FIELD OF THE INVENTION The present invention relates to a method for detecting at least one target analyte. In particular, the invention relates to a method for detecting target analyte(s) at room temperature.

BACKGROUND TO THE INVENTION

Nucleic acid-based and immunological diagnoses are widely employed and have far- reaching impact in the field of medical, environmental, food and/or forensic sciences in particular for the detection of microbial pathogens and determination of disease-related proteins. Usually, the target nucleic acid is present in minute amounts in a sample within a complex biological matrix comprising a relatively large excess of non-target nucleic acids, proteins, and other undefined organic molecules. Due to the detection limits of analytical assays, nucleic acid amplification tests such as polymerase chain reaction (PCR) or nucleic acid sequence-based amplification (NASBA) are often required to increase the target polynucleottde-to-interference ratio before detection steps are carried out. These conventional methods for detection of amplified polynucleotide target sequences are usually based on gel electrophoresis and/or membrane-based probe hybridization methods (Southern blotting, Northern blotting or dot blotting) that provide no quantitative results. Further, several of these approaches are also not 100% specific providing no sequence information and exposing users to hazardous elements such as ethidium bromide and ultraviolet light. The more specific methods require highly-trained personnel, tedious preparation in setting up of detection platforms, are labour-intensive and time-consuming.

Immunological approach for detection of target antibody/antigen is another attractive option for diagnostic purposes. Currently, the enzyme-linked immunosorbent assay (ELISA) and lateral flow tests are amongst the most widely used immunoassay formats available. Despite the high sensitivity of ELISA, it has someJimitations such as the relatively long analysis time and requiring costly instrumentation. The lateral flow technique has the advantages of rapidness and simplicity. However, subjective reading of the colour-based flow test results is a major drawback. Electrochemical biosensing assays exploit elements such as nucleic acids, antibodies, and/or antigens to detect a specific target molecule which is then followed by electrochemical detection of these elements via an appropriate transduction element. These assays have been developed as an ideal alternative to the conventional nucleic acid-based and immunological detection methods because it has the advantages of both types of detection methods known in the art. For example, these assays have inherent specificity and selectivity provided by nucleic acid sequence-specific hybridization and antibody/antigen specific binding as well as the advantages offered by electrochemical- based biosensing technology such as cost effectiveness, rapid and accurate analysis, simplicity, possibility of miniaturization and the capability for point-of-care diagnosis.

Accordingly, there is a need in the art for method of detecting target analytes that is fast, accurate and simple to use and may be capable of improved specificity, sensitivity, rapidity, user-friendliness, portability and affordability compared to the methods known in the art.

SUMMARY OF THE !NVENTION

The present invention is defined in the appended independent claims. Some optional features of the present invention are defined in the appended dependent claims. The present invention is directed towards a simple-to-perform method of detecting at least one target analyte at ambient temperature using reagents comprising at least one carbohydrate polymer.

According to a first aspect, the present invention relates to a method of detecting at least one target analyte in a sample, the method comprising the steps of.

a) contacting the sample with a first capturing reagent comprising at least one thermostabilizing reagent for capturing the target analyte, and a first tagging reagent comprising at least one thermostabilizing reagent for tagging the target analyte, to form a target complex; b) purifying the target complex; and c) detecting the presence of the target complex; wherein the presence of the target complex indicates the presence of the target analyte in the sample, and wherein the capturing reagent and the tagging reagent are stable at ambient temperature. According to a further aspect, the present invention relates to a kit for carrying out the method according to any aspect of the present invention. As will be apparent from the following description, specific embodiments of the present invention allow an optimal use of these test strips in a method of amplifying nucleic acids to take advantage of the specificity, sensitivity and simplicity of the method. This and other related advantages will be apparent to skilled persons from the description below. BRIEF DESCRIPTION OF THE FIGURES

Specific embodiments of the method of the present invention will now be described by way of examples with reference to the accompanying figures in which: Figure 1 is a schematic view of the method of the present invention for quantitative detection of an amplified single-stranded polynucleotide sequences.

Figure 2 is a schematic view of the method of the present invention for quantitative detection of an antigen.

Figure 3 is a schematic view of the method of the present invention for quantitative detection of an antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference. According to a first aspect, the present invention provides a method of detecting at least one target analyte in a sample, the method comprising the steps of:

a) contacting the sample with a first capturing reagent comprising at least one thermostabilizing reagent for capturing the target analyte, and a first tagging reagent comprising at least one thermostabilizing reagent for tagging the target anaiyte, to form a target complex; b) purifying the target complex; and c) detecting the presence of the target complex; wherein the presence of the target complex indicates the presence of the target analyte in the sample, and wherein the capturing reagent and/or the tagging reagent are stable at ambient temperature.

This method may allow for a novel room-temperature-stable reagent based electrochemical biosensing assay system that facilitates on-site quantitative detection of at least one target analyte(s). The method may be used to detect more than one analyte at the same time by comprising more than one capturing and/or tagging reagent. In particular, the method may be an on-site specific nucleic acid-based and/or immunological quantitative detection of different target analytes such as amplified single-stranded polynucleotide sequences, target antigen and/or antibody by using the method according to any aspect of the present invention.

In particular, the method may further comprise the steps of d) providing a control analyte; e) contacting the control analyte with a second capturing reagent comprising a thermostabilizing reagent for capturing the control analyte, and a second tagging reagent comprising a thermostabilizing reagent for tagging the control analyte, to form a control complex; f)„ purifying the control complex; and g) detecting the presence of the control complex; wherein the detection of the presence of the control complex verifies the functionality of the method, and wherein the second thermostabilized capturing reagent and the second thermostabilized tagging reagent are stable at ambient temperature.

According to an example, steps a) to g) may be carried out in a single container. A single -.container may be selected from the group consisting_of,_a test tube, an eppendorttube, a centrifuge tube, a microcentrifuge tube, a beaker, a flask, and the like.

The incorporation of IC detection to the assay platform allows for validation of the results. This method comprises the advantages of the use of thermostabilized, pre-optimized, pre- mixed, pre-aliquoted and ready-to-use biosensing assay reagents that confer simplicity and user-friendliness to the experimental procedures by reducing the number of pipetting steps and subsequently shortens the assay turnaround time. The integrated IC may be analysed using simplified enzyme-based colourimetric scheme by simply observing the colour development due to enzymatic reaction with a naked eye. Such a rapid and simple yes/no response IC detection allows validation of the results of the method according to any aspect of the present invention without any encumbrance or impediment to the electrochemical quantitative detection of the target analyte. As used herein, a thermostabilizing reagent is defined as a reagent capable of stabilizing the capturing reagent and/or the tagging reagent at ambient temperature. The capturing reagent and/or the tagging reagent may be thermostabilized by dehydration by vacuum- drying in the presence of the thermostabilizing reagent. The thermostabilizing reagent may be selected from the group consisting of poly(vinylpyrrolidone) (molecular weight 40,000, PVP) and carbohydrate polymers. In particular, the carbohydrate polymer may be selected from the group consisting of trehalose, sucrose, lactose, maltose, raffinose, inulin, dextran, and the like. More in particular, the carbohydrate polymer may be trehalose and sucrose. The presence of the thermostabilizing reagent in at least one of the reagents allow for these reagents to be considered as room-temperature-stable. These ready-to-use biological and non-biological assay reagents may be used for electrochemical biosensing of different target ana!ytes that can not only be used on the method of the present invention but may also be used in advanced integrated sensing systems with maybe automation mode. These reagents may be pre-optimized, pre-mixed, pre-aliquoted and/or subsequently dehydrated in the presence of the carbohydrate polymer to achieve room temperature stability (thermostabilized). This approach of thermostabilizing biosensing assay reagents confers simplicity to the experimental procedures by eliminating the need of preparation of reaction mixture coupled with the capability of reducing pipetting steps, assay turnaround time and the risk of carry-over contamination. In addition, it enables assay reagents to be transported and stored in non-cold conditions.

The thermostabilizing reagent of the capturing reagent may be the same as the thermostabilizing reagent of the tagging reagent. Alternatively, the thermostabilizing reagent of the capturing reagent may be different from the thermostabilizing reagent of the tagging reagent. In particular, the thermostabilizing reagent of the first capturing reagent may be the same as the thermostabilizing reagent of the second capturing reagent. In one example, the thermostabilizing reagent of the first capturing reagent may be different from the thermostabilizing reagent of the second capturing reagent. More in particular, the thermostabilizing reagent of the first tagging reagent may be the same as the thermostabilizing reagent of the second tagging reagent. In another example, the thermostabilizing reagent of the first tagging reagent may be different from the thermostabilizing reagent of the second tagging reagent.

According to an example of the invention, a reagent may comprise one thermostabilizing reagent. Alternatively, the reagent may comprise more than one thermostabilizing reagent.

In particular, the method comprises the use of non-biological reagents such as but not limited to washing buffer. In particular, the washing buffer may comprise 0.01 M potassium phosphate buffer pH 7.4 containing 0.15 M NaCI, and/or 0.01 % v/v Tween 20. IC detection medium for colorimetric enzyme based detection of IC (etc. TMB/H ₂ 0 ₂ substrate), target detection medium for electrochemical nanoparticle-based detection of target analyte (etc. HBr/Br ₂ dissolution medium) and the like. These non-biological reagents have been empirically proven to be stable for years at room temperature (24-29°C). The first capturing reagent and/or the second capturing reagent may comprise at least one capturing moiety capable of binding to the target analyte to form a capturing reagent-bound complex between the target analyte and the capturing agent. This capturing moiety may be selected from the group consisting of antigen, antibody, and nucleic acids. In particular, the antigen may be capable of being recognized by a target antibody, the antibody may be capable of recognizing a target antigen, and ^" the ^' nucleic acid may be capable of hybridizing to the 5' or the 3'end of a target nucleic acid sequence. More particular, the nucleic acids may be capable of hybridizing to the 3' end of the target nucleic acid sequence.

The first capturing reagent and/or the second capturing reagent may further comprise a magnetic core, such that the capturing reagent-bound complex may be purified by the application of a magnetic field. In particular, the magnetic core may be conjugated with the capturing moiety of the capturing reagent.

In particular, the method according to any aspect of the present invention may comprise a magnetic bead-based sandwich detection scheme. In one example, magnetic beads may be used to prepare the capturing reagent. The magnetic bead may serve as a solid support for a reaction where it may be used for detection and capture of the target and/or IC analyte from the sample. This magnetic bead to which the ana!yte may bind may then be bound to at least one tagging reagent. In an alternative example, the magnetic bead-bound analyte may be bound to at least one tagging reagent. The magnetic bead-bound complexes can be easily separated from the sample using an external magnetic field, without the need for centrifugation.

The first tagging reagent and/or the second tagging reagent comprises a tagging moiety selected from the group consisting of an antigen, an antibody, and nucleic acids. In particular, the antigen may be capable of being recognized by a target antibody, the antibody may be capable of recognizing a target antigen, and the nucleic acid may be capable of hybridizing to the 5' or the 3' end of a target nucleic acid sequence. More in particular, the nucleic acids may be capable of hybridizing to the 5' end of the target nucleic acid sequence.

The first tagging moiety may further comprise a hapten molecule. In particular, the hapten molecular may be selected from the group consisting of fluorescein, biotin, digoxigenin, dinitrophenol, and the like. More in particular, the hapten molecule may be a fluorescein.

The tagging moiety may comprise an antibody specific for the hapten molecule. More in particular, the antibody may be selected from the group consisting of anti-fluorescein, anti- biotin, anti-digoxigenin, anti- dinitrophenol, and the like. According to an example of the invention, the hapten molecule can be provided on a probe, wherein the probe may be capable of hybridizing to the 5'or 3'end of the target nucleic acid. In particular, the probe may be capable of hybridizing to the 5'end of the target nucleic acid. In an alternative example of the invention, the target nucleic acid may be amplified with a pair of primers, one of which may be labeled with the hapten molecule, resulting in the incorporation of the hapten molecule at one end of the amplified nucleic acid.

The first tagging reagent and/or the second tagging reagent may further comprise a signalling moiety selected from the group consisting of an enzyme and an electrochemically active nanoparticle. The nanoparticle may be a gold nanoparticle, a silver nanoparticle, or a semiconductor nanoparticle (called also quantum dots). In particular, the semiconductor narioparticle ^' may be selected from the group corisistin ^" g ^~ of cadmium sulfide, CdS; zinc sulfide, ZnS; and lead sulfide, PbS. The enzyme may be selected from the group consisting of horseradish peroxidase and alkaline phosphate enzyme. In one example, the method may use at least one electrochemical-active nanoparticle as a tagging reagent to enable electrochemical detection of the target analyte. Following the capturing of the target analyte, the magnetic bead-bound target complex may be tagged with the nanoparticle that may chemically dissolve using the target detection medium. The dissolved nanoparticle may then be electrochemically analysed using the differential pulse anodic stripping voltammetry (DPASV) method and from the voltammogram a current value obtained at a fixed potential may be recorded as an analytical signal without searching for peak height. Electroanalysis using nanoparticle allows results to be read in a flexible manner owing to the non-dynamic chemical activity of nanoparticle in detection medium. In particular, the detection medium may be a HBr/Br ₂ dissolution medium.

The electrochemical detection of the target analyte may be conducted on a simplified two- electrode configuration screen-printed electrode (2-SPE) that comprise a carbon working electrode and a Ag/AgCI combined reference and counter electrode or conventional three- electrode configuration SPE. In particular, the method according to any aspect of the present invention may use 2-SPE. The simplified design of 2-SPE makes it simple and quick to make, thus decreasing its production costs. In addition, it also enables small size electrodes to be made, which may significantly reduce the sample volume required. In an alternative example, the target detection medium may be TMB/H ₂ 0 ₂ , which may react with horseradish peroxidase to produce a blue colour, thereby enabling visua!/colourimetry detection of the target analyte. In one example, the target detection medium may be a pNPP (p-Nitrophenul Phosphate) detection medium, which reacts with the alkaline phosphate enzyme to produce a yellow colour.

Detection of the target complex may be by means of colourimetry or electrochemistry. The target analyte and/or control analyte may be an antigen, an antibody, or nucleic acids. As used herein, the term "antibody" refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific epitope. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody. For example, in the method of detection according to any aspect of the present invention, an antibody may be capable of binding to the capture reagent. The term "antibody fragment" as used herein refers to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Fragments of the antibodies according to any aspect of the present invention are encompassed by the invention so long as they retain the desired affinity of the full-length antibody. In particular, it may be shorter by at least one amino acid.

The method according to any aspect of the present invention may from the first step to the last step be conducted at room-temperature without the need for incubation and/or washing steps at elevated temperature.

According to a further aspect, the present invention relates to a kit for carrying out the method according to any aspect of the present invention. The kit comprising: a first capturing reagent comprising a thermalstabilizing reagent for capturing the target anaiyte, and a first tagging reagent comprising a thermostabilizing reagent for tagging the target anaiyte;

wherein the first capturing reagent and the first tagging reagent are stable at ambient temperature.

In one example, the kit may further comprise: a control anaiyte for verifying the functionality of the assay; a second capturing reagent comprising a -thermostabilizing reagent for capturing thecontrol anaiyte, and

a second tagging reagent comprising a thermostabilizing reagent for signalling the presence of the target anaiyte associated therewith;

wherein the second capturing reagent and the second tagging reagent are stable at ambient temperature. The inclusion of a control anaiyte in the kit confers the advantage of instant validation of the assay using the same sample.

In one example, the kit may further comprise a detection medium. In particular, the detection medium may be selected from the group consisting of a HBr/Br ₂ detection medium, a TMB/H ₂ 0 ₂ detection medium and a pNPP detection medium. In an alternative example, the detection medium may be a HBr/Br ₂ dissolution medium. The kit may further comprise a hapten molecule. In particular, the hapten molecular may be provided on a probe. More in particular, the probe is capable of hybridizing to a portion of a target nucleic acid. Even more in particular, the probe is capable of hybridizing to the 5'end of the target nucleic acid.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

A person skilled in the art will appreciate that the present invention may be practised without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books. EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

EXAMPLE 1

Quantitative detection of amplified sing!e-stranded polynucleotide sequences Referring to Figure 1 , amplification of nucleic acid sequences can be carried out using standard molecular biology techniques to generate single-stranded polynucleotide sequences in a liquid sample (Figure 1 , C1). The thermostabilized target and control sequences capturing reagent (A), and the internal control (IC) sequence, which are pre- mixed and pre-aliquoted, are resuspended with the sample followed by incubation at room temperature. The first and the second capturing reagents each comprises a magnetic bead (A1 and A2, respectively) conjugated with a probe capable of hybridizing to the 3'end of the target and the IC polynucleotide sequences, respectively, in the sample to form a magnetic bead-bound complex (C2).

For detection, the IC analyte is labelled with digoxigenin, and the target nucleic acid sequence is labelled with fluorescein. This can be achieved by two means: 1) by the inclusion of a fluorescein-labeled detection probe (A1.1 ) in the capturing reagent, wherein t e probe is capable of hybridizing to the 5'end of the target nucleic acid sequences, or 2) by amplifying the target and the IC nucleic acid with a pair of primers, in which one of the primers is labelled with fluorescein, such that a fluorescein molecule is incorporated into the 5'end of the amplified nucleic acid sequences.

The thermostabilized tagging agent (B) is resuspended in distilled water and added to the magnetic bead-bound complexes (C2). The thermostabilized tagging reagent for the nucleic acid (B1) is a mouse monoclonal anti-fluorescein IgG (a-FITC)-conjugated AuNP (AuNP/a-FITC), which is capable of binding to the fluorescein molecule that has been incorporated into the 5'end of the target nucleic acid, and the tagging reagent for the IC polynucleotide sequences is an anti-Digoxigenin-conjugated horseradish peroxidase (a- Dig/HRP) capable of binding with the Digoxigenin-labelled IC nucleic acid. This step results in the formation of target and IC magnetosandwich complexes (C4). The target and IC magnetosandwich complexes are purified by means of magnetic separation. For validation of the assay, a small volume of the purified sample is extracted from the sample, followed by the addition of an IC detection medium, TMB/H ₂ 0 ₂ to the extracted sample. The HRP on the tagging reagent (B2) produces a blue colour in the presence of TMB/H ₂ 0 ₂ . Thus visual observation of the colour formation indicates the presence of the IC polynucleotide sequence and validates of the assay.

The remaining sample is subjected to electrochemical-quantitative analysis on a simplified two-electrode configuration screen-printed electrode (2-SPE, C6), connected to a potentiostat in the presence of a target detection medium. The 2-SPE (C6) is an in-house fabricated electrode that consists of a carbon working electrode and an Ag/AgCI combined reference and counter electrode.

EXAMPLE 2 Quantitative detection of antigen

Referring to Figure 2, a clinical or environmental sample containing the target antigen is added ^~ to pre-mixed and pre-aliquoted reagent containing the ^~ thermostabilized target antigen capturing reagent (A1), the thermostabilized control analyte capturing reagent (A2), and the control antigen. The sample is then incubation at room temperature to form a magnetic bead-bound complex (C2). The thermostabilized target and control antigen capturing reagent each comprises a magnetic bead (A1 and A2, respectively) conjugated with an antibody that is specific for the target antigen and the IC analyte, respectively.

The thermostabilized tagging agent (B1 and B2) are then resuspended in distilled water and added to sample containing the magnetic bead-bound complexes (C2). The thermostabilized target antigen tagging reagent (B1) comprises a gold nanoparticle conjugated with an antibody specific for the target antigen, while the thermostabilized control analyte tagging reagent (B2) is a horseradish peroxidase conjugated with an antibody specific for the control analyte. This step results in the formation of target and IC magnetosandwich complexes (C4) in the sample.

The target and IC magnetosandwich complexes (C4) are purified by means of magnetic separation. For validation of the assay, a small volume of the purified sample is extracted from the sample, followed by the addition of an IC detection medium, TMB/H ₂ 0 ₂ to the extracted sample. The HRP on the tagging reagent B2 produces a blue colour in the presence of TMB/H ₂ 0 ₂ . Thus, visual observation of the colour formation indicates the presence of the IC antigen and validates of the assay.

The remaining sample is subjected to electrochemical-quantitative analysis on a simplified two-electrode configuration screen-printed electrode (2-SPE, C6), connected to a potentiostat in the presence of a target detection medium. The 2-SPE (C6) is an in-house fabricated electrode that consists of a carbon working electrode and an Ag/AgCI combined reference and counter electrode. EXAMPLE 3

Quantitative detection of antibody

Referring to Figure 3, a clinical sample containing the target antibody is added to pre-mixed and pre-aliquoted reagent containing the thermostabilized target antibody capturing reagent (A1), the thermostabilized control analyte capturing reagent (A2), and the control antibody. The sample is incubation at room temperature to form the magnetic bead-bound complexes (C2). The thermostabilized target antibody and control analyte ^~ capturing reagents each comprises a magnetic bead (A1 and A2, respectively) conjugated with antigens that are capable of being recognized by the target and the IC antibodies, respectively. The thermostabilized tagging reagent (B1 and B2) are then resuspended in distilled water and added to the sample containing the magnetic bead-bound complexes (C2). The thermostabilized target antibody tagging reagent (B1) comprises a gold nanoparticle conjugated with an antigen for which the target antibody is specific, while the thermostabilized control analyte tagging reagent (B2) is a horseradish peroxidase conjugated with an antigen for which the control antibody is specific. This step results in the formation of target and IC magnetosandwich complexes (C4) in the sample.

The target and IC magnetosandwich complexes (C4) are purified by means of magnetic separation. For validation of the assay, a small volume of the purified sample is extracted, followed by the addition of an IC detection medium, TMB/H ₂ 0 ₂ to the extracted sample. The HRP on the tagging reagent B2 produces a blue colour in the presence of TMB/H ₂ 0 ₂ . Thus, visual observation of the colour formation indicates the presence of the IC antibody and validates of the assay.

The remaining sample is subjected to electrochemical-quantitative analysis on a simplified two-electrode configuration screen-printed electrode (2-SPE, C6), connected to a potentiostat in the presence of a target detection medium. The 2-SPE (C6) is an in-house fabricated electrode that consists of a carbon working electrode and an Ag/AgCI combined reference and counter electrode.

REFERENCES

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