JP6495806B2 - Specimen detection method and specimen detection apparatus - Google Patents

Description

  Embodiments relate to a sample detection method and a sample detection apparatus for detecting viruses, bacteria, and the like in a sample.

  In recent years, infectious diseases caused by viruses such as Ebola hemorrhagic fever and dengue fever have spread rapidly throughout the world and have become a social problem. Coping such as proper isolation of patients after onset is very important to prevent pandemics. Going further, it will be important to properly identify patients who have been affected but have not yet developed, and to prevent the spread of the virus from patients.

  The current detection method by immunochromatography is a very inexpensive method and is a powerful method that can obtain results in a relatively short time. However, the detection method does not provide sufficient detection sensitivity even if an antibody corresponding to the virus that is the measurement target substance is developed. In addition, there is a problem that cannot be detected when the amount of virus before the onset is small.

  On the other hand, if genetic testing is performed, the problem of non-detection due to insufficient detection sensitivity can be solved at least. However, genetic testing is expensive for detection devices and reagents, and requires a long test time.

  As a method for solving the above problem, a method for identifying a virus using nanopores is known. This method uses labeled particles set to particles larger than the virus, modified with an antibody that specifically recognizes and binds the virus around it. Even in the early stage of infection with a small amount of virus, the labeled particles and the virus form composite particles in which labeled particles-viruses-labeled particles are combined in this order. When the ion current waveform is observed when the composite particles pass through the micropores, the current drop signal waveform has a double peak shape, so that the virus can be specifically recognized. As a result, it becomes possible to detect viruses one by one, and high sensitivity detection can be realized.

  However, in order to enable observation of the double peak waveform, it is necessary that the composite particles pass through the micropores in the order of labeled particles-viruses-labeled particles. That is, since the passing posture of the composite particles is restricted, the detection efficiency is lowered. Therefore, there is a demand for a method that can detect viruses and bacteria easily in a short time, with high sensitivity and high efficiency.

JP 2014-173936 A Special table 2011-501806 gazette JP 2014-35229 A US Patent Application Publication No. 2007/0202495

  Embodiments provide a sample detection method and a sample detection apparatus that can detect the presence of a measurement target substance such as a virus or bacteria in a sample easily, in a short time, and with high sensitivity.

In order to solve the problem, according to the embodiment,
A measurement cassette, a first chamber and a second chamber formed by partitioning the measurement cassette with a partition, and a through hole provided in the partition and connecting the first chamber and the second chamber to each other A hole, a first electrode provided in the measurement cassette located on the first chamber side, at least a part of which is located in the first chamber, and the measurement cassette located on the second chamber side And a second electrode provided at least in part in the second chamber;
Introducing a sample and a reagent containing a measurement target substance into the first chamber, the reagent is bound to a plurality of tag particles having a size smaller than that of the measurement target substance in the sample, and a surface of each tag particle. A capture substance that specifically binds to the surface of the substance to be measured;
Introducing an energizable liquid into the second chamber;
Applying a voltage between the first electrode and the second electrode;
Passing the substance to be measured in the first chamber through the through-hole, the surface of the substance to be measured being bound and coated with the plurality of tag particles via the capture substance; and 1. observing a change in current flowing between the second electrodes and detecting the presence of the substance to be measured in the specimen;
An analyte detection method is provided.

According to another embodiment,
Body;
First and second chambers formed by partitioning the inside of the main body with partition walls, wherein the first chamber has a flow path;
A through hole provided in the partition wall for connecting the flow path of the first chamber and the second chamber to each other;
Sample inlet provided in the main body portion located at one end of the flow path:
An outlet provided in the body portion located at the other end of the flow path;
A first filter provided in the flow path, the first filter being disposed downstream of the flow of the sample from the sample inlet to the outlet with respect to the through-hole, and in the sample A reagent including a plurality of tag particles having a size smaller than that of the measurement target substance and a capture substance that is bound to the surface of each tag particle and specifically binds to the surface of the measurement target substance is held in advance:
A first electrode provided on the main body portion located on the first chamber side and at least a part of which is located in the first chamber;
A second electrode provided in the main body portion located on the second chamber side, at least a part of which is located in the second chamber; and the measurement target substance in the specimen is contained in the first filter. A desorption section for desorbing the measurement target substance from the first filter to the flow channel upstream of the specimen flow when the plurality of tag particles and the capture substance are combined;
There is provided an analyte detection device comprising:

It is a figure which shows schematic structure of the sample detection apparatus used for the sample detection method which concerns on 1st Embodiment. It is a figure which shows that an electric current signal changes with particle | grain dimensions. It is a figure which shows the example of the reagent used for 1st Embodiment. It is a figure which shows the electric current signal detected in 1st Embodiment. It is a figure which shows the example of the reagent used for 2nd Embodiment. It is a figure which shows the electric current signal detected in 2nd Embodiment. It is a top view which shows schematic structure of the sample detection apparatus which concerns on 3rd Embodiment. It is sectional drawing which follows the VIII-VIII line of FIG. It is a figure which shows frequency distribution of the signal strength in the comparative example 1. It is a figure which shows frequency distribution of the signal strength in Example 1. FIG.

  Hereinafter, the specimen detection method according to the embodiment will be described with reference to the specimen detection apparatus shown in FIG.

(First embodiment)
(Configuration of specimen detection device)
FIG. 1 is a cross-sectional view illustrating a schematic configuration of a sample detection apparatus used in the sample detection method according to the first embodiment.

  The specimen detection apparatus includes a measurement cassette 1 that can be filled with a liquid. The measurement cassette 1 is partitioned vertically by an insulating partition 2. The first chamber 3 is formed above the partition wall 2 inside the measurement cassette 1. The second chamber 4 is formed below the partition wall 2 inside the measurement cassette 1. The support plate 6 is provided on the lower surface of the partition wall 2 and supports the partition wall 2. The frustoconical hole 5 is formed in the center of the support plate 6. The fine through hole 7 is formed in the partition wall 2 corresponding to the hole 5 of the support plate 6. Therefore, the first and second chambers 3 and 4 are connected to each other through the through hole 7 and the hole 5. The diameter of the fine through hole 7 is sufficiently smaller than the hole 5 of the support plate 6.

  For example, the first electrode 8 is provided on the upper wall portion of the measurement cassette 1 in which the first chamber 3 is located, and at least a part of the first electrode 8 is located in the first chamber 3. For example, the first electrode 8 is disposed immediately above the through hole 7 of the partition wall 2. The second electrode 9 is provided on the lower wall portion of the measurement cassette 1 in which the second chamber 4 is located, and at least a part of the second electrode 9 is located in the second chamber 4. For example, the second electrode 9 is located immediately below the through hole 7 of the partition wall 2. The first electrode 8 is grounded. The measurement circuit 10 and the power source 11 are connected to the second electrode 9 in this order.

  The measuring cassette 1 is entirely made of an electrically and chemically inactive material. Alternatively, the measurement cassette 1 is electrically and chemically connected to the inside of the first chamber 3, the inside of the second chamber 4, the contact portion with the first electrode 8, and the contact portion with the second electrode 9. Alternatively, it may be made of an inert material. The measurement cassette is made of, for example, PEEK, polycarbonate, resin such as acrylic resin, glass, sapphire, ceramic, rubber, elastomer, or the like.

Partition wall 2 is formed of glass, sapphire, ceramic, resin, rubber, elastomer, an electrically and chemically inert, such as _{SiO 2, Si 3 N 4,} Al 2 O 3, and the material of the insulating be able to.

  The opening size (for example, the diameter) of the through-hole 7 of the partition wall 2 is preferably 10 times or less the size of the measurement target substance in which a plurality of tag particles are bonded to and coated on the surface via the capture substance as described later. . The length of the through hole 7 (corresponding to the thickness of the partition wall) is equal to or slightly larger than the dimension (for example, diameter) of the measurement target substance whose surface is coated with the plurality of tag particles. It is preferable that

(Detection method and detection signal for specimen alone)
A method for measuring the size and shape of the measurement target substance in the sample using the sample detection apparatus of FIG. 1 will be described below.

  First, the liquid 32 that can be energized is filled in the second chamber 4. At this time, the second electrode 9 is partially immersed in the energizable liquid filled in the second chamber 4.

  Next, a specimen containing a liquid 31 that can be energized and a substance to be measured is introduced into the first chamber 3. At this time, a liquid junction can be taken between the first and second chambers 3 and 4 through the fine through hole 7 of the partition wall 2 and the hole 5 of the support plate 6. In addition, the first electrode 8 is partially immersed in an energizable liquid filled in the first chamber 3. In this state, by applying a voltage between the first electrode 8 and the second electrode 9, current is supplied to the fine through hole 7 of the partition wall 2. The measurement target substance in the energizable liquid in the first chamber 3 passes through the fine through hole 7 by the electric field and moves to the energizable liquid in the second chamber 4.

  FIG. 2 schematically shows a current value measured by the measurement circuit 10 when the measurement target substance passes through the fine through-hole 7. As shown in FIG. 2, the current value changes depending on the size of the measurement target substance 40. Specifically, the current change peak is small as shown by peak A when the measurement target substance 40 is small. The current change peak increases as shown by peaks B and C as the measurement target substance 40 increases. However, when the measurement target substance 40 has the same size and different properties, it is very difficult to identify the measurement target substance only from the change in the current value.

  The energizable liquids 31 and 32 filled in the first and second chambers 3 and 4 are, for example, electrolyte solutions such as KCl aqueous solution and NaCl aqueous solution, Tris Ethylene diamine tetra acetic acid (TE) buffer solution, PBS Buffer solutions such as (Phosphate Buffered Saline) buffer solution, Tris-Buffered Saline (TBS) buffer solution, and N-2-hydroxyetylpiperazine-N′-ethanesulphonic acid (HEPES) buffer solution can be used.

(Detection signal when a reagent is added to the sample)
In the first embodiment, a case where there is one kind of measurement target substance (for example, biomolecule) that may exist in the specimen will be described as an example. As shown in FIG. 3A, the reagent includes a capture substance 42 and tag particles 43. The capture substance 42 is bound to the surface of the tag particle 43 and specifically binds to the surface of the measurement target substance. The tag particle 43 has a smaller dimension (for example, a diameter) than the measurement target substance. This reagent is mixed with the specimen. When the measurement target substance 41 is present in the specimen, a plurality of tag particles 43 are bonded to the surface of the measurement target substance 41 via the capture substance 42 as shown in FIG. Thereby, composite particles 44 are obtained.

  Next, as shown in FIG. 1, a mixed solution 31 of the specimen and the reagent is introduced and filled in the first chamber 3. The second chamber 4 is also filled with a liquid 32 that can be energized. The specimen is a liquid that can be energized and contains a substance to be measured. If the specimen is not a liquid that can be energized, or if the specimen alone is not sufficient to supply the liquid into the first chamber 3, the mixture of the specimen and the reagent is dissolved in the energizable liquid and the first chamber 3 is dissolved. What is necessary is just to introduce.

  Thus, after introducing the mixed solution 31 of the specimen and the reagent into the first chamber 3, a voltage is applied between the first electrode 8 and the second electrode 9, and the minute through hole 7 of the partition wall 2 is applied. Apply current. Particles such as composite particles 44 and the like in which the tag particles 43 are bonded to and coated on the surface of the measurement target substance 41 via the capture substance 42 pass through the fine through-holes 7 from the first chamber 3, and enter the second chamber 4. It moves to the liquid 32 that can be energized. At this time, the current value measured by the measurement circuit 10 changes in a pulse manner.

  The detection signal of the current value changes according to the size and shape of the passing particle as shown in FIG. Specifically, as shown in FIG. 4, the detection signal A appears when the measurement target substance 41 is alone. The detection signal B appears when the particle 41 ′ is separate from the measurement target substance. When the dimension of the measurement target substance 41 is the same as the dimension of the particle 41 ′ separate from the measurement target substance, the amount of change in the current value of the measurement target substance 41 is the same as indicated by the detection signals A and B. . Therefore, the measurement target substance 41 cannot be identified from the separate particles 41 '. The tag particle 43 having the capture substance 42 bound to the surface appears as a detection signal C shown in FIG. The amount of change in the current value of the detection signal C is very small because the size of the tag particle 43 is smaller than that of the measurement target substance 41.

  The detection signal D shown in FIG. 4 is obtained in the case of the composite particle 44. The composite particles 44 are obtained by binding and covering a plurality of tag particles 43 having a size (for example, a diameter) smaller than that of the measurement target substance 41 via the capture substance 42 on the surface of the measurement target substance 41. The amount of change in the current value of the detection signal D shown in FIG. 4 is larger than that in the case of the measurement target substance 41 alone (detection signal A shown in FIG. 4).

  Therefore, even if the measurement target substance 41 and the measurement target substance have the same size and separate particles 41 ′ are present in the specimen, the measurement target substance 41 is bound to the plurality of tag particles 43 via the capture substance 42. The particle size is large compared to the individual particles 41 ′. Therefore, based on the detection signal (detection signal D) from the measurement circuit 10, it is possible to determine only the measurement target substance 41 and detect the presence of the measurement target substance 41 in the sample.

  In addition, the amount of change in the current value measured by the measurement circuit 10 is monitored over a certain period of time, and the number of detection signals D shown in FIG. 4 is counted to measure the concentration of the measurement target substance in the sample. Is also possible.

  For example, an antibody, a peptide, an aptamer, or a hydrocarbon chain can be used as the capture substance 42.

  The tag particles 43 are preferably spherical in shape. As the tag particles, for example, negatively charged spherical polystyrene fine particles or nano-sized gold particles can be used.

  Since the tag particle is bonded to the surface of the measurement target substance via the capture substance and covers the surface, the dimension (for example, diameter) of the tag particle is smaller than that of the measurement target substance. When the dimension (for example, diameter) of the measurement target substance is defined as 100%, the dimension of the tag particle is 10% or more and 70% or less, more preferably 20% or more and 70% or less. Thus, by making the size of the tag particles 10% or more and 70% or less of the size of the measurement target substance, the plurality of tag particles are bonded and coated on the surface of the measurement target substance. Therefore, it is possible to obtain a structure (for example, a composite particle) with appropriately increased dimensions. In particular, it is preferable that the size of each tag particle is selected within the range of 10% to 70%, and the lower limit side is selected, that is, finer tag particles are selected. This makes it possible to reduce the peak height of the detection signal of the current value when a single tag particle is detected. As a result, noise generation during detection can be suppressed.

  Regarding the specific dimensional relationship between the measurement target substance and the tag particle, when the dimension (for example, diameter) of the measurement target substance is about 100 nm, the tag particle preferably has a size (for example, diameter) of 20 to 50 nm. Furthermore, when the dimension (for example, diameter) of the substance to be measured is about 50 nm, the tag particle preferably has a dimension (for example, diameter) of 10 to 30 nm.

When a reagent is mixed with a specimen, the amount of tag particles in the reagent is preferably excessive as compared with the amount of the substance to be measured in the specimen. Specifically, the number of tag particles is preferably 10 to 10 ⁶ times that of the substance to be measured. By setting the number of tag particles to this ratio, the probability of collision and contact of tag particles with the measurement target substance increases. Therefore, the plurality of tag particles can be quickly bonded and coated on the surface of the substance to be measured with the capture substance interposed therebetween. As a result, the surface of each measurement target substance can be covered with a plurality of tag particles before the adjacent measurement target substances are bonded with the tag particles interposed therebetween. Therefore, it is possible to prevent two or more substances to be measured from being bonded to each other with the tag particle interposed therebetween, and to avoid a dumbbell structure or the like. In other words, it is possible to detect independent fine particles in which a plurality of tag particles are bonded to and coated on the surface of each substance to be measured with the above-described sample measuring apparatus. As described above, a voltage is applied between the first and second electrodes to energize the through hole, and a change in current value due to particles passing through the through hole is measured. In the measurement, when the number of tag particles is increased with respect to the number of substances to be measured, the appearance rate of detection signals of current values per unit time due to the tag particles bound to the capture substance increases. In order to suppress the generation of noise by lowering the detection signal peak of the current value, the tag particle size (diameter) is within the range of 10% or more and 70% or less of the measurement target substance size as described above and on the lower limit side. It is preferable to select.

  In a form in which a plurality of tag particles are bonded to the surface of a measurement target substance via a capture substance and coated, the measurement target substance coated with the plurality of tag particles is formed into a shape of the measurement target substance without covering with the plurality of tag particles. It is preferable that they are similar to each other.

  The capture substance is preferably bound so as to localize to the tag particle. That is, it is preferable that the capture substance is locally bound to the tag particle. In such a form, when the tag particle binds to the measurement target substance via the capture substance, the tag particle exhibits binding anisotropy with respect to the measurement target substance. For example, assuming a tag particle having a spherical or substantially spherical shape, a portion corresponding to 30 to 50% of the curvature surface of the tag particle is formed of a material that binds to a capture substance, and the remainder of the tag particle (50 to 70). %) Is formed of a material that does not bind to the trapping substance. Thereby, tag particles having binding anisotropy with respect to the measurement target substance can be produced.

  When tag particles having such binding anisotropy are used, when a plurality of tag particles are bonded to and coated on a measurement target substance via a capture substance, the surface excluding the surface where the tag particles bind to the measurement target substance (the entire surface) The surface showing 50% to 70% of the above (captured substance) is not bound. For this reason, the tag particle binds exclusively to one measurement target substance via a capture substance and does not have a surface that binds to another measurement target substance via a capture substance. As a result, it can be avoided that one tag particle is bonded to two or more substances to be measured to form a dumbbell structure or the like. In other words, it is possible to obtain independent composite particles having a structure in which a plurality of tag particles are bonded and coated on the surface of each measurement target substance. Therefore, it becomes possible to detect independent composite particles with the above-described sample measuring apparatus.

  As described above, according to the first embodiment, a specimen containing a measurement target substance is bound to a plurality of tag particles and a reagent containing a capture substance that specifically binds to the measurement target substance. , Introduced into the first chamber. The tag particle size (for example, diameter) used is smaller than that of the substance to be measured. In addition, an energizable liquid is introduced into the second chamber. In this state, by applying a voltage between the first electrode immersed in the mixed solution in the first chamber and the second electrode immersed in the energizable liquid in the second chamber, The electric current is supplied to the through hole of the partition wall. A specific particle passes through the fine through-hole 7 and observes a pulse-like change in the current value measured by the measurement circuit. This makes it possible to detect the presence of substances to be measured such as viruses and bacteria in the sample simply, in a short time, and with high sensitivity.

  That is, if each tag particle is made larger than the dimension (for example, diameter) of the substance to be measured, the following problem occurs. a) Since the tag particle is large, the probability of contact with the measurement target substance may decrease, and the required reaction time between the measurement target substance and the tag particle may increase. b) The binding state between the measurement target substance and the tag particle is that the two measurement target substances are combined with the capture substance-tag particle-capture substance, and therefore have a dumbbell structure of the measurement target substance-tag particle-measurement target substance. Complex particles are formed, and the detection peak becomes a double peak. As a result, the current waveform needs to be identified for detection. c) In order to identify the current waveform, it is necessary for the composite particles having a dumbbell structure to pass through the through holes in their coupling direction, and the passing posture is restricted. d) Since the tag particles are large, when the tag particles are bound to the measurement target substance via the capture substance, steric hindrance may occur and the binding efficiency may be reduced. e) When the concentration of the substance to be measured in the sample is high, it does not have an ideal dumbbell structure, but has a bunched shape, and when the substance to be measured passes through the through-hole, it becomes difficult to detect the current waveform itself. .

  By using tag particles having a size (for example, a diameter) smaller than the measurement target substance as in the first embodiment, the following benefits can be achieved.

  1) A tag particle having a size (for example, a diameter) smaller than that of the measurement target substance has a more active Brownian motion in the solution than a tag particle having a size larger than that of the measurement target substance. As a result, the collision probability and contact probability of the tag particles with respect to the measurement target substance increase, and the coupling efficiency between them can be increased.

  2) Since the steric hindrance is reduced when the tag particle and the substance to be measured are bound via the capture substance, the binding efficiency between them can be increased.

  3) In detection, it can be judged not by current waveform but by simple signal intensity.

  4) Since tag particles having dimensions smaller than the measurement target substance are bonded to the measurement target substance surface, the steric symmetry by the combined tag particles is maintained, so that the posture passing through the fine through hole is not restricted. . Therefore, since the substance to be measured in the specimen can be discriminated and detected only by simple current signal intensity comparison, the detection efficiency can be improved.

  Therefore, it is possible to provide a specimen detection method that can detect the presence of a measurement target substance such as a virus or bacteria in a specimen simply, in a short time, and with high sensitivity.

(Second Embodiment)
In the second embodiment, an example in which there are two types of measurement target substances (for example, biomolecules) that may exist in the specimen will be described. Note that the specimen detection apparatus shown in FIG. 1 is used for specimen detection.

  As shown in FIG. 5 (a), the reagent binds the first capture substance 42 (for example, the first antibody) that specifically binds to the surface of the first measurement target substance and the capture substance 42, 1st tag particle | grains 43 which have a dimension (for example, diameter) small compared with a 1st measuring object substance. As shown in FIG. 5B, the reagent binds to the second capture substance (for example, the second antibody) 52 that specifically binds to the surface of the second measurement target substance and the capture substance 52. Second tag particles 53. The first and second tag particles 43 and 53 have different dimensions (for example, diameter). For example, the first tag particle 43 has a larger diameter than the second tag particle 53. By mixing this reagent with the specimen, the plurality of first tag particles 43 are bound to the surface of the first measurement target substance 41 via the first capture substance 42 as shown in FIG. 5C. And coat the surface. Further, as shown in FIG. 5D, the plurality of second tag particles 53 are bonded to the surface of the second measurement target substance 51 via the second capture substance 52 to cover the surface.

  Next, a mixed solution of the specimen and the reagent is introduced into the first chamber 3. The second chamber 4 is also filled with a liquid that can be energized. After introducing the mixed solution of the specimen and the reagent into the first chamber 3 in this way, a voltage is applied between the first electrode 8 and the second electrode 9, and a current is passed through the minute through hole 7 of the partition wall 2. Shed. The first composite particles in which the first tag particles in the first chamber 3 are bonded to and coated on the surface of the first measurement target substance 41 via the first capturing substance pass through the fine through-hole 7. The liquid moves to the energizable liquid in the second chamber 4. Further, the second composite particles in which the second tag particles in the first chamber 3 are bound to and coated on the surface of the second substance to be measured via the second capturing substance pass through the fine through-hole 7. Then, the liquid moves to the energizable liquid in the second chamber 4. When these composite particles pass through the fine through-holes 7 respectively, the current value measured by the measurement circuit 10 changes in a pulse manner.

  The detection signal of the current value changes according to the size of the passing particle as shown in FIG. Specifically, as shown in FIG. 6, the detection signal A appears in the case of the first measurement target substance 41 alone. The detection signal B appears when the second measurement target substance 51 is used alone. When the dimensions of the first measurement target substance 41 and the second measurement target substance 51 are the same, the amount of change in the current value is the same as in the detection signals A and B. Accordingly, the first and second measurement target substances 41 and 51 cannot be distinguished from each other. The first tag particles 43 to which the first capture substance 42 is bound and the second tag particles 53 to which the second capture substance 52 is bound appear as detection signals C and D shown in FIG. The amount of change in current value is very slight.

  A plurality of first tag particles 43 having a size (for example, a diameter) smaller than that of the first measurement target substance 41 are bonded to the surface via the first capture substance 42, and the surface is formed by the plurality of first tag particles 43. 6 is detected, that is, the first composite particle described above, the detection signal E shown in FIG. 6 appears. On the other hand, a plurality of second tag particles 53 having a size (for example, a diameter) smaller than that of the second measurement target substance 51 are bonded to the surface via the second trapping substance 52, and the plurality of second tag particles 53. The detection signal F shown in FIG. 6 appears in the case of the second substance 51 to be measured whose surface is covered with, that is, the second composite particles described above. At this time, the dimension (for example, diameter) of the first tag particle 43 is larger than that of the second tag particle 53. Therefore, the amount of change in the current value when the plurality of first tag particles 43 are bonded to the surface of the first measurement target substance 41 and the first composite particles covered are passed through the through holes 7 (see FIG. 6). The detection signal E) is the same amount of change in the current value in the second composite particle in which the plurality of second tag particles 53 are bonded to and coated on the surface of the second measurement target substance 51 (the detection signal shown in FIG. 6). Larger than F).

  In the second embodiment, the size of the first tag particle 43 is different from that of the second tag particle 53. Thereby, the passage of the binding particles in which the first measurement target substance 41 is bound by the first tag particles 43 is caused by the passage of the binding particles in which the second measurement target substance 51 is bound by the second tag particles 53. Can be identified.

  Therefore, the dimension of the single first measurement target substance 41 is essentially the same as that of the single second measurement target substance 51, and their current signals are mutually essential from the detection signal measured by the measurement circuit 10. The current signal in a state where the first measurement target substance 41 is bound to the first tag particle 43 is used as the current signal in the state where the second measurement target substance 51 is bound to the second tag particle 53. It becomes possible to discriminate from the current signal. As a result, it is detected whether the measurement target substance present in the sample is the first measurement target substance 41, the second measurement target substance 51, or both, or neither of them exists. It becomes possible.

  Further, the amount of change in the current value measured by the measurement circuit 10 is monitored over a certain period of time, and the number of detection signals E shown in FIG. 6 is counted to thereby determine the concentration of the first measurement target substance 41 in the sample. Can also be measured. Similarly, by counting the number of detection signals F shown in FIG. 6, it is possible to measure the concentration of the second measurement target substance 51 in the sample.

(Third embodiment)
A sample detection apparatus according to the third embodiment will be described in detail with reference to FIGS.

  FIG. 7 is a plan view of the specimen detection apparatus according to the third embodiment. 8 is a cross-sectional view taken along line VIII-VIII in FIG. For example, the rectangular main body 61 includes a rectangular block 62. The upper groove 64 and the lower groove 65 are provided in the rectangular block 62 with the central wall 63 as a boundary. The upper lid 66 is provided on the upper surface of the block 62 so as to cover the upper groove 64. The lower lid 67 is provided on the lower surface of the block 62 so as to cover the lower groove 65.

  The hollow first chamber 68 is formed in a space defined by the upper groove 64 and the upper lid 66 of the block 62. The hollow second chamber 69 is formed in a space defined by the lower groove 65 and the lower lid 67 of the block 62.

  The first chamber 68 includes an injection port 70 located on the left end side, an outflow port 71 located on the right end side, and a flow path 72 having both ends communicating with the injection port 70 and the outflow port 71. The flow path 72 has a width narrower than the width of each port 70 and 71. The sample inlet 73 is provided in the upper lid 66 on the injection port 70. The sample outlet 74 is provided in the upper lid 66 on the outflow port 71. The partition wall 75 having the same planar shape as the first chamber 68 is provided on the central wall 63 at the bottom of the first chamber 68. The fine through hole 76 is provided in the partition 75 portion below the flow path 72 near the outflow port 71 side. A hole 77 having a diameter sufficiently larger than that of the through hole 76 is opened in the central wall 63 facing the through hole 76. Since the through hole 76 and the hole 77 are respectively provided in the partition wall 75 and the central wall 63, the flow path 72 of the first chamber 68 is communicated with the second chamber 69 through the through hole 76 and the hole 77.

  The first filter 78 is provided in the flow path 72 of the first chamber 68 on the downstream side of the flow of the specimen from the specimen inlet 73 to the outlet 74 with respect to the through hole 76. The flow of the specimen is indicated by an arrow 100 in FIG. The first filter 78 is preferably provided in the flow path 72 close to the through hole 76 at a distance of 0 to 1 mm. The first filter 78 includes, for example, a plurality of nanopillars. Each nanopillar is provided along the length direction of the flow path 72, in other words, the specimen flow direction indicated by the arrow 100. The dry reagent includes a plurality of tag particles and a capture substance that is bonded to the surface of each tag particle and specifically binds to the surface of the measurement target substance, and is held in advance between the plurality of nanopillars. Each tag particle has a size (for example, a diameter) smaller than that of the measurement target substance as described in the first embodiment.

  The second filter 79 is disposed in the flow path 72 between the injection port 70 and the through hole 79. The second filter 79 serves to adsorb contaminants in the sample when the sample flows through the flow path 72 in the direction indicated by the arrow 100 in FIG.

  An energized liquid inlet 80 is provided in the lower lid 67 located on the left end side of the second chamber 69. The liquid discharge port 81 that can be energized is provided in the lower lid 67 located on the right end side of the second chamber 69.

  The first electrode 82 is provided on the upper lid 66 located on the first chamber 68 side. At least a portion of the first electrode 82 is located in the flow path 72 adjacent to the side surface of the first filter 72 downstream of the analyte flow indicated by the arrow 100. The second electrode 83 is provided on the lower lid 67 located on the second chamber 68 side. At least a part of the second electrode 83 is located in the second chamber 68, for example, in the second chamber 68 immediately below the through hole 76. The measurement circuit 84 and the DC power source 85 are connected to the second electrode 83 in this order.

  The third electrode 86 is inserted into the injection port 70 of the first chamber 68 through the inlet 73 of the upper lid 66. The fourth electrode 87 is inserted into the outflow port 71 of the first chamber 68 through the outlet 74 of the upper lid 66.

  The first, third and fourth electrodes 82, 86 and 87 are connected to a DC power source (not shown) through leads 88, 89 and 90, respectively, and via an on-off switch (not shown). ing. The connection of the first, third, and fourth electrodes 82, 86, and 87 to the DC power source is switched in the following process. In this step, the measurement target substance in the specimen is specifically bound to the plurality of tag particles in the first filter 78 via the capture substance, and the measurement target substance is trapped; trapped by the first filter 78 Desorbing a substance to be measured specifically bound to a plurality of tag particles via a capture substance; and a plurality of tags in the first chamber 68 discharged from the first filter 78 to the flow path 72 A step of moving the measurement target substance specifically bound to the particle through the trapping substance through the through-hole 76 to a liquid that can be energized in the second chamber 69.

  The detaching part is constituted by a first electrode 82, a third electrode 86, and a DC power source (not shown) for applying a DC voltage. The DC power source is configured to apply a DC voltage between the first electrode 82 and the third electrode 86 while heating the first filter 78. The desorption unit desorbs the measurement target substance from the first filter 78 to the flow path 72 on the upstream side of the specimen flow indicated by the arrow 100. The substance to be measured is combined with a plurality of tag particles and a trapping substance, and is trapped by the first filter 78 as described later.

  Next, a specimen detection method will be described using the specimen detection apparatus shown in FIGS. 7 and 8 according to the third embodiment. In the third embodiment, a case where there is one kind of measurement target substance (for example, biomolecule) that may exist in the specimen is taken as an example.

  First, a liquid that can be energized is introduced into the second chamber 69 through the inlet 80, and the liquid is filled into the second chamber 69.

  The analyte is then injected through the inlet 70 into the injection port 70 of the first chamber 68. The specimen in the injection port 70 flows in the direction indicated by the arrow 100 in FIG. The specimen passes through the second filter 79 interposed in the flow path 72, where impurities are removed. The specimen further passes through the first filter 78 disposed in the flow path 72 and flows out to the outflow port 71.

  When the specimen flows through the flow path 72, a DC voltage is applied between the first and second electrodes 82 and 83, and current flows through the through hole 76 of the partition wall 75 and the hole 76 of the central wall 63. For example, the substance to be measured in the specimen passes through the through hole 76 and the hole 77 below it by an electric field, and moves to the energizable liquid in the second chamber 69. At this time, the current value measured by the measurement circuit 84 changes in a pulse manner.

  The detection signal of the current value changes according to the size and shape of the particles passing through the through hole 76 as shown in FIG. Specifically, as shown in FIG. 4, the detection signal A appears when the measurement target substance 41 is alone. The detection signal B appears when the particle 41 ′ is separate from the measurement target substance. When the measurement target substance 41 has the same size as the particle 41 ′ separate from the measurement target substance, the amount of change in the current value is the same as that of the particle 41 separate from the measurement target substance as shown in the detection signals A and B. is there. Therefore, the measurement target substance 41 cannot be distinguished from the separate particles 41 '. The tag particle 43 combined with the capture substance 42 appears as a detection signal C shown in FIG. The amount of change in the current value of the detection signal C is very small.

  The substance to be measured in the sample can be identified by the following operation.

  First, a liquid that can be energized is introduced into the second chamber 69 through the inlet 80, and the liquid is filled into the second chamber 69.

  The analyte is then injected through the inlet 70 into the injection port 70 of the first chamber 68. At the same time as or immediately after the injection, the third electrode 86 and the fourth electrode 87 are connected to a DC power source (not shown) through leads 89 and 90. The third and fourth electrodes 86 and 87 are connected to a DC power source so that the third electrode 86 is a negative electrode and the fourth electrode 87 is a positive electrode. Thereafter, a DC voltage is applied between the third and fourth electrodes 86 and 87. At this time, since the substance to be measured in the specimen is generally charged with a negative charge, electrophoresis by applying a DC voltage between the third and fourth electrodes 86 and 87 is performed from the injection port 70 to the flow path. The movement of the measurement target substance in the direction indicated by the arrow 100 toward 72 is promoted. The specimen in the injection port 70 flows through the flow path 72 by capillary action and passes through the second filter 79 arranged in the flow path 72. Thereby, impurities are removed. The specimen further passes from the flow path 72 through the first filter 78 disposed in the flow path 72 and flows out to the outflow port 71. While the specimen passes through the first filter 78 at an appropriate speed, the first filter 78 has a plurality of tag particles and a capture substance that is bound to the surface of each tag particle and specifically binds to the measurement target substance. A dry reagent containing is retained. For this reason, the tag particles are bonded to the surface of the measurement target substance in the specimen via the capture substance, and the plurality of coated composite particles are trapped by the first filter 78. At this time, only the measurement target substance is trapped. Particles separate from the substance to be measured pass through the first filter 78, move to the outflow port 71, and flow out of the outlet 74 and are removed.

  After trapping a plurality of composite particles, the first electrode 82 and the third electrode 86 become a DC power source (not shown) through leads 88 and 89, the first electrode 82 becomes a negative electrode, and the third electrode 86 becomes a positive electrode. Connect as shown. Thereafter, a DC voltage is applied between the first and third electrodes 82 and 86. At this time, the plurality of composite particles trapped in the first filter 78 are generally negatively charged. The first filter 78 is disposed between the first and third electrodes 82 and 86. Therefore, in the first filter 78, the flow in the direction opposite to the flow of the specimen indicated by the arrow 100 in FIG. 7 is electrophoresed (by applying a DC voltage between the first and third electrodes 82 and 86). Caused by). In other words, in the first filter 78, the flow toward the upstream side of the flow of the analyte indicated by the arrow 100 is generated by the electrophoresis. When heat is applied to the first filter 78 by this reverse flow, the plurality of composite particles trapped in the first filter 78 escape toward the upstream side of the specimen flow indicated by the arrow 100. Released. As a result, the composite particles are collected in the flow path 72 adjacent to the through hole 76 and are highly concentrated.

  In the third embodiment, during the separation of the plurality of composite particles trapped in the first filter 78 by electrophoresis by applying a DC voltage between the first and third electrodes 82 and 86. As described above, the first filter 78 may be heated to promote desorption of the composite particles.

  After the plurality of composite particles trapped in the first filter 78 are desorbed and released toward the upstream side of the specimen flow, the lead 88 of the first electrode 82 is connected to ground from a DC power source (not shown). Switch as follows. In this state, a DC voltage is applied from the DC DC power supply 85 between the first and second electrodes 82 and 83 to supply a current to the through hole 76 of the partition wall 75. By applying a DC voltage, the plurality of composite particles have a through-hole 76 and a hole 77 below the first filter 78 that are close to the first filter 78 at a predetermined distance on the upstream side in the flow direction of the specimen. Pass through and move to the energizable liquid in the second chamber 69. At this time, the current value measured by the measurement circuit 84 changes in a pulse manner.

  In this current detection, the detection signal D shown in FIG. 4 described above appears mainly in the case of a composite particle in which a plurality of tag particles are bonded to the surface of the measurement target substance via the capture substance and coated. The amount of change in the current value of the detection signal D is larger than that in the case of the measurement target substance 41 alone (detection signal A shown in FIG. 4).

  Therefore, even if the measurement target substance and the measurement target substance have the same size and separate particles in the specimen, a plurality of tag particles 43 are bound to the surface of the measurement target substance 41 via the capture substance 42. The coated composite particles have a larger particle size than the measurement target substance 41. Therefore, based on the detection signal from the measurement circuit 84, it is possible to determine only the measurement target substance 41 and detect the presence of the measurement target substance 41 in the sample.

  In the sample detection apparatus according to the third embodiment, the first chamber 68 includes an injection port 70, an outflow port 71, and a narrow-width channel 72 that connects these ports 70 and 71. The flow path 72 has a small volume. Therefore, the presence of the measurement target substance in a small amount of specimen can be detected with high frequency.

  Further, the first filter 78 disposed in the flow path 72 holds the reagent in advance. The positional relationship among the first filter 78, the through hole 76 of the partition wall 75, and the first electrode 82 is specified. With these configurations, it is possible to form composite particles in which a plurality of tag particles are efficiently bound to and coated on the surface of the measurement target substance in the specimen via the capture substance. Further, the composite particles can be collected in the flow path 72 located upstream of the specimen flow with respect to the first filter 78. As a result, even when the measurement target substance in the sample is present at a low concentration, the presence of the measurement target substance in the sample can be detected with high frequency.

  In the third embodiment, the first filter may be configured using nanowires instead of a plurality of nanopillars.

  The detachment part is not limited to a case where the first electrode, the third electrode, and a DC power source that applies a DC voltage between these electrodes are used. That is, any structure may be used as long as a plurality of tag particles trapped in the first filter 78 can desorb a measurement target substance (composite particle) specifically bound to the surface via a capture substance. For example, the desorption unit may be configured to apply pressure to the first filter through the outlet, the outflow port, and the flow path to desorb the composite particles from the first filter.

  Hereinafter, an embodiment will be described using the specimen detection apparatus of FIG. 1 described above.

(Comparative Example 1)
As model particles, polystyrene microparticles with a diameter of 1 μm modified with biotin simulating the virus to be measured, and polystyrene microparticles with a diameter of 0.22 μm surface modified with streptavidin (capturing substance) simulating tag particles. Prepared. The sample detection apparatus used for measurement includes a measurement cassette 1 made of polycarbonate. The measurement cassette 1 is partitioned up and down by a quartz partition wall 2 in which a through-hole 7 is opened. The first chamber 3 is placed in the measurement cassette 1 on the upper side, and the first measurement cassette 1 is placed in the measurement cassette 1 on the lower side of the partition wall 2. Two chambers 4 were formed respectively. The through hole 7 had an opening diameter of 2 μm and a length (corresponding to the thickness of the partition wall 2) of 10 μm. The first electrode 8 made of Ag / AgCl was provided on the upper wall portion of the measurement cassette 1, and a part of the first electrode 8 was positioned in the first chamber 3. The second electrode 9 made of Ag / AgCl was provided on the lower wall portion of the measurement cassette 1, and a part thereof was positioned in the second chamber 4.

  First, the second chamber 4 of the specimen detection apparatus was filled with a PBS buffer solution not containing model particles. A PBS buffer solution in which polystyrene fine particles having a diameter of 1 μm and surface-modified with biotin simulating a virus to be measured was suspended was filled in the first chamber 3. Subsequently, a bias voltage was applied between the first and second electrodes 8 and 9 of Ag / AgCl soaked in the PBS buffer solution in the first and second chambers 3 and 4, and the current was monitored.

  In the monitor, a base current of 38 nA was measured when a bias of 100 mV was applied. A relationship of time response-current change in which a spike-like current drop signal accompanying the passage of particles through the through-hole 7 appears was obtained.

  Based on the relationship between time response and current change, the frequency distribution was obtained using the amount of current decrease from the base current value as the signal intensity. The result is shown in FIG.

Example 1
The number ratio of polystyrene microparticles with a diameter of 1 μm modified with biotin that mimics the virus to be measured and polystyrene microparticles with a diameter of 0.22 μm surface-modified with streptavidin (capturing substance) that mimics tag particles is 1 : It mixed so that it might be set to 70. That is, an excess of 0.22 μm diameter polystyrene particles simulating tag particles was added to and mixed with 1 μm diameter polystyrene particles simulating the virus to be measured. After mixing, the sample is allowed to stand for 1 hour. After filling the second chamber 4 of the specimen detection apparatus having the same configuration as that of Comparative Example 1 with a PBS buffer solution not containing model particles, the mixture is suspended in the first chamber 3. Filled with turbid PBS buffer solution. A bias voltage was applied between the first and second electrodes 8 and 9 of Ag / AgCl immersed in the PBS buffer solution in the first and second chambers 3 and 4, and the current was monitored.

  In the monitor, a base current of 38 nA was measured when a bias of 100 mV was applied. A relationship of time response-current change in which a spike-like current drop signal accompanying the passage of particles through the through-hole 7 appears was obtained.

  Based on the relationship between time response and current change, the frequency distribution was obtained using the amount of current decrease from the base current value as the signal intensity. The result is shown in FIG.

  In Comparative Example 1 in which only polystyrene microparticles having a diameter of 1 μm that are surface-modified with biotin simulating a virus are measured, the signal intensity peak is 270 pA as shown in FIG. On the other hand, mixed particles of polystyrene fine particles having a diameter of 1 μm modified with biotin simulating virus and polystyrene fine particles having a diameter of 0.22 μm modified with streptavidin (capturing substance) simulating tag particles are measured. In Example 1, it can be seen that the signal intensity peak is 360 pA as shown in FIG. This is because the surface-modified polystyrene microparticles with a diameter of 1 μm modified with biotin bind to and coat with a plurality of polystyrene microparticles with a diameter of 0.22 μm surface-modified with streptavidin. Since the size (diameter) is increased as compared with the polystyrene fine particle alone, the signal intensity is increased.

  DESCRIPTION OF SYMBOLS 1 ... Measurement container, 2, 75 ... Partition, 3, 68 ... 1st chamber, 4, 69 ... 2nd chamber, 5 ... Hole, 6 ... Support plate, 7, 76 ... Through-hole, 8, 82 ... 1st DESCRIPTION OF SYMBOLS 1 electrode, 9, 83 ... 2nd electrode, 10, 84 ... Measuring circuit, 11, 85 ... DC power supply, 31 ... Mixed solution, 32 ... Liquid which can be supplied with electricity, 41, 51 ... Measuring substance (biomolecule) 42, 52 ... Capture substance (antibody), 43, 53 ... Tag particles, 61 ... Device main body, 62 ... Rectangular block, 63 ... Central wall, 64 ... Upper groove, 65 ... Lower groove, 66 ... Upper lid, 67 ... Lower lid, 70 ... injection port, 71 ... outlet port, 72 ... flow path, 73 ... inlet, 74 ... outlet, 77 ... hole, 78 ... first filter, 79 ... second filter, 80 ... inlet, 81 ... Ejection port, 86 ... third electrode, 87 ... fourth electrode, 88, 89, 90 ... lead.

Claims (13)

A measurement cassette, a first chamber and a second chamber formed by partitioning the measurement cassette with a partition, and a through hole provided in the partition and connecting the first chamber and the second chamber to each other A hole, a first electrode provided in the measurement cassette located on the first chamber side, at least a part of which is located in the first chamber, and the measurement cassette located on the second chamber side And a second electrode provided at least in part in the second chamber;
Introducing a sample and a reagent containing a measurement target substance into the first chamber, the reagent is bound to a plurality of tag particles having a size smaller than that of the measurement target substance in the sample, and a surface of each tag particle. A capture substance that specifically binds to the surface of the substance to be measured;
Introducing an energizable liquid into the second chamber;
Applying a voltage between the first electrode and the second electrode;
Passing the substance to be measured in the first chamber through the through-hole, the surface of the substance to be measured being bound and coated with the plurality of tag particles via the capture substance; and 1. observing a change in current flowing between the second electrodes and detecting the presence of the substance to be measured in the specimen;
A specimen detection method comprising:   The specimen detection method according to claim 1, wherein the tag particles are mixed in excess in the specimen as compared with the substance to be measured therein. The specimen detection method according to claim 2, wherein the number of the tag particles is 10 to 10 ⁶ times the number of the measurement target substances.   The specimen detection method according to claim 1, wherein the specimen and the reagent are introduced into the first chamber together with a liquid that can be energized.   The plurality of tag particles are bound to and coated on the surface of the measurement target substance via the capture substance, and the measurement target substance coated with the plurality of tag particles is the measurement without coating the plurality of tag particles. The specimen detection method according to claim 1, which is similar to the shape of the target substance.   The specimen detection method according to claim 1, wherein a dimension of each tag particle is 10 to 70% of a dimension of the measurement target substance.   The specimen detection method according to claim 1, wherein the plurality of tag particles constituting the reagent include two types of tag particles having different dimensions.   The specimen detection method according to claim 1, wherein each of the tag particles has the capture substance locally bound to the surface of the tag particle.   The analyte detection method according to claim 1, wherein the tag particles are polystyrene particles or nano-sized gold particles. Body;
First and second chambers formed by partitioning the inside of the main body with partition walls, wherein the first chamber has a flow path;
A through hole provided in the partition wall for connecting the flow path of the first chamber and the second chamber to each other;
Sample inlet provided in the main body portion located at one end of the flow path:
An outlet provided in the body portion located at the other end of the flow path;
A first filter provided in the flow path, the first filter being disposed downstream of the flow of the sample from the sample inlet to the outlet with respect to the through-hole, and in the sample A reagent including a plurality of tag particles having a size smaller than that of the measurement target substance and a capture substance that is bound to the surface of each tag particle and specifically binds to the surface of the measurement target substance is held in advance:
A first electrode provided on the main body portion located on the first chamber side and at least a part of which is located in the first chamber;
A second electrode provided in the main body portion located on the second chamber side, at least a part of which is located in the second chamber; and the measurement target substance in the specimen is contained in the first filter. A desorption section for desorbing the measurement target substance from the first filter to the flow channel upstream of the specimen flow when the plurality of tag particles and the capture substance are combined;
A specimen detection apparatus comprising:   The sample detection apparatus according to claim 10, wherein the first filter includes a plurality of nanopillars, and each nanopillar is arranged along a flow direction of the sample.   The specimen detection apparatus according to claim 10, wherein the first filter includes a nanowire.   The second filter according to claim 10, further comprising a second filter disposed in the flow path upstream of the flow of the specimen with respect to the first filter and configured to remove impurities in the specimen. Sample detection device.

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