to neuraminidase immobilised on 50 nm polystyrene beads.

[00135] Figs. 18A and 18B illustrate a preliminary attempt to mimic the virus by immobilising the neuraminidase 40 on a polystyrene bead of the approximate virus size (preferably 50 nm). The visual inspection showed no noticeable colour change, but the image analysis suggests a slight increase in redness. More specifically, Figs. 18A and 18B show silks containing PDA with Zanamivir attached; The 'Immob NA' sample was incubated with 3 mg/mL of 50 nm polystyrene beads containing immobilised neuraminidase (NA) for 30 minutes at 37°C and pH 5; the 'Control' sample was incubated with polystyrene beads only (without enzyme) at the same concentration; the colours red and blue were measured using RBG-tool in ImageJ; SA = Sialic acid (oxidised), OS = Oseltamivir, ZA = Zanamivir.

[00136] Conclusion.

[00137] A system 10 and method 100, 150 to fabricate PDA micelles, functionalise them with relevant bioelement 55 and eventually immobilise the functional micelles into silk fibres 30 has been developed. The obtained biosensors 50 change colour in the presence of neuraminidase 40.

[00138] Fig. 19 shows a flow chart further describing the method 100 for filtering the fluid 15 and detecting neuraminidase 40 in the fluid 15 using impregnated silk fibres 30. The method 100 comprises the steps of providing in step S200 a filter system 10 in the fluid 15, recognizing in step S210 the neuraminidase 40 in the fluid 15 with a bioelement 55 of the silk fibre 30, and reporting in step S220 the presence of the neuraminidase 40 in the fluid 15 using a transducer 60. [00139] Fig. 20 shows a flow chart describing the method 150 for impregnating silk fibres 30 with a biosensor 50. The method comprises reacting in step S300 pre-heated 4MU- NANA substrate with a neuraminidase solution 75. The method further comprises immobilising in step S310 the neuraminidase solution 75 on an amine functionalised polystyrene (PS). The method also comprises the synthesising in step S320 polydiacetylene (PDA) vesicles using a solvent injection method, impregnating in step S330 the silk fibres (30) with the synthesised polydiacetylene (PDA) vesicles, attachment in step S340 of ligands to the impregnated silk fibres 30, and incubating in step S350 the synthesised polydiacetylene (PDA) vesicles in Oseltamivir in at least one of a sodium phosphate buffer, a Zanamivir in sodium phosphate buffer, and a 2,2'-(Ethylenedioxy)bis(ethylamine) in sodium phosphate buffer.

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Reference numerals

10 filter system

15 fluid 16 feed air

20 filter membrane

30 silk fibre

33 droplet

35 particles 40 neuraminidase

45 marker

50 biosensor

55 bioelement

60 transducer 75 neuraminidase solution