ANALYTE DETECTION BY DIELECTROPHORESIS

Abstract

The present invention provides a method for detecting an analyte with high sensitivity. In the present method, a solution is supplied onto a substrate comprising a first electrode and a second electrode. Then, an alternating voltage is applied between the first electrode and the second electrode to aggregate, onto the surface between the first electrode and the second electrode by dielectrophoresis, bioparticles and dielectric particles contained in the solution. The aggregated bioparticles are broken to release the analyte contained in the bioparticles. The released analyte is bound to a first antibody and a second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody. Finally, the analyte is detected through the fluorescent substance contained in the immobilized dielectric particles.

Claims

1. A method for detecting an analyte, the method comprising: (a) supplying a solution onto a substrate comprising a first electrode and a second electrode; wherein the substrate has a first antibody on the surface thereof between the first electrode and the second electrode; the solution contains dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles has a second antibody on the surface thereof; each of the dielectric particles contains a fluorescent substance; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; and the second antibody is capable of binding to the analyte; (b) applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto the surface of the substrate between the first electrode and the second electrode by dielectrophoresis; (c) breaking the bioparticles aggregated in the step (b) to release the analyte contained in the bioparticles; wherein the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody; and (d) detecting the analyte through the fluorescent substance contained in the immobilized dielectric particles.

2. A method for detecting an analyte, the method comprising: (a) supplying a solution to a flow path formed between a substrate comprising a first electrode and a cover comprising a second electrode; wherein the substrate has a first antibody on the surface thereof near the first electrode; the solution contains dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles has a second antibody on the surface thereof; each of the dielectric particles contains a fluorescent substance; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; and the second antibody is capable of binding to the analyte; (b) applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto the surface of the substrate near the first electrode by dielectrophoresis; (c) breaking the bioparticles aggregated in the step (b) to release the analyte contained in the bioparticles; wherein the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody; and (d) detecting the analyte through the fluorescent substance contained in the immobilized dielectric particles.

3. A device for detecting an analyte, the device comprising: a substrate comprising a first electrode and a second electrode; a solution supplying part for supplying, onto the substrate, a solution containing dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles comprising a fluorescent substance, an alternating-current power supply for applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto a surface of the substrate between the first electrode and the second electrode by dielectrophoresis; a resolvent supplying part for supplying, onto the substrate, a resolvent capable of breaking the aggregated bioparticles to release the analyte contained in the bioparticles; and a detector for detecting the analyte through the fluorescent substance contained in the dielectric particles; wherein the substrate has a first antibody on the surface thereof between the first electrode and the second electrode; each of the dielectric particles has a second antibody on the surface thereof; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; the second antibody is capable of binding to the analyte; and the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody.

4. A device for detecting an analyte, the device comprising: a substrate comprising a first electrode; a cover comprising a second electrode; a solution supplying part for supplying, onto the substrate, a solution containing dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles comprising a fluorescent substance, an alternating-current power supply for applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto a surface of the substrate near the first electrode by dielectrophoresis; a resolvent supplying part for supplying, onto the substrate, a resolvent capable of breaking the aggregated bioparticles to release the analyte contained in the bioparticles; and a detector for detecting the analyte through the fluorescent substance contained in the dielectric particles; wherein a flow path is formed between the substrate and the cover; the substrate has a first antibody on the surface thereof near the first electrode; each of the dielectric particles has a second antibody on the surface thereof; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; the second antibody is capable of binding to the analyte; and the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody.

5. A device for detecting an analyte, the device comprising: a substrate comprising a first electrode and a second electrode; a solution supplying part for supplying, onto the substrate, a solution containing dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles comprising a fluorescent substance, an alternating-current power supply for applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto a surface of the substrate between the first electrode and the second electrode by dielectrophoresis; a breaker for breaking the aggregated bioparticles to release the analyte contained in the bioparticles; and a detector for detecting the analyte through the fluorescent substance contained in the dielectric particles; wherein the substrate has a first antibody on the surface thereof between the first electrode and the second electrode; each of the dielectric particles has a second antibody on the surface thereof; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; the second antibody is capable of binding to the analyte; and the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody.

6. A device for detecting an analyte, the device comprising: a substrate comprising a first electrode; a cover comprising a second electrode; a solution supplying part for supplying, onto the substrate, a solution containing dielectric particles and at least one kind of bioparticles selected from the group consisting of viruses and cells; each of the dielectric particles comprising a fluorescent substance, an alternating-current power supply for applying an alternating voltage between the first electrode and the second electrode to aggregate the bioparticles and the dielectric particles onto a surface of the substrate near the first electrode by dielectrophoresis; a breaker for breaking the aggregated bioparticles to release the analyte contained in the bioparticles; and a detector for detecting the analyte through the fluorescent substance contained in the dielectric particles; wherein a flow path is formed between the substrate and the cover; the substrate has a first antibody on the surface thereof near the first electrode; each of the dielectric particles has a second antibody on the surface thereof; each of the bioparticles contains the analyte; the first antibody is capable of binding to the analyte; the second antibody is capable of binding to the analyte; and the released analyte is bound to the first antibody and the second antibody to cause the dielectric particles to be immobilized onto the substrate through formation of a sandwich structure composed of the first antibody, the analyte, and the second antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows a schematic view of a step (a) included in a method according to a first embodiment of the present invention.

[0042] FIG. 2 shows, subsequently to FIG. 1, a schematic view of a step (b) included in the method according to the first embodiment.

[0043] FIG. 3 shows a schematic view after completion of the step (b).

[0044] FIG. 4 shows, subsequently to FIG. 3, a schematic view of a step (c) included in the method according to the first embodiment.

[0045] FIG. 5 shows a schematic view after completion of the step (c).

[0046] FIG. 6 shows, subsequently to FIG. 5, a schematic view of a step (d) included in the method according to the first embodiment.

[0047] FIG. 7 shows a schematic view of a step (a) included in a method according to a second embodiment of the present invention.

[0048] FIG. 8 is a duplicate of a part of FIG. 1 included in Japanese Patent laid-open Publication No. Hei 4-080657.

[0049] FIG. 9 is a duplicate of FIG. 1 included in Japanese Patent laid-open Publication No. Hei 11-127846.

[0050] FIG. 10 is a duplicate of FIG. 2 included in Japanese Patent laid-open Publication No. Sho 61-272663.

[0051] FIG. 11 is a duplicate of FIG. 4 included in Japanese Patent laid-open Publication No. Sho 61-272663.

[0052] FIG. 12 shows a schematic view of a device for detecting an analyte in a case where bioparticles are broken chemically.

[0053] FIG. 13 shows a schematic view of a device for detecting an analyte in a case where bioparticles are broken physically.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment

[0055] (Step (a))

[0056] First, as shown in FIG. 1, a solution 23 is supplied onto a substrate 16 comprising a first electrode 22a and a second electrode 22b on the surface thereof. A flow path 12 is formed between the substrate 16 and a cover 15. The solution 23 flows through the flow path 12. The cover 15 need not be provided.

[0057] The substrate 16 has first antibodies 21 on the surface thereof between the first electrode 22a and the second electrode 22b. The solution 23 contains at least one kind of bioparticles 26 selected from the group consisting of viruses and cells. The solution 23 contains dielectric particles 24. Desirably, the solution 23 is an aqueous solution.

[0058] Each of the dielectric particles 24 has second antibodies 25 on the surface thereof. Each of the dielectric particles 24 contains a fluorescent substance (not shown). Specifically, the dielectric particles 24 may have the fluorescent substance on the surface thereof; however, it is desirable that the dielectric particles 24 contain the fluorescent substance. Each of the bioparticles 26 contains an analyte 33. An example of the analyte is a protein, a nucleic acid, or a sugar chain.

[0059] The first antibody 21 is capable of binding to the analyte 33. The second antibody 25 is also capable of binding to the analyte 33. It is desirable that a part of the analyte 33 to be bound to the first antibody 21 is different from a part of the analyte 33 to be bound to the second antibody 25. In other words, it is desirable that the analyte 33 comprises a first epitope and a second epitope (both of which are not shown) to be bound to the first antibody 21 and the second antibody 25, respectively.

[0060] (Step (b))

[0061] The step (b) is conducted after the step (a).

[0062] As shown in FIG. 2, after the flow path 12 is filled with the solution 23, an alternating voltage is applied between the first electrode 22a and the second electrode 22b from an alternating-current power supply 40. An example of the applied alternating voltage and its frequency are 1 Vpp-20 Vpp and 0.1 MHz-1 MHz, respectively.

[0063] In this way, the bioparticles 26 and the dielectric particles 24 aggregate onto the surface of the substrate 16 between the first electrode 22a and the second electrode 22b by dielectrophoresis. As a result, the bioparticles 26 and the dielectric particles 24, both of which have been dispersed in the solution 23, are located with high density on the surface of the substrate 16 between the first electrode 22a and the second electrode 22b. As just described, as shown in FIG. 3, the bioparticles 26 and the dielectric particles 24 are located with high density by dielectrophoresis.

[0064] (Step (c))

[0065] The step (c) is conducted after the step (b).

[0066] In the step (c), as shown in FIG. 4, the bioparticles 26 aggregated in the step (b) are broken. As a result, the analyte 33 is released to the solution 23. Since the bioparticles 26 also contain substances 34 other than the analyte 33, the substances 34 are also released to the solution 23 after the breaking.

[0067] A method for breaking the bioparticles 26 is roughly divided into a physical method and a chemical method. An example of the physical method is to heat the solution 23, to apply an ultrasonic wave to the bioparticles 26, or to apply a high voltage pulse to the bioparticles 26. An example of the chemical method is to add a bacteriolysis agent to the solution 23, to change the salt concentration of the solution 23, or to change the pH of the solution 23. It is desirable to apply a high voltage pulse to the bioparticles 26. When the chemical method is used, as shown in FIG. 12, a device for detecting the analyte according to the present embodiment may comprise not only the substrate 16,the alternating-current power supply 40, and a detector 41 (which will be described later), but also a solution supplying part 66 and a resolvent supplying part 67. The solution 23 is supplied from the solution supplying part 66 onto the substrate 16 . A resolvent such as a bacteriolysis agent, a salt, an acid, or an alkali is supplied onto the substrate 16 from the resolvent supplying part 67. An example of the solution supplying part 66 is a syringe. An example of the resolvent supplying part 67 is also a syringe. The solution supplying part 66 may comprise a valve. The resolvent supplying part 67 may also comprise a valve. When the physical method is used, as shown in FIG. 13, a device for detecting the analyte according to the present embodiment may comprise not only the substrate 16,the alternating-current power supply 40, and the detector 41 (which will be described later), but also the solution supplying part 66 and a breaker 69. An example of the breaker 69 is a heater, an ultrasonic wave generator, or a high-voltage pulse generator. The alternating-current power supply 40 may double as a heater and a high-voltage pulse generator. The device for detecting the analyte according to the present embodiment may comprise a controller (not shown). The controller transmits a control signal to the alternating-current power supply 40, the detector 41, a light source 42 (which will be described later), the solution supplying part 66 and the resolvent supplying part 67 (or the breaker 69) in accordance with the method for detecting the analyte according to the present embodiment. The valves included in the solution supplying part 66 and the resolvent supplying part 67 may be controlled by the controller.

[0068] As shown in FIG. 5, the released analyte 33 is bound to both of the first antibody 21 and the second antibody 25. In this way, a sandwich structure 36 composed of the first antibody 21, the analyte 33 and the second antibody 25 is formed. Since the dielectric particles 24 having the second antibodies 25 are located with high density on the surface of the substrate 16 between the first electrode 22a and the second electrode 22b, the sandwich structures 36 are also formed with high density. Needless to say, the analyte 33 is located between the first antibody 21 and the second antibody 25. The dielectric particles 24 are immobilized onto the substrate 16 through the sandwich structure 36.

[0069] During the period between the end of the step (b) and the end of the step (c), it is desirable that the solution 23 remains in the flow path 12 so as not to flow through the flow path 12. In other words, it is desirable that the solution 23 has a flow rate of approximately 0 cm/minute. When the solution 23 flows through the flow path 12, the analyte 33 released from the bioparticles 26 would flow with the solution 23 through the flow path 12 without being bound to the first antibody 21.

[0070] (Step (d))

[0071] The step (d) is conducted after the step (c).

[0072] As shown in FIG. 6, excited light 43 emitted from a light source 42 travels through the sandwich structure 36 to excite the fluorescent substance contained in the dielectric particles 24. In this way, fluorescent light 44 is emitted from the dielectric particles 24. This fluorescent light 44 is received by the detector 41. As just described, the analyte 33 is detected. Since the sandwich structures 36 are formed with high density, the analyte 33 is detected with high sensitivity. It is desirable that the substances 34 other than the analyte 33 are removed after the step (c) and before the step (d).

Second Embodiment

[0073] Next, the second embodiment of the present invention will be described. The difference of the second embodiment from the first embodiment will be listed below.

[0074] (I) The substrate 16 has the first electrode 22a; however, the cover 15 has the second electrode 22b.

[0075] (II) The substrate 16 has the first antibodies 21 on the surface thereof near the first electrode 22a.

[0076] See FIG. 7. Desirably, the substrate 16 has the first antibodies 21 on the surface of at least a part of the circumference of the first electrode 22a. More desirably, the first antibodies 21 are adjacent to the first electrode 22a. In the second embodiment, the bioparticles 26 and the dielectric particles 24, both of which have been dispersed in the solution 23, are located near the first electrode 22a on the surface of the substrate 16 with high density.

INDUSTRIAL APPLICABILITY

[0077] The present invention can be used for a device for collecting or detecting a virus.

REFERENTIAL SIGNS LIST

[0078] 12 Flow path

[0079] 15 Cover

[0080] 16 Substrate

[0081] 21 First antibody

[0082] 22a First electrode

[0083] 22b Second electrode

[0084] 23 Solution

[0085] 24 Dielectric particle

[0086] 25 Second antibody

[0087] 26 Bioparticle

[0088] 33 Analyte

[0089] 34 Substance other than analyte 33

[0090] 36 Sandwich structure

[0091] 40 Alternating-current power supply

[0092] 41 Detector

[0093] 42 Light source

[0094] 43 Excited light

[0095] 44 Fluorescent light

[0096] 66 Solution supplying part

[0097] 67 Resolvent supplying part

[0098] 69 Breaker