probes

Abstract

Disclosed herein are probes for detecting the presence of one or more target proteins, the probe comprising a structure I1 and a structure I2. Also disclosed herein are methods of using the same.

Description

PROBESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application No. 10202303556Y, filed 18 December 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology. In particular, the present invention relates to the use of nucleic acid-based detection probes.BACKGROUND

[0003] Proteins are ubiquitous biomolecules responsible for regulating biological processes and is an important class of disease biomarkcr, often present in pg / mL or lower concentrations. Unlike its nucleic acids counterpart which can easily be evaluated even from minute quantity due to the compatibility with numerous DNA amplification techniques such as polymerase chain reaction, the evaluation of protein targets relies on the conversion of a target recognition event into intermediary' for signal amplification. One approach is to tag specific protein binders with DNA barcodes, which are highly programmable based on Watson Crick base pairing rule, to enable the use of DNA exponential amplification chemistries.

[0004] Other methods known in the art typically have less detection sensitivity than that for DNA / RNA detection due to the inherent inability to amplify protein targets. Furthermore, these methods are often related to limitations such as labour-intensive and lengthy procedure.

[0005] Thus, there is an unmet need for protein detection assays with improved sensitivity.SUMMARY

[0006] In one aspect, the present disclosure refers to a probe for detecting the presence of one or more target proteins, the probe comprising a structure II and a structure 12:wherein 11 comprises domains p*, s*, and a*; wherein domain s* is a spacer; wherein domain p* is a first agent that binds to a first binding site on the one or more target proteins; and wherein domain a* is at least 4 nucleotides in length; wherein 12 comprises domains p, s, a, t, n, and k; wherein domain p is a second agent that binds to a second binding site on the one or more target proteins, wherein the second binding site is different from the first binding site of domain p*; wherein domain s is a spacer; wherein domain a is at least 4 nucleotides in length, wherein domain a* is complementary to domain a; wherein domain t is a nucleotide sequence which is present or absent; wherein domain n is a nucleotide sequence which comprises a nicking endonuclease site, wherein the length of domain n is dependent on a nicking endonuclease used; and wherein domain k is a nucleotide sequence comprises a self-copying template sequence; wherein domain t, if present, domain k, and domain n are each single stranded nucleotide sequences.

[0007] In another example, the present disclosure refers to a method for detecting one or more target proteins in a sample, the method comprising: i. adding a probe as defined herein to the sample; ii. allowing binding of the probe to the one or more target proteins, wherein upon binding, domains a* of 11 and domain a of 12 form a duplex structure, resulting in domain k in a single stranded form with a free 5’ overhang and a 3’ end at domain a*; iii. allowing binding of a polymerase or fragment thereof to domain k, which elongates the 3’ end of domain a* using the 5’ overhang of domain k as a template to result in an elongated arm; iv. nicking the elongated armusing a nicking enzyme to obtain a linearly amplified amplicon; v. exponentially amplifying the amplicon using a self-repeating template; and vi. detecting the amplicon of step v.

[0008] In yet another example, the present disclosure refers to a method for detecting one or more target proteins in a sample, the method comprising: vii. adding a probe as defined herein to the sample; viii. allowing binding of the probe to the one or more target proteins, wherein upon binding, domains a* of II and domain a of 12 form a duplex structure, resulting in a 5’ overhang at domain k and a 3’ end at domain a*; ix. allowing binding of a polymerase or fragment thereof to domain k, which elongates the 3’ end of domain a* using the 5’ overhang of domain k as a template to result in an elongated arm; x. nicking the elongated arm using a nicking enzyme to obtain a linearly amplified amplicon; xi. exponentially amplifying the amplicon using a selfrepeating template; and xii. detecting the amplicon of step xi.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0010] Fig, 1 provides at Fig. 1A schematics of the Proximity-activated Exponential Amplification Reaction (PEAR), where the proximity activation module was facilitated by a pair of initiator probes (Il and 12), and a pair of DNA polymerase and nicking endonuclease to provide a constant source of amplicon for the downstream exponential amplification reaction (EXPAR) mediated by the same pair of enzymes and template strand (the self-repeating units were indicated with black dotted box). Fig. IB shows signals generated when different combinations of the PEAR reactants were reacted.

[0011] Fig, 2A and Fig 2B show the limit of detection for probes of similar concepts as known in the art. This data shows that the probes in the art are only capable of reaching limits of detection (LODs) in the picomolar range. However, the probes disclosed herein have limits of detection in the femtomolar range.

[0012] Fig, 3 shows in Fig. 3A a schematic illustration of the comparison using linear and hairpin initiator probe structures. The hairpin form of 12 was obtained by changing a few nucleotides upstream of domain a2 to be complementary to the 5’-end. The association length was defined by the domain a for the linear probe and combined length of domains ai+a for the hairpin lock probe. A split target (ST) was used to mimic proximity binding events. Fig. 3B shows the time difference between negative (“-” refers to 0 nM ST) and positive (“+” refers to 1 nM ST) controls, A, was strongly dependent on the association length. The performance of the hairpin lockprobes (black) consistently outperformed that of the linear probes (grey). This was due to the former delaying the onset of non-specific background without compromising on the rate of signal generation by comparing the kinetics trace for 6 nucleotide (nt) association length (see Fig. 3C), 7 nt association length (Fig. 3D) and 8 nt association length (Fig. 3E).LOO 13 J Fig. 4 shows robustness of PEAR to key system parameters tested. Fig. 4A shows data indicating negligible template-induced background amplification observed by comparing the use of desalted template strand (solid line) and HPLC-purified template strand with 3’-phoshorylation modification (dotted line). Fig. 4B shows that the kinetics of signal evolution was invariant to the template strand concentration which again demonstrated the lack of signal artefacts from template- induced amplification. Fig. 4C shows data demonstrating the feasibility of warm start with PEAR. Identical experimental sets were run in parallel - one was left at 25 °C for an hour before ramping to 37°C for another hour (grey); while the other set was directly incubated at 37°C for an hour (black). There was minimal difference between the two sets of signal evolution profile. Fig. 4D shows data indicating the feasibility of streamlining the reagent preparation step using a single Mastermix formulation (black) compared to the conventional approach of pre-incubating DNA and enzyme mixture separately (grey) with one more mixing step before sample addition.

[0014] Fig. 5 shows implementation of PEAR on protein systems. Fig. 5A shows results of signal generation contributed by the respective PEAR components - template, II, 12 and target. Fig. 5B shows data indicating that both signal and background generation of PEAR were affected by increasing % of human serum. However, the differential of the two characteristic times remained relatively invariant up to 10% human scrum. Fig. 5C to Fig. 5E show signal generation profiles for when different concentrations of (from left to right) interferon-gamma (IFN-y), tumour necrosis factor alpha (TNF-a) and Neprilysin were titrated with their specific pairs of PEAR probes. Fig. 5F to Fig. 5H show data indicating that PEAR exhibited strong target dosage dependence with a wide dynamic range spanning 4 to 5 orders of magnitude. Each data point represents mean ± S.D. (n = 3). The breakthrough time of the negative control (txc) was denoted as grey solid line, while the cut-off for the limit of detection (INC subtracted by three S.D.) was denoted as grey dotted lines. Fig. 51 shows results of different combinations of 100 pM protein inputs (shown in table below the graph) when mixed with the three respective sets of PEAR probes. Only the right combination of probe and protein inputs (both pure and mixtures) generated high time differential (A) which could be clearly discerned from the cases with “wrong” combinations. For clarity of presentation across different PEAR systems, the A was normalized by dividing the individual A values by that generated in presence of pure protein input for each set of PEAR probes.

[0015] Fig. 6 shows effect of initiator probe concentration on proximity activated exponential amplification reaction (PEAR) signal generation. Fig. 6A shows a signal profile when low concentration of initiator probes was reacted. There was little-to-no variability in the negative control up to 1 nM probe concentration. However, the rate of signal generation in presence of 10 pM split target (ST) was lower at lower probe concentration. Fig. 6B shows a signal profile when high concentration of initiator probes was reacted. The breakthrough time for the negative control was much faster beyond 2 nM probe concentration. There was no additional target binding advantage from using too high a probe concentration as evident from the relatively consistent breakthrough time observed in presence of 10 pM ST. Fig. 6 C shows results of an probe concentration, which was determined to be 1 nM for the design reported in the present disclosure based on the difference in breakthrough time in presence and absence of ST, A.

[0016] Fig. 7 provides reaction schematics for the opening of hairpin lock initiator probe. The original association length is split into domains ai (exposed) and a2 (protected in hairpin lock). A few nucleotides upstream of domain ar on initiator 2 (12) were mutated to obtain the hairpin structure. Upon proximity activation, initiator 1 (II) binds to 12 at domain ai and strand displaces domain a2*. The remaining hairpin stem was designed to be only 2 nucleotides (nt) and spontaneously dissociates into a single stranded 12 which then serves as the template for EXPAR.

[0017] Fig. 8 shows a schematic of exemplary probes as described herein.

[0018] Fig. 9 shows schematics of the DNA-programmed reaction to evaluate specific IgE (slgE) antibody against Der p 2, a dust mite allergen. Fig. 9A shows the slgE antibody being converted to an oligonucleotide (oligo) barcode for isothermal exponential amplification reaction. Fig. 9B shows a signal evolution profile when different combinations of DNA reactants were reacted in the Mastermix. Where relevant, 0.25 nM II, 0.25 nM 12 and 100 nM template strand was used. Fig. 9C shows a regression graph indicating a strong target-signal dependence which was observed for a dynamic range of 3 orders of magnitude. The data are shown as mean ± S.D. (n = 3). The average S / N of the NC was denoted as grey solid line, while the cutoff for the limit of detection (based on 3 S.D. difference) was denoted as grey dotted line.

[0019] Fig. 10 shows the results of visualising output of slgE PEAR on lateral flow. Fig. 10A shows a schematic showing single stranded amplicon outputs were continuously generated from the template duplex by the combined activity of the polymerase and nicking enzyme which could be utilized for lateral flow visualization. Fig. 10B shows a schematic depicting the incorporation of biotin-dCTP in the amplification reaction and a F AM-labelled probe in the post-reaction hybridization enabled the biotinylated amplicon to form a sandwiched streptavidin-gold nanoparticle complex. Fig. 10C shows a graph showing signal profile generated in absence (NC)and presence of 15 pM Der p 2 IgE using the modified Mastermix. The molar amount of dCTP:biotin-dCTP was adjusted to achieve the stated % of biotin-dCTP while keeping the total amount of dCTP (both with and without biotin modifications) constant at 5 LIM. For example, 20% biotin-dCTP meant that a mixture of 1 LI M biotin-dCTP and 4 pM dCTP was used. Fig. 10D shows images visualizing PEAR signal on lateral flow using either 10% or 50% biotin-dCTP at various Der p 2 concentrations. A higher % of biotin-dCTP was advantageous for capturing amplicons generated at low target input concentration.

[0020] Fig. 11 shows the specificity of slgE PEAR assay as disclosed herein. Fig. 11A shows a schematic of the PEAR initiator probes serving as an AND gate to generate a YES signal only when the input fulfilled both criteria of binding specificity to (i) Der p 2 allergen and (ii) is of IgE isotype. Fig. 1 IB shows a column graph showing the S / N quantification of 0.9 pM Der p 2 slgE was robust against 0.125 nM - 4 nM of Der p 2 slgG. The data are shown as mean ± S.D. (n = 3). The mean S / N of the reference 0.9 pM Der p 2 slgE test is denoted as grey solid line while the grey dotted line denotes 1.96 S.D. below this mean value. Fig. 11C shows the signal evolution profile when 15 pM of slgE for other allergens (i.e. Ara h 2 for peanut allergy and Gal d 2 for egg white allergy) was reacted with anti-Der p 2 slgE probes. Fig. 1 ID shows that distinguishable S / N was obtained only in presence of 15 pM Der p 2 slgE. Other inputs of slgE against other allergens or Der p 2 slgG returned a negative output. The data are shown as mean ± S.D. (n = 3). The truth table of the AND gate is shown below the graph.

[0021] Fig. 12 shows the results of the evaluation of clinic samples using the slgE PEAR method described herein. Fig. 12A shows a comparison of the Der p 2 slgE quantification outcome from PEAR assay and the ImmunoCap reference values for n = 18 volunteers with known dust mite allergy. The data are shown as mean ± S.D. (n = 2). The cut-off value for classifying the test outcome, which in this work was taken to be the LOD of the PEAR assay, is denoted as dotted line. Fig. 12B is a column graph depicting the outcome of PEAR quantification for three negative clinical samples. The data are shown as mean ± S.D. (n = 2). The limit of detection (LOD) of PEAR assay is denoted as dotted line. Note that all values shown in A and B have been corrected for the dilution factor of 20. Fig. 12C shows a two-by-two table representing the concordance of outcome from PEAR test and ImmunoCap test - (left) PEAR in its assay format wherein real-time fluorescence measurement was used to quantify Der p 2 slgE level and (right) PEAR in its lateral flow (LF) format wherein the presence or absence of test line was used to qualitatively assess the status of dust mite allergy. All clinical samples were provided with reference values quantified on the ImmunoCap platform which was taken as the ground truth for comparison. Fig. 12D shows images of readouts from one run of the PEAR lateral flow format which was quenched after 24minutes of in-tube incubation at 37°C and ran for 5 minutes on the lateral flow strip. The qualitative outcome, determined based on visual interpretation, was labelled below each sample.

[0022] Fig. 13 shows results of the effect of magnesium chloride (MgCh) on the speed of assay. The graphs are signal profiles generated in absence (NC) and presence of 15 pM Der p 2 slgE using different MgCh concentration in the assay diluent.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0023] DNA is an unsurpassed programming material for executing bio-computing logics due to its predictable Watson Crick base pairing property. DNA-programmed bio-computers are often designed to operate autonomously, i.e. DNA logic gates are triggered by pre-defined input to return predictable output, which can be cascaded into a processing system of varying complexities. This can range from a single gain function for signal amplification to a system-level molecular classifier capable of evaluating multiple inputs simultaneously.

[0024] The ability to perform bio-computation autonomously practically implies the potential for such DNA-programmed reactions to be executed as a simple one-pot format which is highly suitable for point-of-care applications. Recent progress in the DNA design toolbox has extended its utility beyond evaluating nucleic acids inputs, and with improved computation speed and sensitivity by driving the reactions with highly efficient enzymes. This has resulted in the translating DNA-programmed biosensing concepts into a broader range of physiologically relevant biomarkers

[0025] DNA isothermal amplification techniques have been applied for evaluating nucleic acids inputs but cannot be implemented directly on other types of biomolecules. Disclosed herein is a proximity activation mechanism that allowed proteins to function as an input trigger for the DNA exponential amplification reaction, also referred to herein as the proximity-activated exponential amplification reaction “PEAR” system. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via NUPACK design tool, and the implementation of hairpin lock on the proximity probe structure.

[0026] Due to the constraints imposed for the intended protein evaluation, the PEAR system described herein deviates from commonly reported DNA-based exponential amplification reaction configuration, and is shown to be more robust towards non-specific DNA amplification. The PEAR describe here exhibits good target responsiveness for three exemplary protein models with a dynamic range of 4 to 5 orders of magnitude down to femtomolar input concentration.

[0027] Overall, the proximity activation mechanism described herein led to the development of a stable and robust configuration of DNA exponential amplification reaction, and enabled the use of protein as inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

[0028] Recent advances in isothermal nucleic acid amplification techniques, especially those which operate optimally near 37 °C, offer signal amplification toolboxes which can co-exist with the protein evaluation step at a single temperature and even one-pot reaction step. In particular, the exponential amplification reaction (EXPAR) offers design versatility due to its compatibility with short amplicon lengths (and hence more straightforward DNA barcode sequence design) while achieving amplification factors of between 106to 109within a few minutes. Due to the two abovementioned properties, EXPAR and its modified detection schemes are most commonly used for the ultrasensitive DNA / RNA detection application especially for microRNA (miRNA) targets.

[0029] Despite the advances of EXPAR in applications involving nucleic acids inputs, its implementation for non-nuclcic acids targets had largely been fragmented. The general approach is to incorporate a trigger probe which converts the recognition event into an active oligo form for initiating the downstream EXPAR. Current trigger strategies are limited to targets with specific properties, such as aptamer structure switching induced by specific protein target binding, proteins with binding affinity to specific DNA sites, e.g. transcription factors, or small molecule moieties which can be modified to the oligo terminals to block exonuclease digestion and enzymes directly involved in detectable DNA modifications.

[0030] In one example, the present disclosure describes the use of antibody for interrogating protein inputs. There had only been two antibody-based EXPAR schematics which either utilizes EXPAR to replace the enzymatic amplification moiety in traditional sandwich immunoassay or involves elaborate design with magnetic beads used in competitive ELISA format known thus far. However, there was no clear improvement to the limit of detection (pM to pg / mL range) which remained to traditional ELISA methods, at best.

[0031] The present disclosure describes a proximity activation mechanism facilitated by antibody-oligo probe to enable protein responsiveness for DNA isothermal exponential amplification reaction (PEAR). The present disclosure describes a pair of DNA-based proximity probes which primes signal amplification via a combined polymerase and nicking enzyme reaction when bound to the target protein. The technology lies in the sequence design of the DNA probes (for transducing target binding event into signal initiating barcode) and template strand (for signal amplification), such that the polymerase / nicking enzyme reaction can proceed stably with distinct signal-to-noise ratio down to sub-femtomolar (or fg / mL) protein concentration.

[0032] In one example, there is disclosed a probe for detecting the presence of one or more target proteins, the probe comprising a structure II and a structure 12:wherein 11 comprises domains p*, s*, and a*; wherein domain s* is a spacer; wherein domain p* is a first agent that binds to a first binding site on the one or more target proteins; and wherein domain a* is at least 4 nucleotides in length; wherein 12 comprises domains p, s, a, t, n, and k; wherein domain p is a second agent that binds to a second binding site on the one or more target proteins, wherein the second binding site is different from the first binding site of domain p*; wherein domain s is a spacer; wherein domain a is at least 4 nucleotides in length, wherein domain a* is complementary to domain a; wherein domain t is a nucleotide sequence which is present or absent; wherein domain n is a nucleotide sequence which comprises a nicking endonuclease site, wherein the length of domain n is dependent on a nicking endonuclease used; and wherein domain k is a nucleotide sequence comprises a self-copying template sequence; wherein domain t, if present, domain k, and domain n are each single stranded nucleotide sequences.

[0033] In one example, all domains except p, p*, s and s*, are nucleic acid sequences.

[0034] In one example, domain s and domain s* each have a length of up to 60 nm (nanometres). In another example, each of domains s and s* are, but are not limited to, nucleotides,linkers, molecules occupying physical space, such as, poly ethylene glycol, beta-alanine, 4- aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5 -amino valeric acid (Ava), 6- aminohexanoic acid (Ahx), PEG2 Spacer or AEEA (8-amino-3,6-dioxaoctanoic acid), PEG3 Spacer (12-amino-4,7,10-trioxadodecanoic acid), and combinations thereof. In one example, the molecules occupying physical space (or spacers) are selected from the group consisting of polyethylene glycol (PEG), PEG2 spacer, PEG3 spacer, 18-atom hexa-ethyleneglycol, and combinations thereof.

[0035] In one example, domain s, if comprising nucleotides, the nucleotides have a length of at least 3 nucleotides.

[0036] In one example, domain a is between 4 to 12 nucleotides in length. In another example, domain a* comprises domain ai* and domain ai*, wherein domains ai* and a2* are complementary to domain a. In a yet another example, domain ai* is between 4 to 12 nucleotides in length, and wherein domain a2* is between 0 to 8 nucleotides in length.

[0037] In another example, domain t, if present, is between 1 nucleotide to 10 nucleotides in length.

[0038] In one example, domain k is at least 5 nucleotides in length. In another example, domain k is at least 8 nucleotides in length. In a further example, domain k comprises domain ki and domain 1Q. In another example, the sum of the number of nucleotides in ki and l<2 is between 5 to 200 nucleotides in length. In yet another example, domain k2 is complementary to domain t. In another example, domain k2 is complementary to domain t and domain a2. In another example, domain k2 is at least 3 nucleotides in length, hi yet another example, domain l<2 is between 3 nucleotides and 10 nucleotides in length. In a further example, wherein domain k is between 5 to 200 nucleotides in length, optionally wherein domain k is between 5 to 20 nucleotides in length, optionally wherein domain k is between 10 to 20 nucleotides in length.

[0039] In one example, domain n is 9 nucleotides in length.

[0040] In one example, the nicking endonuclease is active at a temperature between 35 °C to 65°C. In another example, the nicking endonuclease is active at a temperature between 35°C to 45°C. In yet another example, the nicking endonuclease is active at a temperature between 45°C to 55°C. In one example, the nicking endonuclease is active at a temperature between 55°C to 65°C. In yet another example, the nicking endonuclease is active at a temperature between 37°C to 65 °C. In one example, the nicking endonuclease is active at 37 °C. In another example, the nicking endonuclease is active at 45°C. In yet another example, the nicking endonuclease is active at 55°C. In another example, the nicking endonuclease is active at 65°C.

[0041] In another example, the nicking endonuclease is, but is not limited to, Nt.Alwl, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtSI, Nt.BstNBI, and variants thereof.

[0042] In one example, a polymerase or fragment thereof binds to domain k, or, where present, domain ki .

[0043] In another example, the probe is as follows:

[0044] In one example, the target proteins are, but are not limited to, immune markers or inflammatory markers, cardiac markers, infectious disease markers, cancer markers, prenatal markers or development markers, neurodegenerative markers, and allergy markers or autoimmune markers. In one example, the immune markers or inflammatory markers can be, but are not limited to, IL-2, IL-6, procalcitonin (PCT), c-reactive protein (CRP), IFN-y and TNF-a. In another example, the immune markers can be, but are not limited to, IFN-y and TNF-a. In a further example, the cardiac markers can be, but arc not limited to, neprilysin, troponin, N-tcrminal pro b-type natriuretic peptide (NT-proBNP), and soluble suppression of tumorigenesis-2 (sST2). In another example, the cardiac marker is neprilysin. In one example, the infectious disease markerscan be, but are not limited to, Dengue NS 1, coronavirus, influenza antigens, SARS-CoV-2 antigens and SARS-CoV-2 antibody. In another example, the allergy markers or autoimmune markers are selected from the group consisting of Specific IgE (slgE) against Der p 2, Ara h 2 and Gal d 2. In another example, the markers can be, but are not limited to, exemplary markers as shown in the table below:Table 6. Exemplary markers

[0045] In one example, the first and second agents are each, independently, agents that have a specific binding affinity for the one or more target proteins.

[0046] In another example, each of the first and second agents are, but are not limited to, an antibody, an antigen, an affibody, a nanobody, and a peptide. In one example, the antibody is but is not limited to, anti-IFN-y, anti-TNF-a, or anti-neprilysin. In another example, antibody is polyclonal or monoclonal. In another example, the antibody is human, murine, leporine, caprine, bovine, ovine, asinine (donkey), or avian (chicken).

[0047] In this disclosure, a design framework towards a general proximity activation mechanism, which enabled the use of protein as input trigger for the DNA exponential amplification reaction, herein termed PEAR (Fig. 1), has been established. This general activation mechanism combines the use of ubiquitous antibody as specific protein binders, carefully designedproximity initiator probe sequences to convert the protein recognition event into DNA amplicons with high signal- to-background ratio and the high amplification factor of EXPAR for sensitive protein evaluation.

[0048] The present disclosure describes a probe for detecting the presence of one or more target proteins, as disclosed herein. The design framework resulted in the identification of a working combination of enzyme choice, template strand and initiator probe sequences, which were refined using NUPACK design tool and experimentally verified. In one example, the concept of hairpin lock was implemented to further improve the signal-to-background ratio achieved by the proximity initiator probes. Overall, the as-designed PEAR system achieved improved robustness against common EXPAR issues such as non-specific DNA amplification. Clear target responsiveness over a dynamic range of 4 - 5 orders of magnitude for the three protein systems tested, for example, but not limited to, interferon-gamma (IFN-y), tumour necrosis alpha (TNF-a) and neprilysin, down to femtomolar input concentration was observed.

[0049] In this disclosure, antibody-based proximity activation mechanism has been developed to enable protein responsiveness for EXPAR (Fig. 1). This general activation mechanism combines the use of ubiquitous antibody as specific protein binders, carefully designed initiator oligonucleotide (oligo) probe sequences to convert the protein recognition event into DNA amplicons and the high amplification factor of EXPAR for femtomolar protein detection limit. A short complementary region was included in II and 12 such that minimal association occurred in free solution while the event of protein target binding brought them to close proximity and stabilize the duplex formation. The 3 ’-end of II then primed the polymerase extension using 12 as a template strand containing the nicking endonuclease cut site. The antibody-11-12 complex served as a constant linear source of amplicons for the downstream isothermal EXPAR.

[0050] Furthermore, the present disclosure implemented the concept of hairpin lock to further improve the signal-to-background ratio of the proximity initiator probes. Overall, the proximity activation mechanism disclosed herein improved system robustness against common EXPAR issues such as self-priming of multiple template strands. In one example, clear protein concentration responsiveness over a dynamic range of between 4 to 5 orders of magnitude had been observed for the three protein systems tested, i.e. interferon-gamma (IFN-y), tumour necrosis alpha (TNF-a), and neprilysin, with femtomolar detection limit in 10% human serum matrix.Overview of Proximity-Activated Exponential Amplification Reaction (PEAR)

[0051] To achieve the goal of protein responsiveness, a proximity activation mechanism was incorporated into the isothermal exponential amplification reaction (EXPAR), herein denoted PEAR, which converted dual proximity recognition events into oligo barcodes for downstreamDNA amplification (Fig. 1). A pair of initiator probes with short association region (domain a) was designed such that there was minimum hybridization in free solution, i.e. melting temperature below room temperature and usually below 0°C. Upon target binding, the probes were brought into proximity which increased their local concentrations and stabilised the association region. The polymerase then extended the 3 ’-end of initiator 1 (11) which served as the primer against the template sequences presented by initiator 2 (12). A nicking endonuclease recognition site was included in the 12 sequence to constantly release one amplicon strand per cycle of polymerase elongation and nicking from the target recognition complex. The released amplicons then participated in the downstream EXPAR consisting of self-repeating catalytic units separated by the nicking enzyme recognition site for doubling reaction per cycle.Identification of enzyme pair[0052 J The Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein was performed with enzyme pairs comprising nicking endonuclease and DNA polymerase. The nicking endonuclease used in the Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein can be, but is not limited to, Nt.AlwI, Nt.BstNBI, Nt.BspQI, Nt.CviPII, Nb.BsrDI, Nb.BtsI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.BsmAI, and variants thereof. In one example, the nicking endonuclease disclosed herein is a type of enzyme that cleaves only one strand on a double- stranded DNA molecule. In one example, the nicking endonuclease disclosed herein recognizes specific sequences in the DNA and cleave one of the two strands at a particular site, leaving the other strand intact. In another example, the specific point of cleavage for the nicking endonuclease Nt.AlwI is sequence-independent. The method disclosed herein provides design flexibility for the initiator probe sequence.

[0053] The DNA polymerase used in the Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein can be, but is not limited to, Bst DNA Polymerase, Bsu DNA Polymerase, Large (Klenow) Fragment, phi29 DNA Polymerase, and variants thereof, such as Bst 2.0 and Bst 2.0 WarmStart.

[0054] In one example, the Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein is performed with enzyme pairs that operate within a temperature range of 37 °C to 65°C. In another example, the Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein is performed with enzyme pairs that operate within a temperature range of 45 °C to 65°C. In one example, when the enzyme pair of Nt.AlwI and Klenow fragment is used in the Proximity-activated Exponential Amplification Reaction (PEAR), the reaction is performed at 37°C. In another example, when the enzyme pair of Bst DNA polymerase and Nt.BstNBI nicking endonuclease is used in the Proximity -activated Exponential Amplification Reaction (PEAR), thereaction is performed at 55°C. In yet another example, the Proximity-activated Exponential Amplification Reaction (PEAR) disclosed herein is performed at 65 °C.Sequence Design for Proximity Initiator Probes and Template Strands

[0055] After selecting the enzyme pair, the next step was to design the initiator probe sequences using NUPACK web server to minimize the occurrence of secondary structures. The presence of secondary structure, especially those folding back to hybridize at the 3’ end, is not acceptable due to the generation of non-specific background. For the first design iteration, an association region of 6 nucleotides (nt) and amplicon length of 10 nt was defined. The sequence of 12 probe defined the amplicon sequence, which together with the Nt.AlwI cut site, defined the EXPAR template sequence and the entire sequence design space was fully specified.Feasibility Testing of PEAR Concept

[0056] Next, the feasibility of PEAR was tested using a synthetic DNA split target (ST) to mimic the proximity event of II and 12 binding to a protein target. In this case, a recognition sequence partially complementary to split target was used as the binder of the initiator probes. The proximity activation module contributed negligibly to non-specific amplification as evident from the breakthrough time of the reactions with II and / or 12 and template strands being largely similar to the reaction consisting of only the template strand (Fig. IB). Without being bound by theory, it is thought that the concentration of the initiator probes should be pre-determined for a new sequence design or protein system, due to the trade-off between slower target binding kinetics at lower probe concentration and the increased probability for II and 12 to associate transiently (at domain a) at higher probe concentration (Fig. 6). On the other hand, the EXPAR module was critical for generating rapid signal amplification as no appreciable signal growth was observed from the reaction consisting of solely the proximity activation module (Il and 12; template strand was omitted) with a split target. The only reaction setup generating signal with clearly faster break! h rang h time was when all components, i.e. Il, 12 and template, were added to the reaction in presence of a split target.Design Considerations for the Proximity Initiator Probes

[0057] With a functional EXPAR template sequence at hand, the proximity initiator probe design was further improved while keeping the amplicon sequence fixed. Two design parameters were investigated - the length of the association region (domain a) and the use of hairpin lock which had been reported to improve the turn-on signal-to-background ratio of a split proximity circuit (Fig. 3A). The difference in the breakthrough time between the negative (0 nM split target, split target (ST)) and positive (1 nM split target (ST)) control, A, was used as the quantification parameter to compare the signal above background across different designs.

[0058] For the linear initiator probe design, there was a slight increase in the A as the length of domain a increased from 5 nucleotides to 6 nucleotides due to the less stable 5 nucleotide ST-I1- 12 duplex formed at 37°C (Fig. 3B). A decreasing trend was observed for longer association lengths until A fell markedly below 10 minutes at 8 nucleotides. The drop in A was mainly due to the faster breakthrough time of the negative control whereby 11 and 12 hybridized more effectively in absence of target at longer association length. This corroborates with the increasingly negative Gibbs free energy, and hence enhanced stability, of the 11-12 duplex.

[0059] Next, a hairpin lock was implemented on the existing 6 to 8 nucleotide linear initiator probe through slight modification of the 12 sequence (located immediately upstream of the newly denoted domain a:*) to facilitating the folding back of the 5’ end of 12 upon itself to form a hairpin structure. This effectively reduced the length of the exposed association region responsible for non-specific activation from domain a to ai (Fig. 3A). Note that the hairpin stem can include a short region (denoted in light green) not complementary to the association region of II to counter transient hybridization events between domain ai + a2. The detailed reaction mechanism is elaborated in Fig. 7. By way of an example, the process outlined in Fig. 7 can be identical to steps ii to vi when using, for example, a linear probe (including the description of the overhangs involved), except that when using a hairpin lock, an additional hairpin opening step is involved between step i to ii. In this hairpin opening step, domain a2* of II partially displaces domain 1Q (denoted in grey in Fig. 7) of 12, which spontaneously opens to reveal the linear version of the probe.

[0060] For all association lengths, the inclusion of a hairpin lock increased the A and hence signal differentiation from the negative control (Fig. 3B). The improvement was more evident for longer association length probes, i.e. 17.3% for 6 nucleotides (nt), 31.0% for 7 nucleotides and 362% for 8 nucleotides, due to the delay in the breakthrough time of the negative control (Figs. 3C-E). However, a much larger CV% of 28.8% was calculated for the 8 nucleotides hairpin lock design, compared to < 10% for the other two association lengths, demonstrating the difficulty in achieving controlled amplicon generation at longer association length. Based on the best A, the 6 nucleotides hairpin lock design, comprising of 4 nucleotides exposed association region and 2 nucleotides locked domain 32*, was selected for further characterisation.

[0061] In one example, Il is, but is not limited to, SEQ ID NO: 4, 6, 9, 12, 15 and / or 17. In another example, 12 is, but is not limited to, SEQ ID NO: 5, 7, 8, 10, 11, 13, 14, 16 and / or 18. In yet another example, Il and 12 are but are not limited to the combinations of SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 6 and 8, SEQ ID NOs: 9 and 19; SEQ ID NOs: 9 and 11;SEQ ID NOs: 12 and 13; SEQ ID NOs: 12 and 14; SEQ ID NOs: 15 and 16; and SEQ ID NOs: 17 and 18.Identifying System Operating Parameters

[0062] After demonstrating the design process for the initiator probes and EXPAR template sequences, the operating parameters affecting the performance of PEAR have been identified in this disclosure. EXPAR is known for its often-uncontrollable background amplification for which one of the main contributors is believed to be the non-specific interaction of the template strand, including spurious self-priming of multiple template strands, secondary structure and other unintended hybridization with other nucleic acids in the sample. As such, the parameters related to the template strand quality and workflow processes known to affect EXPAR performance have been prioritised. Without being bound by theory, it is thought that the EXPAR, being the exponentially amplified module, controlled the overall stability of the amplification reaction, which its lineally amplified proximity activation counterpart played only an incremental role in.

[0063] The use of template strand was first compared with 3 ’-end phosphorylated modification (with HPLC purification), a strategy for blocking polymerase extension from spurious self -priming of template pair, and that without any modification or purification beyond basic desalting (Fig. 4A). There was negligible difference between the two in terms of signal generation profile. Without being bound by theory, there are thought to be two possibilities - (i) there was only negligibly low level of spurious template self -priming or (ii) the non-specific background arising from a small fraction of template strands not successfully phosphorylated at the 3 ’-end during synthesis grew quickly nonetheless due to the high amplification factor of EXPAR.

[0064] In order to determine which one of the two possibilities above was the more probable scenario, different concentrations of template strand were titrated. The breakthrough time and rate of signal generation from both the negative and positive controls were found to be invariant of the template concentration (Fig.4B). Only the endpoint fluorescence intensity depended on the template concentration, which was expected due to the increased amount of template duplexes and amplicons generated. Without being bound by theory, this is thought to indicate that the first scenario of good template sequence design was the most likely explanation, since the latter scenario of spurious hybridization arising from synthesis defect should increase with higher template concentration from a kinetics perspective.

[0065] As Nt.AlwI nicking endonuclease has minimal activity below 37 °C, it was tested to see if PEAR was suitable for warm start operation, thereby eliminating the need for pre-incubation on ice commonly done for EXPAR preparation. One set of reaction mixture was first reacted at 25°C for 1 hour. This was followed by the ramping up of reaction temperature to 37°C with the additionof another set of identical reaction mixture for direct comparison. The breakthrough time and rate of signal generation was observed to be similar between the two reaction sets (Fig. 4C). This indicates that the reaction preparation can be done at room temperature for up to 1 hour without compromising on the enzyme activity or fidelity. Though the Klenow fragment does operate efficiently at 25°C, it was unable to initiate premature signal generation without supporting from its nicking endonuclease counterpart since exponential amplification had yet to commence. The feasibility of a warm start protocol enabled all PEAR operations disclosed herein is to be prepared under room temperature condition before reacting at 37°C.

[0066] Given the robustness of PEAR demonstrated thus far, PEAR was challenged by seeing if it was feasible to bypass a common practice in the art to prepare the oligo and enzyme portion of the EXPAR reaction separately and mixing just before the reaction to minimize any non-specific amplification. Performing a side-by-side comparison of the conventional two-portion preparation versus the bulk preparation of a single Mastermix stored as a frozen aliquot and thawed before use, showed no significant variability in the signal evolution (Fig. 4D).

[0067] All the above observations indicate that the design choices made due to the constraints imposed for protein analysis (as outlined above) also resulted in an EXPAR system configuration that behaved fundamentally different from the commonly reported format, especially in terms of the improved robustness against non-specific amplification. The following system operating conditions were applied for the implementation of PEAR for protein analysis in the next section - desalted template strand was used with warm start and single Mastennix operation.

[0068] In one example, the polymerase or fragment thereof possessing strand displacement properties. In another example, the polymerase or fragment thereof is selected from the group consisting of Klenow fragment, Bst DNA polymerase, Bsu DNA polymerase, phi29 DNA Polymerase, and fragments or variants thereof. In one example, such a fragment can be, but is not limited to fragments of DNA polymerase I subtilisin cleavage (i.e., Klenow Fragment (3'— >-5' exo- ), and the smaller fragment).

[0069] In one example, the combination of deoxynucleotide triphosphates (dNTPs) or variants thereof can be, but is not limited to, dcoxyadcnosinc triphosphate (dATP), dcoxycytidinc triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and variants thereof. In another example, the deoxynucleotide triphosphates (dNTPs) are labelled for detection. The term “label” or “tag” is used interchangeably herein and refers to any chemical moiety attached to a nucleotide or nucleic acid, wherein the attachment may be covalent or non- co valent. In one example, the label renders nucleotide or nucleic acid detectable using technology known in the ait. Examples of label are, but not limited to, a fluorescent label, a colorimetric label,a chemical label, a chemiluminescent label, an electro-chemiluminescent label, an enzymatic label, a radioactive label, a gold label, a biotin label, and combinations thereof. In one example, the biotin-labelled deoxynucleotide triphosphates (dNTPs), such as biotin-labelled deoxyadenosine triphosphate (dATP), biotin-labelled deoxycytidine triphosphate (dCTP), biotin-labelled deoxyguanosine triphosphate (dGTP), biotin-labelled deoxythymidine triphosphate (dTTP), or combination thereof is enzymatically incorporated into the nucleic acid as substitute for its natural counterpart. In one example, the molar ratio of dcoxycytidinc triphosphate to biotin-labelled deoxycytidine triphosphate is no more than 1:1. The resulting biotin-labelled nucleic acid are subsequently detected using streptavidin conjugated with detection agents including, but not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), a fluorescent dye, or agarose / magnetic beads.Characterisation of the Protein Responsiveness of PEAR

[0070] In this disclosure, the protein responsiveness of PEAR was exemplified using intcrfcron-gamma (IFN-y) as an exemplary, model protein system. IFN-y is a cytokine involved in inflammatory pathways and a common disease biomarker. The II and 12 initiator probes were conjugated to a pair of IFN-y antibodies validated for immunoassay specificity using an antibody- oligo preparation workflow. The system specificity was tested using different combinations of reactants (Fig. 5A). Contrary to the observation made using DNA-only probe strands (Fig. IB), the reaction consisting of template and 11 initiator probe resulted in faster breakthrough time compared to the base case involving only the template strand. Since the difference between the protein and DNA-only system was the use of the antibody binder, it is thought that this faster breakthrough time was possibly due to the increased non-specific binding or complexation of II (noted to have a free 3 ’ end) in the presence of antibody, which increased the frequency of transient hybridization leading to faster background amplification. While this resulted in a faster breakthrough time for the negative control of the protein system, a clear signal distinction was nonetheless observed upon proximity activation with 100 pM IFN-y target.

[0071] In one example, serum, which is a commonly used sample matrix for evaluating protein biomarkcr, had been tested for the matrix interference effect at different % of human scrum spiked with 100 pM IFN-y (Fig. 5B). While PEAR was accelerated at higher human serum %, the time difference (A) between the two cases was largely invariant in 1% to 10% human serum (Fig. 5B). Using 10% human serum as the sample matrix, varying concentrations of IFN-y (10-fold dilution from 100 pM) were titrated with clear protein responsiveness down to a limit of detection (LOD) of 0.244 fM and inter-assay CV% of 4.3% (Figs. 5C and F).

[0072] To test the general applicability of PEAR for evaluating different protein systems, two other protein inputs were tested namely, tumour necrosis factor alpha (TNF-a) which is another cytokine biomarker (Figs. 5D and G) and neprilysin (Figs. 5E and H) which is a cardiac enzyme biomarker. Similar to IFN-y, PEAR generated signal with clear concentration dependence (Figs. 5G and 5H). For all protein systems evaluated, the target responsiveness spanned a dynamic range between 4 to 5 orders of magnitude and with sensitivity down to femtomolar target concentration (Table 1).

[0073] The target specificity of each set of PEAR probes was further tested in this disclosure by reacting with different combinations of 100 pM protein targets (Fig. 51). Positive signal was generated only when the correct protein target specific for the set of initiator probes was present in the mixture without crosstalk with other proteins inputs. This example allows complex protein evaluation in a multiplexed format.

[0074] While proximity activation using antibody-oligo probes is already widely used in several immunoassay designs, such as PEA, nicking endonuclease signal amplification (NESA), proximity CRISPR Casl2a assay, and DNA proximity assay (DPA), these designs either fall short of the detection limit achievable by EXPAR or require multiple reaction steps each requiring its own set of enzymes to achieve protein responsiveness at low levels. As the sequences of the template and proximity probes designed in this disclosure were able to co-exist in a single reaction mixture, PEAR was able to leverage on the high amplification factor of EXPAR to achieve sub- femtomolar to femtomolar limit of detection (LOD).

[0075] In one example, there is described a method for detecting one or more target proteins in a sample, the method comprising: i. adding a probe as described herein; ii. allowing binding of the probe to the one or more target proteins, wherein upon binding, domain a* of II and domain a of 12 form a duplex structure, resulting in domain k in a single stranded form with a free 5’ overhang and a 3’ end at domain a*; iii. allowing binding of a polymerase or fragment thereof to domain k, which elongates the 3’ end of domain a* using the 5’ overhang as a template to result in an elongated arm; iv. nicking the elongated arm using a nicking enzyme to obtain a linearly amplified amplicon; v. exponentially amplifying the amplicon using a self-repeating template; and vi. detecting the amplicon of step v.

[0076] In another example, there is described a method for detecting one or more target proteins in a sample, the method comprising: vii. adding a probe as defined herein; viii. allowing binding of the probe to the one or more target proteins, wherein upon binding, domain a* of II and domain a of 12 form a duplex structure, resulting in a 5’ overhang at domain k and a 3’ end at domain a*; ix. allowing binding of a polymerase or fragment thereof to domain k, which elongates the 3’ endof domain a* using the 5’ overhang of domain k as a template to result in an elongated arm; x nicking the elongated arm using a nicking enzyme to obtain a linearly amplified amplicon; xi. exponentially amplifying the amplicon using a self -repeating template; xii. detecting the amplicon of step xi.

[0077] As used herein, the term “self-repeating unit” refers to a nucleic acid sequence that copies itself such that the number of the sequence is doubled per round of reaction.

[0078] In one example, the detection in step vi is performed using a colorimetric or fluorescent detection method. In another example, the fluorescent detection method is selected from the group consisting of intercalating dye, such as EvaGreen, and a fluorophore quencher reporter pair. In yet another example, the colorimetric method comprises pH sensitive methods, such as but not limited to, Phenol red.

[0079] In a further example, the method described herein has a sub -femtomolar to femtomolar limit of detection (LOD).

[0080] In another example, the method described herein is performed under isothermal conditions. In a further example, the method is performed in one or more reaction vessels.

[0081] In one example, the method disclosed herein is performed for detection of a single target. In another example, the method disclosed herein is performed for detection of multiple targets (also referred to as multiplexed detection). Such a multiplexed detection can take place using a single sample, or multiple samples. This detection of multiple targets can be performed sequentially or concurrently. When performing the method disclosed herein for multiplexed detection (which is in active development), for example, detecting multiple targets from a single sample, one form of the workflow would be to perform the addition of the probe to the sample, the binding of the probe to the sample, and the binding of the polymerase to the probe in a single reaction vessel (steps i to iii, or steps vii to ix). Following that, the resulting product would be split into a separate number of reaction vessels for nicking, obtaining a linearly amplified amplicon, exponentially amplifying the amplicon using a self-repeating template, and detecting the amplicon (steps iv to vi or steps x to xii). By way of an example, if there were five targets of interest, five reaction vessels will be involved, each with their own set of self-repeating templates (step v or xi) and signal reporter reagents (step vi or xii).

[0082] Also disclosed herein is a kit comprising the probe as described herein. In one example, the kit further comprises buffers, the deoxynucleotide triphosphates (dNTPs) or variants thereof, the polymerase or fragment thereof as defined herein, the nicking endonuclease as defined herein, and the self-repeating unit as defined herein. In another example, the kit further comprises anintercalating dye and / or a reporter strand. In yet another example, the probe is provided separately, in the form of a lyophilised powder, or as a concentrated stock solution.

[0083] Discussed herein is a design framework towards a general antibody-based proximity activation mechanism to enable protein triggered EXPAR, which has been termed PEAR in the present disclosure. Several design parameters were tested and characterized, including the choice of enzyme, the use of NUPACK to facilitate the sequence design of proximity probes and template strand, and the implementation of hairpin lock on the proximity probe structure. These design choices for PEAR were shown to result in a fundamentally different implementation of the EXPAR system which exhibited robustness against non-specific amplification. By conjugating the initiator probes onto validated pair of antibody binders, PEAR was evaluated in three protein models and achieved protein responsiveness with a dynamic range spanning 4 to 5 orders of magnitude down to a femtomolar detection limit. Overall, the PEAR system disclosed herein enabled the use of a wide concentration range of protein input for more complex molecular evaluation.

[0084] Below is provided a non-limiting and exemplary use of the PEAR system disclosed herein.

[0085] Allergy is a chronic condition affecting more than 20% of the global population with higher prevalence in urbanized area. Two types of conventional tests are available, i.e. a skin prick test which is fast but often inconclusive, or a lab blood test for specific IgE (slgE) with up to a week’ s turnaround time. The increasing acceptance of using molecular allergens as reagents to measure allergen epitope-based IgE antibody level has enabled more accurate quantification of allergic response and for guiding immunotherapy. House dust mite allergy is one of the most common conditions affecting approximately 130 million people and is a major risk factor for asthma. The Der p 2 allergen component, accounting for 63% to 97% of dust mite allergy patients, is used as the model target input in the present disclosure to exemplarily demonstrate the utility of a DNA-programmed reaction and the method disclosed herein as an allergy point-of-care test.

[0086] The DNA-programmed reaction as mentioned above is exemplarily based on the proximity-activated exponential amplification reaction (PEAR) system disclosed herein, had been to be responsive to specific IgE (slgE) antibody input and generate an output cither as quantifiable fluorescence signal or for direct visualization on lateral flow. Critically, the input-to-oligo conversion module was tailored as an AND logic gate to recognize target input that meets two criteria of binding specificity to Der p 2 antigen and is an IgE isotype. In addition, the exponential amplification reaction has been modified to generate biotinylated amplicons which can be readily adapted for use with off-shelf lateral flow products without compromising on the detection speed. Concept of PEAR to Evaluate Specific IgE Antibody

[0087] The DNA-programmed reaction disclosed herein had been designed to react autonomously in a single tube and at a single temperature of 37°C (Fig. 9A). It comprised of five molecular components to execute two modular programmes. The overall reaction was driven by a single pair of enzymes, i.e. Klenow fragment and Nt.AlwI nicking enzyme, which operates harmoniously under the same buffer condition and reaction temperature. The other three oligobased components encoded the proximity-activated exponential amplification reaction (PEAR) to convert the slgE antibody input into oligo barcode with high signal gain.

[0088] In the first molecular step, the slgE antibody input was converted to its corresponding oligo barcode using a pair of proximity initiator probes (Il and 12) conjugated with anti-IgE nanobody and Der p 2 antigen respectively (Fig. 9 A), which recognized target molecules fulfilling both criteria of having binding affinity to Der p 2 and is of IgE isotype. Upon binding with the Der p 2 slgE target which held them in proximity, their local concentration was greatly enhanced to form a stable target-11-12 complex, from which the polymerase can extend from the free 3’-end of Il using 12 as the template. Oligo barcodes were linearly generated with each round of polymerase extension and nicking.

[0089] In the next molecular step, the oligo barcode undergoes exponential amplification using a self-repeating template strand. The amount of oligo barcodes doubled with each round of polymerase extension and nicking until all the template strands were exhausted, after which the system switched to linear generation of an amount of amplicons instead from the saturated template duplexes.Fluorescence Signal Output to Quantify slgE Level

[0090] Signal amplification was rapidly achieved by the exponential formation of template duplex which was measured via the fluorescence generated from an intercalating dye (EvaGreen Plus) whose intensity increased with an increasing amount of template duplex which was interpreted via real-time fluorescence measurement followed by calculating the point of inflection (POI) for quantification.

[0091] Different combinations of the DNA component strands were reacted, with each additional DNA strand contributing incrementally to the background signal (Fig. 9B). The signal generated significantly faster in the presence of the Der p 2 slgE target only when all the molecular components, i.e. Il, 12 and template, were present. The PEAR assay was responsive to the slgE target concentration wherein the POI varied inversely with the target concentration, i.e. higher concentration generated faster signal with a smaller POI value (Fig. 9C).Visualizing Output of DNA-Programmed Reaction on Lateral Flow

[0092] Once all the template strands were exhausted as duplexes, the reaction then switched to linear generation of an amount of amplicon from the saturated template duplex, i.e. 100 nM per round of polymerase extension and nicking. Without being bound by theory, it is thought that the single stranded amplicons can be directly visualized on a lateral flow strip by incorporating commonly used tags, e.g. biotin, digoxin or FITC as an easy-to-interpret readout for point-of-care applications (Fig. 10A). This is typically achieved via primer modification for several isothermal amplification methods such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP).

[0093] The use of a self-copying template in the exponential amplification reaction (EXPAR) technique, while effective for amplifying short oligo barcodes rapidly, meant that the amplicons were produced fully by the polymerase using the deoxynucleotide supplied in the Mastermix. The short amplicon length of 10 nucleotides (nt) also meant that a sandwiched hybridization design, which required the amplicon to hybridize with both a capture and detector strand, was infeasible. Existing efforts to introduce lateral flow visualization for EXPAR include a reporter degradation method and two-stage EXPAR to generate a longer second-stage amplicon to form a sandwiched hybridized complex. The former is based on the extent of signal turn-off which, while simple, is a less sensitive approach (limit of detection (LOD) in the picomolar range). The latter approach compromised the overall turnover efficiency of EXPAR, presumably due to the longer amplicon length involved, with an overall workflow time exceeding 70 min.

[0094] To overcome the design constraints while retaining the core advantage of EXPAR amplifying short oligo rapidly, disclosed herein is an approach of incorporating biotin-dCTP in the reaction Mastermix as a means to directly introduce a biotin tag to the amplicon output (Fig. 10B) was implemented. In this design, the biotinylated amplicon was captured by the streptavidin immobilized on the test line and a fully complementary FAM-labelled probe facilitated the formation of a sandwiched gold nanoparticle complex for direct visualization. Klenow fragment, a family-A type DNA polymerase, is known to be able to incorporate modified deoxynucleotide in reactions such as a primer extension and nick translation. In one example, the full replacement of dCTP with biotin-dCTP has resulted in no product formation within 1 hour of reaction (Fig. 10C). In another example, the overall PEAR reaction was tolerant of up to 50% biotin-dCTP (or 1:1 dCTP:biotin-dCTP molar ratio) as evident from the consistently distinguishable signal in presence of 15 pM Der p 2 slgE over the negative control where no target input was added (Fig. 10C). It was demonstrated in this disclosure that the degree of substitution of dCTP with biotin- dCTP did not affect the overall reaction speed based on the similar' signal evolution profileobtained with Mastermixes modified with 10%, 20% and 50% biotin-dCTP. In one example, the molar ratio of deoxycytidine triphosphate to biotin-labelled deoxycytidine triphosphate is no more than 1:1.

[0095] The reaction output based on the two extremes of 10% and 50% biotin-dCTP were visualized on lateral flow strips (Fig. 10D). A lower biotin-dCTP % compromised the signal formation at a lower Der p 2 slgE concentration, as evident from the lack of a distinct test line for 0.9 pM input. In contrast, when 50% biotin-dCTP was used, a clear test line was visible for 0.9 pM input and a faint test line still remained for an even lower 0.45 pM input. Since the amplicon sequence has only a single cytosine site (Fig. 10B), the % biotin-dCTP directly affected the binary outcome of whether an amplicon strand was labelled with one biotin tag. As a high 50%> biotin- dCTP amount did not negatively impact the polymerase progressivity (Fig. 10C) while enabling improved detection sensitivity to be realized on the lateral flow platform, it was used for all studies disclosed herein.

[0096] In one example, a lateral flow assay (LFA) can be used for nucleic acid detection. The lateral flow assay (LFA) is known in the art for the detection of analytes (such as proteins, nucleic acids, pathogens, or small molecules) in a liquid sample, where the sample is placed at one end of a strip or membrane and diffuses across the test strip or membrane without the assistance of external forces (driven by capillary action), to the other end of the strip or membrane. During this process, the sample interacts with various reagents or markers embedded or immobilized along the strip. The lateral flow strip is typically made of materials such as, but not limited to, glass fiber, cellulose, and polyesters. The test strip typically contains immobilized reagents that specifically bind to the target analyte (such as proteins, nucleic acids, pathogens, or small molecules). The labelled detection reagents used in lateral flow assay are, but not limited to, colloidal gold, coloured latex, or fluorescent particles, which are used for visual detection. The results of lateral flow assay are often displayed as colour changes, typically forming one or more visible lines or regions on the strip. For example, a control line that indicates the test is working properly (i.e., the sample has flowed through the strip), and a line that appears if the target analyte is present.

[0097] Disclosed herein is the visualization of nucleic acid carried out on a lateral flow strip. In one example, the method carried out on the lateral flow strip comprises: (a) contacting the sample comprising the amplicon with the lateral flow strip; (b) allowing binding of the amplicon to the lateral flow strip; and (c) detecting the amplicon. In another example, the method carried out on the lateral flow strip comprises: (a) contacting the sample comprising the biotin-labelled amplicon with a lateral flow strip, wherein the lateral flow strip comprises a testing zone withimmobilised streptavidin; (b) allowing binding of the biotin-labelled amplicon to the immobilised streptavidin to form a complex; and (c) detecting the amplicon. slgE PEAR Assay Exhibited Good Analytical Sensitivity and. Specificity

[0098] For point-of-care applications, it is desirable for the detection time to be rapid which was defined as a goal of 30 minutes. The reaction speed of PEAR was modulated by direct modulation of the MgCh concentration, wherein a reduction in MgCh concentration from 10 mM to 2.5 mM almost halved the reaction time (Fig. 13) and the latter was used for all assay characterisation work disclosed herein.

[0099] The PEAR assay as described herein was titrated with 0.096 pM to 300 pM of recombinant Der p 2 slgE with clear target-signal dependence within a dynamic range spanning 3 orders of magnitude (Fig. 9C). A limit of detection of 0.16 pM was calculated based on a cut-off of 3 S.D. above below the mean of the S / N of the negative control (Fig. 9C).

[0100] The PEAR initiator probes disclosed herein were tailored to function as an AND logic gate to generate signal only if the input possessed both criteria of (i) having a binding affinity for Der p 2 antigen and (ii) being an IgE isotype (Fig. 11 A). Since IgE is present in ca. 2000-fold lower amount than IgG in serum, 30 the ability of the PEAR assay to recognize the Der p 2 slgE isotype specifically over a large excess of Der p 2 specific IgG (sig G) was tested. The presence of about 100-fold to 5000-fold higher concentrations of Der p 2 slgG, which approximately spans the reference specific IgG interval for common allergens, did not significantly interfere with the S / N quantified by PEAR for a low Der p 2 slgE input concentration of 0.9 pM (Fig. 1 IB).

[0101] To test the other design criteria of binding specificity for Der p 2 allergen, the anti-Dcr p 2 PEAR probes were reacted with 15 pM of two other allergens with high allergy prevalence, i.e. Ara h 2 for peanut allergy and Gal d 2 for egg white allergy (Fig. 11C). Distinguishable S / N was reproducibly obtained only when the correct input of 15 pM Der p 2 slgE was reacted (Fig. 1 ID). All other inputs, i.e. slgE against Ara h 2 and Gal d 2 allergens or Der p 2 slgG returned a negative output. The positive outcomes from the two interference challenges using Der p 2 IgG and slgE of other allergens confirmed that the PEAR initiator probes functioned specifically as an AND gate to generate signal only for Der p 2 slgE input. It is noted that all experiments were performed in 5% human serum matrix, known for being a source of potential interference with more complex composition. As shown herein, the use of 5% human serum matrix did not deter the PEAR assay from achieving the displayed analytical sensitivity and specificity.Evaluating Clinical Samples Using Der p 2 slgE PEAR Assay

[0102] As shown herein, 21 clinical samples, consisting of 18 positive samples and 3 negative samples based on reference values measured by the ImmunoCap gold standard method, wereevaluated using the PEAR assay or system as disclosed herein. The Der p 2 slgE level in the samples were first quantified using the real-time fluorescence measurement method wherein the POI value was used to calculate the signal-to-noise (S / N) ratio from which the slgE level was determined using a standard calibration curve based on recombinant Der p 2 slgE (Fig. 9C). Within the scope of this work, the cut-off value to classify the test outcome as positive or negative was taken to be the limit of detection (LOD) of the PEAR assay described herein.

[0103] Eighteen (18) samples were determined to be positive for Der p 2 slgE and hence dust mite allergy, with their individual quantified slgE level presented in Fig. 12A. Three samples returned a negative test outcome due to the quantified Der p 2 slgE level falling below the cut-off value of the PEAR assay (Fig. 12B). Within this small clinical sample set, a clinical sensitivity and specificity of 100% was preliminary determined for the PEAR real-time fluorescence measurement assay format (Fig. 12C).

[0104] The direct visualization of the PEAR lateral flow test was used to qualitatively classify the samples as cither positive or negative (Fig. 12D). No further image processing or optical quantification was performed, in line with the intended use of the lateral flow format as a quick and simple yes / no test. 20 samples generated the expected test outcome while one positive sample (P12, based on ImmunoCap quantification) was not reproducibly detected, which was annotated as a false negative. As such, a clinical sensitivity of 95.2% and specificity of 100% was determined for the PEAR lateral flow format. It is further noted that the test outcome of PEAR was compared to the ImmunoCap reference values provided for the samples.

[0105] Disclosed herein is therefore a DNA-programmcd reaction based on the proximityactivation exponential amplification reaction (PEAR) technology to exemplarily evaluate specific IgE (slgE) against Der p 2 implicated in dust mite allergy. By tailoring the molecular component of the input-to-oligo conversion module and the exponential amplification reaction, the versatility of PEAR to be programmed based on specific input criteria and the desired output signal, be it quantification via real-time fluorescence measurement or direct visualization on lateral flow, was demonstrated. The Der p 2 slgE PEAR assay was fast (about 30 min), sensitive (sub-picomolar slgE level) and specific, with potential utility to analyse clinical samples in point-of-carc setting.

[0106] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0107] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value,more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0108] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.[00109J Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form pail of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0110] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0111] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTIONMaterials

[0112] All DNA oligonucleotides (oligo) used in this disclosure were designed using NUPACK web server and purchased from Integrated DNA Technologies (IDT), either in desalted format or with HPLC purification. The DNA sequences are listed in (Table 2). The lyophilized oligo was reconstituted in lx Tris EDTA buffer (lx TE, pH 8.0) to give 100 pM stock with desalting and stored at 4°C. Initiator strands modified with amine ends were ordered with HPLC purification and in lyophilized form (initiator oligo probes for conjugation with antibody) were reconstitutedas 1 mM stock solution in nuclease free water (IDT) and stored at 4°C. The proteins and antibodies used in this disclosure were sourced from various suppliers and listed in (Table 3). Oligo modified with fluorophore were ordered as 100 pM in lx Tris-EDTA buffer (lx TE, pH 8.0) with HPLC purification and stored in the dark at 4°C. The DNA sequences are listed in Table 2.L00113 J All enzymes-related reagents used in the experiments disclosed herein were purchased from New England Biolab: lOx NEBuffer™ 2 buffer (#B7002S), lOx rCutSmart™ buffer (#B6004S), 10 mM dcoxynuclcotidc (dNTP) (#N0447), 100 mM dNTP solution set (dATP, dCTP, dGTP and dTTP) (#N0446S), recombinant albumin (#B9200S), 10 U / pL Nt.AlwI nicking enzyme (#R0627S) and 5 U / pL Klenow fragment (3'— >5' exo-) (#M0212). All buffers and dNTP were aliquot as single-use vial. All items were stored at -20°C until use.

[0114] The following chemicals were used as received: sodium chloride (NaCl, > 99.5%, # S3014), sodium acetate (#S8625), absolute ethanol (#E7023), acetonitrile (#34851), disuccinimidyl suberate (#S1885), dimethylformamide (#D4551), triethylamine (#T0886) and human scrum (#H3667) were purchased from Sigma Aldrich. NHS-PEG-NHS, Ik (# H0023023- 1K) was purchased from BioPharma PEG. Phosphate buffer was prepared using sodium phosphate monobasic dehydrate and sodium phosphate dibasic anhydrous purchased from Acros Organics and Fisher Scientific, respectively. lOx Tris-Borate-EDTA (TBE) Buffer pH 8.3 and 0.5 M ethylenediaminetetraacetic acid (EDTA) was purchased from 1st Base. microBCA assay (#23235), Qubit™ ssDNA Assay Kit (# Q10212), SYBR gold nucleic acid gel stain (#S 11494) and 10% normal goat serum (#50062Z) was purchased from ThermoFisher Scientific. 20x EvaGreen® Plus Dye (#31077) was purchased from Biotium. EvaGreen® Plus Dye arc covered under US patent nos. 7,803,943 and 7,776,567. Biotin- l l-dCTP(#2715) was purchased from Lumiprobe. The HybriDetect - Universal Lateral Flow Assay Kit (#MGHD 1) was purchased from Milenia Biotec. [001 15] The following protein and antibody reagents were purchased: CaptureSelect™ biotin anti-IgE conjugate (#7103542500) from ThermoFisher Scientific. Recombinant Der p 2 (#RP- DP2A-1), human IgE monoclonal antibody 2G1 anti Der p 2 (#E-2G1), human IgE monoclonal antibody 11F10 anti Ara h 2 (#E-llF10), human IgE monoclonal antibody 11B6 Gal d 2 (#E- 11B6) and anti-Dcrp 2 Human IgGl (#2B12-IgGl) were purchased from Indoor Biotechnologies. Antibody-Oligonucleotide ( Oligo ) Preparation Workflow

[0116] The initiator probes (11 and 12) modified with 5’- and 3’-amine groups were used to conjugate with antibody using NHS-PEG-NHS (Ik) as a linker. Briefly, the oligo (reconstituted to 200 uM in nuclease free water) was first mixed with equi-volume amount of acetonitrile, linker (25 mM, dissolved in dimethylformamide) and 1:800 (volume basis) of triethylamine. After 15 minutes of activation at room temperature, the activated probes were purified using ethanolprecipitation using 85% ethanol as wash solution. The activated oligo was reacted with 3 molar equivalent amount of antibody in 50 mM phosphate buffer (pH 7.2) for at least 2 hours at room temperature.

[0117] The antibod -oligo conjugates were purified using anion exchange chromatography (1EX) using Agilent Bio SAX NP3 (4.6x50mm) column on Agilent 1260 Infinity HLPC system. The salt gradient for elution was started with 100% buffer A (50 mM phosphate buffer, pH 7.2), followed by a step up to 70% buffer and 30% buffer B (50 mM phosphate buffer, pH 7.2 and 1.0 M NaCl) from which the gradient was gradually increased to 35% buffer A and 65% buffer B over 13 minutes. The collected fractions with protein-oligo conjugates were concentrated using the Amicon ultrafiltration columns with 50 kDa MWCO. The antibody-oligo conjugates were quantified on oligo basis using Qubit™ ssDNA Assay Kit. The purified conjugates were stored in lx phosphate buffered saline and 1 mM EDTA (pH 7.4) at 4°C until use.

[0118] In the examples disclosed herein, the initiator probes (Il and 12) modified with 5’- and 3 ’-amine group were conjugated with the binders, i.c. Dcr p 2 antigen and anti-IgE nanobody, using DSS as a linker Briefly, the oligo (200 pM in nuclease free water) was first mixed with equi-volume amount of acetonitrile, DSS (25 mM, dissolved in dimethylformamide) and 1:800 (volume basis) of triethylamine for 15 minutes at room temperature, followed by purification via ethanol precipitation. The activated oligo was reacted with a 2-molar equivalent amount of binders in 50 mM phosphate buffer (pH 7.2) for at least 2 hours at room temperature.

[0119] The antibody-oligo conjugates were purified with anion exchange chromatography (IEX) using an Agilent Bio SAX NP3 (4.6x50mm) column on an Agilent 1260 Infinity HLPC system. The collected fraction of binder conjugated with one oligo strand was concentrated using the Amicon ultrafiltration columns with 10 kDa MWCO. The antibody-oligo conjugates were quantified on oligo basis using Qubit™ ssDNA Assay Kit. The purified conjugates were stored in lx phosphate buffered saline and 1 mM EDTA (pH 7.4) at 4°C until use.Performing Proximity Activated EX.PAR

[0120] The assay reagents were prepared as a 2x Mastermix consisting of 0.5x NEBuffer™ 2 buffer, 0.5x rCutSmart™ buffer, 100 pM dNTP, 0.2 U / pL Nt.AlwI nicking enzyme and 0.05 U / pL Klenow fragment (3'— >5' exo-), aliquot and stored at -20 °C. Batches of Mastermix prepared with the same lot of enzymes had acceptable inter-assay CV% of less than 15% and consistent breakthrough time for the negative control. Larger variations were observed with different enzyme lots, especially that of the nicking enzyme. The trends observed for breakthrough time remained consistent. All quantification studies requiring precise breakthrough times were carried out using Mastermix prepared with the same lot of enzymes.

[0121] For each reaction, 1.0 pL of sample was added to 4.0 pL of assay buffer consisting of 2.5 nM initiator probes (Il and 12) diluted in lx rCutSmart™ buffer and incubated in PCR tube. The sample diluent used was 10% normal goat serum during the initial design phase (using synthetic split target strand) and human serum for protein system. At least two negative controls were included per experimental set where 1.0 p L sample diluent without any target was added. Next, one aliquot vial of 2x Mastermix was thawed at room temperature for 5 minutes, followed by the addition of 100 nM template strand and lx EvaGrccn plus. Finally, 5.0 pL of the prepared 2x Mastermix was added to each reaction and mixed thoroughly. All operations were performed at room temperature.

[0122] A typical final reaction condition was 0.75x rCutSmart™, O.25x NEBuffer™ 2, 50 pM dNTP, 0.1 U / pL Nt.AlwI, 0.025 U / pL Klenow fragment (31— >5' exo-), 1.0 nM initiator probes, 50 nM template strands, 0.5x EvaGreen Plus dye and 10% sample matrix. The reaction tubes were transferred to an isothermal real-time fluorescence reader (Allsheng Gene-8C) for reaction at 37°C with fluorescence measurement (excitation = 470 nm, detection = 525 nm) every 15 seconds over 1 hour.Performing DNA-Programmed PEAR Assay for slgE Detection

[0123] During the initial concept development, the assay reagents were prepared as a 2x Mastermix consisting of lx rCutSmart buffer, 5 pM dNTP, 0.2 U / pL Nt.AlwI nicking enzyme and 0.05 U / pL Klenow fragment (3'— >5' exo-), aliquot as single-use vial and stored at -20 °C. During assay development for lateral flow visualization, the assay reagents were prepared as a 2x Mastermix consisting of lx rCutSmart buffer, 5 pM dNTP (for dATP, dTTP and dGTP; 2.5 pM dCTP and biotin-dCTP), 0.2 U / pL Nt.AlwI nicking enzyme and 0.05 U / pL Klenow fragment (3'— >5' exo-), aliquot as single-use vial (good for 10 reactions) and stored at -20 °C.

[0124] For each reaction, 1 .0 pL of target (either recombinant slgE spiked in human serum or volunteer serum samples) was added to 0.625 nM of II and 12 probes prepared in 4.0 pL of lx rCutSmart buffer (during initial concept development) or 4.0 pL of 2.5 mM MgCh and 2 mg / mL recombinant albumin (during assay characterisation) and incubated in PCR tube. Next, one aliquot vial of 2x Mastermix was thawed at room temperature for 5 minutes, followed by the addition of 200 nM template strand and lx EvaGreen plus. Finally, 5.0 pL of the prepared 2x Mastermix was added to each reaction and mixed thoroughly. All preparations were done at room temperature.

[0125] For real-time fluorescence measurement, the reaction tubes were transferred to a Allsheng Gene-8C (excitation = 470 nm, detection = 525 nm) to measure the fluorescence signal every 30 seconds over up to 2 hours at 37°C. For lateral flow visualization, the reaction tubes were similarly incubated at 37°C in the Allsheng Gene-8C reader. After 24 minutes incubation, 0.1 MEDTA was added to quench the reaction. This was followed by hybridization with 25 nM FAM- labelled probes for 10 minutes at room temperature. The reaction volume was then transferred to a 1.5 mL tube pre-filled with the as-provided running buffer to achieve a final volume of 100 pL, followed by the direct dipping of the lateral flow strip.Data Analysis

[0126] For quantification, each data point was expressed as the breakthrough time, defined as the time at which the rate of signal development grows beyond a threshold value determined by default algorithm of the Allsheng Gene-8C reader. During the design evaluation phase, the difference in breakthrough time, denoted as A, between the test and negative control was used.

[0127] When applied on protein systems, calibration curves were obtained by plotting the breakthrough time against the target concentrations. The dataset disclosed herein is best described by the linear regression fitting of a semi-log plot which all calibration curves are presented in. The limit of detection (LOD) was defined to be 3 standard deviations (S.D.) of the breakthrough time of negative control (INC). All data shown reflects the average value from at least triplicate experiments and error bar describing the standard deviation was plotted where relevant (this was omitted for most real-time signal plots to avoid cluttering). Coefficient of variation (CV), which is the ratio of standard deviation to the average signal, was calculated for the protein quantification experiments.

[0128] In the example disclosed herein, the raw data was first normalized by taking the difference between each data point and the minimum value, and dividing it by the difference between the maximum and minimum value for each kinetics curve obtained. The normalized fluorescence kinetics curve was then used to derive the point of inflection (POI). Data normalization was used only for the graphical representation of the kinetics curve and did not affect the POI calculation which was independent of the absolute value of the raw data. A signal - to-noise (S / N) ratio was used to quantify the measured fluorescence signal which was defined as the POI of the test setup (i.e. with target added) divided by the POI of the negative control (i.e. no target added, and abbreviated as NC).TABLESTable 1 provides a summary of the limit of detection (LOD), calculated based on three S.D. below the mean breakthrough time of the negative control, and inter-assay coefficient of variation (CV%) for the three proteins tested, (n = 3)Table 2: List of Oligonucleotide SequencesTable 3: List of protein and antibodies.Note: mAb refers to monoclonal antibody; pAb refers to polyclonal antibody.Table 4: Overview of exemplary linear and hairpin structures

Claims

Claims1. A probe for detecting the presence of one or more target proteins, the probe comprising a structure II and a structure 12:wherein II comprises domains p*, s*, and a*; wherein domain s* is a spacer; wherein domain p* is a first agent that binds to a first binding site on the one or more target proteins; and wherein domain a* is at least 4 nucleotides in length; wherein 12 comprises domains p, s, a, t, n, and k; wherein domain p is a second agent that binds to a second binding site on the one or more target proteins, wherein the second binding site is different from the first binding site of domain p*; wherein domain s is a spacer; wherein domain a is at least 4 nucleotides in length, wherein domain a* is complementary to domain a;wherein domain t is a nucleotide sequence which is present or absent; wherein domain n is a nucleotide sequence which comprises a nicking endonuclease site, wherein the length of domain n is dependent on a nicking endonuclease used; and wherein domain k is a nucleotide sequence comprises a self-copying template sequence; wherein domain t, if present, domain k, and domain n are each single stranded nucleotide sequences.

2. The probe of claim 1, wherein domain a is between 4 to 12 nucleotides in length.

3. The probe of any one of the preceding claims, wherein domain t, if present, is between 1 nucleotide to 10 nucleotides in length.

4. The probe of any of the preceding claims, wherein domain k is at least 5 nucleotides in length.

5. The probe any one of the preceding claims, wherein domain k comprises domain ki and domaink26. The probe of claim 5, wherein domain k2 is complementary to domain t.

7. The probe of any one of the preceding claims, wherein domain a* comprises domain ai* and domain a2*, wherein domains ai* and a2* are complementary to domain a.

8. The probe of claims 1 to 5, and 7, wherein domain k2 is complementary to domain t and domain a2.

9. The probe of any one of claims 7 to 8, wherein domains ai* is between 4 to 12 nucleotides in length, and wherein domain a^* is between 0 to 8 nucleotides in length.

10. The probe of any one of claims 5 to 9, wherein domain k2 is at least 3 nucleotides in length.

11. The probe of any claim 10, wherein domain 1Q is between 3 nucleotides and 10 nucleotides in length.

12. The probe of any one of the preceding claims, wherein domain n is 9 nucleotides in length.

13. The probe of any one of the preceding claims, wherein domain k is between 8 to 200 nucleotides in length, optionally wherein domain k is between 8 to 20 nucleotides in length, optionally wherein domain k is between 10 to 20 nucleotides in length.

14. The probe of any one of the preceding claims, wherein domain s and domain s* each have a length of up to 60 nm (nanometres).

15. The probe of any one of the preceding claims, wherein the probe is:

16. The probe of any one of the preceding claims, wherein domain s is selected from the group consisting of nucleotides, linkers, molecules occupying physical space, such as, poly ethylene glycol, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5 -amino valeric acid (Ava), 6 -aminohexanoic acid (Ahx), PEG2 Spacer or AEEA (8-amino-3,6-dioxaoctanoic acid), PEG3 Spacer (12-amino-4, 7, 10-trioxadodecanoic acid), and combinations thereof.

17. The probe of claim 16, wherein domain s, if comprising nucleotides, the nucleotides have a length of at least 3 nucleotides.

18. The probe of any one of the preceding claims, wherein the nicking endonuclease is active at a temperature between 37°C to 65°C.

19. The probe of any one of the preceding claims, wherein the nicking endonuclease is selected from the groups consisting of Nt.Alwl, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtSI, Nt.BstNBI, and variants thereof.

20. The probe of any one of the preceding claims, wherein a polymerase or fragment thereof binds to domain k, or, where present, domain ki.

21. The probe of any one of the preceding claims, wherein all domains except p, p*, s and s*, are nucleic acid sequences.

22. The probe of any one of the preceding claims, wherein II is selected from the group consisting of SEQ ID NO: 4, 6, 9, 12, 15, and 17.

23. The probe of any one of the preceding claims, wherein 12 is selected from the group consisting of SEQ ID NO: 5, 7, 8, 10, 11, 13, 14, 16, and 18.

24. The probe of any one of the preceding claims, wherein II and 12 are selected from the group consisting of SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 6 and 8, SEQ ID NOs: 9 and 19; SEQ ID NOs: 9 and 11; SEQ ID NOs: 12 and 13; SEQ ID NOs: 12 and 14; SEQ ID NOs: 15 and 16; and SEQ ID NOs: 17 and 18.

25. A method for detecting one or more target proteins in a sample, the method comprising: i. adding a probe as defined in any one of claims 1 to 4, 7, and 9 to 24 to the sample; ii. adding a polymerase or fragment thereof to the sample; iii. adding a combination of deoxynucleotide triphosphates or variants thereof to the sample; iv. allowing binding of the probe to the one or more target proteins, wherein upon binding, domain a* of II and domain a of 12 form a duplex structure, resulting indomain k in a single stranded form with a free 5’ overhang and a 3’ end at domain a*; v. allowing binding of the polymerase or fragment thereof to domain k, which elongates the 3’ end of domain a* using the 5’ overhang of domain k as a template to result in an elongated arm; vi. nicking the elongated arm using a nicking enzyme to obtain a linearly amplified amplicon; vii. exponentially amplifying the amplicon using a self-repeating template; and viii. detecting the amplicon of step vii.

26. A method for detecting one or more target proteins in a sample, the method comprising: ix. adding a probe as defined in any one of claims 5, 6, and 8 to the sample; x. adding a polymerase or fragment thereof to the sample; xi. adding a combination of dcoxynuclcotidc triphosphates and / or variants thereof to the sample; xii. allowing binding of the probe to the one or more target proteins, wherein upon binding, domain a* of II and domain a of 12 form a duplex structure, resulting in a 5’ overhang at domain k and a 3’ end at domain a*; xiii. allowing binding of the polymerase or fragment thereof to domain k, which elongates the 3’ end of domain a* using the 5’ overhang of domain k as a template to result in an elongated arm; xiv. nicking the elongated arm using a nicking enzyme to obtain a linearly amplified amplicon; xv. exponentially amplifying the amplicon using a self-repeating template; and xvi. detecting the amplicon of step xv.

27. The method of any one of claims 25 to 26, wherein the polymerase or fragment thereof possessing strand displacement properties.

28. The method of any one of claims 25 to 27, wherein the polymerase or fragment thereof is selected from the group consisting of Klenow fragment, Bst DNA polymerase, Bsu DNA polymerase, phi29 DNA Polymerase, and fragments or variants thereof.

29. The method of any one of claims 25 to 28, wherein the deoxynucleotide triphosphates is selected from the group consisting of deoxy adenosine triphosphate, deoxycytidinetriphosphate, deoxygu ano sine triphosphate, deoxythymidine triphosphate, and combinations thereof.

30. The method of any one of claims 25 to 29, wherein the deoxynucleotide triphosphates or variants thereof are labelled for detection.

31. The method of any one of claims 25 to 30, wherein the detection in step viii or step xvi is performed using a colorimetric or fluorescent detection method.

32. The method of claim 31, wherein the fluorescent detection method is selected from the group consisting of intercalating dye, such as EvaGreen, and a fluorophore quencher reporter pair.

33. The method of claim 31, wherein the colorimetric method comprises phenol red.

34. The method of any one of claims 25 to 33, wherein the method is carried out on a lateral flow strip.

35. The method of claim 34, wherein the method carried out on the lateral flow strip comprises:(a) contacting the sample comprising the amplicon of step vii or xv with the lateral flow strip;(b) allowing binding of the amplicon of step vii or xv to the lateral flow strip; and(c) detecting the amplicon.

36. The method of any one of claims 25 to 35, wherein the method is performed under isothermal conditions.

37. A kit comprising the probe according to any one of claims 1 to 24.

38. The kit of claim 37, wherein the kit further comprises buffers, the polymerase or fragment thereof as defined in claims 27 to 28, the deoxynucleotide triphosphates or variants thereof as defined in any one of claims 29 to 30, the nicking endonuclease as defined in claim 18 to 19, and the self-repeating unit as defined in claim 23 or 24.

39. The kit of any one of claims 37 to 38, further comprising a lateral flow strip.

40. The kit of any one of claims 37 to 39, further comprising an intercalating dye and / or a reporter strand.

41. The kit of any one of claims 37 to 40, wherein the probe is provided separately, in the form of a lyophilised powder, or as a concentrated stock solution.

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