Abstract
Disclosed herein is a method of detecting the presence of a target analyte in a sample. Disclosed herein are also a system, an article, and a kit for detecting the presence of a target analyte in a sample. Disclosed herein is also the use of the system, or the article, or the kit for biomolecule, bioorganelle, bioparticle, cell and microorganism detection.
Claims
1. A method of detecting the presence of a target analyte in a sample comprising the steps of: a) incubating the sample with a surface coated with a first sensing element, wherein the first sensing element is capable of specific binding with the target analyte, and wherein when the target analyte is present in the sample, the target analyte binds to the first sensing element present on the coated surface, concurrently with or followed by; b) incubating the coated surface of step (a) with a plurality of probe particles, wherein the probe particles are coated with a second sensing element capable of specific interaction with the target analyte bound on the coated surface or capable of specific interaction with the first sensing element on the coated surface; c) applying a mechanical force to separate the non-specifically bound probe particles of step (b) from the coated surface; and d) measuring a property that reflects the amount of specifically bound probe particles on the coated surface, wherein the property is selected from the group consisting of particle density, colour density, aggregate size and combinations thereof, or measuring a property that reflects the amount of non-specifically bound probe particles, wherein the property is selected from the group consisting of colour density, aggregate size and combinations thereof.
2. The method according to claim 1, wherein the first sensing element is the same or different as the second sensing element, and wherein the first sensing element and second sensing element are independently selected from the group consisting of biomolecule, bioparticle, material or product derived from microorganisms, or wherein the first sensing element and second sensing element are independently selected from the group consisting of carbohydrate, polysaccharides, lipid, protein, peptide, nucleic acid, antibody, antigen, hormone, enzyme, or chemical compounds.
3. The method according to claim 1, wherein the target analyte is soluble in an aqueous medium or solvent, and wherein the target analyte is dispersed in a liquid medium prior to incubating with the coated surface.
4. The method according to claim 1, wherein the probe particles are non-magnetic, magnetic or superparamagnetic particles, and wherein the size of the probe particles is in the range of 8 nm to 100,000 nm.
5. The method according to claim 1, wherein the mechanical force is generated from a permanent magnet, an electric magnet, a centrifuge, an acoustics, an ultrasonic wave, a laser beam, fluid motion, fluid buoyancy, or gravity, and wherein the mechanical force is in the range of 0.01 pN to 100 pN.
6. The method according to claim 1, wherein when the property measured is colour density, or aggregate size, or colour density and aggregate size, the step (d) further comprises a step (d0) of condensing the specifically bound probe particles on the coated surface into an aggregate before step (d) by applying a magnetic force on the other side of the coated surface.
7. The method according to claim 1, wherein the step (c) further comprises providing a second surface to contact the non-specifically bound probe particles separated from the coated surface; and condensing the non-specifically bound probe particles into an aggregate on one side of the second surface by applying the mechanical force on the other side of the second surface; wherein the mechanical force is a magnetic force.
8. The method according to claim 7, further comprising measuring the colour density, or aggregate size, or colour density and aggregate size of the aggregate.
9. The method according to claim 6, wherein the magnetic force is generated from a permanent magnet or an electric magnet in the form of a magnetic needle, and wherein the magnetic force is in the range of 0.01 pN to 100 pN.
10. The method according to claim 1, wherein when steps (a) and (b) are carried out concurrently, a mixture of the sample and the plurality of probe particles is incubated with the coated surface.
11. A system for detecting the presence of a target analyte in a sample comprising: a) a surface coated with a first sensing element, wherein the first sensing element is capable of specific binding with the target analyte; b) a plurality of probe particles, wherein the probe particles are coated with a second sensing element capable of specific interaction with the target analyte when present or capable of specific interaction with the first sensing element on the coated surface, thereby forming specifically bound probe particles on the coated surface; c) a mechanical force capable of separating non-specifically bound probe particles from the coated surface; and d) a measurement means to measure a property of the specifically bound probe particles, wherein the property is selected from the group consisting of particle density, colour density, aggregate size of the specifically bound probe particles, or a property of the non-specifically bound probe particles, wherein the property is selected from the group consisting of colour density, aggregate size of the non-specifically bound probe particles.
12. The system of claim 11, further comprising a second surface for contacting the non-specifically bound probe particles; and a measurement means to measure a property of the non-specifically bound probe particles, wherein the property is selected from the group consisting of colour density, aggregate size and combinations thereof.
13. An article for detecting a presence of a target analyte in a sample, the article comprising: at least one test well comprising a bottom surface coated with a first sensing element configured to bind with the target analyte in the sample, wherein the test well is configured to receive the sample and probe particles that are contained in one or more liquid mediums, the probe particles being coated with a second sensing element configured to bind with one selected from the group consisting of the first sensing element and the target analyte, such that the probe particles are specifically bound to the coated surface depending on the presence of the target analyte in the sample; and a channel connected to the test well at a distance from the bottom surface to allow fluid communication between the channel and the test well, wherein the channel is configured to receive non-specifically bound probe particles in the test well upon an exertion of an external force on the probe particles which move the non-specifically bound probe particles away from the bottom surface.
14. The article according to claim 13, wherein the channel: (i) comprises a probe particles trap configured to contain the non-specifically bound probe particles received by the channel after a removal of the external force exerted on the probe particles; or (ii) the channel is connected to the test well adjacent an opening of the test well, the opening being formed at a top portion of the test well, optionally further comprising: an enclosure including an inner surface configured to face the test well and wherein the channel comprises a space defined between the inner surface of the enclosure and a wall extends outwardly from the opening of the test well.
15. The article according to claim 13, further comprising: a system control well including a bottom surface coated with the target analyte, wherein the system control well is configured to receive the sample and probe particles; a blank control well including a bottom surface coated with non-bait molecules that prohibit binding with the target analyte, wherein the blank control well is configured to receive the sample and probe particles; or a negative control well including a bottom surface coated with the first sensing element, wherein the negative control well is configured to receive the one or more liquid mediums in the absence of the target analyte.
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. A kit for detecting a presence of a target analyte in a sample, the kit comprising: an article according to claim 13; the probe particles coated with a second sensing element, the probe particles being contained in a solution; and an instrument configured to exert the external force on the probe particles.
21. The kit according to claim 20, wherein the instrument comprises a magnetic needle and the probe particles comprises magnetic or superparamagnetic particles.
22. The kit according to claim 20, further comprising a syringe for introducing the sample into a dropper bottle for mixing with the solution containing the probe particles to form the one or more liquid mediums and transferring the one or more liquid mediums into the article.
23. A method of using the kit according to claim 20, the method comprising: introducing the sample and the probe particles contained in the one or more liquid mediums into the test wells of the article; exerting the external force on the probe particles to move the non-specifically bound probe particles away from the bottom surface of the test well and into the channel; analysing the probe particles in at least one selected from the group consisting of the test well and the channel, thereby determining a presence of the target analyte in the sample.
24. The method of using the kit according to claim 23, wherein the step of exerting the external force on the probe particles comprises: turning the article upside down; and controlling a movement of the non-specifically bound probe particles into the channel using a magnetic needle.
25. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0201] The accompanying drawings illustrate a disclosed embodiment and serve to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.
[0202] FIG. 1A is a schematic illustration of the general concept of the detection method of the present disclosure, where (1) denotes the sensing element, (2) denotes the target analyte, and (3) denotes the probe particle.
[0203] FIG. 1B is a schematic illustration of the detection method of the present disclosure where the specific target analyte-probe particle interaction results in cross-linking of the probe particle to the coated surface in the presence of the target analyte, leading to a positive result as shown in (a). The absence of the target analyte will lead to a negative result as shown in (b). The label (7) denotes the target analyte, (8) denotes the sensing element, and (9) denotes the probe particle.
[0204] FIG. 1C is a schematic illustration of the detection method of the present disclosure where the specific target analyte-probe particle interaction results in un-linking of the probe particle from the coated surface, leading to a positive result as shown in (a). The absence of the target analyte will lead to a negative result as shown in (b). The label (11) denotes the target analyte, (12) denotes the sensing element, and (13) denotes the probe particle.
[0205] FIG. 2 is a schematic illustration of the proof-of-concept experimental procedure for detection of a mock virus target analyte. The label (15) denotes the ACE2-coated surface, (16) denotes the target analyte (RBD-coated mock virus), and (17) denotes the superparamagnetic probe particle (ACE2-coated).
[0206] FIG. 3 is an image depicting the experimental results of the procedure of FIG. 2, where the specifically bound superparamagnetic probe particles are found at the bottom of the multi-well plates, for various mock virus target analyte concentrations.
[0207] FIG. 4 is the data illustrating the experimental results of the number of specifically bound superparamagnetic probe particles with respect to mock virus target analyte concentrations in the procedure described in FIG. 2, where: (a) two example analysing images of the resultant coated surface in the conditions of different mock virus target analyte concentrations, (b) ten example trials of the experiments with different mock virus target analyte concentrations, (c) an example trial of the experiment for 100 fM and 1 pM mock virus analyte concentrations in the sample, (d) two example trials to determine the level of cross-reactivity of the method.
[0208] FIG. 5 is a schematic illustration of a proof-of-concept experimental procedure for detection of an antibody target analyte (CR3022, an antibody to SARS-CoV-2).
[0209] FIG. 6 is a set of images depicting the experimental results of the procedure of FIG. 5.
[0210] for various antibody analyte concentrations.
[0211] FIG. 7 is the data illustrating the experimental results of the number of specifically bound superparamagnetic probe particles with respect to antibody target analyte (CR3022, an antibody to SARS-CoV-2) concentrations in the experiment to detect antibody target analyte. Data for experimental result of control experiment with no antibody target analyte (i.e. 0 M) used is presented for comparison.
[0212] FIG. 8A is an image depicting visualization and subsequent quantification of the colour density of specifically bound probe particles on a coated surface. The label “+” indicates a well containing a positive result while the label “−” indicates a well containing a negative result.
[0213] FIG. 8B is an image depicting visualization and quantification of the particle density of specifically bound probe particles on a coated surface using a microscope.
[0214] FIG. 8C is an image depicting visualization and quantification of the aggregate size of the aggregates formed by specifically bound probe particles on a coated surface. The arrows indicate the aggregates formed as a spot on the coated surface.
[0215] FIG. 8D is an image depicting visualization and quantification of the aggregate size of the aggregates formed by non-specifically bound probe particles on a second surface (position of the aggregates indicated by the white arrows). The black arrows indicate the position of the specifically bound probe particles on a coated surface.
[0216] FIG. 9A is the data illustrating the clinical validation of the method in anti-SARS-CoV-2 receptor binding domain (anti-SARS-CoV-2 RBD) antibody detection, with quantification results of antibody levels detected in the plasma samples of COVID-19 and dengue convalescent patients.
[0217] FIG. 9B is the data illustrating the clinical validation of the method in anti-SARS-CoV-2 receptor binding domain (anti-SARS-CoV-2 RBD) antibody detection, where the results obtained in the method of the present disclosure as described in FIG. 9A are directly compared against the quantification results using ELISA titer assay on the same set of plasma samples of COVID-19 and dengue convalescent patients.
[0218] FIG. 10A is an image illustrating the preliminary results of detecting the IgG antibody against SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD). The label “w/o” denotes the absence of target analyte as a negative control.
[0219] FIG. 10B is the data illustrating the corresponding quantification results of FIG. 10A.
[0220] The label “w/o” denotes the absence of target analyte as a negative control.
[0221] FIG. 11A is an image illustrating the results of specifically bound probe particles on the coated glass surface of the wells for the detection of SARS-CoV-2 nucleocapsid antigen (target analyte). The label “w/o” denotes the absence of target analyte as a negative control.
[0222] FIG. 11B is an image illustrating corresponding microscope imaging results of FIG. 11A for certain samples. The label “w/o” denotes the absence of target analyte as a negative control.
[0223] FIG. 11C is the data illustrating the corresponding quantification results of FIG. 11B. The label “w/o” denotes the absence of target analyte as a negative control.
[0224] FIG. 11D is an image illustrating the SARS-CoV-2 nucleocapsid antigen (target analyte) detection using commercially available PANBIOTM COVID-19 Ag Rapid Test Device for the indicated target analyte concentrations. The label “w/o” denotes the absence of target analyte as a negative control.
[0225] FIG. 12 shows a diagram illustrating a perspective view of a kit for detecting a presence of a target analyte in samples in accordance with an example embodiment.
[0226] FIG. 13A shows a side view of the well plate upon an introduction of the samples and the probe particles into the test wells.
[0227] FIG. 13B shows a side view of the well plate during the incubation step inside the test wells.
[0228] FIG. 13C shows a side view of the well plate enclosed in the water box.
[0229] FIG. 13D shows a side view and a perspective view of the well plate enclosed in the water box, with both the well plate and water box facing down.
[0230] FIG. 13E shows a side view and a perspective view of the well plate enclosed in the water box, with the aggregates being moved through the channel using the magnetic needles array.
[0231] FIG. 13F shows a side view and a top view of the well plate enclosed in the water box, with both the well plate and water box facing up.
[0232] FIG. 14A shows a smartphone camera image of a well plate after the non-specifically bound beads have been separated by force and relocated outside the test wells. Before the force exertion step, the wells had been incubated with liquid mediums at different concentrations of nucleocapsid proteins in different solution conditions (top four wells: working buffer; middle four wells: mid-turbinate sample; bottom four wells: saliva samples).
[0233] FIG. 14B shows an enlarged image of a test well visualized under a microscope.
[0234] FIG. 14C shows a graph illustrating the relation between normalized fraction of probe particles quantity and normalised probe particles aggregate size.
[0235] FIG. 15 shows a diagram illustrating a perspective view of a kit for detecting a presence of a target analyte in a sample in accordance with a further example embodiment.
[0236] FIG. 16A shows three images illustrating the steps involved in the preparation of the liquid mediums including the sample and probe particles.
[0237] FIG. 16B shows a side view of the microfluidic plate upon an introduction of the liquid medium into the test well.
[0238] FIG. 16C shows a side view of the microfluidic plate during the incubation step inside the test well.
[0239] FIG. 16D shows a side view of the microfluidic plate facing down.
[0240] FIG. 16E shows a side view of the microfluidic plate facing down, with the aggregates being moved through the removal channel using the magnetic needles array.
[0241] FIG. 16F shows a side view of the microfluidic plate facing up for magnetic aggregation step to be conducted in the test wells.
DETAILED DESCRIPTION OF THE DRAWINGS
[0242] FIG. 1A is a schematic general concept of the detection method of the present disclosure. Firstly, a first sensing element (1) is pre-coated on a surface, such as a coverslip. The coated surface (4) is then incubated with a sample potentially containing the target analyte. If the target analyte (2) is present in the test sample, the target analyte (2) would bind onto the coated surface (4). Thereafter, probe particles (3) which are coated with a second sensing element are loaded onto the coated surface (4) and incubated, to allow specific interaction to occur between the target analyte (2) bound onto the coated surface and the probe particles (3). Finally, a mechanical force is applied to the probe particles (3) as a selection step, to separate the non-specifically bound probe particles from the coated surface (4). To determine the presence of target analyte (2), the number of specifically bound probe particles on the coated surface is measured. The selected specific target analyte-bound surface can be used for other downstream analysis such as sequencing.
[0243] FIG. 1B is a schematic illustration of the detection method of the present disclosure where the specific target analyte-probe particle interaction results in cross-linking of the probe particle to the coated surface in the presence of the target analyte. Firstly, a first sensing element (8) is pre-coated on a surface, such as a coverslip. The coated surface is then incubated with a sample potentially containing the target analyte. If the target analyte (7) is present in the test sample, the target analyte (7) would bind onto the coated surface. Thereafter, probe particles (9) which are coated with a second sensing element are loaded onto the surface and incubated, to allow specific interaction to occur between the target analyte (7) bound onto the coated surface and the probe particles (9). In this instance, if the target analyte (7) is present in the test sample (positive result), the probe particles (9) can be cross-linked to the coated surface via specific target analyte-probe particle interaction as shown in (a). In the absence of the target analyte (negative result) (7), the probe particles (9) will be non-specifically bound to the coated surface as shown in (b). Thereafter, a mechanical force is applied to the probe particles (9) as a selection step, to separate the non-specifically bound probe particles from the coated surface. In the absence of the targeted analyte (7) as in (b), the probe particles (9) are weakly bound the coated surface due to non-specific binding, and thus will be removed from the coated surface upon the application of the mechanical force. In contrast, for probe particles (9) that are cross-linked to coated surface via specific target analyte-probe particle interaction as in (a), they are able to withstand a certain mechanical force and remain bound to the coated surface. To determine the presence of target analyte (7), the number of specifically bound probe particles on the coated surface is measured.
[0244] FIG. 1C is a schematic illustration of the detection method of the present disclosure where the specific target analyte-probe particle interaction results in un-linking of the probe particle from the coated surface. Firstly, a first sensing element (12) is pre-coated on a surface, such as a coverslip. The coated surface is then incubated with a test sample potentially containing the target analyte (11). If the target analyte (11) is present in the test sample, the analyte (11) would bind onto the coated surface. Thereafter, probe particles (13) which are coated with a second sensing element are loaded onto the surface and incubated, to allow specific interaction to occur between the target analyte (11) bound onto the coated surface and the probe particles (13). In this instance, if the target analyte (11) is present in the sample, the target analyte (11) would prevent the cross-linking of the probe particles (13) to the coated surface via specific target analyte-probe particle interaction as shown in (a), thus the target analyte (11) results in un-linking of the probe particles (13) from the coated surface. In the absence of the target analyte (11), the probe particles (13) will be cross-linked to the coated surface as shown in (b). Thereafter, a mechanical force is applied to the probe particles (13) as a selection step, to separate the non-specifically bound probe particles from the coated surface. In the absence of the targeted analyte (11) as in (b), the probe particles (13) are strongly bound onto the coated surface due to cross-linking of the probe particles (13) with the coated surface, and thus the probe particles (13) will not be removed from the coated surface upon the application of the mechanical force. In contrast, for probe particles (13) that are un-linked from the coated surface due to the presence of the intervening target analyte (11) as in (a), the probe particles (13) are thus unable to withstand the mechanical force and will be removed from the coated surface. To determine the presence of target analyte (11), the number of remaining probe particles on the coated surface is measured.
[0245] FIG. 2 is a schematic illustration of the proof-of-concept experimental procedure for detection of a mock virus target analyte, where the specific mock virus target analyte-superparamagnetic probe particle interaction results in cross-linking of the superparamagnetic probe particle to the coated surface in the presence of the mock virus target analyte, leading to a positive result as shown in (a). The absence of the mock virus target analyte will lead to a negative result as shown in (b). The label (15) denotes the ACE2-coated surface, (16) denotes the target analyte (RBD-coated mock virus), and (17) denotes the superparamagnetic probe particle (ACE2-coated).
[0246] FIG. 3 is an image depicting the experimental results of the procedure of FIG. 2, where the specifically bound superparamagnetic probe particles are found at the bottom of the multi-well plates, after applying a mechanical force in the form of a magnetic force to remove the non-specifically bound superparamagnetic probe particles in the experiment to detect mock virus target analyte, for various mock virus target analyte concentrations: 0 pM (control), 100 fM, 1 pM and 10 pM. The results can be directly visualized by the naked human eyes and distinguished by the intensity change of the surfaces, in this case, the increase in the opaque region at the bottom of the wells (shown by the full arrows (.fwdarw.)), due to different density of specifically bound superparamagnetic probe particles on surface with different concentrations of mock virus target analyte. The area without any superparamagnetic probe particles is shown by the dashed arrows (---->).
[0247] FIG. 4 is the data illustrating the experimental results of the number of specifically bound superparamagnetic probe particles with respect to mock virus target analyte concentrations in the procedure described in FIG. 2, where: (a) two example analysing images of the resultant coated surface in the conditions of 0 pM (control, left) and 10 pM (right) mock virus target analyte, (b) ten example trials of the experiments with 0 pM (control) and 10 pM mock virus analyte. For each pair of box-plot, as represented by the grey area under “Assembled” and the areas bounded by the dotted lines for “Trial 1” to “Trial 10”, the box-plot on the left (which is lower) represents the control, and the box-plot on the right (which is higher) represents the test value. All trials clearly show the detection of 10 pM mock virus target analyte in the samples, (c) an example trial of the experiment clearly detecting 100 fM and 1 pM mock virus target analyte in the sample. Similar experiments were repeated for more than 5 times, (d) two example trials to determine the level of cross-reactivity of the method. The results clearly show that the method can specifically detect the target mock virus analyte from other non-targeted analyte. “NA” denotes neutravidin coated paramagnetic beads.
[0248] FIG. 5 is a schematic illustration of a proof-of-concept experimental procedure for detection of an antibody target analyte (CR3022, an antibody to SARS-CoV-2), where the specific antibody target analyte-superparamagnetic probe particle interaction results in un-linking of the superparamagnetic probe particle from the coated surface in the presence of the antibody target analyte, leading to a positive result as shown in (a). The absence of the antibody target analyte will lead to a negative result as shown in (b).
[0249] FIG. 6 is a set of images depicting the experimental results of the procedure of FIG. 5, where the specifically bound superparamagnetic probe particles after applying mechanical force in the form of magnetic force to remove non-specifically bound superparamagnetic probe particles in the experiment to detect the antibody target analyte (CR3022, an antibody to SARS-CoV-2), for various antibody target analyte concentrations: (a) 0 M, (b) 100 pM, (c) 1 nM, (d) 10 nM, (e) 100 nM and (f) 1 μM.
[0250] FIG. 7 is the data illustrating the experimental results of the number of specifically bound superparamagnetic probe particles with respect to antibody target analyte (CR3022, an antibody to SARS-CoV-2) concentrations in the experiment to detect antibody target analyte. Data for experimental result of control experiment with no antibody target analyte (i.e., 0 M) used is presented for comparison.
[0251] FIG. 8A is an image depicting visualization and subsequent quantification of specifically bound probe particles on a coated surface suitable in a point-of-care testing, where the coated surface has multiple-wells allowing simultaneous testing of sample(s), positive and negative controls. Since the specifically bound probe particles forms a coloured patch on the coated surface, qualitative analysis of test results by measurement of colour density by the naked human eyes is possible during visualization. The label “+” indicates a well containing a positive result while the label “−” indicates a well containing a negative result.
[0252] FIG. 8B is an image depicting visualization and quantification of specifically bound probe particles on a coated surface using a microscope. The particle density of the specifically bound probe particles per unit area may be quantified. This is a form of quantitative analysis of test results by measurement of particle density that is highly accurate and suitable for central laboratory-based testing.
[0253] FIG. 8C is an image depicting visualization and quantification of aggregates formed by specifically bound probe particles on a coated surface that is suitable in a central laboratory-based testing or point-of-care testing when used together with smartphone-based imaging, where the coated surface has multiple-wells allowing simultaneous testing of sample(s), positive and negative controls. This is a form of quantitative analysis by measurement of aggregate size or colour density of the aggregates, where the aggregate size and color density is positively or negatively correlated with the target analyte concentration in the sample when a cross-linking/linking assay or a blocking/un-linking assay is used, respectively. The arrows indicate the aggregates formed as a spot on the coated surface.
[0254] FIG. 8D is an image depicting visualization and quantification of aggregates formed by non-specifically bound probe particles on a second surface that is suitable in a central laboratory-based testing or point-of-care testing when used together with smartphone-based imaging, allowing simultaneous testing of sample(s), positive and negative controls. This is a form of quantitative analysis by measurement of aggregate size or colour density of the aggregates (white arrows denoting the aggregates), where the aggregate size and color density are positively or negatively correlated with the target analyte concentration in the sample when a blocking/un-linking assay or a cross-linking/linking assay are used, respectively. As the second surface may be in the form of a transparent enclosure enclosing the first surface, the specifically bound probe particles (black arrows denoting the specifically bound probe particles in the wells of the coated multi-well plate) may also be visible through the second surface, thus the visualization and quantification of the specifically bound probe particles may also be measured.
[0255] FIG. 10A is an image illustrating the preliminary results of detecting the IgG antibody against SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD), where (top row) images are obtained for specifically bound probe particles on coated surface at various concentrations of CR3022 antibody (target analyte), and (bottom row) images are obtained for various levels of serially diluted human serum containing CR3022 antibody (target analyte). The label “w/o” denotes the absence of target analyte as a negative control.
[0256] FIG. 10B is the data illustrating the corresponding quantification results of FIG. 10A, where the specifically bound probe particles are detected per 100×100 μm.sup.2 area of coated surface in the experiment of IgG antibody against SARS-CoV-2 receptor binding domain (SARS-CoV-2 RBD), where (left) particle density data obtained for specifically bound probe particles on coated surface at various concentrations of CR3022 antibody (target analyte), and (right) particle density data obtained for various levels of serially diluted human serum containing CR3022 antibody (target analyte). The label “w/o” denotes the absence of target analyte as a negative control.
[0257] FIG. 11A is an image illustrating the results of specifically bound probe particles on the coated glass surface of the wells for the detection of SARS-CoV-2 nucleocapsid antigen (target analyte), where SARS-CoV-2 nucleocapsid antigen are spiked-in at the indicated concentrations in a homogenizing buffer (top four wells), mid-turbinate sample pre-treated with the homogenizing buffer (middle four wells), and saliva sample pre-treated the homogenizing buffer (bottom four wells). The label “w/o” denotes the absence of target analyte as a negative control.
[0258] FIG. 11B is an image illustrating corresponding microscope imaging results (using 4×magnifying lens) of specifically bound probe particles of FIG. 11A for (top row) mid-turbinate sample, and (bottom row) saliva sample in the experiment for the detection of SARS-CoV-2 nucleocapsid antigen (target analyte). The label “w/o” denotes the absence of target analyte as a negative control.
[0259] FIG. 11C is the data illustrating the corresponding quantification results of FIG. 11B, where specifically bound probe particles are detected per 100×100 μm.sup.2 area of coated surface for the detection of SARS-CoV-2 nucleocapsid antigen (target analyte), where SARS-CoV-2 nucleocapsid antigen are spiked-in at the indicated concentrations in (left) mid-turbinate sample, and (right) saliva sample. The label “w/o” denotes the absence of target analyte as a negative control.
[0260] FIG. 11D is an image illustrating the SARS-CoV-2 nucleocapsid antigen (target analyte) detection using commercially available PANBIOTM COVID-19 Ag Rapid Test Device for the indicated target analyte concentrations. The label “w/o” denotes the absence of target analyte as a negative control.
EXAMPLES
Example 1: Detecting Mock SARS-CoV-2 Virus Analyte
[0261] To demonstrate the feasibility of detection, mock SARS-CoV-2 virus particles prepared by coating tiny spherical polystyrene particles with a layer of receptor-binding domain protein (RBD) of SARS-CoV-2 were used as target analyte (16) for this experiment as shown in FIG. 2, to mimic actual virus bioparticles. The size of the mock virus particle was ˜200 nm in diameter, which is within the range of actual virus bioparticle of 20 nm to 400 nm. The coated surface (15) used was a coverslip coated with a layer of receptor protein ACE2 as the sensing element. The superparamagnetic probe particles (17) were likewise coated with a layer of receptor protein ACE2 as the sensing element. The receptor protein ACE2 was chosen as it is known to be capable of binding with the RBD which was coated on the mock virus particles.
[0262] The coated coverslip (15) was incubated with 10 μl of liquid saliva samples containing RBD-coated mock virus particles at concentrations of 100 fM, 1 pM and 10 pM for 30 minutes at room temperature to allow the RBD-coated mock virus particles (16) to bind onto the coated coverslip (15). The volume of liquid sample used was sufficient to fully cover the surface of the coated coverslip (15). Control experiment was conducted where coated coverslip (15) was incubated with 10 μl of liquid saliva sample that does not contain the coated mock virus particles (i.e. 0 M RBD-coated mock virus particle).
[0263] Thereafter, sufficient ACE2-coated superparamagnetic probe particles (17) were loaded onto the coverslip and incubated at room temperature for 10 minutes. The coverslip was exposed to a selective mechanical force of pN-scale using superparamagnetic arrays to separate the non-specifically bound ACE2-coated superparamagnetic probe particles from the ACE2-coated coverslip. For the above-mentioned duration of the sample incubation, the level of mechanical forces applied and the duration of the applied forces, are calibrated for the mock virus target analyte and the corresponding sensing elements demonstrated by the example. These parameters may change when different sensing elements are used, or different analytes are targeted. For each system, the values should be pre-calibrated before applications.
[0264] The coated coverslip (15) that had target mock virus target analyte (16) bounded, that is the RBD-coated mock virus particle, would cross-link with the superparamagnetic probe particles (17), resulting in a much higher density of the specifically bound probe particles on the surface compared to the control, when exposed to the selective mechanical force as shown in (a) of FIG. 2.
[0265] The sample wells on the coverslips can be directly visualized by naked human eyes as shown in FIG. 3 or visualized under a microscope using 4×objective lens to record high resolution images. With the images recorded in light microscope, the remaining superparamagnetic probe particles on the coated surface can be quantified by automated counting the number of particles using proprietary written software, as shown in FIG. 4. Based on the results, the number of superparamagnetic probe particles for target analyte concentrations of 100 fM to 10 pM show significant difference from that obtained from the control. Further, the results of FIG. 4 (d) show that the method can specifically detect the mock virus target analyte from other non-targeted analyte.
[0266] The results serve as an initial proof-of-concept for the present disclosure, demonstrating the limit of detection to be possible for low concentrations of target analyte as low as sub-picomolar level. Based on this result, the method of the present disclosure may be applied in the clinical setting, to generate accurate diagnosis of virus in human sample.
Example 2: Detecting Antibody Analyte
[0267] The capability of the present disclosure in antibody detection was also tested. In this experiment, CR3022 which is an antibody that can bind SARS-CoV-2 receptor-binding domain protein (RBD), was used as the target analyte (21) as shown in FIG. 5. The coated surface (20) used was a coverslip coated with a layer of RBD as its sensing element. The superparamagnetic probe particles (22) were coated with a layer of receptor protein ACE2 as the sensing element. The receptor protein ACE2 was chosen as it is known to be capable of binding with the RBD. Since the target antibody analyte CR3022 (21) is also known to be capable of binding with the RBD, the target analyte (21) would prevent the binding of the superparamagnetic probe particles (22) on the coated coverslip (20).
[0268] The coated coverslip (20) was incubated with 10 μl of liquid serum samples containing CR3022 antibodies at concentrations of 100 pM, 1 nM, 10 nM, 100 nM and 1 μM at room temperature to allow the CR3022 (21) to bind onto the RBD-coated coverslip for 60 minutes (20). The volume of liquid sample used was sufficient to fully cover the surface of the coated coverslip (20). Control experiment was conducted where coated coverslip (20) was incubated with 10 μl of liquid serum sample that does not contain the antibody CR3022 (i.e. 0 M CR3022).
[0269] Thereafter, the ACE2-coated superparamagnetic probe particles (22) were loaded onto the coverslip and incubated at room temperature for 10 minutes. The coverslip was exposed to a selective mechanical force of pN-scale using a magnets array to separate the non-specifically bound ACE2-coated superparamagnetic probe particles from the RBD-coated coverslip.
[0270] For the above-mentioned duration of sample incubation, the level of mechanical forces applied and the duration of the applied forces are calibrated for the specific sensing elements used in serum. These parameters may change when different sensing elements are used, or when different medium is used. For each system, the values should be pre-calibrated before applications.
[0271] The coated coverslip (20) that had target analyte CR3022 (21) bounded would prevent the cross-linking of the superparamagnetic probe particles (22) with the RBD-coated coverslip (20), hence resulting in lower amount of the superparamagnetic probe particles remaining on the coated surface after application of the mechanical force. Hence, the target analyte CR3022 (21) resulted in suppressing the stable binding of superparamagnetic probe particles (22) on the coated coverslip (20). Upon exposure to the selective mechanical force as shown in (a) of FIG. 5, the superparamagnetic probe particles (22) were removed, leading to a positive result indicated by decreased number of remained superparamagnetic probe particle on the surface. In the control experiment where the coated coverslip (20) had no target analyte (21) bounded, the superparamagnetic probe particles (22) would be stably bounded to the coated surface (20), leading to a negative result indicated by more superparamagnetic probe particles remained on the surface after being exposed to the selective mechanical force as shown in (b) of FIG. 5.
[0272] The coverslips were put under a microscope using 4×objective lens to visualise the specifically bound superparamagnetic probe particles on the coated surface as shown in FIG. 6. Based on the results, the number of specifically bound superparamagnetic probe particles on the coated surface for target analyte concentrations of 1 nM to 1 μM (FIG. 6 (c) to FIG. 6 (f)) were significantly lower than the number of specifically bound superparamagnetic probe particles on the coated surface of the control (FIG. 6 (a)).
[0273] The detailed quantification of the specifically bound superparamagnetic probe particles on the coated surface is shown in FIG. 7. The result clearly shows that the number of specifically bound superparamagnetic probe particles on the coated surface for target antibody analyte concentrations of 1 nM to 1 μM were significantly lower than the number of specifically bound superparamagnetic probe particles on the coated surface of the control, hence serving as an initial proof-of-concept for the present disclosure, demonstrating the limit of detection to be possible for low concentrations of target antibody analyte as low as nanomolar level in undiluted serum.
[0274] Based on this result, the method of the present disclosure may be applied in the clinical setting, to generate accurate diagnosis of viral antibodies in human sample.
Example 3: Detecting Anti-SARS-CoV-2 RBD Antibody in Clinical Samples
[0275] Antibody detection: The method of the present disclosure has been applied to the detection and quantification of anti-receptor-binding domain protein (RBD) IgG antibody (target analyte) in human plasma (sample). A total of 22 convalescent plasma samples were used for the validation experiment. Of these samples, 18 plasma samples were collected from 17 patients recovered from COVID-19. One patient has donated two samples, which are samples #10 and #11, on separate dates. The sample #11 was collected 90 days after the patient's hospital admission. All other samples were collected between 29 days to 59 days post hospital admission. Besides the COVID-19 convalescent patient samples, anti-RBD antibody level in 4 plasma samples (samples #19 to #22) from patients presenting dengue fever symptoms and confirmed to be SARS-CoV-2 negative were examined using the method of the present disclosure. Although patients providing sample #20 and #22 were tested to be negative for dengue fever, these 4 samples (samples #19 to #22) serve as negative controls.
[0276] The quantification of anti-RBD antibody levels across all samples are shown in FIG. 9A. The particle density of the specifically bound probe particles on the coated surface for the samples measured are compared against commercially available SARS-CoV-2-negative human serum (Sigma Aldrich, US) of result set at 0, and against a serum with 1 μM SAD-S35 anti-RBD antibody spike-in (Acro Biosystems) of result set at 100. Based on the result shown in FIG. 9A, dengue patient samples #19 to #22 were found to be consistent, producing signals at around 0, demonstrating the absence of anti-RBD antibody in these patient samples. For COVID-19 patient samples, as seen in results of FIG. 9A for samples #1 to #18, considerably different levels of anti-RBD antibody were measured. For instance, the antibody in patient sample #5 is found to top the strength of 1 μM SAD-S35 by giving a reading of ˜127, while many others like #3, #7-11, #13 etc. give readings comparable to negative controls, suggesting the absence of such specific antibody in the patient plasma.
[0277] The antibody level in the same COVID-19 patient samples was measured using the current gold standard ELISA titer assays. The generated quantification from ELISA titer assays were represented by the half maximum absorbance at optical density at 450 nm wavelength. The data generated from the method of the present disclosure as in FIG. 9A are plotted against the results generated from the ELISA titer assays as shown in FIG. 9B. Based on the result shown in FIG. 9B, it is observed that the large the value of the tire, the higher the signal generated from the method. The method of the present disclosure when used to quantify anti-RBD antibody in human plasma is shown to be at similar accuracy to ELISA titer assays.
[0278] Quantitative titre antibody assay: The method of the present disclosure may be used to perform classic quantitative titre assay, wherein the sample is subjected to several dilutions until the signal generated falls below certain pre-set value. The method may include a standard antibody serving as a reference signal for additional level of quantification.
[0279] In this experiment, a well characterized RBD-binding IgG antibody CR3022 (Creative Biolabs) as target analyte was dissolved in a pre-diluted human serum sample (Sigma Aldrich, US) at different concentrations. A pre-diluted human serum was prepared by mixing a commercially purchased human serum and a lab-prepared homogenizing working buffer solution at a ratio of 1:49 (i.e., the human serum was diluted by 50 times). Probe particles (superparamagnetic microbeads) coated with RBD (second sensing element) were incubated with 50 μl of the pre-diluted human serum/CR3022 mixture for 15 minutes in a test tube, to allow the probe particles to link with the CR3022 target analyte in the pre-diluted human serum. Thereafter, the pre-diluted human serum was separated from the probe particles, by trapping the superparamagnetic probe particles in the test tube using a magnet and removing the pre-diluted human serum solution using a micropipette. Thereafter, the probe particles were re-suspended in 50 μl lab-prepared homogenising working buffer in a test tube, and 30 μl of the re-suspended mixture was loaded into the test well, followed by incubation for 30 minutes before analysing.
[0280] Based on the results shown in FIG. 10A, the method of the present disclosure is shown to be capable of detecting and quantifying the presence of CR3022 target analyte at concentrations as low as about 10 pM, which is about 1000 times below the dissociation constant of CR3022 target analyte to RBD sensing element. Considering that the human serum was pre-diluted by 50 times, it corresponds to about 500 pM CR3022 target analyte (equivalent to about 0.1 μg/mL CR3022 target analyte). As shown in FIG. 10B, the method of the present disclosure may be used to perform titer assay as well. A test performed using the method of the present disclosure requires approximately 45 minutes, while a typical quantitative ELISA assay requires 2 to 3 hours since it involves multiple rounds of buffer exchange. Further, a test performed using the method of the present disclosure only requires 30 μl of pre-diluted human serum solution (50 times diluted); hence only requiring a small volume of the original human serum which can be conveniently obtained by finger-prick method to extract a droplet. Although the data obtained is based on a particular RBD-binding IgG antibody (target analyte) dissolved in commercially purchased human serum, the same principle is expected to work for other RBD-binding IgG antibodies, or other antibodies against different antigens as target analyte, where a sample may be drawn from finger-prick derived whole blood sample.
Example 4: Detecting SARS-CoV-2 Nucleocapsid Antigen in Human Saliva and Mid-Turbinate Spike-In Samples
[0281] In this experiment, a pair of antibodies recognizing different epitopes of SARS-CoV-2 nucleocapsid antigen (target analyte) was adopted as the first sensing element and second sensing element and coated on the surface and on the probe particles respectively. An homogenizing buffer was formulated to homogenize both the saliva samples and mid-turbinate samples that were pre-mixed with the antigen. Hereafter the samples mixed with the homogenizing buffer are referred to as the pre-treated sample The homogenizing buffer can also facilitate the release of the nucleocapsid antigen from the SARS-CoV-2 viral particles. The experiment was performed based on both saliva and mid-turbinate samples obtained from healthy human donors with different concentrations of nucleocapsid antigen spiked into the samples.
[0282] The results shown in FIG. 11A is derived when the experiment was conducted in a 12-well plate where the bottom glass surface was coated with the antibody belonging to the anti-SARS-CoV-2 nucleocapsid antibody pair as mentioned. Probe particles (superparamagnetic microbeads) (Invitrogen, US) were coated with the other antibody of the pair were used as the probe particles. The probe particles were mixed with homogenizing buffer, the saliva sample or the mid-turbinate sample pre-treated with the homogenizing buffer, and 30 μl of the mixture was loaded into each well. After 30 minutes of incubation, a mechanical force was exerted on the probe particles using an array of magnet to separate the non-specifically bound probe particles from the coated surface. Thereafter, the specifically bound probe particles on the coated surface were detected using a smartphone-based camera as shown in FIG. 11A, or a microscope as shown in FIG. 11B. For the microscope detection, the particle density of the specifically bound probe particles was quantified per 100×100 μm.sup.2 area of the coated surface.
[0283] The results of FIG. 11A show that the specifically bound probe particles can be directly observed using a smartphone-based camera to detect the yellowish light scattered from the specifically bound probe particles, where the specifically bound probe particles is visualized as a yellow patch. The intensity of the scattered yellowish light can indicate the relative amount of the specifically bound probe particles contained in the wells. The results of FIG. 11A show that there is visually perceptible difference in the colour density for different target analyte concentrations of 10 pM, 1 pM, and 0 pM (labelled as “w/o”, negative control) in the homogenizing buffer (top 4 wells), mid-turbinate sample (middle 4 wells) pre-treated with the homogenizing buffer, or saliva sample (bottom 4 wells) pre-treated with the homogenizing buffer.
[0284] The results of FIG. 11B show the microscopic images of the coated glass surfaces of the test wells using a 4× magnifying objective lens. The specifically bound probe particles were observed as tiny, dark dots in the microscopic images, where the particle density of the specifically bound probe particles decreased as the nucleocapsid antigen (target analyte) concentration decreased. The corresponding quantification of the particle density of the specifically bound probe particles of FIG. 11B is seen in FIG. 11C, based on the particle density per 100×100 μm.sup.2 area of coated surface. For both the pre-treated saliva and turbinate samples, the observed signal-to-noise ratios (the ratio of the density of the specifically bound probe particles in a sample well compared to the density of the specifically bound probe particles in the negative control well i.e., “w/o” labelled well) were more than 3, demonstrating the sensitivity of the method of the present disclosure. Similar results were obtained for the nucleocapsid antigen of a SARS-CoV-2 variant (UK variant, B1.1.7) (data not shown).
[0285] The results of FIG. 11A to 11C demonstrate that the method of the present disclosure can be used to detect the presence of ≤1 pM SARS-CoV-2 nucleocapsid antigen, when visualization and quantification is done using a smartphone-based camera or microscope for both saliva and mid-turbinate samples for ultralow viral load (i.e. target analyte concentration), as low as about 1 fM (about10.sup.5/mL) assuming most of the nucleocapsid antigen was released by the homogenizing buffer. This ultralow viral load achievable is comparable to the limit of detection using the current RT-qPCR detection method, where in comparison the method of the present disclosure is faster, cheaper, and easier to operate.
[0286] In another comparison shown in FIG. 11D, commercially available PANBIOTM COVID-19 Ag RAPID TEST DEVICE was observed to only detect>10 pM nucleocapsid antigen (target analyte) spiked into the working buffer of the PANBIOTM antigen test kit. Hence, the sensitivity of nucleocapsid antigen detection based on the method of the present disclosure is at least 10 times greater than that of the PANBIOTM antigen test kit. While the data presented were obtained from saliva and mid-turbinate samples, it is expected that similar detection sensitivity could be obtained for nasopharyngeal swap samples.
INDUSTRIAL APPLICABILITY
[0287] The method, system, article and kit as disclosed herein may be used in a wide variety of diagnostic applications such as clinical testing, environmental monitoring, central laboratory testing and home-based self-diagnostic, for the of detection of the presence of a target analyte in a sample. The method, system, article and kit offer single target sensitivity of detection using specific target analyte-probe particle interaction, such that low concentrations of the target analyte, in the range of nanomolar to femtomolar, may be detectable, thus requiring reduced sample size which may in turn enable early-stage diagnosis of diseases in clinical testing.
[0288] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.
