FIELD OF THE INVENTION

The present disclosure relates to the field of detection of biological molecules. More particularly, the present invention relates to methods of detecting target biological molecules in a sample with the use of catalytic nucleic acid molecules and gold nanoparticles.

BACKGROUND OF THE INVENTION

The development of simple, cost-effective, sensitive, and specific point-of-care (POC) diagnostics represents a major challenge in the 21 st century. [1 ] The spread of infectious diseases has caused significant losses in global economy and life. A method to control the spread of diseases is to detect the pathogens early at POC, and to administer proper treatment or quarantine. [2] Detection of DNA as biomarkers is currently a widely used technique for pathogenic detection. [3] Quantitative PCR (qPCR) is the method of choice, since it includes an enzymatic signal amplification step to achieve highly sensitive and specific detection of genetic targets. [4] However, qPCR requires expensive equipment and highly trained personnel, limiting the technique use to medical laboratories. There is a strong need to develop cheaper and simpler methods for detecting DNA targets and/or other biological targets. [5]

DNAzyme is a synthetic DNA enzyme that can catalyze the cleavage of another nucleic acid molecule. [6-8] Since the catalysis is carried out with multiple turnover, DNAzyme introduces an enzymatic amplification step into the experimental setup. [9] This amplification is performed without the need for protein components, which are costly and have low thermal and storage stability. While initially designed to detect Pb ²⁺ and other divalent cations, [10- 19] a number of modifications have been reported that allow DNAzyme to detect biomolecular targets. [9,20-27] Two main approaches were used to implement detection of genetic targets. The first method utilizes competitive activation of peroxidase-mimicking DNAzyme, which can catalyze production of colorimetric or chemiluminescent product. [28] In the second, more sensitive approach, a similar strategy was followed by a number of groups to implement detection of genetic targets by splitting the DNAzyme into inactive components, which could be reactivated by the target binding. [9, 23-25, 29] However, these studies used fluorescence as the readout, which is not an optimal detection modality for POC applications since it requires access to fairly complex fluorometer apparatus. An alternative method using the colorimetric readout of gold nanoparticles (GNPs) has been reported for detection of metal ions, adenosine and cocaine. [10-14, 16-18, 20, 21 ] The wavelength at which GNPs absorb light is dependent on whether they are in a monodispersed or aggregated state. [30] Since the GNP solutions in monodispersed or aggregated states are easily distinguishable by their respective red or purple colors, this approach provides clear colorimetric results that can be visualized by the naked eye. [31 ]

As such, an object of the invention is to overcome the above limitations by combining DNAzyme technology with GNPs to engineer a simple point-of- care diagnostic platform that can be used in remote settings. [32, 33]

Further and other objects of the invention will be realized from the following Summary of the Invention, the Discussion of the Invention and the embodiments and Examples thereof.

SUMMARY OF THE INVENTION

The present invention relates to the combined colorimetric coupling of surface plasmons of gold nanoparticles with DNAzyme signal amplification technology to engineer a fast and simple detection platform for genetic targets with a simple colorimetric readout. The inventiveness of this disclosure includes the integration of these two emerging technologies for detection of biological targets that may be used for point-of-care analysis of infectious disease targets.

As such, in one embodiment, the present invention provides for a method of detecting the presence of a biological target in a sample. The method, in one embodiment, includes: (a) contacting the sample with: (i) an un-catalyzed nucleic acid substrate, (ii) a catalytic nucleic acid, the catalytic nucleic acid configured for catalyzing the substrate solely in the presence of the target, and (iii) GNPs, the GNPs functionalized with a linking moiety for assembling with the un-catalyzed nucleic acid substrate; and (b) analyzing the sample to determine whether the GNPs are in a substantially dispersed form or in an assembled form with the un-catalyzed nucleic acid substrate, wherein the GNPs being in the substantially dispersed form is indicative that the target is present in the sample.

In one embodiment of the method for detecting a biological target in a sample, (i) and (ii) are first added to the sample for a sufficient amount of time at a suitable temperature, and then (iii) is added to the sample.

In another embodiment of the method for detecting a biological target in a sample, the catalytic nucleic acid are provided as pre-catalytic nucleic acid subunits, each pre-catalytic nucleic acid subunit including a sensor domain configured for binding to at least a portion of the target, and a catalytic domain which catalyzes the un-catalyzed nucleic acid substrate solely when the target is bound to the sensor domain.

In another embodiment of the method for detecting a biological target in a sample, the GNPs are functionalized with nucleic acid strands that hybridize with a 3' end and a 5' end of the un-catalyzed nucleic acid substrate for assembling.

In another embodiment of the method for detecting a biological target in a sample, in the assembled form the GNPs turn the sample to a first color, and in the substantially dispersed form the GNPs turn the sample to a second color, and wherein step (b) of the method includes determining the color of the sample. Detecting the second color is indicative of the presence of the target in the sample.

In another embodiment of the method for detecting a biological target in a sample, the un-catalyzed nucleic acid substrate, the catalytic nucleic acid and the GNPs are functionalized to be target specific.

In one embodiment, the present invention provides for a method of simultaneously detecting the presence of a number of different biological targets of interest in a sample. In one embodiment, the method includes: (a) mixing the sample suspected of having the number of different biological targets with target-specific sets of (i) target-specific un-catalyzed nucleic acid substrates and (ii) target-specific catalytic nucleic acids, the catalytic nucleic acids in each target-specific set configured for catalyzing the un-catalyzed substrate solely in the presence of the target; (b) contacting the mixture of step (a) with separate target-specific populations of GNPs, the GNPs in each target-specific population functionalized with a linking moiety for assembling with its corresponding target-specific un-catalyzed nucleic acid substrate; and (c) determining for each separate target-specific population of GNPs whether the target-specific GNPs are in an assembled form with the corresponding target specific un-catalyzed substrate or in a substantially dispersed form, wherein the target-specific GNPs being in the substantially dispersed form in one population is indicative that the target corresponding to this population of target-specific GNP is present in the sample.

In one embodiment of the method of simultaneously detecting the presence of a number of different biological targets of interest in a sample, the catalytic nucleic acid are provided as pre-catalytic nucleic acid subunits, each pre- catalytic nucleic acid subunit including a sensor domain for specifically binding to one of the number of different targets, and a catalytic domain configured for catalyzing its corresponding target-specific un-catalyzed nucleic acid substrate solely when the corresponding target is bound to the sensor arm.

In another embodiment of the method of simultaneously detecting the presence of a number of different biological targets of interest in a sample of the present invention, in the assembled form the GNPs turn the sample to a first color, and in the substantially dispersed form the GNPs turn the sample to a second color, and wherein step (c) of the method includes determining the color of the sample for each population of GNPs. Detecting the second color in one population of GNPs is indicative of the presence of the target of this population of GNPs in the sample.

In one embodiment of the method of any the previous embodiments, the method further includes spotting the sample onto thin layer chromatography (TLC) plates for visual detection. In one aspect of this embodiment, the method further includes storing the TLC for later viewing. In another embodiment of the method of any the previous embodiments, the method further comprises a spectroscopic measurement of the UV-visible spectroscopic shift of peak absorbance.

In another embodiment of the method of any the previous embodiments, the catalytic nucleic acid and the un-catalyzed nucleic acid substrate are provided as a lyophilized mixture.

In another embodiment of the method of any the previous embodiments, the catalytic nucleic acid, the un-catalyzed nucleic acid substrate and the GNPs are provided lyophilized.

In yet another embodiment, the present invention provides for a kit for detecting a biological target of interest in a sample. The kit, in one embodiment, includes: (a) an un-catalyzed nucleic acid substrate, (b) catalytic nucleic acid capable catalyzing the un-catalyzed substrate solely in the presence of the target, and (c) GNPs functionalized with a linking moiety to assemble with the un-catalyzed nucleic acid substrate.

In one embodiment of the kit, the catalytic nucleic acid are provided as pre- catalytic nucleic acid subunits, each pre-catalytic nucleic acid subunit including a sensor domain for binding to at least a portion of the target, and a catalytic domain which catalyzes the un-catalyzed nucleic acid substrate solely when the target is bound to the sensor domain.

In another embodiment of the kit, GNPs assembled with the un-catalyzed nucleic acid substrate turn the sample to a first color, and GNPs in a substantially dispersed form turn the sample to a second color.

In another embodiment of the kit, the un-catalyzed nucleic acid substrate and the catalytic nucleic acid are in a lyophilized mixture.

In another embodiment of the kit, the GNPs, un-catalyzed nucleic acid substrate and the catalytic nucleic acid are in a lyophilized mixture.

In another embodiment of the kit, the kit further includes ingredients necessary for optimal activity of the catalytic arm.

In another embodiment of the kit, the kit further includes a TLC. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, with reference to the drawings, in which:

Figure 1 : Panel A - Mechanism of MNAzyme catalysis. Adapted with permission from [9]. Copyright 2009 American Chemical Society. Panel B - GNP aggregate formation. Linker hybridizes to DNA strands A and B, cross- linking the two sets of GNPs and turning solution purple. Panel C - MNAzyme assay outline. Target activated MNAzyme degrades Linker DNA, preventing GNP aggregate formation. In the absence of the target Linkers remain intact and cross-link GNPs (as in B); the solution turns purple. Panel D - Schematic depicting how the assay will be conducted at point-of-care to analyze multiple targets in parallel.

Figure 2: Panel A - Optimization of Linker! concentration. Panel B - Sensitivity of detecting AF-1 genetic target. For panels A and B - Top: colorimetric readout on TLC plate. Bottom: shift in the peak absorbance. Data based on 3 replicates; error bars are standard errors. Panel C - Parallel detection of 5 targets: AF-1 , genetic sequences from N. gonorrhoeae (Gon) and T. pallidum (Syph) bacteria, malarial parasite P. falciparum (Mai), and HBV virus. Detection of all 5 targets (top row), 3 out of 5 targets (center row), or no-target control (bottom row). Spot color indicates target presence (large grey spots) or absence (small black spots). Images were adjusted for contrast and brightness.

Figure 3: Graph illustrating DNAzyme signal amplification combined with surface plasmon properties of gold nanoparticles to achieve colorimetric detection of biological targets in accordance to one embodiment of the present invention.

Figure 4: Top: colorimetric readout on TLC plate. Bottom: shift in the SPR peak absorbance. Data based on 3 replicates; error bars are standard errors. DETAILED DESCRIPTION OF THE INVENTION

Definitions

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the meanings below. All numerical designations, e.g. , dimensions and weight, including ranges, are approximations that typically may be varied ( + ) or ( - ) by increments of 0.1 , 1 .0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term "about".

The singular form "a", "an", and "the" includes plural references unless the context clearly dictates otherwise.

The term "comprising" means any recited elements are necessarily included and other elements may optionally be included. "Consisting essentially of" means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of" means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

"Catalytic nucleic acid molecule", "catalytic nucleic acid", and "catalytic nucleic acid sequence" are equivalent, and each shall mean a DNA molecule or DNA-containing molecule (also known in the art as a "DNAzyme") or an RNA or RNA-containing molecule (also known in the art as a "ribozyme") which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate. The nucleic acid bases in the DNAzymes and ribozymes can be the bases A, C, G, T and U, as well as derivatives thereof known in the art [PCR Systems, Reagents and Consumables. Perkin Elmer Catalogue 1996-1997. Roche Molecular Systems, Inc., Branchburg, N.J., USA.]. Each catalytic nucleic acid molecule may be divided into pre-catalytic nucleic acid subunits that are not capable of catalyzing the chemical modification of the substrate.

"GNP" or "GNPs" refer to gold nanoparticle(s).

"GNP-A" refers to a GNP functionalized with a linking moiety A, which may be a nucleic acid strand A, as explained bellow. "GNP-B" refers to a GNP functionalized with a linking moiety B, which may be nucleic acid strand B, as explained bellow. "GNP-AB" refers to a GNP functionalized with both linking moieties A and B, which may be nucleic acid strand A and nucleic acid strand B, as explained bellow. Accordingly, at least three populations of GNP may exist: GNP-A, GNP-B and GNP-AB. Other linking moieties may also be used.

"Reporter substrate", "chemical substrate" and "substrate" are equivalent, and each refers to any molecule which is specifically recognized and modified by a catalytic nucleic acid molecule.

"Target" refers to a biological target, including, nucleic acid sequences (DNA and RNA), peptides, proteins, polysaccharides, fatty acids, toxins, phages, antibodies and so forth. The target of interest to be detected or measured by the instant invention, which comprises a sequence that hybridizes with a target domain, also referred to as sensor domain, of the catalytic nucleic acid molecule when contacted therewith in this method, and that can be either an entire molecule or a portion thereof.

In one embodiment, the present invention relates to a method of detecting a molecular target in a sample. The method may include: (a) contacting the sample with: (i) an un-catalyzed nucleic acid substrate, (ii) a catalytic nucleic acid, the catalytic nucleic acid configured for catalyzing the substrate solely in the presence of the target, and (iii) GNPs, the GNPs, which may be provided in a substantially dispersed form (for the purpose of this document, "substantially dispersed" includes "dispersed"), may be functionalized with a linking moiety for assembling with the un-catalyzed nucleic acid substrate; and (b) analyzing the sample to determine whether the GNPs remain substantially dispersed or are assembled with the un-catalyzed nucleic acid substrate, wherein the GNPs being in the substantially dispersed form is indicative that the target is present in the sample.

The GNPs may be functionalized with a nucleic acid strand A and/or a nucleic acid strand B. For example, 5'-thiol-modified Agg/A (Strand A) or 3'- thiol-modified Agg/B (Strand B) DNA. [34] A linker nucleic acid strand may include a substrate region for a catalytic nucleic acid molecule, and it may be designed to have its 5' -end complementary to 3' of Agg/A, and its 3'-end to complement 5' of Agg/B. The linker in its un-catalyzed form may then serve to cross-link GNPs through hybridization with strands A and B, forming GNP aggregate or assembled network. When the linker is cleaved, it may no longer serve to cross-link GNPs.

Figure 1 illustrates an embodiment of an experimental setup. In Figure 1 B, two sets or populations of GNPs, GNP-A and GNP-B are functionalized with a nucleic acid strand A and strand B, respectively. However, as explained in the previous paragraph, one population of GNPs having strands A and B may be used. For example, 5'-thiol-modified Agg/A (Strand A) or 3'- thiol-modified Agg/B (Strand B) DNA, respectively. A linker nucleic acid strand may be designed to have its 5'-end complementary to 3' of Agg/A, and its 3'- end to complement 5' of Agg/B. The linker may serve to cross-link GNP-A and GNP-B through hybridization, forming GNP aggregate or assembled network (Figure 1 -B).

Close proximity of linked GNPs may cause coupling of their individual localized plasmon fields, leading to the shift in their peak absorbance to a longer wavelength, and solution color changes from red to purple. In addition, the linker strand may also include a substrate sequence that may be cleaved by a catalytic nucleic acid molecule, for example a multicomponent nucleic acid enzyme (MNAzyme), which is one of the reported DNA-responsive DNAzymes. MNAzyme uses Mg2+ as the divalent cation, and has optimal activity at 50°C. Among the three reported DNA-responsive bipartite DNAzymes, the inventors chose to work with the MNAzyme, since it has the highest catalytic rate. [9, 24, 25, 29] In this document, MNAzymes are used as an example, however, it should be understood that other catalytic nucleic acid molecules, including other DNA-responsive DNAzymes, and DNAzymes that respond to metal ions, such as lead and uranyl, or small molecule targets, such as adenosine, FMN and ATP, may be used as well. [46-48]

As illustrated in Figure 1 B, the catalytic nucleic acid molecule initially comprises of two inactive parts or pre-catalytic subunits, Part A and Part B. Each one of Part A and B may include a domain or arm complementary to at least a different portion of a target of interest (target or sensor domain) and an domain capable of interacting with at least a portion of a substrate to be cleaved (substrate domain). Initially in the inactive state, target's binding to the sensor domains bridges pre-catalytic subunits A and B, thereby bringing parts A and B together and forming the active catalytic structure, which can then hybridize to a substrate sequence and catalyze its cleavage (Figure 1 -A).

[9] For MNAzymes, the substrate sequence may comprise DNA bases with two RNA nucleotides, which are required for cleavage, imbedded in the middle. [6]

The catalytic nucleic acid molecule may be made responsive to any biological target simply by changing the nucleotides of the sensor domains or sensor arms. The sequence of the substrate arms ensures specificity to a particular substrate, so that multiple MNAzyme-substrate pairs may be used in the same assay without cross-reactivity. Aptamers are nucleic acid molecules that bind with high specificity and sensitivity to biological targets. Aptamers may be created basically for any biological target [See U.S. Pat. No. 5,670,637]. The aptamer sequences may be incorporated in the sensor arm to bind any biological target of interest.

Referring to Figure 1 C, the sensing assay of the present invention may be implemented as a two-step system. In the first "amplification" step, a mix of catalytic nucleic acid molecules, such as MNAzymes, having a sensor domain for a desired target, and linkers (the MNAzyme substrate) may be added to a sample solution, which may be suspected of having the target of interest. The mix MNAzyme/linker may be buffered. The sample with the mix may be incubated at a suitable temperature and for a suitable time. The mix may be done under conditions suitable for permitting catalytic nucleic acid activity. The temperature and time of incubation of the mix may depend on the DNAzyme being used. If the target is present in the sample, the target may bind the sensor domains of the MNAzyme subunits and activate the MNAzymes. Each active MNAzyme may in turn catalyze cleavage of multiple linkers, which effectively translates into signal amplification. In a second "detection" step, a mix of GNP-A and GNP-B (or, alternatively GNP-AB) may be added to the sample solution. In the absence of the target intact linkers interact with the GNPs to form aggregate or assembled network, turning solution purple. In contrast, presence of a target results in linker degradation, the GNP-A and GNP-B (or GNP-AB) do not get cross-linked (i.e. remain in unassembled form), and the solution remains red. A more sensitive visual detection may be achieved by spotting the solution onto thin layer chromatography (TLC) plates. [31 ] TLC plates intensify the color differences allowing for a more sensitive visual detection, and may be stored for later viewing. A measurement of the UV-vis spectroscopic shift of peak absorbance may also be used for confirmation. [35]

For a given GNP concentration there may be enough linker concentration able to crosslink the GNPs sufficiently to cause visual aggregation. With such a linker/GNP ratio, even if a small proportion of linker strands get cleaved by the MNAzymes, this should have an effect on GNP crosslinking state and color of the solution. Therefore, minimizing the linker concentration may effectively maximize assay sensitivity. Optimization results are presented in Figure 2-A. Assay setup matched the experimental condition of the MNAzymel assay, but lacked the genetic target to prevent Linken degradation. As the amount of Linker is increased from 0 to 100 nM, there is a sigmoidal shift in the peak absorbance, APeak, from 527 nm to 546 nm, indicating increase in GNP aggregate size. The TLC spot color follows a similar, but steeper sigmoidal trend. The spots appear red up to 20 nM, then gradually shift to dark purple-black at 50 nM, and remain unchanged for higher concentrations. Based on both color change and peak shift we chose 40 nM as the optimal Linker concentration.

The MNAzyme assay outlined in Figure 1 was carried out to detect AF-

1 as the biological target (in this case a DNA target). As previously determined, the linker at 40 nM was used, and AF- 1 was assayed from 0 pM to 1 nM (Figure 2-B). Similar to Figure 2-A, spectroscopic data demonstrates a sigmoidal, but decreasing trend in APeak. Peak absorbance shifts from 533 nm for the sample without AF-1 to 523 nm for 1 nM of target. The color of TLC spots follows similar trend, remaining purple from 0 pM to 25 pM AF-1 , then gradually shifting to reach red at 500 pM and 1000 pM. Both spectroscopic and spot color data establish the limit of detection of this assay at about 50 pM. This sensitivity is slightly lower than 5 pM reported for the fluorescence readout. [9]

If the linker is chosen as the genetic detection target instead of AF-1 , the experiment of Figure 2-A matches the original MNAzyme-free direct DNA detection assay reported by Mirkin group (the inventors confirmed that the 50°C, 1 hour incubation step does not affect assay results, Figure 4). [31 ] Based on Figure 2-A, our implementation of this assay has detection sensitivity of -30 nM. Therefore, inclusion of MNAzyme enzymatic signal amplification step increases detection sensitivity by a factor of 600.

The detection methods of the present invention may be used to detect a single target or to detect multiple targets in parallel (Figure 1 -D). The inventors tested with 4 additional targets corresponding to genetic sequences for gonorrhea (Gon)[36] and syphilis (Syph)[37] bacteria, malaria parasite (Mai), [38] and hepatitis B virus (HBV). [39] These infectious diseases are prevalent in both the developed and developing world, and can cause severe health problems and death in patients that are not properly diagnosed and treated. For example, to detect four targets, four MNAzyme-Linker-GNP sets may be developed, each specific for one of the targets. For example, for malaria, MNAzymeMal may include substrate arms specific for LinkerMal, and sensor arms complementary to Mai target, but not other linker sequences or targets. LinkerMal, in turn, could aggregate GNP-AMal and GNP-BMal, but not other GNP pairs. Likewise, MNAzymeGon for gonorrhea, MNAzymeHBV for hepatitis B, MNASyph for syphilis, and so forth.

In the first step of the assay, a solution suspected of containing more than one biological targets may be added to a reaction tube containing a mixture of different sets of MNAzymes/linker pairs, each set being specific for a target of interest. For example, if the targets of interest include AF-1 , gonorrhea, syphilis, malaria and HBV, then 5 sets of MNAzyme-Linker pairs (MNAzymeAF-1 , MNAzymeGon, MNAzymeSyph, MNAzymeMal and MNAzymeHBV) may be used. The mixture and the sample solution may then be incubated at a determined temperature and for a sufficient amount of time. Assuming AF-1 and Gon targets are present in the reaction tube, they activate MNAzymeAF-1 and MNAzymeGon, which in turn degrade LinkerAF-1 and LinkerGon. The other 3 linkers (i.e. LinkerSyph, LinkerMal and LinkerHBV) remain intact. In the second step, the MNAzyme-Linker-target solution is transferred to 5 detection tubes, each containing target-specific GNP pair. Any linkers that remain un-cleaved aggregate the corresponding GNP pairs, turning solution purple. In contrast, Linkers cleaved by target- activated MNAzymes are not able to cross-link associated GNP pairs, and solution remains red. In the example above, only mixtures in AF-1 and Gon- specific detection tubes would remain red. Following this protocol all 5 targets were detected simultaneously (Figure 2-C). In addition, the assay with lyophilized components was used to correctly identify AF-1 , Mai and HBV sequences in sample mixture contained 3 out of 5 targets.

The present invention also provides for a kit for detecting a biological target of interest in a sample. In one embodiment, the kit may include: (a) an un-catalyzed nucleic acid substrate, (b) catalytic nucleic acid capable catalyzing the un-catalyzed substrate solely in the presence of the target, and (c) GNPs functionalized with a linking moiety to assemble with the un- catalyzed nucleic acid substrate. The GNPs may be provided substantially dispersed. The kit may include GNP-A and GNP-B or the kit may include GNP-AB. The kit may also include other ingredients that may be necessary for optimal activation of the catalytic nucleic acid molecule. The kit may also include a TLC. The ingredients (for example the linker, pre-catalytic subunits with or without the populations of GNPs) may be provided as a lyophilized mixture. In aspects of the invention, the kit may also include two or sets of un-catalyzed nucleic acid substrate, catalytic nucleic acid and GNPs that are target specific, that may be used in methods to simultaneously detect more than one target of interest in a sample.

In another embodiment, the present invention provides for lyophilization of assay components. In this form reagents remain functionally stable during storage and transport, making them well suited for POC testing.

[40] The ingredients used in the methods of the present invention may conveniently be dehydrated or lyophilized for storage, transportation and extended shelf-life. For example, the catalytic nucleic acid molecules and the linkers may be lyophilized and provided as a freeze-dried mixture that may be added to a sample being tested. The GNPs may also be lyophilized and added to the freeze-dhed mixture.

Advantages

DNAzyme-GNP assay of the present invention provides a simple and fast colorimetric scheme for detection of biological targets, including genetic targets of bacterial, viral and fungal origins with high sensitivity without the need for purification and separation steps. Results may be archived when spotted on TLC plates. To the knowledge of the applicants, this is the first report of combining the multi-component DNA-responsive DNAzyme with gold nanoparticle colorimetric platform.

The assay of the present invention may detect multiple genetic sequences in parallel and is easily translatable to new targets.

Color-based readout that does not require any complex equipment, and uses stable and cost-effective reagents make this experimental approach particularly suitable for POC testing. Furthermore, it is difficult to identify specific requirements for analytical sensitivity of a pathogen as this may vary based on the treatment strategy and whether one analyzing a single or co- infection.

Improved sensitivity may be achieved by modifying the nanoparticle size and chemistry, varying the length of MNAzyme/Linker or target sequences, [41 ] or by combining the fast catalytic rate of MNAzyme and colorimetric plasmonic readout with the sensitive protein-free autocatalytic DNAzyme approach demonstrated by the Willner group. [23, 42] Furthermore, in a complete point-of-care system, extra components may be added that can extract the genetic targets of interest. Such technologies have been developed for isolating targets from blood and stool samples. [43-45]

Freeze-drying of the components used in the methods of the present invention, provides storage, transportation and shelf-life advantages, not seen and not possible in DNAzyme methods of the prior art.

In order to aid in the understanding and preparation of the present invention, the following illustrative, non-limiting examples are provided. EXAMPLES

Materials

HAuCI4, sodium citrate tribasic, sodium phosphate monobasic, sodium phosphate dibasic, magnesium chloride and potassium chloride were purchased from Sigma-Aldrich. Sodium chloride, Tween-20 and Tris were purchased from BioShop (Burlington, ON, Canada). Hydrochloric acid and nitric acid were purchased from Caledon Laboratories (Georgetown, ON, Canada). Thiolatedmethoxyl polyethylene glycol MW=1000 Da (mPEG- SH-iooo) was purchased from Laysan BIO (Arab, AL). RPSF reversed phase hydrocarbon impregnated silica gel TLC plates were purchased from Analtech (Newark, DE). All RNA-containing Linker DNA strands were purchased from Integrated DNA Technologies (IDT) in HPLC-purified lyophilized form. All other DNA sequences were purchased from BioBasic (Markham, ON, Canada) and purified (thiol-functionalized sequences were purified by HPLC and non-functionalized ones by HAP purification) and lyophilized by the company. All lyophilized DNA sequences were re-suspended in UltraPure distilled water (Invitrogen) to 100 μΜ concentration and stored at -20°C. DNA sequences used in the study are outlined in Table 1.

Table 1. List of DNA se uences

NAME SEQ SEQUENCE (5'-to-3') NOTES ID

rGrU GTA TAG GAT TTA CTA CAT GCC CTA A, and

CA bolded

region binds Agg strand B.

3. MNAzyme subunits A and B

ΜΖΊ/Α" 16 AGC TGC TGC CCG TGC TGG TGA CAA CGA For all

GAG GAA ACC TT MNAzyme

(Mz)

MZGon/A' 1 7 TGC CAA TAT CGG CGG CCG ATG ACA ACG

sequences,

AATAGT GAG T

bolded

Mz _Ma/ /A' 1 8 AAC TCA ATC ATG ACT ACC CGA CAA CGA

region binds

TAT GGT TTG CG

the target

MZsyph/A' 1 9 GTC CAT CGG CAA ACA CGT CAA CAA CGA DNA and

ATA GTG TCA CA italicized

20 CAG GAG GTT GGT GAG TGA TTA CAA CGA region binds

MZHBIZ A ¹

TCT GTC CAG CA the

appropriate

ΜΖΊ/Β" 21 TGC CCA GGG AGG CTA GCT CCA TTG CCC

Linker

CAT GTG AAG TCA

sequence.

MZG _OH B ¹ 22 CAT CTC TTC CAG GCT AGC TAC GGT ACC Underlined

TGA AGA ATA AGC region forms

MZMa B' 23 CAC GTG TTC TCA GGC TAG CTT CTG TTA half of the

TGA ACA CTT AAT TTT catalytic core.

MZsyph/B' 24 CAA GAT CGC TAG GCT AGC TAC TGC AGC

ATC CAT CAG AGT CT

MZHBIZ B ¹ 25 AAT CCTATA CAG GCT AGC TGG AGG TTG

GGG ACT GCG AAT TT

4. Target sequences NAME SEQ SEQUENCE (5'-to-3') NOTES ID

AF-1 ^J 26 CAG TGA CTT CAC ATG GGG CAA TGGCAC For all target

CAG CAC GGG CAG CAG CTG GC sequences,

bolded

Gon ^K 27 TGC TTA TTC TTC AGG TAC CGTCAT CGG

region is CCG CCG ATA TTG GCA AC

bound by the

Mat 28 AAA ATT AAG TGT TCA TAA CAG ACG GGT appropriate

AGT CAT GAT TGA GTT CAT TGT GT Mz part A,

Syph* 29 GGA GAC TCT GAT GGA TGC TGC AGTTGA and

CGT GTT TGC CGA TGG ACA G underlined region is

ΗΒ 30 GCC AAA ATT CGC AGT CCC CAA CCT CCA

bound by ATC ACT CAC CAA CCT CCT GTC CTC CAA

Mz part B.

Sequences adapted from (i).

Sequences adapted from (2).

Sequences were randomly generated.

dMNAzyme-cleavable sequences (underlined) adapted from (3).

eMNAzyme-cleavable sequences (underlined) adapted from (4).

fMNAzyme-cleavable sequences (underlined) adapted from (5).

⁹ MNAzyme-cleavable sequences (underlined) adapted from (6).

hMzi subunit sequences taken from (3).

'Μζ-ι catalytic core sequence (underlined) used for all Mz. Linker and target

binding sequences adjusted to match corresponding Linker/target

sequences.

Sequence taken from (3).

ksequence designed to target 16 S ribosomal RNA gene from Neisseria

gonorrhoeae(7). sequences were chosen based on: Mal:(8), Syph:(9), andHBV:(10).

Specifically, the authors indicated the PCR primers that they used for detection of themicroorganisms by PCR method. We obtained the microorganisms' genomic DNA from GenBank (Mai: 16S ribosomal RNA not in asexual parasites, GenBank accession M19173; Syph: Treponema pallidum subsp. pallidum str. Nichols chromosome, complete genome, GenBank accession NC_000919; HBV: Hepatitis B virus isolate RS1 , complete genome, GenBank accession HQ236014) and identified the specific regions that these PCR primers would amplify. The DINAMelt Web Server Two-state melting was used to identify the 46-54 base pair long regions within these ampliconscontaining the least amount of secondary structure [Markham, N. R. & Zuker, M. (2005) DI NAMelt web server for nucleic acid melting prediction. Nucleic Acids Res., 33, W577- W581 ; the server can be found at

http://mfold.rna. albany.edu/?q=DINAMelt/Two-state-folding1. These regions were chosen as MNAzyme targets.

Instrumentation:

Spectroscopic measurements were performed using a SHIMADZU UV- Visible spectrophotometer UV-1601 PC (Kyoto, Japan). DLS measurements were performed on MALVERN Nano-ZS Zetasizer (Worcestershire, UK).

Freeze drying was performed in LABCONCO (Kansas City, MO) LYPH-LOCK 6 Freeze Dry System. TLC spots were photographed with Canon PowerShot S95 camera.

Experimental Details:

Preparation of 13nm gold nanoparticles (GNPs):

1 mL of 25 nM HAuCI ₄ was added to 98 mL of nanopure water in 250 mL flask prewashed with 100 mL of aqua regia (3 parts hydrochloric acid, 1 part nitric ac\d)(11). After bringing solution to a rapid boil, stirring was initiated on a benchtop stir plate, and 1 mL of 33 mg/mL of sodium citrate tribasic was added. The mixture was incubated for 10 min under boiling and stirring conditions (at which point solution color turned red), and then cooled to room temperature. DLS measurement was used to verify nanoparticles size and monodispersity (only particles with polydispersity index < 0.1 were used). Tween-20 was then added to a final concentration of 0.01 % v/v and GNPs were concentrated by 1X centrifugation at 12,000g for 30 minutes. GNP concentration was measured by UV-vis spectroscopy by using absorbance at λ=520 nm (assuming extinction coefficient of 2.33x10 ⁸ M ^"1 cm ^"1 and GNP diameter of 13 nm), then adjusted to 100 nM using nanopure water with 0.01 % v/v Tween-20. The extinction coefficient for gold was calculated using the formula from^). There was a small error in the published version; the corrected formula is as follows:

Extinction Coefficient = 10 ⁽¹ 0643*LOG[3/2*3.141592654*(diameter)*3] ₊ 4.0935)

Adsorbing thiolated AggDNA onto GNPs

We followed a protocol described previously by Mirkin group(i3) with a few modifications. 60 μΙ_ of 100 nM 13nm GNPs (final concentration 10 nM) were mixed with 60 μΙ_ of 32 μΜ of one of thiol-functionalized Agg strands (final concentration 3.2 μΜ, or 320 Agg/GNP) in 600 μΙ_ of 10 mM phosphate buffer (PB, pH 7.2) with 0.01 % v/v Tween-20. Solution was incubated at room temperature for 20 minutes. 3.43 M NaCI solution in 10 mM PB with 0.01 %v/v Tween-20 was then added to the reaction mixture in 10 steps according to the schedule of Table 2 to reach final concentration of 0.88M NaCI. Between each addition step and after the last addition step the mixture was incubated for 20 minutes at room temperature. Salt is required to block the negative charges on DNA molecules to allow high density loading of DNA on GNP surface; slow aging process is used to prevent salt-induced aggregation of GNPs. Next,30 μΙ_ of2 mMmPEG-SH ₁₀ oowere added (10,000 mPEG-SH ₁₀₀ o molecules per GNP), and reaction mixture was incubated for 30 min at 60°C. mPEG-SH-iooo adsorption step was included to block any DNA-free areas on GNP surface, thus improving the stability of DNA-functionalized GNPs against aggregation in salts during the MNAzyme 50°C cleavage step (see below), centrifugation, and storage. Finally, DNA-functionalized GNPs were purified by 3X centrifugation at 16,000g for 30 min, resuspended in nanopure water with 0.01 % v/v Tween-20 at 1 1 nM concentration, and stored at 4°C. Table 2. Salt addition schedule for loading thiolated AggDNA onto 13 nm GNPs

* Initial reaction volume contains 10 nM of 13nm GNPs and 3.2 μΜ of Agg DNA in 10 mM PB with 0.01 % v/v Tween-20.

** Salt Mix contains 3.43 M NaCI in 10 mM PB with 0.01 % v/v Tween-20.

Optimizing Linkeri concentration (Figure 2A)

Twelve 0.6 ml_ tubes were set up, each containing 2 μΙ_ of nanopure water, 2 μΙ_ of 10X MNAzyme buffer (0.1 M Tris-HCI, 0.5 M KCI, pH 8.3), 2 μΙ_ of 200 mM MgCI ₂ , and 2 μΙ_ of 2 μΜ [Mzi/A + Mzi/B] solution. In addition, each tube contained 2 μΙ_ of one of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM of Linker-,. The 12 ^th tube was included as negative control and contained 2 μΙ_ of nanopure water instead of Linkeri . The tubes were incubated at 50°C for 1 hour to mimic MNAzyme cleaving step. To each tube 5 μΙ_ of 1 1 nM GNP A (13 nm GNPs functionalized with Agg^A DNA) and 5 μΙ_ of 1 1 nM GNP B (13 nm GNPs functionalized with Agg^B DNA) were added. The samples (20 μΙ_ final volume) were incubated at 50°C for 20 minutes, then cooled to room temperature for 5 minutes to allow GNPs to be cross-linked by Linker-i. 3 μΙ_ of the mixture were then spotted on TLC plates to visualize the degree of aggregation. Remaining mixture was used to measure the location of the surface plasmon resonance (SPR) absorbance peak. This experiment was performed in triplicate.

Referring to Figure 4, the assay experimentally matches the experiment described in Figure 2A, with the exception that in this case the samples were not incubated for 1 hr at 50°C before colorimetric detection. Similar to Figure 2A, the sensitivity of Linker detection is -30 nM. This experiment was performed to confirm that inclusion of 1 hr/50°C incubation step does not affect the sensitivity of the MNAzyme-free direct detection assay.

Photographs of TLC spots were adjusted for contrast and brightness

(differences in spot color could be easily distinguished by a naked eye).

Determining MNAzyme colorimetric assay sensitivity for detection of AF- 1 genetic target (Figure 2B)

Eight 0.6 ml_ tubes were set up, each containing 2 μΙ_ of 10X MNAzyme buffer, 2 μΙ_ of 150mM MgCI ₂ , 2 μΙ_ of 2 μΜ [Mzi/A + Mz^B] solution, and 2 μΙ_ of 400 nM Linker-i. In addition, each tube contained 2 μΙ_ of one of 100, 250, 500, 1000, 2500, 5000, or 10000pM of AF-1 . The 8 ^th tube was included as negative control and contained 2 μΙ_ of nanopure water instead of AF-1 . The samples were incubated for 1 hour at 50°C to allow AF-1 -activated

MNAzymes to cleave Linker^ To each tube 5 μΙ_ of 1 1 nM GNPi-A and 5 μΙ_ of 1 1 nM GNPrB were then added (20 μΙ_ final volume), and the samples were incubated at 50°C for 20 minutes followed by cooling to room

temperature for 5 minutes. 3 μΙ_ of the mixture were then spotted on TLC plates to visualize the degree of aggregation. Remaining mixture was used to measure the location of the SPR absorbance peak. This experiment was performed in triplicate. Detecting multiple DNA targets in parallel (Figure 2C)

MNAzyme mix was made by mixing 100 μΙ_ of each of the following: 2 μΜ [Mzi/A + Mzi/B], 2 μΜ [Mz _Gon /A + Mz _GON /B], 2 μΜ [Mz _Ma //A + Mz _MA //B], 2 μΜ [Mzsyph/A +Mzsyph/ ], and 2 μΜ [MZHBV A +MZ _H BV/B] solutions. Linker mix was made by mixing 100 μΙ_ of each of: 2 μΜ Linker^ 2 μMLinkerGoπ, 2

μMLinkerMa , 2 To detect all 5 target sequences in parallel, two solutions were set up in 0.6 mL tubes, each containing 10 μΙ_ of 10X MNAzyme buffer, 1 0 μΙ_ of 400 mM MgCI ₂ , 10 μΙ_ of MNAzme mix, 40 μΙ_ of Linker mix, and 20 μΙ_ of nanopure water. To the first reaction solution, 2 μΙ_ each of 1 μΜ AF-1 , 1 μΜΘοη, 1 \MMal, 1 \MSyph, and 1 μΜ/-/Β \ targets were added. In contrast, 1 0 μΙ_ of nanopure water were added to the second solution, which was used as a negative control solution. In addition, 5 detection mixes were set up in 0.6 mL tubes in duplicate, each containing one of the following GNP pairs: (i) [5 μΙ_ of 1 1 nM GNPi-A and 5 μΙ_ of 1 1 nM GNPi-B], or (ii) [5 μΙ_ of 1 1 nM GNP _Ma rA and 5 μΙ_ of 1 1 nMGNP _Ma r B], or (iii) [5 μΙ_ of 1 1 nM GNP _Go n-A and 5 μΙ_ of 1 1 nMGNP _Go n-B], or (iv) [5 μΙ_ of 1 1 nM GNPsyph-A and 5 μΙ_ of 1 1 nMGNP _SyP h-B], or (v) [5 μΙ_ of 1 1 nM GN and 5 μΙ_ of 1 1 nM GN The reaction and control solutions were incubated for 1 hour at 50°C to allow activated MNAzymes to cleave their corresponding linkers. 10 μΙ_ from reaction solution were then transferred to the 5 tubes of the first detection mix set, and 10 μΙ_ from control solution to the 2 ^nd 5 detection mix tubes. All samples were incubated for 20 minutes at 50°C, then for 5 minutes at room temperature to allow GNP aggregate formation. 3 μΙ_ from each tube were spotted on TLC plates for color readout.

The scheme for detecting 3 (AF-1 , Ma/, and HB V) out of 5 possible targets in parallel included lyophilization of the components. Reaction solution was premade by mixing 6 μΙ_ of 10X M NAzyme buffer, 6 μΙ_ of 400 mM MgCI ₂ , 6 μΙ_ of MNAzyme mix, 24 μΙ_ of Linker mix, and 12 μΙ_ of nanopure water in 1 .5 mL tube. I n addition, 5 GNP-containing detection mix tubes were set up as described above. All of the samples were frozen at -80°C for 30 minutes, freeze-dried overnight, and stored at room temperature. On the day of the experiment, lyophilized reaction solution was resuspended in 54 μΙ_ of nanopure water, and 1 .2 μΙ_ of 2 μΜ AF-1 , 1 .2 μΙ_ of 2 μΜ Ma/, 1 .2 μΙ_ of 2 μΜ HBV targets and 2.4 Ml of nanopure water were added. The reaction solution was then incubated for 1 hour at 50°C. Next, 10 μΙ_ of the solution was transferred to each lyophilized detection mix, and the detection tubes were incubated for 20 minutes at 50°C and cooled to room temperature for 5 minutes. 3 μΙ_ from each tube were spotted on TLC plates for readout.

As many changes can be made to the embodiments described above without departing from the scope of the invention, it is intended that all material contained herein be interpreted as illustrative of the invention and not in a limiting sense.

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