Abstract
Disclosed herein is a method of detecting the presence or absence of an analyte in a test sample, based on the use of motion resistant particle for example non-magnetic particle coated with first sensing element for the analyte and force driven particle for example superparamagnetic particle coated with a second sensing element for the analyte so as to form a motion resistant particle-analyte-force driven particle conjugate. A viscous medium is added to the mixture and a force such as magnetic force is applied to provide separation of the conjugate for quantification. Disclosed herein are also a sensing kit, a system for detecting the presence or absence of an analyte in a test sample and the use of the kit or the system thereof.
Claims
1. A method of detecting a presence or absence of an analyte in a test sample comprising the steps of: a) incubating a plurality of motion-resistant particles and a plurality of force-driven particles with said test sample to form a test mixture, wherein said motion-resistant particles are coated with a first sensing element and said force-driven particles are coated with a second sensing element, both first and second sensing elements are capable of specific binding with the analyte, wherein when the analyte is present in the test sample, said analyte binds with said first and second sensing elements on said motion-resistant particles and said force-driven particles, respectively, to form bound motion-resistant/analyte/force-driven particle conjugates; and b) adding the test mixture of step a) to a viscous medium and applying a force to the force-driven particles to separate said bound motion-resistant/analyte/force-driven particle conjugates, when present, from the unbound motion-resistant particles and unbound force-driven particles.
2. The method of claim 1 further comprising step c) quantification of the bound motion-resistant/analyte/force-driven particle conjugates after separation of the bound motion-resistant/analyte/force-driven particle conjugates from the unbound motion-resistant particles and unbound force-driven particles.
3. The method of claim 1, wherein a size of said motion-resistant particles is in a range of 1 μm to 100 μm.
4. The method of claim 1, wherein said motion-resistant particles are selected from the group consisting of polystyrene (PS), poly(lactic-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), poly(c-caprolactone) (PCL), polyacrylate (PA), polylactide (PLA), poly(Glycidyl Methacrylate) (PGMA), melamine resin, silica, hydrogel and lipid membrane vesicle.
5. The method of claim 1, wherein a size of said force-driven particles is in a range of 10 nm to 50 μm.
6. The method of claim 1, wherein when said force-driven particles are magnetic particles said magnetic particles are selected from the group consisting of iron, cobalt, nickel, alloy thereof, oxide thereof and combinations thereof.
7. The method of claim 1, wherein said viscous medium has a viscosity in a range of 0.01 Pa.Math.s to 10 Pa.Math.s; and wherein said viscous medium is selected from the group consisting of glycerol, olive oil, linseed oil, motor oil, syrup, ferrofluid, a solution of crowding agents, a base liquid with one or more thickening agents, and mixture thereof.
8. The method of claim 1, wherein said analyte has multiple equivalent binding sites for binding with said first sensing element and said second sensing element; and wherein said binding sites are different for the first sensing element and second sensing element.
9. The method of claim 1, wherein step a) comprise the steps of a1) incubating said motion-resistant or said force-driven particles with said test sample first to form a first incubation solution, and a2) further incubating said first incubation solution with the other force-driven particles or other motion-resistant particles to form the bound motion-resistant/analyte/force-driven particle conjugates.
10. The method of claim 9, further comprising, after step a1) and before step a2), a step of vortexing said first incubation solution.
11. The method of claim 9, wherein an incubation time for step a1) is in a range of 10 minutes to 18 hours; and wherein an incubation time for step a2) is in a range of 10 minutes to 2 hours.
12. The method of claim 11, wherein the incubation time for step a1) is longer than the incubation time for step a2).
13. The method of claim 1, wherein said force is applied vertically or horizontally or both vertically and horizontally across said viscous medium.
14. The method of claim 1, wherein said force is applied for a duration of 10 seconds to 60 minutes.
15. The method of claim 1, further comprising a step of collecting said bound motion-resistant/analyte/force-driven particle conjugates from said viscous medium after step b).
16. A sensing kit comprising: a plurality of motion-resistant particles; and a plurality of force-driven particles, wherein said motion-resistant particles and the force-driven particles are coated with a first sensing element and a second sensing element respectively, that can specifically bind to a target analyte in a test sample.
17. A system for detecting a presence or absence of an analyte in a test sample comprising: a) a plurality of motion-resistant particles and a plurality of force-driven particles, wherein said motion-resistant particles are coated with a first sensing element and said force-driven particles are coated with a second sensing element, wherein both first and second sensing elements are capable of specific binding with the analyte, when present, to form bound motion-resistant/analyte/force-driven particle conjugates; and b) a separation means comprising a viscous medium and a force that can separate said bound motion-resistant/analyte/force-driven particle conjugates, when present, from unbound motion-resistant particles and unbound force-driven particles.
18. (canceled)
Description
BRIEF DESCRIPTION OF DRAWINGS
[0090] 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.
[0091] FIG. 1A is a schematic illustration depicting the general concept of the detection method of the present disclosure, where (1) denotes a sensing element, (2) denotes an analyte, (3) denotes a coated force-driven particle (in this case, a superparamagnetic bead coated with sensing element (1)), (4) denotes a coated motion-resistant particle (in this case, a non-magnetic bead coated with sensing element (1)), (5) denotes bound motion-resistant/analyte/force-driven particle conjugate, (6) denotes a highly viscous medium and (7) denotes a pair of magnets.
[0092] FIG. 1B is a schematic illustration depicting two-dimensional detection method of the present disclosure for analyte detection and collection, which is based on the general concept of FIG. 1A coupled with the flow of viscous medium at a velocity v. The labelling of the components are as follows: (1) denotes a sensing element, (2) denotes an analyte, (3) denotes a coated force-driven particle (in this case, a magnetic bead coated with sensing element (1)), (4) denotes a coated motion-resistant particle (in this case, a non-magnetic bead coated with sensing element (1)), (5) denotes bound motion-resistant/analyte/force-driven particle conjugate and (6) denotes a highly viscous medium.
[0093] FIG. 2 is a schematic illustration of a proof-of-concept experimental procedure using double-stranded DNA with biotin and digoxigenin labelling as analyte. The labelling of the components are as follows: (100) denotes a double-stranded DNA with biotin and digoxigenin labelling as analyte, (200) denotes a 10 μm polystyrene bead coated with streptavidin, (300) denotes a 3 μm superparamagnetic bead coated with streptavidin, (400) denotes a buffer solution, (500) denotes a glycerol viscous medium, (600) denotes an interface between the buffer solution and the glycerol viscous medium, (700) denotes the motion-resistant/analyte/force-driven particle conjugate, (800) denotes a pair of magnets, and (900) denotes a microscope.
[0094] FIG. 3 is a schematic illustration depicting the detection of digoxigenin-coated 200 nm mock virus as analyte (1000) with motion-resistant, large anti-digoxigenin coated polystyrene bead (2000) and small force-driven, anti-digoxigenin coated superparamagnetic bead (3000). The analyte (1000) thus dimerizes with the coated large anti-digoxigenin polystyrene bead (i.e. coated non-magnetic bead) (2000) and the coated small anti-digoxigenin coated superparamagnetic bead (3000) to form bound motion-resistant/analyte/force-driven particle conjugate (4000).
[0095] FIG. 4 is the video imaging results of the proof-of-concept experiment of FIG. 2 where double-stranded DNA with biotin and digoxigenin labelling is the analyte to be detected.
[0096] FIG. 5 is the experimental results comparing the number of polystyrene beads detected using different concentrations of analyte (0.4 pM and 4 fM) for the proof-of-concept experiment of FIG. 3 where double-stranded DNA with biotin and digoxigenin labelling is the analyte to be detected. The number of polystyrene beads counted in experiment Trial 1 and 2 showed 121 times increase and 0.9 times increase for the 0.4 pM and 4 fM analyte concentrations as compared to without analyte respectively.
[0097] FIG. 6 is the experimental results comparing the number of polystyrene beads detected using different concentrations of analyte (6 pM, 60 fM and 600 fM) in standard buffer for the experiment of FIG. 3, with additional results for using the 600 fM analyte in 10 times diluted human serum. The number of polystyrene beads counted showed 3 times increase, 32 times increase, and 156 times increase for 60 fM, 600 fM and 6 pM analyte concentrations in standard buffer as compared to without analyte respectively. When 10 times human serum is used instead of standard buffer, under 600 fM analyte concentration, the number of polystyrene beads counted showed 39 times increase as compared to without analyte.
DETAILED DESCRIPTION OF DRAWINGS
[0098] FIG. 1A is a schematic illustration depicting the general concept of the detection method of the present disclosure. Without the analyte (2), the beads would not dimerize to form the bound motion-resistant/analyte/force-driven particle conjugate (5). After a certain period of incubation, the mixture was subjected to highly viscous medium (6) with magnets (7) placed aside. All force-driven particles (superparamagnetic beads) (3) in the mixture were then attracted and travel towards the direction where the force is induced. When the superparamagnetic bead dimerized with the motion-resistant particle (non-magnetic bead in this case) in the presence of the analyte (5), due to larger size of dimerized beads, the dimerized beads experienced larger drag force in the viscous medium (6) and travelled slower than the unbound superparamagnetic beads (3). Unbound non-magnetic beads (4) on the other hand stayed still in the medium. That created separation of different species of beads for easy quantification of the analyte (2). The drag force between the dimerized beads also provided a gating mechanism to screen off the non-specific bindings. As non-specific binding is generally much weaker than specific interactions, beads dimerized based on non-specific binding would dissociate before the dimer travels far. Only specific interaction-based bead dimer (5) could achieve the separation.
[0099] FIG. 1B is a schematic illustration depicting two-dimensional detection method of the present disclosure for analyte detection and collection, which is based on the general concept of FIG. 1A coupled with the flow of viscous medium at a velocity v, such that the analyte (2) was pre-incubated with the sensing elements (1) coated motion-resistant (non-magnetic) bead (4) and force-driven (magnetic) bead (3) to allow forming specifically bound motion-resistant/analyte/force-driven particle conjugates (5), which were then loaded into a sorting and collecting channel containing a viscous medium (6) where a force F (in this case a magnetic force) was applied in the vertical direction to separate the specifically bound motion-resistant/analyte/force-driven particle conjugates (5) from the individual non-bound beads. An additional flow of the viscous medium is introduced in a different direction from the direction of the induced force (i.e., magnetic field direction) and at a velocity of v to facilitate analyte detection and collection (in this case along the direction labelled as “positive direction” towards to the end of the separator).
EXAMPLES
[0100] Non-limiting examples of the invention will be further described in greater details by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
Example 1: Detecting Dual-Labelled Double-Stranded DNA
[0101] To demonstrate the feasibility of detection, a proof-of concept experiment was performed as shown in FIG. 2 where double-stranded DNA with biotin and digoxigenin labelled at two ends (ThermoFisher, US) as the analyte (100) was used to induce bead dimerization. The analyte (100) was designed to dimerize the anti-digoxigenin antibody (Pierce, US)-coated 10 μm polystyrene beads (Polysciences, US) (200) and the streptavidin (Pierce, US)-coated 3 μm superparamagnetic beads (Invitrogen, US) (300). Here, the motion-resistant particle is the polystyrene bead (which is non-magnetic) and the force-driven particle is the superparamagnetic bead (which is magnetic).
[0102] Firstly, as shown in the “1. Incubation” step of FIG. 2, the analyte (100) at 0.4 picomolar (pM) and 4 femtomolar (fM) concentrations were incubated with motion-resistant polystyrene beads (200) for 30 minutes and 10 hours respectively to allow binding of the analyte (100) to the polystyrene beads (200). Then excessive streptavidin-coated superparamagnetic beads (300) were added into the buffer and incubated a further 30 minutes to maximize the chance of bead dimerization.
[0103] Secondly, as shown in the “2. Sedimentation” step of FIG. 2, the resultant mixture from step 1 containing the bound motion-resistant/analyte/force-driven particle conjugate (700) was added atop a viscous medium (500), such as glycerol (Fisher Chemical, US), in the channels, forming a transient interface (600) between the buffer solution (400) of the resultant mixture and glycerol (500). Here, the bound motion-resistant/analyte/force-driven particle conjugate is bound polystyrene/analyte/superparamagnetic bead conjugate. Due to the high viscosity of the medium and small gravitational force, both bead species are kept near the interface.
[0104] Following that, a pair of magnets (Supermagetman, US) (800) is placed beneath the glycerol channels for approximately 10 minutes. Magnetic force drove separation of bead species as shown in the “3. Segregation” step of FIG. 2.
[0105] The channels were then put under a microscope (Olympus IX41) (900) using 10× and 50× objective lens as shown in the “4. Detection” step of FIG. 2. By adjusting the focus height of objective, large polystyrene beads at bottom region of the glycerol, which are in fact the bound motion-resistant/analyte/force-driven particle conjugate (700), can be observed and counted.
[0106] Following the previous procedures, videos of scanning through the glycerol were generated. Several frames of the video are listed in FIG. 4, to demonstrate the successful separation of bead species and therefore the detection of analyte in the buffer. In the control experiment without any analyte added, under 10× magnification, 3 μm superparamagnetic beads were seen scattering over the bottom region of glycerol, with only one 10 μm polystyrene bead visible. Moving up the focus, neither superparamagnetic beads nor polystyrene beads can be observed. Further focusing up to the glycerol top region, a layer of polystyrene beads can be observed. Compared to the control experiment without analyte, in the presence of only 0.4 pM analyte, over a hundred polystyrene beads can be observed in the glycerol bottom and middle regions. Under 50× magnification, the large polystyrene beads can be always seen companied by one or more small magnetic beads, in a dimerization or oligomerization form.
[0107] The detailed quantifications of polystyrene bead number in the glycerol bottom and middle regions are shown in FIG. 5 for both experimental trials in the presence of 0.4 pM analyte (with 30 min incubation time) and 4 fM analyte (with overnight incubation). Stark contrast can be seen from their respective control experiments, where the analyte was absent. This experiment serves as the initial proof-of-concept for the method in the present disclosure and demonstrates the limit of detection in said method can be as low as femtomolar concentration level.
Example 2: Detecting Digoxigenin-Coated Mock Virus
[0108] The capability of the present method in virus detection was also tested. Procedures followed were similar to what has been described in Example 1, except that the type of analyte was replaced with digoxigenin-coated 200 nm latex beads (Bangs Lab, US) as mock virus particles (1000), and accordingly, the large polystyrene beads (2000) and small superparamagnetic beads (3000) were all anti-digoxigenin coated, as shown in FIG. 3. The analyte (1000) was thus capable of dimerizing the anti-digoxigenin coated large polystyrene beads (2000) and the anti-digoxigenin coated small superparamagnetic beads (3000), to form bound motion-resistant/analyte/force-driven particle conjugate (4000). Here, the motion-resistant particle is the polystyrene bead (which is non-magnetic), the force-driven particle is the superparamagnetic bead (which is magnetic) and the bound motion-resistant/analyte/force-driven particle conjugate is bound polystyrene/analyte/superparamagnetic bead conjugate.
[0109] Similar to the results for the dual-labelled DNA analyte detection, when 6 pM, 600 fM and 60 fM mock virus (analyte, (1000)) in PBS standard buffer were included in the sample for detection, sharp increase of signal can be observed, giving 156 times, 32 times, and 3 times increase respectively compared to the signal from the control where the mock virus analyte was absent, as shown in FIG. 6. The result further strengthens the reliability of the detection method. In addition, to mimic the actual diagnosis, the detection was also performed in 10 times diluted human serum. It is clear that the signal generated in such condition does not deviate from the sample with the same concentration of mock virus, that is 600 fM concentration, in PBS standard buffer, demonstrating the robustness of detection method in the complex environment like human serum. Based on this result, the method disclosed in the present invention may be easily applied in the clinical setting, to generate accurate diagnosis of virus in human biological samples.
INDUSTRIAL APPLICABILITY
[0110] The method, sensing kit and system as disclosed herein may be used in a wide variety of diagnostic applications such as environmental monitoring, clinical testing and home-based self-diagnostic, for the of detection of the presence or absence of an analyte in a test sample. The method, sensing kit and system offer single-particle sensitivity and enhanced specificity based on mechanically gated selection of specific interactions. Further, the method allows collection or concentration of the targeted analyte for downstream processing.
[0111] 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.
