WO2023287363A2

WO2023287363A2 - High sensitivity lateral flow immunoassay for detection of analyte in samples

Info

Publication number: WO2023287363A2
Application number: PCT/SG2022/050500
Authority: WO
Inventors: Jian Hang Danny TNG; Khee Chee Soo; Eng Eong Ooi; Lee Kiang Melvin CHUA; Yong Zhang; Guek-Hong Jenny LOW
Original assignee: National University Of Singapore; Singapore Health Services Pte Ltd
Priority date: 2021-07-16
Filing date: 2022-07-15
Publication date: 2023-01-19
Also published as: WO2023287363A3

Abstract

A method and device for detecting an analyte in a liquid sample, including applying a liquid sample on a permeable material defining at least a first channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other, said first portion being the site for application of the liquid sample, and for a set of conjugates movably supported therein, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, wherein the first analyte binding molecule or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte and/or analyte binding molecule.

Description

HIGH SENSITIVITY LATERAL FLOW IMMUNOASSAY FOR DETECTION OF ANALYTE

IN SAMPLES

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority to Singapore patent application No. 10202107837T, filed on 16 July 2021 , the contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates generally to lateral flow immunoassay devices and methods for detecting analytes in liquid samples.

BACKGROUND

[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

[0004] Lateral flow immunoassays (LFIA), also known as lateral flow tests, lateral flow immunochromatography assays or rapid tests, are simple devices intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment. These tests are widely used in medical diagnostics for home testing, point of care testing, or laboratory use. As depicted in Figure 1 , a liquid sample is placed along the surface of a sample pad embedded with reactive binding molecules that show a visual positive or negative result. A conventional LFIA as described in Figure 1(A), has a sample pad that would absorb the liquid sample from the patient which contains the analyte. The liquid sample will then flow via capillary action to a conjugation pad, which contains gold nanoparticle, or another dye or detectable particle conjugated to a binding molecule such as an antibody against the analyte. These conjugates are visible to the naked eye when they bind to the analyte, thereby tagging the analyte with a colour. This forms a conjugate-analyte complex which flows through the nitrocellulose membrane until the complex encounters the test line and is isolated at that region. Isolation is achieved by the impregnation of an antibody at the test line which have shown to have a strong affinity with the analyte. This forms a sandwich structure as seen in LFIA sandwich assays in Figure 1(D). As more of the complexes get isolated at the test line, a red band develops indicating the binding of the conjugates to the analyte and thus indicating the presence of the analyte in the sample. The remainder of unbound conjugates which did not form a sandwich structure flow past the test line and get isolated at the control line that contains a secondary antibody against the antibody conjugate, showing up as another red line. This confirms the test is working correctly and that the conjugation of the antibodies to the nanoparticles were successful. A positive test would thus have both test line and control line showing up as coloured bands as seen in Figure 1(B), whereas a negative test would only have a control line band and no test line band as seen in Figure 1 (C). One of the major drawbacks of LFIA is that when the target analyte is in low concentration it becomes difficult to detect the analyte, resulting in a false negative result.

[0005] Traditional LFIAs have a relatively low limit of detection, particularly when detecting viral analytes in saliva samples, producing a weak optical signal when low concentrations of the target protein are encountered. In the realm of LFIAs using salivary samples, this is coupled with the difficulties in sampling, where the activities of the patient such as food intake, may influence the concentration of the virus in the saliva sample. Traditional LFIAs have some success in testing fasting samples. However, it has been noted that their performance is poor with saliva samples obtained directly after food in which the virus concentration is dilute/depleted. This produces difficulties in isolating the virus at the test line and as well as producing a discernable detection signal. This is often a problem when detecting viral analytes using saliva. A method of providing signal enhancement utilizing nanoparticle probe enhancement through the use of linker antigens has been previously described. While there is an increase in the limit of detection this is not sufficient for some analytes. Besides these methods, there are many other strategies which are used for signal amplification. These can be divided into two broad categories, nanoparticle-based methods and device-based methods. Nanoparticle-based methods revolve around making modifications to the traditional nanoparticles conjugated with antibodies against the viral antigens in order to produce a signal enhancement. In remains difficult to increase the limit of detection where the analyte is not sufficient for current detection methods.

[0006] The COVID-19 virus has caused a global health crisis that has deeply affected all countries, not only putting strain on their healthcare systems, but also impacting economies and the ways of life. The virus is highly contagious and susceptible individuals can develop severe complications resulting in on going health issues or death. It is currently over a year into the pandemic although vaccines have begun to roll out, the world is far from reaching the minimum level of protection. There are still various challenges facing their worldwide adoption and more time is needed before restrictions can be removed. The recent outbreaks in Europe and Taiwan are strong reminders that a vaccination program must be paired with equally effective surveillance through the use of effective tools, identifying potential clusters before they grow out of control. There are 3 main ways of which to detect the virus. The first method is genomic testing which consists of methods such as Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) - the current gold standard of viral detection. The second method is through serologic testing which consists of testing for the various immunoglobulins present in a patient and determining if the patient is developing acute phase antibodies to an existing COVID-19 infection. The final method is antigenic testing, where the proteomic components of the virus are detected, thereby confirming the presence of the virus in the tested sample.

[0007] The problem with detection of the COVID-19 virus with existing LFIA devices is the inherent low limit of detection. This is complicated by the issue of viral loads declining when oral intake by patients is factored in. Available test kits result in a detection sensitivity of 40-50% which is not sufficient for COVID-19 screening. Although there are some commercially available salivary LFIAs, they do not disclose the issue of subjects having to abstain from oral intake to achieve adequate sensitivity. As a result, the sensitivities from these manufacturers may be misleading to non-healthcare professionals and will not accurately rule out COVID-19 infection.

[0008] There exists a need to increase the detection limit and alleviate at least one of the aforementioned problems.

SUMMARY

[0009] Alternative lateral flow immunoassay devices and methods to increase the detection limit are envisaged.

[0010] Accordingly, an aspect of the invention refers to a device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other said first portion being the site for application of the liquid sample, and for a set of conjugates movably supported therein, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

[0011] According to another aspect there is a method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining the first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present, the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second conjugate movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second conjugate comprises a set of amplification analyte binding molecules each coupled to a detectable particle capable of binding to the first analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

[0012] According to another aspect there is a device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel and a second and/or subsequent channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other, said first portion being the site for sequential application of the liquid sample, and for a set of conjugates movably supported therein, wherein a first set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle and a second set of conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle, a disruption between the first, second and/or subsequent channel, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte.

[0013] According to another aspect there is a method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication therebetween; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second set of conjugates movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second set of conjugates each comprises an amplification analyte binding molecule coupled to a detectable particle capable of binding to the first analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion.

[0014] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present invention,

[0016] Figure 1: Prior art Lateral Flow Immuno-Assay (LFIA): (A) Schematic diagram showing the view from above and the cross-section, (B) Positive test showing both control line and test line, (C) Negative test showing only control line, (D) Sandwich mechanism at the test line using the prior art method, (E) Quantitative analysis of test results using image processing with mobile phone photography, (F) Standard viral isolation at test line.

[0017] Figure 2: Embodiments including: (A) a schematic diagram of Parallel Lateral Flow Immuno-Assay (P-LFIA) with amplification, showing individual components channel 1 containing anti-spike probes 9 which tag the virus 8 and channel 2 containing amplification nanoparticles 10 which amplify the signal; (B) a schematic diagram of a conventional Lateral Flow Immuno-Assay (LFIA) with multiple binding sites 13 of the analyte at the test line 5 (C) Quantitative analysis of test results using image processing with mobile phone photography 14.

[0018] Figure 3: Parallel Lateral Flow ImmunoAssay (P-LFIA) with amplification: (A) prototype P-LFIA with two parallel flow channels, with casing (above) and without casing (below), (B) schematic diagram of P-LFIA showing individual components channel 1 contains anti-spike probes which tag the virus and channel 2 contains amplification nanoparticles which amplify the signal, (C) schematic diagram of the amplification nanoparticles, (D) sandwich assay formed at test line after the activation of channel 1 - faint test line signal is obtained and (E) sandwich assay after activation of channel 2, 15 minutes later - test line signal amplified showing positive result.

[0019] Figure 4: P-LFIA used to perform cascade signal amplification: (A) example showing 4 channels, (B) In-vitro characterization of cascade amplification with inset showing correlated amplification schematic at test line in parts: (I) after activation of 1 channel, (II) after activation of 2 channels, (III) after activation of 3 channels and (VI) after activation of 4 channels - process can be repeated many times to enhance signal further as required.

[0020] Figure 5: Applications of multisite antigen binding: (A) multisite antigen binding at test line with larger region of binding, (B) example 3 channel parallel flow with different nanoparticle probe conjugates - channel 1 with viral binding probe, channel 2 with fluorescent probe, channel 3 with signal enhancement probe and (C) multisite antigen binding showing binding of different probes at different locations on the target antigen allowing for both antigen amplification and other functions such as multimodal imaging with fluorescent nanoparticles.

[0021] Figure 6: Results of Single Enhanced Parallel-LFIA. (A) In-vitro testing of nanoparticle probes with model virus showing detection up to 1 E7 viral particles/ml, (B) In- vitro testing of LFIA showing the multisite antigen binding technique to perform signal enhancement at different model virus dilutions. Horizontal dotted line denotes the detection thresholds, positive values indicate in-vitro samples tested at that concentration are detectable by the assay.

[0022] Figure 7: Results from case-control clinical study using conventional LFIA: (A) Comparison of sensitivity and specificity when tested for RBD antigen alone, S1 antigen alone and RBD with S1 antigen together, (B) Effect of oral intake on test sensitivity using the multiantigen RBD with S1 antigen test which is unamplified compared against the amplified P-LFIA at the 1 hr mark, (C) Sensitivity of other anterior nasal swab rapid antigen tests using saliva samples obtained within 1 hour of oral intake (Sensitivity of one other test, Arista 2.0, was 0% and not shown).

[0023] Figure 8: Results from case-control clinical study using cascade amplified P-LFIA to detect the S1 protein sub-unit: (A) Distribution of the relative intensities of tested PCR confirmed cases pre-amplified and post amplification (Horizontal line indicates detection threshold, intensities above 0 indicate successful detection of the virus), (B) Signal amplification of the P-LFIA showing each tested case before and after amplification also showing the capability of the P-LFIA to detect variants (with Y = 0 indicating detection threshold, intensities above 0 indicate successful detection of the virus), (C) Improvement in sensitivity and specificity after amplification, (D) Receiver operator characteristics (ROC) curve of unamplified and amplified test, (E) Mean normalized intensity of the test line for all patients tested according to day of illness (Horizontal line Y =0 indicates the threshold, intensities above the line indicate successful detection) and (F) Mean reciprocal CT number of all patients tested according to day of illness tested. (Horizontal line indicates the CT value of 35 which is the criteria for de-isolation for confirmed cases used in Singapore).

[0024] Figure 9: Results from case-control clinical study using cascade amplified P-LFIA to detect the RBD protein sub-unit: (A) Distribution of the relative intensities of tested PCR confirmed cases pre-amplified and post amplification (Horizontal line indicates detection threshold, intensities above 0 indicate successful detection of the virus), (B) Signal amplification of the P-LFIA showing each tested case before and after amplification also showing the capability of the P-LFIA to detect variants (with Y = 0 indicating detection threshold, intensities above 0 indicate successful detection of the virus), (C) Improvement in sensitivity and specificity after cascade amplification, (D) Receiver operator characteristics (ROC) curve of unamplified and amplified test, (E) Mean normalized intensity of the test line for all patients tested according to day of illness (Horizontal line Y =0 indicates the threshold, intensities above the line indicate successful detection) and (F) Mean reciprocal CT number of all patients tested according to day of illness tested. (Horizontal line indicates the CT value of 35 which is the criteria for desolation for confirmed cases used in Singapore).

[0025] Figure 10: LFIA with a stacked/series flow attempting to implement multiple site antigen binding. (A) Schematic diagram showing 3 conjugate pads lined up in series ahead of the sample pad, with each conjugate pad having a different nanoparticle conjugated with antibodies directed against different sites of the same antigen and (B) Inset showing the test line sandwich assay where there is competitive binding between the 3 nanoparticle conjugates which are trying to bind to the antigen at the same time.

[0026] Figure 11: LFIA with nanoparticle conjugate signal amplification. (A) Schematic diagram showing 2 conjugate pads lined up in series ahead of the sample pad, one containing the nanoparticle conjugated with antibody against the virus antigen and another containing a signal amplification nanoparticle conjugated with antibody against the nanoparticle conjugate that targets the viral antigen. (B) Reduced binding of the nanoparticle against the target antigen due to the presence of linker antigens on the nanoparticle surface needed to perform amplification, (C) schematic diagram of the nanoparticle for binding to the target antigen where antibody conjugation sites are taken up by linker antigens, (D) schematic diagram of amplification nanoparticles with antibodies against the linker antigen and (E) Schematic diagram of linker-free amplification nanoparticles as used in this disclosure that do not require linker antigens for enhancement. [0027] Figure 12: Serial LFIA unfeasible with cascade amplification and also linker-free amplification probes: (A) schematic diagram showing serial LFIA with amplification nanoparticles on one pad and viral probe nanoparticles on another conjugate pad, (B) linker- free amplification probes binding to viral probe nanoparticles causing blockage of viral binding site and (C) no probe binding to the virus at the test line due to steric hinderance by the amplification nanoparticles, no further amplification possible due to stacked array.

[0028] Figure 13: Limit of detection studies of the P-LFIA without amplification with various nanoparticle probes against a model virus constructed using the antibody’s known binding proteins represented as (antibody)x(protein) according to the manufacture’s catalog number: (A) 40591-MM42 (S1 MAb), (B) 40591-T62 (S1 PAb), (C) 40589-T62 (S1+S2 Ab), (D) 40150-T62 SARS (S1 Ab), (E) Ab01680-10.0 (S1/RBD), (F) 40592-T62 (RBD Ab), (G)

40150-T30 SARS (RBD Ab), (H) Average COVID-19 RBD Ab performance and (I) Average COVID-19 S1 Ab performance. Model virus concentration is represented as number of particles/ml. [0029] Figure 14: Limit of detection studies of the P-LFIA without amplification with various nanoparticle probes against a model virus constructed a common spike protein (40589- V08B1). (A) 40591-MM42 (S1 Ab), (B) 40591-T62 (S1 Ab), (C) 40589-T62 (S1 Ab), (D) 40150-T62 SARS (S1 Ab), (E) Ab01680-10.0 (S1/RBD), (F) 40592-T62 (RBD Ab), (G) 40150-T30 SARS (RBD Ab), (H) Average COVID-19 RBD Ab performance and (I) Average COVID-19 S1 Ab performance. Model virus concentration is represented as number of particles/ml.

DETAILED DESCRIPTION

[0030] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0031] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0032] Throughout the description, it is to be appreciated that the term ‘analyte’ and its plural form may include target molecule, target biomarker, target protein or target antigen.

[0033] Throughout the description, it is to be appreciated that the term ‘analyte binding molecule’ and its plural form may include or refer to one or more molecule which is capable of binding to a target antigen or target analyte, and encompasses an aptamer, a protein such as receptor proteins, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s) or target analyte.

[0034] Unless defined otherwise, all othertechnical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs.

[0035] According to various embodiments there is a device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other said first portion being the site for application of the liquid sample, and for a set of conjugates movably supported therein, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

[0036] As used herein a liquid sample comprises a biological sample. In various embodiments liquid sample comprise saliva samples. In various embodiments liquid sample comprise urine samples. In various embodiments liquid sample comprise sweat and tear samples. In various embodiments liquid sample comprise breast milk samples. In various embodiments liquid sample comprise pus or fluid samples from cysts or blisters. In various embodiments liquid sample comprise lacrimal fluid samples. In various embodiments liquid sample comprise nasopharyngeal mucous, nasal or oral pharyngeal samples. In various embodiments liquid sample comprise faecal samples. In various embodiments liquid sample contain or are suspected of containing microorganisms. In various embodiments liquid sample contain or are suspected of containing infectious microorganisms. In various embodiments liquid sample comprise medical samples. In various embodiments liquid sample comprise veterinary samples. In various embodiments liquid sample comprise samples from food. In various embodiments liquid sample comprise water samples. In various embodiments liquid sample comprise sewage samples. The methods and device are particularly suitable to remote sampling or field sampling and testing.

[0037] The highly sensitive antigenic test device may be used for many implications and applications. In various embodiments the applications may include detection of infectious diseases such as viral detection including COVID-19 testing. Besides infectious diseases, the use of such technology would also have many applications in other areas of medicine where frequent tests for the surveillance of acute conditions or for the management of chronic conditions are required in areas such as in oncology, emergency medicine and also rheumatology.

[0038] Frequent testing is often required to decide on the titration of medication. In the area of oncology, the device could be used for frequent monitoring of certain oncologic markers, where a quick point of care test would be useful for the guidance of treatment such as the measurement of biomarkers after each chemotherapy cycle to determine the effectiveness of the cycle and for further modifications to the regime. For instance, this is seen in the area of gastric cancer where carcinoembryonic antigens and carbohydrate antigens are monitored to direct the chemotherapy regime.

[0039] The monitoring of other organ specific biomarkers with the device would be useful to determine toxicity as well, minimizing treatment related complications.

[0040] Other areas of oncology in which the device would be useful would be in the monitoring of recurrence, such as the monitoring of Epstein Bar Virus DNA titers in nasopharyngeal samples or the monitoring of thyroid related antigen titers in thyroid cancer.

[0041] In the area of emergency medicine, the immediate evaluation of crucial biomarkers to determine organ function using the device would be instrumental for the management of many diseases. Biomarkers such as tryptase have known to be tied to anaphylaxis and the quick screening of such biomarkers would be useful in emergency situations to direct treatment as delay would spell worse clinical outcomes.

[0042] As used herein, the term ‘detectable particle’ refers to a particle able to detect the presence on the test analyte. In various embodiments, the detectable particle may be detected in a variety of ways. In various embodiments, the detectable particle may be detectable by eye in visible light providing an optical signal. In various embodiments, the detectable particle may provide a fluorescent signal. In various embodiments, the detectable particle may provide an infrared signal. In various embodiments, the detectable particle may provide a plasmonic signal. This enables target analyte detection with different modalities.

[0043] In various embodiments, the detectable particle may be an enzyme such as horseradish peroxidase which catalyzes a reaction to produce an end product with optical properties.

[0044] In various embodiments, the detectable particle may be particles which have other types of signals such as an electromagnetic signal which thereby confers multimodal detection.

[0045] As used herein ‘where the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites’ refers to one of the 3 permutations: (i) conjugates each comprises a first analyte binding molecule directed to one site of the analyte, coupled to a detectable particle and another first analyte binding molecule directed to a different site of the analyte, coupled to the detectable particle; (ii) a second analyte binding molecule directed to one site of the analyte immobilized in the second portion and another second analyte binding molecule directed to another site of the analyte immobilized in the second portion; or (iii) conjugates each comprises a first analyte binding molecule directed to one site of the analyte, coupled to a detectable particle and another first analyte binding molecule directed to a different site of the analyte, coupled to the detectable particle and a second analyte binding molecule directed to one site of the analyte immobilized in the second portion and another second analyte binding molecule directed to another site of the analyte immobilized in the second portion.

[0046] In various embodiments, the first portion comprises the said first channel, a second and/or subsequent channel with a second set of conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle, and a disruption between the first, second and/or subsequent channel. This has the advantage of amplification of detection or signal enhancement in the second portion at the test line in order to increase the limit of detection.

[0047] As used herein, the term ‘amplification analyte binding molecule’ refers to a linker free amplification analyte binding molecule.

[0048] In various embodiments the first portion comprises 1 , 2, 3, 4, 6, 7, 8, 9, or 10 channels wherein each channel includes a set of second or subsequent conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle. For signal enhancement purposes, multiple sets of conjugates are made. Each set of conjugates may comprise an analyte binding molecule that targets a different binding site of the same target analyte, another amplification analyte binding molecule or conjugate coupled to a detectable particle. This results in multiple detectable particles being bound to a single analyte, allowing each target analyte to have a larger summated signal for enhancement.

[0049] In various embodiments, the detectable particles are identical with the same detection properties used which thereby provides an enhanced detection signal through summation.

[0050] In various embodiments, the disruption between the first and second and/or subsequent channel is a gap in the permeable material. [0051] In various embodiments, the disruption between the first and second and/or subsequent channel is a feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel.

[0052] In various embodiments, the disruption between the first and second and/or subsequent channel comprises the first set of conjugates being situated at different regions along the first channel compared to the location of the second and/or subsequent set of conjugates along the second and/or subsequent channels.

[0053] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises the second or subsequent channel having a narrower width than the width of the first channel.

[0054] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises the second or subsequent channel having a thinner channel compared to the thickness of Z axis dimension of the first channel.

[0055] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises the second or subsequent channel having a serpentine channel path.

[0056] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises the second or subsequent channel having an inert material such as another blank conjugation pad or absorption pad partially blocking the path of liquid flow along the second or subsequent channel.

[0057] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises a frustrater with a liquid input linking to a series of outputs at each channel allowing the application of the liquid to each channel to be applied at different thereby the liquid sample is first added to the first channel through a first output without much delay then the liquid sample moves to the second output at the second channel but it is delayed and the subsequent outputs at the subsequent channels are further delayed by the frustrater in the liquid path. [0058] In various embodiments, the feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel comprises the second and subsequent channels being interconnected to one another before being connected to the second portion.

[0059] In various embodiments, the second analyte binding molecule comprises two or more different analyte binding molecules capable of binding the same analyte at different sites on the analyte. This has the advantage of improved analyte immobilization or isolation in the second portion at a test line the multisite antigen binding technique.

[0060] In various embodiments, the two or more different analyte binding molecules capable of binding the same analyte at different sites on the analyte comprise a host receptor protein that binds the analyte and an antibody. In various embodiments, the two or more different analyte binding molecules capable of binding the same analyte at different sites on the analyte comprise two different antibodies. In various embodiments, multiple sites on the same target analyte or target antigen are utilized for binding, allowing increased binding to the test line in the second portion and/or increased binding of the first and second conjugates, both contributing to signal enhancement at the test line. In various embodiments, multiple antibodies which bind to different sites on the analyte or target antigen are immobilized onto the permeable material in the second portion at a test line. This allows an increased number of the target analyte to be isolated at the test line though the increased binding of the target analyte to the test line.

[0061] In various embodiments, the permeable material comprises sorbent material defining a flow path extending from the first portion to a test site in the second portion.

[0062] In various embodiments the second portion comprises a nitrocellulose membrane.

[0063] In various embodiments, the detectable particle is a coloured particle; whereby accumulation of coloured particles at the test site produces a colour visible to the unaided eye indicative of the presence of the analyte in the liquid sample.

[0064] In various embodiments, the first and/or second conjugate is disposed in the flow path upstream of the test site and is mobilizable along the flow path with passing liquid.

[0065] The In various embodiments, the conjugate is in dry form.

[0066] In various embodiments, the first and/or second conjugate is transported along the flow path by liquid wicking or wetting through the sorbent material. [0067] In various embodiments, the first analyte binding molecule binds to a coronavirus protein.

[0068] In various embodiments, the second analyte binding molecule binds to a coronavirus spike protein.

[0069] In various embodiments, the coronavirus comprises a SARS-CoV-2 whole virus. In various embodiments, the SARS-CoV-2 comprises a single component of the virus or combinations of SARS-CoV-2 individual components.

[0070] In various embodiments, the second analyte binding molecule comprises two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule is capable of binding to S1 subunit of the SARS-CoV-2 spike protein, or is capable of binding to S2 subunit of the SARS-CoV-2 spike protein and the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein the same analyte at different sites on the analyte.

[0071] In various embodiments, the second analyte binding molecule comprises three analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule capable of binding to S1 subunit of the SARS-CoV-2 spike protein, the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein and the third SARS-CoV-2 spike protein binding molecule capable of binding to the S2 subunit of the SARS-CoV-2 spike protein with the same analyte at different sites on the analyte.

[0072] In various embodiments, one of the analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises Angiotensin Converting Enzyme 2 (ACE2) protein. ACE2 is the host receptor protein that binds to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises an antibody. This has the advantage of being able to detect emerging variants that have a higher affinity to ACE2 protein, thereby making ACE2 protein suitable candidate for use in various embodiments. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises a monoclonal antibody. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises polyclonal antibody. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises or a combination of both monoclonal and polyclonal antibody. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises an antibody to the S1 region of the SARS-CoV-2 spike protein. In various embodiments, one of the two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites comprises an antibody to the S2 region of the SARS-CoV-2 spike protein. In various embodiments, one of the two analyte binding molecules capable of binding to SARS- CoV-2 spike protein at different sites comprises an antibody to the RBD region of the SARS- CoV-2 spike protein.

[0073] In various embodiments, the second analyte binding molecule is capable of binding to SARS-CoV-2 spike protein comprising or consisting of the amino acid sequences set forth in SEQ ID NO.: 1 or 2. In various embodiments, the different sites on the spike SARS-CoV-2 spike protein are selected from analyte binding sites capable of binding to SARS-CoV-2 spike protein S1 , RBD, or S2. In various embodiments the SARS-CoV-2 spike protein S1 subunit comprises or consists of any one of the amino acid sequences set forth in SEQ ID NO.: 3, 4, 5 or 14. In various embodiments the SARS-CoV-2 spike protein RBD subunit comprises or consists of any one of the amino acid sequences set forth in SEQ ID NO.: 6, 7 or 13. In various embodiments the SARS-CoV-2 spike protein S2 subunit comprises or consists of any one of the amino acid sequences set forth in SEQ ID NO.: 8, 9 or 15. In various embodiments, the ACE2 protein comprises or consists of any one of the amino acid sequences set forth in SEQ ID NO.: 10, 11 , 12, 16, 17, or 18. In various embodiments, the second analyte binding molecule capable of binding to SARS-CoV-2 spike protein comprises any protein or antibody known to bind specifically with affinity to a site of the SARS-CoV-2 spike protein. In various embodiments, the second analyte binding molecule is capable of binding to SARS-CoV-2 nucleocapsid protein comprising or consisting of the amino acid sequences set forth in SEQ ID NO.: 19.

[0074] In various embodiments, the first analyte binding molecule comprises an antibody. In various embodiments, the first analyte binding molecule comprises a monoclonal antibody. In various embodiments, the first analyte binding molecule comprises a polyclonal antibody.

[0075] In various embodiments, the first analyte binding molecule comprises a protein which are known to bind to the target analyte.

[0076] In various embodiments, the coloured particle is a metal sol particle. [0077] In various embodiments, the coloured particles are nanoparticles which are conjugated with proteins which have specific binding ability to the protein to be detected, the “target analyte, target protein or target antigen”.

[0078] In various embodiments, the metal sol particle is colloidal gold.

[0079] Various embodiments specifically address antigenic testing by using a Lateral Flow ImmunoAssay (LFIAs), which employs nanoparticles that are conjugated with proteins or antibodies which specifically bind to COVID-19 antigens, thereby producing a detectable signal.

[0080] In various embodiments, the permeable material comprises a nitrocellulose membrane.

[0081] In various embodiments, the second portion further comprises a control site comprising a third analyte binding molecule immobilized therein capable of binding to the first analyte binding molecule.

[0082] According to various embodiments there is a method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining the first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second set of conjugates movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second set of conjugates each comprises an amplification analyte binding molecule coupled to a detectable particle capable of binding to the first analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

[0083] The method has the potential to overtake PCR as a choice of community screening for infectious diseases such as COVID-19. Salivary testing with such rapid kits does not require sophisticated laboratory infrastructure like PCR and trained staff are not required to extract samples. Moreover, PCR has a limitation in terms of screening as noted in some studies, PCR sensitivity remains only at around 90% for the first 5 days of SARS CoV-2 infection, before gradually decreasing to between 80 - 70% from days 8 -10 before dropping below 50% by day 18.5. This could be due to the decrease in viral load which was also reflected in other studies that used RT-PCR to detect other respiratory illnesses where concordant results of RT-PCR and virus culture positivity had reduced viral load in tandem with increase days since onset of symptoms.

[0084] In various embodiments the method allows a parallel flow technique. This is where multiple parallel lateral flow channels are used to perform sequential reactions on the lateral flow assay for signal enhancement. Each flow channel contains different sets of conjugates that bind either to the target analyte or to perform signal enhancement. Each flow channel is activated sequentially to allow one reaction to occur at a time. This ensures that each set of conjugates not only have optimal time to bind to the binding site on the target analyte but also reduces the effect of steric hinderances and competitive binding experienced with using multiple nanoparticle conjugates at the same time. In various embodiments, the parallel flow method may be used with novel linker-free amplification nanoparticles to perform cascade amplification, which theoretically would allow multiple amplification of the signal, allowing it to perform similar to other methods such as Polymerase Chain Reaction (PCR), where the amplification step is repeated multiple times until a signal is shown. In various embodiments the linker-free amplification nanoparticles bind to the first analyte binding conjugates. In various embodiments the linker-free amplification nanoparticles bind to the target antigen but at a different site.

[0085] In various embodiments, when the analyte is present the analyte binds to the conjugate and the conjugate binds to the second analyte binding molecule immobilized in the second portion, immobilizing the entire complex comprising the conjugate and the second analyte binding molecule in the second portion for detection.

[0086] In various embodiments, the modified LFIA method has the advantages of i) testing for multiple antigens to increase sensitivity, and ii) employs sequential reactions via the parallel flow technique to perform signal enhancement with the use of linker-free amplification nanoparticles. In the context of this invention, the technology is used to create a rapid, point-of-care antigen test for COVID-19.

[0087] In various embodiments, the method may be used to detect proteins in the areas of other diseases, such as oncologic, hematological, rheumatological, and other infectious biomarkers.

[0088] In various embodiments, the detectable particle of the first set of conjugates is different from the detectable particle of the second set of conjugates. In this way the method can also be used to generate multiple different optical signals, for example, one fluorescent and one visible optical signal.

[0089] In various embodiments, the method further comprises: e.1 ) applying the liquid to a subsequent channel in the first portion of the device; and e.2) allowing the liquid to flow to a subsequent set of conjugates movably supported in the subsequent channel of the first portion then to the second portion of the permeable material, wherein said subsequent set of conjugates each comprise a subsequent amplification analyte binding molecule coupled to a detectable particle capable of binding to the second analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein and the second conjugate is bound to the first conjugate, the subsequent conjugate will bind to the second conjugate and amplify the detection.

[0090] In various embodiments, second analyte binding molecule comprising two or more different analyte binding molecules capable of binding to the same analyte at two different sites on the analyte. In various embodiments, this has the advantage of utilizes multisite antigen binding for increased immobilization or isolation of the analyte.

[0091] In various embodiments, determining the presence of the analyte comprises: g) capturing the image of the second portion to analyse intensity of binding; and h) calculating the quantity of the analyte based on the analysis of the intensity of binding. In various embodiments, the image is captured using an image capture device such as a mobile phone, camera, fluorometer or any other similar device that permits densitometric analysis that can be correlated with concentration of the quantity of the analyte present. In various embodiments a processor calculates the quantity of the analyte present. In various embodiments the processor is part of the image capture device. In various embodiments the processor is located separately from the image capture device and the image is converted to a signal prior to being communicated from the image capture device to the processor located separately. [0092] In various embodiments, the liquid sample comprises saliva.

[0093] In various embodiments, the detectable particle is a coloured particle and determining the presence of the analyte comprises observing visually the test result at a test site wherein accumulation of coloured particles produces a colour indicative of the presence of the analyte in the liquid sample.

[0094] In various embodiments, the method comprises the additional step of mixing the conjugate with the liquid sample prior to applying the sample to the device.

[0095] In various embodiments, in use the first analyte binding molecule binds to a coronavirus protein.

[0096] In various embodiments, in use the second analyte binding molecule binds to a coronavirus spike protein.

[0097] In various embodiments, the coronavirus comprises a SARS-CoV-2 whole virus. In various embodiments, the SARS-CoV-2 comprises components of the virus or a combination of components of the virus.

[0098] In various embodiments, the implementation of such techniques in an LFIA would be able to produce a highly sensitive, rapid, point of care test which would also be self- administrable. A negative result would help to exclude COVID-19 infection with a high degree of confidence and those with a positive result would then be quarantined for further testing with a PCR nasal swab. The implementation of such a test would serve a “health visa”, to be performed before various activities such as mass gatherings or travel. The use of such a test would be an important step for the return to normalcy as full vaccination of the entire world population is not expected to be completed within the next 1 to 2 years.

[0099] In various embodiments, in use the second analyte binding molecule comprises two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule is capable of binding to S1 subunit of the SARS-CoV-2 spike protein or to S2 subunit of the SARS-CoV-2 spike protein and the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein the same analyte at different sites on the analyte.

[00100] In various embodiments, in use the second analyte binding molecule comprises three analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule capable of binding to S1 subunit of the SARS-CoV-2 spike protein, the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein and the third SARS-CoV-2 spike protein binding molecule capable of binding to the S2 subunit of the SARS-CoV-2 spike protein with the same analyte at different sites on the analyte.

[00101] In various embodiments, the first analyte binding molecule comprises a monoclonal antibody. In various embodiments, the first analyte binding molecule comprises a polyclonal antibody.

[00102] In various embodiments, in use the coloured particle is a metal sol particle.

[00103] In various embodiments, in use the metal sol particle is colloidal gold.

[00104] In various embodiments, the coloured particles are gold nanoparticles. In various embodiments, the binding of these conjugated nanoparticles to the target protein produces an optical signal, which confirms the presence of the target protein.

[00105] In various embodiments, in use the second portion further comprises a control site comprising a third analyte binding molecule immobilized therein capable of binding to the first analyte binding molecule. This confirms the test is working correctly and that the conjugation of the antibodies to the nanoparticles were successful.

[00106] In various embodiments, the method demonstrates that two or more binding sites can be detected simultaneously in order to increase sensitivity. In various embodiments, two spike protein antigens, namely the Receptor Binding Domain (RBD) subunit and the S1 subunit were tested, making this the first multiple antigen point-of-care test for COVID-19. In various embodiments, through the use of the multisite antigen binding method, antibodies and proteins which bind to different sites of the spike protein were used at the test line for increased viral isolation. In various embodiments, the parallel flow method, which can enable control of sequential reactions on the test site, permits signal amplification. In various embodiments, the virus is initially labelled by a nanoparticle probe conjugated with antibodies against the viral Spike RBD or Spike S1 protein in the first flow channel. Subsequently, the preceding second and/or subsequent flow channel, which contains signal enhancement detection particles that bind to the first conjugate viral-nanoparticle probe complex is activated to perform signal amplification. These second conjugate amplification nanoparticles do not require any additional linker antigens on the nanoparticle probes that bind with the target viral antigens. In various embodiments these second conjugate amplification nanoparticles are conjugated to antibodies which bind to the antibodies against viral Spike RBD or Spike S1 protein. Additional flow channels can be tagged on in order to repeat this reaction infinitely, allowing the detection of virus at low concentrations. Clinical studies of various embodiments have been performed with salivary samples to compare the performance of assay device using a multiantigen binding viral isolation technique alone and when the assay is enhanced with parallel flow enhancement. When various embodiments are combined, even more effective detection of the COVID-19 virus was detectable from after food salivary samples with a sensitivity and specificity of 95% and 100% respectively. The test shows 95% agreement with PCR testing even at lower CT values of 35 - 40. A head-to-head comparison of currently available antigen tests are also included which show that various embodiments of the method demonstrated superior in salivary testing.

[00107] In various embodiments the methods and devices mentioned herein above may be further enhanced by using other known means to increase detection together with the device and methods listed above. There are many other nanoparticle-based methods for signal enhancement with various degrees of improvement in detection performance. The nanoparticle can be modified from its traditional spherical configuration to other structures such as nanostars™ or nanopopcorns™, which show a 5 to 10 times enhancement in detection performance. Larger nanoparticles also increase the optical signal and there have been several methods used to stably increase the size of the nanoparticle conjugates that have shown to have up to 400-fold improvement in detection. Hybrid nanoparticles which are stable at larger sizes such as gold-silica nanoparticles have also shown similar improvements in detection performance of up to 30 times. Similar ways of stabilizing the nanoparticles include polyethylene glycol coating which also shows an increase in detection performance of up to 12.5 times.

[00108] Controlled antibody conjugation has also shown to improve detection performance as well, ensuring the Fab region of the conjugated antibodies are oriented outwards, allowing a larger amount of binding sites to bind to the antigenic target. The nanoparticles can also be modified at the test line, where additional reagents are added to the test line to enhance the signal. One popular method is silver enhancement where the reduction of silver lactate by hydroquinone is catalysed by gold, which would be present at the test line after successful binding of the nanoparticle conjugates to the viral antigens. This has shown an improvement of up to 15 times in detection performance. The nanoparticle conjugates can also be modified with enzymes such as horseradish peroxidase, where Tetramethylbenzidine (TMB) is added to the test line and the reduction of TMB produces a more intense color change, enhancing the optical signal at the test line indicating the presence of the nanoparticle conjugates binding to the viral antigens. Magnetism can also be used to increase the amount of nanoparticle conjugates at the test line, which have shown up to 20 times improvement in detection.16 Methods can be employed to introduce optical energy from external sources to improve the signal intensity as well. Popular examples would be to use fluorescent nanoparticles which would produce a fluorescent signal upon being excited via an external light source. Other methods could also rely on infrared heating which would utilize an external infrared light source to head the nanoparticle on the test line with subsequent measurement of their emission thereby improving the signal strength at the test line.

[00109] According to various embodiments there is a device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel and a second and/or subsequent channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other said first portion being the site for sequential application of the liquid sample, and for a set of conjugates movably supported therein, wherein a first set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle and a second set of conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle, a disruption between the first, second and/or subsequent channel, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte.

[00110] According to various embodiments there is a method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication therebetween; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second set of conjugates movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second set of conjugates each comprises an amplification analyte binding molecule coupled to a detectable particle capable of binding to the first analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion.

[00111] Device based signal amplification involves making modifications to the LFIA itself, such as the nitrocellulose membrane, sample pad, conjugate pad and absorption pad. These modifications change the flow of the reagents on the LFIA, thereby allowing certain reactions to occur or making available other types of reagents at a higher concentration in order to produce a signal enhancement. The Parallel-LFIA presented in this disclosure is an example of a device-based signal amplification and there are also other methods. One other common method is to introduce flow slowing, where the sample flow speed is reduced in order to allow more time for the reagents to react, such as by using wax pillars in the pathway of the sample flow. Such methods have shown to improve detection by up to 3-fold. Another method of achieving this is through the use of a stacking pad, which is an additional membrane between the conjugate and test pad which also delays the flow of the sample to the test line, allowing for more time for the nanoparticle binding to occur, this has shown to improve detection by 2-fold. Concentrators have also been effective at removing excess water, thereby allowing the sample to be more concentrated with the antigen of interest. One such method is to use a semi-permeable membrane at the sample pad which wicks away excess moisture from the sample, thereby allowing it to become more concentrated. This approach has shown up to 10 times enhancement in detection.

[00112] Sequences

[00113] Examples

[00114] Many countries are currently operating at “limited capacity” due to the COVID- 19 pandemic, with restrictions on many activities such as mass gatherings, working fully at office and limitations on non-essential travel. This is also true for Singapore, where its 'Disease Outbreak Response System Condition (DORSCON) is currently still at orange and there is moderate disruption to daily activities. The strategy worldwide is to build resistance through mass vaccination programs and surveillance testing to identify potential clusters to prevent outbreaks. The world cannot afford to work on this limited capacity forever and rapid testing procedures with high sensitivity are required in order to provide a form of a “health visa” to rule out infection and resume normal daily activities.

[00115] To fulfill this demand, rapid antigen Lateral Flow ImmunoAssays (LFIA) have been gaining attention due to their ability to produce results in minutes, costing only several dollars, with the ability to be implemented at the Point-Of-Care (POC) and without requiring any additional equipment for results interpretation. In recent times, despite having an average specificity of 99.5%, LFIA test kits have received negative press as clinical reviews of the test kit performance only yielded an average sensitivity of 56.2% (Dinnes et al. Cochrane Database Syst Rev. 2020.). Early LFIA test kits focused primarily on specificity to diagnose COVID-19 infection and not as a high sensitivity tool to rule out infection. In the current stage of the pandemic, screening tools to rule out infection are now greatly needed to resume normal life.

[00116] In this example, a highly sensitive LFIA is outlined, which utilizes 3 key inventive steps to improve the sensitivity of the conventional LFIA: 1) Multisite antigen binding and 2) parallel flow technique to enable sequential reactions and 3) cascade signal amplification using linker free amplification nanoparticles. This is coined as a “Parallel LFIA (P-LFIA)” and Preliminary clinical studies have been conducted to evaluate its performance. Salivary samples from COVID-19 patients were used as well as negative salivary samples with no known exposure to COVID-19 were used. The test performance was compared against the PCR nasal swab for COVID-19. The study showed that the proposed device had a sensitivity of 96.7% and specificity of 100%, even detecting the virus in serial saliva samples collection from confirmed patients with CT values of even more than 35, which is the criteria for deisolation. This performance was significantly better when compared head-to-head with 5 other commercially available anterior nasal swab antigen kits tested with saliva. The best performing test only had a sensitivity of 43% (Abbot Panbio™).

[00117] A conventional LFIA is as described in Figure 1 (A). It has sample pad that would absorb the salivary specimen from the patient which contains the virus. The salivary sample will then flow via capillary action to the conjugation pad, which contains gold nanoparticle tagging the virus with a red colour. This forms a conjugate-viral complex which flows through to the nitrocellulose membrane until the complex encounters the test line and is isolated at that region. Isolation is achieved by the impregnation of protein or antibodies at the test line which have shown to have a strong affinity with the surface antigens of the COVID-19 virus. This forms a sandwich structure as seen in LFIA sandwich assays in Figure 1(D). As more of the complexes get isolated at the test line, a red band develops indicating the binding of the conjugates to the virus and thus indicating the presence of the virus. The remainder unbound conjugates which did not form any conjugate-viral complex flow past the test line and get isolated at the control line that contains a secondary antibody against the spike antibody which is an anti-species antibody against the spike antibody being tested, showing up as another red line. This confirms the test is working correctly and that the conjugation of the antibodies to the nanoparticles were successful. A positive test would thus have both test line and control line showing up as red bands as seen in Figure 1(B), whereas a negative test would only have a control line band and no test line band as seen in Figure 1 (C). [00118] In Figure 2 a device is depicted that uses two or more different analyte binding molecules directed to the same analyte target either in two different channels (Figure 2A) or at the test line (figure 2B). This enhances the detection limit of analytes. To objectively compare the results, the intensity of the test line from each test strip is compared to a negative control and both are recorded though the use of mobile phone imaging (Figure 2C). The image intensities from each test line are then compared using image processing software for objective measurement. This allows for correlation of the test line intensities when testing the for the limit of detection using model virus as well as when testing with the patient samples.

[00119] In order to improve the sensitivity of the LFIA, the use of two or more different analyte binding molecules directed to the same analyte target was used as a parallel flow method. To improve the test line signal intensity and also expand the applications of the LFIA for other roles such as multimodal imaging. The parallel flow technique allows the LFIA to perform sequential reactions, thereby enabling it to have more complex functions such as signal enhancement. An example Parallel LFIA (P-LFIA) is as described in Figure 2(A) and Figure 3 both with 2 parallel flow channels. One prototype of the device is shown in Figure 2(A). The first parallel flow channel 1 , contains nanoparticles conjugated with anti-spike protein antibodies 9, and the second parallel flow channel 2, contains signal enhancement nanoparticle conjugates 10. Both these nanoparticles are contained in conjugation pads 4. To perform detection, the sample which contains the COVID-19 Virus 8 is first added to the sample pad 3 on to the first parallel flow channel 1. The sample will then travel upwards to the conjugation pad 4 and interact with the nanoparticles with anti-spike protein antibodies 9, tagging the virus with a color. This complex will continue to flow upwards to the test line 5, which contains virus binding proteins such as ACE protein 12, which will immobilize the virus at the test line 5 forming a visible color band. The unbound nanoparticles conjugated with anti-spike protein antibodies will then move towards the control line 6, which contains antibodies against the anti-spike protein antibodies 11. This forms another band at 6 which will confirm the correct working of the test. Finally, the remainder of the solution will be absorbed by the absorption pad 7. In the second step, more saliva sample or water is added to the second parallel flow channel 2. The fluid would then dislodge the amplification nanoparticles 10 from the conjugation pad. Thereafter, the amplification nanoparticles 10 would interact with the bound virus with the nanoparticles conjugated to anti-spike protein antibodies at 5.

[00120] In one form, these signal enhancement nanoparticles 10 can be conjugated with antibodies against the anti-spike protein, but binding to a different site on spike protein than the antibody conjugated with anti-spike protein 9, from the first parallel channel 1. In another form, these signal enhancement nanoparticles 10 can be conjugated with antibodies against the anti-spike protein antibody, binding to the nanoparticles conjugated with anti-spike protein antibodies 16. In both cases, the amplified signal consists of the color signal from both conjugates 9 and 10 at the test line 5. To record the results, a mobile phone 14, is then used the capture the test strip’s test line 5 and control line 6. The picture is analyzed with an image processing algorithm which compares areas C1 and C2 for a negative/control sample and S1 and S2 for a positive sample. A positive number when taking S2 - S1 is considered as a positive test line indicating a positive result. A negative number or 0 number when taking C2 - C1 is considered as a negative test indicating a negative result.

[00121] Another prototype of the device is shown in Figure 3(A) and its schematic is shown Figure 3(B). Parallel flow channel 1 contains nanoparticle conjugated with anti-spike protein antibodies and parallel flow channel 2 contains signal enhancement nanoparticle conjugates with antibodies against the anti-spike protein antibodies as shown in Figure 3(C). To achieve an amplified signal, the salivary sample is first put into channel 1 using a dropper and stood up vertically. After a duration of 10 minutes, a sandwich assay is formed as shown in Figure 3(D), where a faint line may or may not be seen on the test line. In channel 2, either water or saliva is added, which then transports signal enhancement nanoparticles to the test line. These signal enhancement nanoparticles then bind to the sandwich assay as shown in Figure 3(E). This results in a more visible line as seen in the inset of Figure 3(E). This amplification process can be repeated multiple times to get a stronger and stronger signal as shown in Figure 4.

[00122] In order to improve the sensitivity of the LFIA, the use of two or more different analyte binding molecules directed to the same analyte target was used as a multisite antigen binding technique. This is where multiple sites of the target antigen are used for increased isolation to the test line or used for binding nanoparticle conjugates in the sandwich assay to enhance the signal. Multisite antigen binding for increased viral isolation is depicted in Figure 2(B), and Figure 5A where 3 different antibodies are used, each capable of binding to the S1 spike protein subunit, S2 spike protein subunit and the RBD spike protein subunit. This greatly expands the available region the virus can bind to the test line, resulting in increased chance of viral isolation at the test line 5 shown in Figure 2(B). It can be seen that the test line 5 contains two different binding molecules 13 are used at the test line for multisite antigen binding. As opposed to conventional viral isolation techniques as shown in Figure 1(E), where a single antibody is used which is capable of binding to only one site at the target antigen. This is a comparatively smaller region of binding and this reduces the chance the virus will bind to the test line. The multisite antigen binding technique can also be used in conjunction with the parallel flow method outlined above.

[00123] The device and method can be adapted to form a cascade signal amplification (See Figures 4 and 5). As an example, 4 parallel flow channels are shown in Figure 4(A) and more flow channels can be added to increase the degree of amplification. The assay described in Figure 2A and Figure 3 is a cascade amplified P-LFIA with a single amplification step, as shown in the inset of Figure 4B part Ί” and “II". With the addition of more flow channels, the signal is enhanced even further as show the inset of Figure 4B part “III” and “VI”. This signal enhancement can be appreciated in the in-vitro testing of the P-LFIA with the use of a virus model at different concentrations as shown in Figure 4(B). In comparison with Figure 6(A), it can be seen that the P-LFIA has at least a 10-fold improvement in detection limit with it being able to still produce a signal at 1 E6 and 1 E5 particles/ml. It is also noted that with the use of the parallel flow channels and signal enhancement nanoparticles, the intensity increases with each successive flow as seen in Figure 4(B). Theoretically, this process can be repeated indefinitely for more and more signal enhancement. Cascade enhancement is made possible with the use of parallel flow channels as the sequential flow of these conjugates are required to make the sandwich assays as described. In comparison with 6(A), it is also noted that even at lower concentrations of 1 E6 and 1 E5 particles/ml, the unamplified signal is greater, showing the importance of multisite antigen binding for viral isolation. It is important to note that unlike currently available techniques, the binding performance of the nanoparticle conjugated with the antibodies against the target antigens is unaffected. This is due to the use of linker free amplification nanoparticles which do not need any linker antigens as illustrated in Figure 11. This will be further discussed below. Other than the different numbers of channels, several other variations may exist as would be understood by a person skilled in the art.

[00124] For the purposes of COVID-19 viral detection from saliva, using the LFIA with 2 channels. The entire length of the LFIA is 80mm. Both channels are 20mm in length. The width of each channel is 4mm with a 2mm gap in between, making the entire LFIA 10 mm wide. The length of the common section (after the joining of both channels) is 40mm. The parallel flow technique can also be combined with the multisite antigen binding technique to achieve additional functions such as signal enhancement, multimodal imaging, etc. This is where the parallel flow channels are loaded with nanoparticle conjugates with antibodies such that they bind to the same antigen of interest but at different sites. An example is shown in Figure 5(C), where there are 3 parallel flow channels. [00125] When flowed sequentially as shown in Figure 5(C), each nanoparticle probe binds to a different part of the spike protein, which is the targeting antigen. In this example, nanoparticles from channel 1 and 3 provide a visible signal whilst nanoparticles from channel 2 provide a fluorescent signal. Other variations of this embodiment are envisaged.

[00126] In-Vitro studies and Proof-of-concept

[00127] A prototype was made as a conventional LFIA as described in Figure 1 and as well as the P-LFIA as depicted in Figure 3 with 2 channels. For the P-LFIA, the multisite antigen binding technique was utilized at the test line for increased viral isolation and the cascade amplification technique was used for 1 cycle amplification. A model virus was created using colourless silica nanoparticles which is the same size as the coronavirus (100 nm) and surface functionalizing it with the target antigen (COVID-19 Spike Protein). As a proof of concept, the conventional LFIA tested using nanoparticle conjugated with antibodies targeting the Receptor Binding Domain (RBD) subunit of the COVID-19 spike protein. The results are shown in Figure 6(A), where the intensity of the test line was recorded using mobile phone imaging (y-axis) at different model virus concentrations (x-axis). Any value which is positive indicates that the tested model virus concentration produced a visible line intensity, meaning it is a positive result showing that the viral model is detected. From the testing, it is shown that a conventional LFIA has a detection limit of 1E7 particles/ml and thereafter at 1 E6 particles/ml, no appreciable positive intensity difference was noted. Next, as a proof of concept of multisite antigen binding, the experiment was repeated using 2 different nanoparticle conjugates. One set of nanoparticles were conjugated with antibodies against the Receptor Binding Domain (RBD) subunit of the spike protein and the other set of nanoparticles were conjugated with antibodies against the S1 spike protein subunit. The stock model virus solution was diluted ten-fold serially until no detection was possible with the nanoparticles conjugated to the RBD subunits alone (Figure 6(B)). This was observed to be at 0.001 x dilution, where using nanoparticle probes with only antibodies against the RBD subunit demonstrated no appreciable detection. When another set of nanoparticles with the antibodies against the S1 subunit were added, detection was now observed at 0.001 x and 0.0001X dilution, showing an improvement in detection of 2 orders of magnitude. These suggest that there are available binding sites on the viral proteins in the virus models where the multisite antigen binding technique could be used for the purposes of signal enhancement and viral isolation, resulting in an improvement of test sensitivity.

[00128] Clinical study Design [00129] A prototype was then further evaluated clinically in a case-control clinical study.

[00130] Positive cases were recruited from patients who had confirmed exposure to COVID-19 and as well as a positive PCR test from nasopharyngeal swabs. Conversely, negative cases recruited had no documented exposure to COVID-19 and also had a negative PCR test from nasopharyngeal swabs.

[00131] Salivary samples were obtained from recruited cases using the spitting or passive drooling technique (according to subject preference). The study was conducted with 2 kits, the first version is a conventional LFIA which tests for S1 Spike protein sub-unit and RBD Spike protein sub-unit separately to show the use of multiple antigens in diagnosis of a disease such as COVID-19. In the second kit, a prototype that had the S1 Spike protein subunit was selected and implemented in a P-LFIA to show how parallel flow and cascade amplification can be used to overcome the difficulties faced in conventional LFIA.

[00132] To further investigate the effect of oral intake on test results, a series of different samples were taken from positive cases: i) more than 8 hours after oral intake, ii) 3 hours after oral intake and iii) within 1 hour after oral intake. Finally, 5 commercially available kits were also compared head-to-head with the P-LFIA with the same clinical saliva samples to further illustrate the increase in sensitivity using the P-LFIA with the outlined 3 inventive steps.

[00133] In total 150 subjects were recruited to the study. All test results were captured via mobile phone imaging using similar lighting conditions. Images were then analyzed objectively using image processing algorithms to determine the appearance of a positive test line using pixel intensity measurements.

[00134] Clinical Study Results

[00135] In the first part of the clinical study, a conventional LFIA was used to test for the COVID-19 RBD protein sub-unit and S1 protein sub-units separately. The results shown in Figure 7(A) show that when RBD and S1 protein subunits were tested separately, the tests yielded identical performance of 56.3% sensitivity and 100% specificity. This shows that both antigenic targets are viable targets for an LFIA to detect COVID-19 infection. In order to boost the sensitivity of the assay, both tests for the S1 protein subunit and RBD subunits were evaluated in unison for each case. It can be seen that this resulted in a significant increase in performance, with 75.0% sensitivity, whilst maintaining the same specificity of 100%. As an additional point of the spike protein being suitable for the detection of COVID- 19, it was also noted that the B1617.2 and E31.160 SARS-CoV2 variants were detectable using the prototype device and method. This shows that a multi-antigen approach can be effective in increasing LFIA test sensitivity. This is the rapid antigen first test to show the use of multiple antigens in this manner.

[00136] From several studies, timing of saliva sample collection after food intake has known to be a possible issue affecting rapid antigen test performance. In two independent studies conducted by Agullo et al. (J. Infect, 2021 :82(5)) and Schlidgen et al. (Pathogens. 2021 :10(1), 1-7), salivary samples were collected with no limitation in relation to food intake. These samples were then tested using the Abbot Panbio™ rapid antigen test kit and it was shown to have sensitivities of 23.1% and 50% respectively. This is similar to other rapid antigen test kits using nasopharyngeal swab samples which had an average sensitivity of 56.2%. Therefore, despite the conventional LFIA using spike protein for detection of the virus showing higher sensitivity of 75.0% when using both S1 and RBD protein subunit detection, the multiantigen LFIA’s performance was also evaluated in relation to oral intake. It was used to test salivary samples from patients 8 hours, 3 hours and within 1 hour after oral intake and the results are shown in Figure 7(B). It was observed that oral intake was a significant factor influencing test performance. Sensitivity of the assay was 75.0% in patients who did not have any oral intake for at least 8 hours and sensitivity dropped to as low as 40% when patients were tested within an hour after oral intake. This is similar to the results seen by both Agullo and Schlidgen et al. As a matter of further investigation, the same test kit, the Abbot Panbio™ as well 4 other COVID-19 test kits were tested with salivary samples obtained within 1 hour of oral intake to determine of a similar effect was observed. The 4 other test kits were the Becton Dickinson (BD) Veritor™, MyBiosource, Arista™ version 1.0 and Arista™ version 2.0 test kits. The results as seen in Figure 7(C), show that the sensitivity of the Panbio™ was 47.8%, similar to the studies performed by Schlidgen et al. The 4 other test kits had poorer sensitivities than the Abbot Panbio™ with mybiosource having the second highest sensitivity with 35.7% and the BD veritor™ with 25% sensitivity. According to the manufacturer, the Arista™ test kits were kits which were shown to be compatible with sputum samples and were chosen to see if such kits yielded any different performance when dealing with such samples. As seen in Figure 7(C), the Arista™ kit did not perform any better than the BD Veritor™, with its version 1 test kit yielding 25% sensitivity and its version 2 test kit yielding no detection at all for all saliva samples. From these results, it is apparent that conventional LFIA kits do not have sufficient detection sensitivity and are significantly affected by oral intake. [00137] It was postulated that despite there being no discernable signal at the test line, sandwich assays as depicted in Figure 3(D) were indeed being formed, albeit at a level so low that it is not visible to the naked eye. Therefore, a modified Parallel LFIA (P-LFIA) test kit was produced using the abovementioned inventive steps. It utilizes advanced viral capture techniques using multisite antigen binding and signal amplification though the combined application of parallel flow with cascade amplification techniques as shown in Figure 3. To illustrate the inventive steps, the P-LFIA was implemented with the S1 Spike sub-unit antigen as the target and used to test samples exclusively obtained within 1 hour of oral intake to determine if the described inventive steps address the issues highlighted above. Theoretically, this same concept can be extended to include the RBD Spike sub-unit as well to create a multiantigen P-LFIA with even higher sensitivity. The results of the P-LFIA using the S1 Spike sub-unit as the target antigen are shown in Figure 8. From Figure 8(A), which shows the distribution of the test line intensity of samples tested, it can be seen that preamplification intensities were largely at 0 a.u., which correlates to a negative result for approximately 50% of the known positive cases tested. Post amplification, the mean intensity increased to 5, with almost all the positive cases above the detection threshold (only 1 case was missed). The amplification effect is better appreciated on a per-patient level in Figure 8(B), which shows the test line signal intensity pre-amplification and post-amplification. It can be seen that a large proportion of confirmed cases who would otherwise test negative with such samples obtained 1 hour after food intake were now correctly identified using the P- LFIAwith only 1 case being missed.

[00138] Similar to the previous versions of the test kit, the P-LFIA is also able to detect SARS-CoV2 variants and it was noted that the B1617.2 and B1617.3 SARS-CoV2 variants were successfully detected. Both the variants were seen to be only discovered and tested positive after amplification, suggesting the improved detection performance of the P-LFIA. Figure 8(C) further illustrates the effect of cascade amplification on the increase in test sensitivity. With only one cycle of cascade amplification used, the sensitivity of the assay was increased from 42.9% to 96.7%. This was while maintaining the test kit performance of 100% specificity pre and post amplification. It is possible to create even higher sensitivity variations for healthcare profession uses with two or more cycles of cascade amplification being used. Figure 8(D) further illustrates the improvement in sensitivity though the comparison of the receiver operator characteristics of the amplified and unamplified test. The Area Underthe Curve (AUC) for the amplified test is 0.92 compared to 0.73 as seen in the unamplified version. Finally, Figure 8(E) and 8(F) both illustrate the clinical sensitivity of the test, where confirmed patients were followed up longitudinally and repeatedly tested to determine the test performance in correlation to the viral load as represented by the PCR Cycle Threshold (CT). The horizontal line in Figure 8(F) represents the CT value of 35, which is the de-isolation criteria in Singapore. Correlating Figure 8(E) and Figure 8(F), it can be seen that the P-LFIA is able to successfully test patients even at higher CT values above the de-isolation criteria of CT = 35, as seen from day 27, 31 and 34, where there is still a normalized intensity signal. These results highlight the use of P-LFIAs in the current pandemic setting where high sensitivity point of care diagnostics is required. Furthermore, the approach not only applies to COVID-19, but can be expanded for the detection of other proteins of low concentration as well, with application in other infectious diseases, oncology, hematology, rheumatology, etc.

[00139] A similar prototype using the RBD subunit was also made and the preliminary results of such a prototype using the RBD subunit are shown in Figure 9. Similarly, it is able to amplify line intensities which would otherwise test negative as shown in Figures 9A and 9B. However, the sensitivity and specificity achieved are only 60% (Figure 9(C)) and interpretation is tricky due to its non-zero threshold value as shown in Figure 9(E). However, this version also has potential as it is able to detect samples at higher CT values (Figure 9(F)). More development is required to address the issues with the prototype.

[00140] Embodiments of the device and method discussed above specifically addresses antigenic testing by using the Lateral Flow Immunoassays (LFIAs), which employs nanoparticles that are conjugated with proteins/antibodies which specifically bind to COVID- 19 antigens, thereby producing an optical signal. Various embodiments demonstrate that two or more antigens can be detected simultaneously in order to increase sensitivity. Two spike protein antigens, namely the Receptor Binding Domain (RBD) subunit and the S1 subunit were tested, making this the first multiple antigen point-of-care test for COVID-19. Various embodiments demonstrate that through the use of the multisite antigen binding technique, antibodies and proteins which bind to different sites of the spike protein were used at the test line for increased viral isolation. Various embodiments demonstrate that parallel flow technique, which can control sequential reactions on a LFIA device, may be used to perform signal amplification. Various embodiments demonstrate that the virus is initially labelled by the nanoparticle probe conjugated with antibodies against the viral Spike RBD or Spike S1 protein in one flow channel. Subsequently, the second and/or subsequent flow channel, which contains signal enhancement particles that bind to the viral-nanoparticle probe complex is activated to perform signal amplification. These amplification nanoparticles do not require any additional linker antigens on the nanoparticle probes that bind with the target viral antigens. Additional flow channels can be tagged on in order to repeat this reaction infinitely, allowing the detection of virus at low concentrations.

[00141] Clinical studies mentioned above have been performed with salivary samples to compare the performance of assay using the multiantigen binding viral isolation technique alone and when the assay is enhanced with parallel flow enhancement. These techniques, when combined, allow the detection of the COVID-19 virus to be detectable from after food salivary samples with a sensitivity and specificity of 95% and 100% respectively. The test shows 95% agreement with PCR testing even at lower CT values of 35 - 40. A head-to-head comparison of currently available antigen tests were also included which showed that the demonstrated method is superior in salivary testing.

[00142] To utilize multisite antigen binding, an appropriate target viral antigen must be selected. The protein should be specific to the virus, assessable to binding, be large enough to have multiple binding sites and be sufficiently immunogenic to enable the production of antibodies against its binding sites. In this embodiment, the COVID-19 SARS CoV2 viral spike protein is used as an example, but it is important to note that any viral antigen which fits these conditions can be used Spike protein is a complex, highly immunogenic protein which contains 3 subunits, the S1 , S2 and Receptor Binding Domain (RBD). To provide increased viral isolation, antibodies and proteins binding to different sites of the spike protein are used at the test line. This results in improved viral isolation at the test line, thereby increasing the detection limit of the LFIA. In the same vein, nanoparticle probe conjugates which bind to different sites on the spike protein can be used to bind to increase the number of nanoparticle probes per virus, thereby showing an increase in signal. This is demonstrated in-vitro and has shown up to 2 orders of magnitude improvement in limit of detection performance.

[00143] In the clinical study two different antigens were detected separately in order to increase test sensitivity. It is important to note that commercial COVID-19 LFIAs target the nucleocapsid antigen which is not as accessible, as spike protein is located on the viral surface. The presented embodiment is the first COVID-19 test evaluated clinically to use the spike protein as its target antigen. Each subunit by itself is highly immunogenic and multiple antibodies are available to specifically target each subunit. With this enhanced viral detection, a test device was created which detected both the RBD spike protein and the S1 spike protein. Individually, these test devices had similar performance, with sensitivity of 56% and specificity of 100%. When the two antigens were investigated together, this multi-antigen antigen approach boosts the sensitivity to 75%, whilst maintaining the specificity of 100%. This is the first device of its kind to show multiple antigens being used to boost sensitivity.

[00144] It was noted during the study that the test performance varied with the duration the patient was tested after oral intake. If the patient was tested within 1 hour of oral intake, test sensitivity dropped to only 41%. A head-to-head comparison was also performed with 5 other available brands of antigen test kits which showed similar performance to the best performer, the Panbio™ COVID-19 from Abbot, which was 43.5%. To overcome this issue, signal amplification was used to boost the weakened signal intensity at the test line, allowing the test function even when presented samples with lower viral loads due to oral intake as close as 1 hour within testing. To achieve this, multiple parallel flow channels were used to flow different nanoparticle conjugates to the same test line. As each channel can be initiated independently, the wetting of each of the parallel channels allows sequential deployment of the nanoparticle conjugates to the test line. This allows one set of conjugates to interact with the target antigen at a time, allowing for optimal conditions for each nanoparticle antibody conjugate to fulfill its function. The binding sites of the enhancement nanoparticles are located close to the binding sites of the nanoparticle probes used to bind with the target antigen. Therefore, there are significant steric hinderances if these nanoparticles are deployed at the same time, resulting in a reduced signal instead of amplification. In this disclosure, a sample device is presented with 2 parallel flow channels, the first parallel flow channel is used to label the spike proteins of the virus, whilst subsequent parallel flow channels are used to increase the signal at the test line. This device was then tested with samples within 1 hour of patient’s oral intake, and it was shown that the device was not affected by oral intake of the patient. Using S1 spike protein detection with parallel flow amplification, a sensitivity of 96.4% and specificity of 100% was achieved and the study shows the test is able to detect the virus in serai saliva sampling from confirmed patients at CT values of even more than 35, which is the criteria for deisolation.

[00145] Using a previously described signal enhancement method utilizing nanoparticle probe enhancement though the use of linker antigens initial adaptation to increase the limit of detection was attempted. Figure 10(A) illustrates the series flow LFIA with the proposed multisite antigen binding for enhancement. In this instance, the sequence of the nanoparticle binding cannot be controlled as the entire channel must flow at the same time as soon as the sample is added. The 3 sets of nanoparticles conjugates interact with the virus at the same time as seen in Figure 10(B). As a result, the nanoparticle conjugates, albeit targeting different sites of the same target antigen, compete with one another for binding to the antigen. Additionally, unlike the parallel flow methods described above, the conjugates have 1/3 less interaction time with the virus as compared to the parallel flow. As an estimate, if the affinity for all the 3 conjugates’ antibodies were the same, there would be an equal distribution of antigens where only 1/3 of the antigens are bound to a single nanoparticle conjugate each, resulting in no signal amplification at all as the summation of the total nanoparticles bound would be the same as if a single conjugate were used.

[00146] Presently, signal enhancement has been described through the use of a linker antigen bound to the viral nanoparticle probe’s surface. Another nanoparticle which will bind to this linker antigen will then bind to this antigen using the serial arrangement. This is described in Figure 11 (A). One conjugate pad contains the nanoparticle conjugate that has the antibody against the viral antigen with linker antigens on the surface (Figure 11 (C)) while another conjugate pad contains a signal enhancement nanoparticle which functions like a secondary antibody that binds to the linker antigens nanoparticle conjugate (Figure 11(D)). The major disadvantage of this approach is seen in Figure 11 (B). As valuable space on the nanoparticle probe is taken up by antigen linker antigens, this would result in lower binding to the viral antigen of interest, of which is already in low concentration due to the patient’s possible oral intake. This is opposed to the method used above as depicted in Figure 11 (E), where the amplification nanoparticle binds directly to the antibody targeting the viral antigen, allowing the viral nanoparticle probe to conserve valuable space for more antibodies against the target antigen. As will be apparent in Figure 12, this approach is only possible due to the parallel flow technique as the order of binding can be controlled. Otherwise, the amplification nanoparticle will bind to the probe nanoparticle antibodies as shown in Figure 12(B), blocking the site for binding to the virus and binding will not occur at the test line (Figure 12(C)). Furthermore, as is already apparent from Figures 9 to 11 , no cascade amplification can occur as all the flow is serial, there can be no subsequent flow of amplification conjugates so the test line.

[00147] In various embodiments, the detection of proteins are not limited to these SARS- CoV-2 sequences. As shown in Figure 13 (A) to (G), multiple antibodies and model virus constructed using different target proteins have been tested in the LFIA format and each showed a varying degree of detection to their known protein targets. Enhancement of the detection of each of these viruses can be made by using two or more different analyte binding molecules directed to the same analyte at different sites on the test line and/or by using P- LFIA without amplification. Figure 13(H) and (I) shows the average performance of the LFIA for the detection of RBD and S1 proteins. Figure 14 (A) to (G) shows these same sets of antibodies tested against model virus constructed using a common target spike protein (40589-V08B1) for cross comparison of the performance of different antibodies without amplification. Figure 14(H) and (I) shows the average performance of the LFIA for the detection of the common spike protein.

[00148] In various embodiments, a system in which the conjugation pads can be replaced with new conjugation pads loaded with detectable particles for signal enhancement. [00149] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.

[00150] As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

Claims

1 . A device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other said first portion being the site for application of the liquid sample, and for a set of conjugates movably supported therein, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

2. The device according to claim 1 , wherein the first portion comprises the said first channel, a second and/or subsequent channel with a second and/or subsequent set of conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle, and a disruption between the first, second and/or subsequent channel.

3. The device according to claim 2, wherein the disruption between the first and second and/or subsequent channel is a gap in the permeable material.

4. The device according to claim 2, wherein the disruption between the first and second and/or subsequent channel is a feature that reduces the flow rate of liquid in the second and/or subsequent channel compared to the flow rate of the liquid in the first channel.

5. The device according to any one of claims 1 - 4, wherein the second analyte binding molecule comprises two or more different analyte binding molecules capable of binding to the same analyte at different sites on the analyte.

6. The device according to any one of claims 1 - 5, wherein the permeable material comprises at least one sorbent material defining a flow path extending from the first portion to a test site in the second portion.

7. The device according to any one of claims 1 -6, wherein the detectable particle is a coloured particle; whereby accumulation of coloured particles at the test site produces a colour visible to the unaided eye indicative of the presence of the analyte in the liquid sample.

8. The device according to claim 6, wherein the first and/or second conjugate is disposed in the flow path upstream of the test site and is mobilizable along the flow path with passing liquid.

9. The device according to any one of claims 1 -6, wherein the conjugate is present in dry form.

10. The device according to claim 6, wherein the first and/or second conjugate is transportable along the flow path by liquid wicking or wetting through the sorbent material.

11. The device according to any one of claims 1 - 10, wherein the first analyte binding molecule binds to a coronavirus spike protein.

12. The device according to any one of claims 1 - 11 , wherein the second analyte binding molecule binds to a coronavirus spike protein.

13. The device according to claim 11 and 12, wherein the coronavirus comprises SARS-CoV-2 virus.

14. The device according to claim 13, wherein second analyte binding molecule comprises two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule is capable of binding to S1 subunit of the SARS-CoV-2 spike protein or to S2 subunit of the SARS-CoV-2 spike protein, and the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein the same analyte at different sites on the analyte.

15. The device according to any one of claims 1 - 14, wherein the first analyte binding molecule comprises a monoclonal antibody.

16. The device according to any one of claims 1 - 14, wherein the first analyte binding molecule comprises a polyclonal antibody.

17. The device according to any one of claims 7 - 16, wherein the coloured particle is a metal sol particle.

18. The device according to claim 17, wherein the metal sol particle is colloidal gold.

19. The device according to any one of claims 1 - 18, wherein the permeable material comprises a nitrocellulose membrane.

20. The device according to any one of claims 1 - 19, wherein the second portion further comprises a control site comprising a third analyte binding molecule immobilized therein capable of binding to the first analyte binding molecule.

21. A method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication therebetween; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second set of conjugates movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second set of conjugates each comprises an amplification analyte binding molecule coupled to a detectable particle capable of binding to the first analyte binding molecule or directly to the analyte, whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion, wherein the first analyte binding molecule and/or the second analyte binding molecule comprises two or more different analyte binding molecules directed to the same analyte at different sites.

22. The method according to claim 21 , further comprising: e.1) applying the liquid to a subsequent channel in the first portion of the device; and e.2) allowing the liquid to flow to a subsequent set of conjugates movably supported in the subsequent channel of the first portion then to the second portion of the permeable material, wherein said set of subsequent conjugates each comprises a subsequent amplification analyte binding molecule coupled to a detectable particle capable of binding to the first amplification analyte binding molecule whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein and the second conjugate is bound to the first conjugate, the subsequent conjugate will bind to the second conjugate and amplify the detection.

23. The method according to claim 21 or 22, wherein the second analyte binding molecule comprises two or more different analyte binding molecules capable of binding to the same analyte at two different sites on the analyte.

24. The method according to any one of claims 21 to 23, wherein determining the presence of the analyte comprises: g) capturing the image of the second portion to analyse intensity of binding; and h) calculating the quantity of the analyte based on the analysis of the intensity of binding.

25. The method according to any one of claims 21 to 24, wherein the liquid sample comprises saliva.

26. The method according to any one of claims 21 to 25, wherein the detectable particle is a coloured particle and determining the presence of the analyte comprises observing visually the test result at a test site wherein accumulation of coloured particles produces a colour indicative of the presence of the analyte in the liquid sample.

27. The method according to any one of claims 21 to 26, comprising the additional step of mixing the conjugate with the liquid sample prior to applying the sample to the device.

28. The method according to any one of claims 21 to 27, wherein the first analyte binding molecule binds to a coronavirus spike protein.

29. The method according to any one of claims 21 to 28, wherein the second analyte binding molecule binds to a coronavirus spike protein.

30. The method according to claim 28 or 29, wherein the coronavirus comprises SARS- CoV-2 virus.

31 . The method according to claim 30, wherein second analyte binding molecule comprises two analyte binding molecules capable of binding to SARS-CoV-2 spike protein at different sites, the first SARS-CoV-2 spike protein binding molecule is capable of binding to S1 subunit of the SARS-CoV-2 spike protein or to S2 subunit of the SARS-CoV-2 spike protein, and the second SARS-CoV-2 spike protein binding molecule capable of binding to receptor binding domain (RBD) of the SARS-CoV-2 spike protein the same analyte at different sites on the analyte.

32. The method according to any one of claims 21 to 31 , wherein the first analyte binding molecule comprises a monoclonal antibody.

33. The method according to any one of claims 21 to 31 , wherein the first analyte binding molecule comprises a polyclonal antibody.

34. The method according to any one of claims 21 to 33, wherein the coloured particle is a metal sol particle.

35. The method according to claim 34, wherein the metal sol particle is colloidal gold.

36. The method according to any one of claims 21-35, wherein the second portion further comprises a control site comprising a third analyte binding molecule immobilized therein capable of binding to the first analyte binding molecule.

37. A device for detecting an analyte in a liquid sample, the device comprising a permeable material defining at least a first channel and a second and/or subsequent channel in a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication with each other said first portion being the site for sequential application of the liquid sample, and for a set of conjugates movably supported therein, wherein a first set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle and a second set of conjugates each comprising an amplification analyte binding molecule coupled to a detectable particle, a disruption between the first, second and/or subsequent channel, said second portion being the site for detecting the presence of the detectable particle, said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte.

38. A method for determining the presence of an analyte in a liquid sample comprising: a) applying a liquid sample to a first channel in a first portion of a device comprising a permeable material defining a first portion and a second portion, the portions being in the same plane so as to permit capillary flow communication therebetween; b) allowing the liquid sample to flow to a set of conjugates movably supported in the first channel, wherein said set of conjugates each comprises a first analyte binding molecule coupled to a detectable particle, whereby when the analyte is present the conjugate binds to the analyte; c) allowing the liquid sample to flow to the second portion of the permeable material said second portion comprising a second analyte binding molecule immobilized therein which specifically binds to the analyte, whereby when the analyte is present the analyte bound to the conjugate binds to the second analyte binding molecule immobilized therein; d) applying a liquid to a second channel in the first portion of the device; e) allowing the liquid to flow to a second set of conjugates movably supported in the second channel of the first portion then to the second portion of the permeable material, wherein said second set of conjugates each comprises an amplification analyte binding molecule coupled to a detectable particle capable of binding to the first analyte binding molecule or directly to the analyte, whereby when the analyte is bound to the first conjugate and the second analyte binding molecule immobilized therein, the second conjugate will bind to the first conjugate and amplify the detection; and f) determining the presence of the analyte in the liquid sample by detection of the first and second conjugate at the second portion.