JP2020046284A - Biosensor, target particle detection method, and separation method - Google Patents

Abstract

To provide a method for detecting or separating a target particle specifically with high sensitivity and a biosensor used therefor.SOLUTION: Provided is a biosensor for detecting a target particle in a sample. The biosensor includes a sensor array provided with a plurality of sensor elements. The plurality of sensor elements include at least two electrodes 9, 10 buried in a first insulation layer 7 by being spaced apart from each other, a channel 12a connected to both electrodes, a second insulation layer 13 provided in the first insulation layer and on the channel and having an opening 15 for causing a channel corresponding to an interval to be exposed, and a second electrode 3 provided upward of the opening.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a biosensor, a target particle detection method, and a separation method.

  Exosomes are a kind of extracellular vesicles and are minute vesicles having a particle size of about 100 nm contained in the living body. It is known that extracellular vesicles have a lipid bilayer structure containing nucleic acid and the like inside, and have the function of moving between distant organs and transporting substances. It has been reported that lipid bilayer membranes contain proteins or glycoproteins, and that the results suggest that cfDNA is attached to the periphery. It is thought that they include information on cells in which extracellular vesicles have been produced, information on cells to which metastasis has occurred, and the like.

  Extracellular vesicles have been reported to be deeply involved in cancer metastasis. Alternatively, it has been reported that Alzheimer's disease, Parkinson's disease, dementia such as Lewy body dementia, neurodegenerative diseases, and diseases other than cancer such as renal impairment are involved in transport of causative substances. Therefore, extracellular vesicles are attracting attention as biomarkers for use in early detection of disease, monitoring of recurrence, and the like. In addition, there are many unclear points about the structure and function thereof, so that it is attracting attention as a research target, and development of a method for detecting and separating extracellular vesicles is desired.

  An object of the present invention is to provide a method for detecting or separating target particles with high specificity and high sensitivity, and a biosensor used for the method.

  The biosensor according to the embodiment is a sensor for detecting a target particle in a sample. The biosensor includes a sensor array in which a plurality of sensor elements are arranged on a first substrate. Each of the plurality of sensor elements includes a first insulating layer provided on the first substrate, at least two electrodes embedded in the first insulating layer with a gap therebetween, and at least two electrodes and And a channel connected to at least two electrodes, a second insulating layer provided on the first insulating layer and the channel, and having an opening exposing a channel corresponding to the gap; And a second electrode provided above. When the biosensor is viewed in a plan view, the area of the second electrode is larger than the area of the channel.

FIG. 1 is a cross-sectional view illustrating an example of the biosensor according to the embodiment. FIG. 2 is a plan view illustrating an example of the biosensor according to the embodiment. FIG. 3 is a flowchart illustrating the target particle detection method according to the embodiment. FIG. 4 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 5 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 6 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 7 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 8 is a graph showing a current value between the source and the drain. FIG. 9 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 10 is a cross-sectional view illustrating an example of the biosensor according to the embodiment. FIG. 11 is a plan view illustrating an example of the biosensor according to the embodiment. FIG. 12 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 13 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 14 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 15 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 16 is a flowchart illustrating the target particle detection method according to the embodiment. FIG. 17 is a cross-sectional view illustrating an example of the biosensor according to the embodiment. FIG. 18 is a flowchart illustrating a target particle detection method and a separation method according to the embodiment. FIG. 19 is a flowchart illustrating a target particle detection method and a separation method according to the embodiment. FIG. 20 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 21 is a diagram illustrating a state when the biosensor of the embodiment is used. FIG. 22 is a diagram illustrating a state when the biosensor of the embodiment is used.

  Hereinafter, various embodiments will be described with reference to the drawings. Each drawing is a schematic diagram for promoting the understanding of the embodiment and its understanding, and there are places where the shape, dimensions, ratio, etc. are different from the actual ones, but these may be changed as appropriate in consideration of the following description and known techniques. can do.

  The biosensor according to the embodiment is a sensor for detecting a target particle in a sample. The biosensor includes a sensor array in which a plurality of sensor elements are arranged on a first substrate. Each of the plurality of sensor elements includes a first insulating layer provided on the first substrate, at least two electrodes embedded in the first insulating layer with a gap therebetween, and at least two electrodes and A second insulating layer provided on the first insulating layer and the channel, the second insulating layer having an opening for exposing the channel corresponding to the gap; A second electrode provided above. When the biosensor is viewed in a plan view, the area of the second electrode is larger than the area of the channel. According to a further embodiment, there is provided a biosensor further comprising a probe in a channel exposed from the opening. According to a further embodiment, a method for detecting target particles and a method for separating target particles using a biosensor are provided.

  Hereinafter, a biosensor, a target particle detection method, and a target particle separation method of the embodiment will be described.

(First embodiment)
-Biosensor Fig. 1 is a sectional view showing an example of the biosensor of the first embodiment. The biosensor 1 includes a sensor array 2 and an upper electrode 3.

  The sensor array 2 includes a plurality of sensor elements 5 formed on a first substrate 6. The plurality of sensor elements 5 are arranged in an array, for example, in a matrix direction when viewed in a plan view.

  Each of the plurality of sensor elements 5 includes a drain electrode 9, a first source electrode 10, and a second source electrode 11 embedded in a first insulating layer 7 provided on a first substrate 6. For example, the drain electrode 9, the first source electrode 10, and the second source electrode 11 are embedded so as to be flush with the surface of the first insulating layer 7. The drain electrode 9, the first source electrode 10, and the second source electrode 11 are arranged in a straight line in this order. The drain electrode 9 and the first source electrode 10 are arranged with a first gap 8a. The first source electrode 10 and the second source electrode 11 are arranged with a second gap 8b.

  A channel 12 is provided on the drain electrode 9, the first gap 8 a, the first source electrode 10, the second gap 8 b, and the second source electrode 11. The channel 12 is connected to the drain electrode 9, the first source electrode 10, and the second source electrode 11.

  A second insulating layer 13 is provided on a region of the first insulating layer 7 which is not covered with the channel 12 and on the channel 12. The second insulating layer 13 has an opening 15 exposing a region of the channel 12 (channel 12a) corresponding to the first gap 8a. The shielding electrode 14 is provided on the second insulating layer 13 except for the opening 15.

  The upper electrode 3 is provided above the sensor array 2 with a space 4 therebetween. The upper electrode 3 is arranged on one surface of the second substrate 16 so as to face the shield electrode 14.

  For example, the first substrate 6 and the second substrate 16 are connected by a wall (not shown) located on the outer periphery thereof, thereby supporting the upper electrode 3. In addition, the sensor array 2, the upper electrode 3, the second substrate 16, and the liquid-tight space 4 surrounded by the wall are formed by the walls. The sample to be analyzed by the biosensor 1 is accommodated in the space 4. For example, the second substrate 16 has an inlet (not shown) for injecting the sample and an outlet (not shown) for discharging the sample. Alternatively, a column connecting the first substrate 6 and the second substrate 16 can be formed as necessary to reinforce a portion other than the outer peripheral portion.

  In the biosensor 1, the switch circuit 50 is connected to the second source electrode 11, and the second source electrode 11 can be connected to either the ground line 20 or the power supply line. The power supply line 54 can be connected to either the alternating current (AC) power supply 18 or the direct current (DC) power supply 56 by the switch circuit 52. Further, the drain electrode 9 is connected to a ground line 20 via a series resistor 21. The switch circuit 50 and the series resistor 21 are respectively formed in the individual sensor elements 5, and the switch circuit 50 can switch individually for each sensor element 5. The series resistor 21 has a sufficiently large resistance value as compared with the sensor element 5 or a variable resistor whose resistance value can be varied according to the resistance of the sensor element 5. Thus, the potential of the channel 12a can be controlled by the potential of the power supply line 54. For example, if the resistance of the sensor element is about 10 to 20 kΩ, the channel 12a can be controlled to a potential of about 90% of the potential of the power supply line 54 by setting the resistance to 100 to 200 kΩ.

  In the biosensor 1, a switch 53 is connected to the shield electrode 14 and can be connected to either the ground line 20 or the DC power supply 57.

  The upper electrode 3 is connected to a switch 53, and can be connected to either the ground line 20 or the DC power supply 57.

  Each sensor element 5 includes an ammeter 19 and a voltmeter 58. The ammeter 19 is connected in series with the channel 12a, for example, between the drain electrode 9 and the series resistor 21. The voltmeter 58 is connected to the drain electrode 9 and the first source electrode 10, and is connected to the channel 12a in parallel.

  Therefore, the resistance value of the channel 12a can be read from the current value flowing through the channel 12a measured by the ammeter 19 and the potential difference of the channel 12a measured by the voltmeter 58.

  FIG. 1 shows a part of the biosensor 1 for convenience and shows two sensor elements 5, but the number of sensor elements 5 is not limited to this. For example, 10 to 1,000,000, preferably 500 to 10,000 sensor elements 5 are arranged in one biosensor 1.

  FIG. 2 shows a plan view of the biosensor 1 according to the first embodiment. Here, a sensor array 2 including four sensor elements 5A, 5B, 5C, and 5D is shown for convenience. Note that the cross-sectional view of FIG. 1 corresponds to a cross section of the biosensor of FIG. 2 cut along B-B ′. The second insulating layer (not shown) and the shield electrode 14 have substantially the same planar shape, and have openings 15A, 15B, 15C, and 15D above the sensor elements 5A, 5B, 5C, and 5D, respectively. Prepare. Here, the area of the shielding electrode 14 is larger than the area of each of the channels 12A, 12B, 12C, 12D exposed from the openings 15A, 15B, 15C, 15D. The shape of the upper electrode 3 is, for example, a flat plate that covers the entire upper surface of the sensor element.

  Hereinafter, each configuration will be described in detail.

  Each of the first substrate 6 and the second substrate 16 is, for example, a rectangular plate. Each of the substrates is provided with, for example, an insulating film on a surface on which the electrodes are arranged. The insulating film is made of, for example, silicon oxide, silicon nitride, aluminum oxide, a polymer material, or the like. The substrate itself is, for example, silicon, glass, ceramics, a polymer material, or a metal. When a gate electrode is formed below the channels 12A, 12B, 12C, and 12D with an insulating film interposed therebetween, the thickness of the insulating film in the same portion is preferably as thin as possible without impairing the insulating property. It is preferable to set it to about several nm. Such a high-quality thin film can be formed by, for example, an ALD (Atomic Layer Deposition) method. The gate voltage can be applied to the channels 12A, 12B, 12C, and 12D from the shield electrode 14 and, if necessary, the upper electrode 3 via a sample solution. In this case, it is not necessary to reduce the thickness of the insulating film below the channel as described above.

  The size of the first substrate 6 is not limited, but may be, for example, 1 to 16 mm × 1 to 16 mm × 0.1 to 1 mm (width × length × thickness). The size of the second substrate 16 is not limited, but may be, for example, 1 to 16 mm × 1 to 16 mm × 0.001 to 1 mm (width × length × thickness).

  When the first substrate 6 is, for example, silicon, a semiconductor circuit can be formed, and the above-described switch circuit, power supply circuit, voltmeter, ammeter, series resistor, and the like can be formed. . Further, input / output terminals for the semiconductor circuit may be formed on the outer peripheral portion of the first substrate 6.

  The second substrate 16 is formed by forming a sensor element, a second insulating film, a shielding electrode, and the like on the first substrate 6, and then forming a sacrificial layer thereon, together with the upper electrode 3. After the film is formed, it can be manufactured by removing the sacrificial layer. When this method is used, the thickness of the sacrificial layer can be made thin and uniform, so that the thickness of the space 4 to which the sample solution is supplied and the distance between the upper electrode 3 and the shield electrode 14 can be made highly accurate. it can.

  Note that the second substrate 16 and the upper electrode 3 do not necessarily have to be flat plates without holes, and for example, holes for removing the sacrificial layer may remain. When this hole is used as a supply port or a discharge port of the sample solution, the hole may be left as it is, or when the supply port and the discharge port of the sample solution are formed separately, and the space 4 is to be a closed channel, Alternatively, the sacrificial layer removing hole can be closed. As a method of closing the sacrificial layer removing hole, various methods such as attaching another flat plate and forming a high-viscosity coating film can be used.

  The first insulating layer 7 is made of, for example, an insulator such as silicon oxide, silicon nitride, aluminum oxide, or a polymer material. The thickness of the first insulating layer 7 can be, for example, 50 nm to 500 nm.

  The drain electrode 9, the first source electrode 10, and the second source electrode 11 are, for example, gold (Au), silver (Ag), copper (Cu), palladium (Pd), platinum (Pt), and nickel (Ni), respectively. ), Metal such as titanium (Ti), chromium (Cr), tungsten (W), ruthenium (Ru), or aluminum (Al), or zinc oxide (ZnO), indium tin oxide (ITO), IGZO, conductive It is made of a conductive material such as a polymer. The size of each electrode can be set to 100 × 100 nm to 5 × 50 μm. When the detection target is an extracellular vesicle, the size is preferably set to 100 × 100 nm to 1 × 1 μm. The thickness of each electrode may be, for example, the same thickness as the first insulating layer 7 or may be slightly thicker than the first insulating layer 7.

  1 and 2, the resistance value of the channel 12a is read by the three-terminal method. However, the resistance value of the channel 12a can be read by the four-terminal method with two drain electrodes. In this case, a second drain electrode is formed in series on the side of the drain electrode 9 opposite to the channel 12a, an ammeter is installed in series between the second drain electrode and the series resistor 21, and By installing a voltmeter in parallel with the first source electrode 10, the resistance of the channel 12a can be measured. In this case, the influence of the parasitic resistance of the drain electrode 9 can be reduced, so that the resistance value of the channel 12a can be read with higher accuracy. Alternatively, for example, a two-terminal method including two of the drain electrode 9 and the first source electrode may be adopted.

Channel 12 is, for example, graphene, a carbon nanotube, or MoS ₂ and the like. The channel 12 has a film-like or wire-like shape. The size of the channel 12 is not limited, but may be, for example, 0.1 to 100 μm × 0.1 to 100 μm (width × length). Practically, 20 to 200 nm x 300 to 5,000 nm is suitable for capturing extracellular vesicles.

  The second insulating layer 13 is formed of, for example, an insulator such as silicon oxide, silicon nitride, aluminum oxide, or a polymer material. The thickness of the second insulating layer 13 can be, for example, 50 nm to 2 μm, and preferably 50 nm to 300 nm when extracellular vesicles are to be detected.

  The opening 15 formed in the second insulating layer 13 is a hole into which target particles to be analyzed by the biosensor 1 enter, which will be described later in detail. The opening 15 is preferably sized to allow only one target particle to enter. For example, when the target particles are extracellular vesicles, the opening 15 is preferably 50 × 50 nm to 1 × 1 μm. Also, the size of the channel 12a exposed from the opening 15 can be 50 × 50 nm to 1 × 1 μm, or the channel width can be reduced to, for example, 30 × 50 nm to 0.5 × 1 μm (channel width × exposed channel). Length).

  The shielding electrode 14 is made of, for example, gold (Au), silver (Ag), copper (Cu), palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), or aluminum (Al). ) Or a conductive substance such as zinc oxide (ZnO), indium tin oxide (ITO), IGZO, or a conductive polymer. The thickness of the shielding electrode 14 can be, for example, 50 nm to 2 μm, and preferably 50 nm to 300 nm when extracellular vesicles are to be detected. The opening 15 has, for example, a taper that extends from the channel 12a side to the shielding electrode 14 side. This makes it easier for the target particles to enter the opening 15.

  The upper electrode 3 is made of, for example, gold (Au), silver (Ag), copper (Cu), palladium (Pd), platinum (Pt), nickel (Ni), titanium (Ti), chromium (Cr), or aluminum (Al). ) Or a conductive substance such as zinc oxide (ZnO), indium tin oxide (ITO), IGZO, or a conductive polymer. The thickness of the upper electrode 3 can be, for example, 50 nm to 5 μm.

  The sensor array 2 and the upper electrode 3 can be formed by, for example, a semiconductor process or a MEMS process.

  The space 4 preferably has a thickness (height) of 1 to 20 μm, for example. For example, the space 4 may contain a liquid such as water, physiological water, an ionic liquid, an organic solvent, or PBS.

  It is said that about 1% of extracellular vesicles in blood are derived from cancer. For example, when the sensor array 2 includes 500 sensor elements, the probability of detecting cancer-derived extracellular vesicles contained at a rate of 1% is 1−0.99 ＾ 500, which is 99% or more. The detection rate can be expected. Furthermore, it is known that cancer cells have diversity within a single tumor, and it is said that extracellular vesicles secreted therefrom are also diverse. When the sensor array 2 includes 10,000 sensor elements, approximately 100 extracellular vesicles derived from cancer contained at a rate of 1% can be captured, so that even if there is diversity, detection can be performed without overlooking. can do. In a further embodiment, the biosensor 1 may not include the upper electrode 3. In that case, the upper part of the space 4 is open, and the sample is directly dropped into the space 4 on the sensor array 2.

-Target particle detection method Hereinafter, a target particle detection method using the biosensor of the first embodiment will be described. This method is a method for detecting target particles in a sample.

  A sample is an analyte that can include target particles therein. The sample is, for example, a biological liquid sample. The sample is, for example, blood, serum, plasma, blood cells, urine, stool, sweat, saliva, sputum, lymph, cerebrospinal fluid, tear, breast milk, amniotic fluid, semen, cell extract or tissue extract, or a mixture thereof. is there. The sample also includes a waste liquid (cleaning liquid) generated when the living body is washed at the time of treatment such as surgery or an examination. Alternatively, the sample may be a sample from the environment, such as soil, river water, seawater, or a mixture thereof.

  Alternatively, the sample may be a preparation of the above material. The preparation may be, for example, one that has been subjected to any known pretreatment such as dilution, concentration, purification, and extraction in order to use any of the above materials as a sample according to the present embodiment. Good.

  The target particle is, for example, an object derived from a living body. For example, the target particle is a microvesicle having a structure surrounded by a lipid bilayer having a diameter of 20 nm to 50 μm and existing in a living body. For example, the target particle is a specific extracellular vesicle, for example, a specific exosome. Alternatively, the target particle may be a fragment or a coagulate of a protein or the like, a cell, a bacterium, a fungus, a virus, or the like.

  Here, the dielectric constant of the target particles is higher than that of a liquid that is a medium of the target particles in the sample (hereinafter, referred to as “sample liquid”). For example, the target particles include a biological membrane and have a negative charge therefrom. When using a sample in which the dielectric constant of the target particles is smaller than that of the sample solution, alcohol may be mixed with the sample to reduce the dielectric constant of the sample solution.

  FIG. 3 is a diagram illustrating a schematic flow of the target particle detection method according to the first embodiment. The target particle detection method includes the following steps. (S1) A sample solution is accommodated in the space of the biosensor. (S2) The upper electrode and the shield electrode are connected to a ground line, each sensor element is connected to an AC power supply, and dielectrophoresis is caused to cause target particles to reach each sensor. Capturing at the opening on the element, (S3) washing the sample solution and discharging excess target particles, (S4) adding a capturing body that specifically binds to the target particles into the space, (S5) measuring a current value flowing through each sensor element and a potential difference between at least two electrodes (source / drain); and (S6) determining the presence or absence or amount of target particles in the sample according to the result of the measurement.

  Hereinafter, the principle that target particles can be detected by performing the above steps will be described with reference to FIGS. FIG. 4 and FIG. 5 are views showing a state when the biosensor 1 is used.

  Prior to the step (S1), it is preferable that a liquid is previously stored in the space 4 of the biosensor 1. The liquid is, for example, water, physiological water, ionic liquid, organic solvent, PBS, or the like.

  In the step (S1), the sample solution is stored in the space 4.

  In the step (S2), as shown in FIG. 4, the switch 53 is connected to the ground side, and the shield electrode 14 is grounded. When the upper electrode 3 is used, the upper electrode 3 is also grounded by the switch 53. On the other hand, the switch 52 is connected to the AC power supply side, and the switch 50 connected to each sensor element 5 is connected to the switch 52. As a result, an AC voltage is applied to each sensor element 5. If the resistance of the series resistor 21 is sufficiently larger than the resistance of the channel 12, a voltage substantially equal to the AC power supply is applied to the entire channel 12. Is done. As a result, an electric field is applied between the channel 12 and the shielding electrode 14, and an electric field is also applied between the channel 12 a of the opening 15 and the upper electrode 3. When the upper electrode 3 is not used, the electric field immediately above the channel 12a of the opening 15 is applied toward infinity. These electric field intensity distributions are not uniform in the space 4. That is, since the area of the channel 12a is smaller than the area of the upper electrode 3 and the shield electrode 14, the electric field intensity generated near the channel 12a becomes stronger, and the electric lines of force indicated by broken lines in the drawing become denser. On the other hand, the intensity of the electric field generated near the upper electrode 3 and the shield electrode 14 is weakened, and the lines of electric force are sparse.

Therefore, if particles having different dielectric constants are present in the sample solution, migration by dielectrophoresis occurs. According to the Clausius-Mossotti equation shown below, if the dielectric constant of the object is higher than the dielectric constant of the medium, the object migrates in the direction in which the electric field intensity is strong.

_Where “ _FDEP ” is the dielectrophoretic force, “ε _p ” is the dielectric constant of the particle, “ε _m ” is the dielectric constant of the medium, “r” is the particle radius, and “E "Is the electric field strength. Therefore, when the target particles 22 are present in the sample, as shown in FIG. 4, an object having a higher dielectric constant than the sample liquid (for example, the target particles 22) is attracted to the channel 12a where the electric field is concentrated and is opened. Enter the part 15. Until the target particles 22 enter the opening 15, for example, it is left for several minutes. Alternatively, after the detection by the following sensor elements, the apparatus is allowed to stand until a desired number of sensor elements indicate the capture of target particles.

  The potential difference between the drain electrode 9 and the first source electrode 10 and the current were measured using the ammeter 19 and the voltmeter 58 of each sensor element 5 to confirm whether the target particles 22 have entered the opening 15. The resistance of the channel 12a (hereinafter, also referred to as “source-drain resistance” or “channel resistance”) may be measured from the value.

  As shown in FIG. 4A, in the sensor element 5a in which the target particles 22 enter the opening 15, the current value changes. Since an AC voltage is applied between the electrodes, the current value swings at a specific cycle. For example, the change in the current value is a shift of a position where the current value is amplitude. For example, after the target particle 22 enters the opening 15, the position of the amplitude may shift to a position having a lower current value (hereinafter, referred to as “lower”) as shown in FIG. Alternatively, depending on the configuration of the biosensor 1 and the type of the sample, the amplitude may shift to a position where the current value is higher (hereinafter, referred to as “upper side”).

  On the other hand, in the sensor element 5b in which the target particles 22 have not entered the opening 15, the current value does not change as shown in FIG. Although the case where the voltage is constant is described here for convenience, the voltage value actually fluctuates slightly because the channel resistance changes. Therefore, by measuring not only the current value but also the potential difference and measuring the change in the channel resistance, the capture of the target particles can be detected more accurately.

  However, in this step, an object (non-target particle 23) other than the target particle 22 having the same shape as the target particle 22 may enter the opening 15. Also in that case, the current value changes.

  Next, the step (S4) and the subsequent steps will be described with reference to FIG. After the sample solution is washed in step (S3) to wash away excess target particles, a capture body is added into the space 4 in step (S4). Since the capturing body 24 is a substance that specifically binds to the target particle 22, it binds to the target particle 22 that has entered the opening 15, for example. In order to bind the capturing body 24 to the target particles 22, it is preferable to leave the capturing body 24 for about one hour, for example. On the other hand, the capturing body 24 does not bind to the non-target particles 23. The step (S4) is performed while maintaining the dielectrophoresis generated in the step (S2) so that the target particles are not released from the openings 15.

  In the step (S5), the resistance between the source and the drain is measured. It is desirable that the measurement is performed continuously immediately after the step (S3) immediately before the step (S4), but may be performed intermittently as needed. For example, if the capturing body 24 is labeled with a labeling substance 25 having a negative charge, when the capturing body 24 binds to the target particles 22 in the opening 15, the resistance between the source and the drain in the sensor element 5a is reduced. It changes compared to before. If it is described as a constant voltage for convenience, the current value decreases or increases as shown in FIG. On the other hand, in the sensor element 5b in which the non-target particles 23 enter the opening 15 or the sensor element 5 (not shown) in which the target particles 22 do not enter the opening 15, as shown in FIG. There is no change in the current value and the amplitude is at the same position. Although the change in the current value has been described for the sake of convenience, the actual change is the resistance value, and the accurate measurement can be performed by detecting the change in the resistance value, as described above.

  In step (S6), the presence or absence or amount of target particles 22 is determined from the measurement result in step (S5). For example, when there is the sensor element 5 whose resistance value has changed, it can be determined that the target particles 22 exist in the sample. Conversely, when there is no sensor element 5 whose resistance value has changed, it may be determined that the target particle 22 does not exist in the sample. Further, the sensor element 5 whose resistance value has changed can be regarded as the target particle 22 existing in the opening 15. Conversely, a sensor element 5 whose resistance value does not change can be regarded as having no target particle 22 in its opening 15.

  Alternatively, when there is a sensor element 5 in which the amount of change in the resistance value is equal to or greater than a preset threshold value, it is determined that the target particle 22 exists in the opening 15 or that the target particle 22 exists in the sample. Is also good. Such a threshold value can be determined by subjecting a sample in which the target particles 22 are known to be present to detection by the biosensor and measuring the amount of change in the resistance value.

  Alternatively, the abundance of the target particles 22 in the sample can be determined from the number of sensor elements 5 whose resistance value has changed or the number of sensor elements 5 whose change amount is greater than or equal to a threshold value.

The change in the resistance will be described using an example in which graphene is used for the channel 12. Since graphene has bipolar characteristics, there are a state in which electrons are carriers and a state in which vacancies are carriers. Therefore, as shown in FIG. 8, the relationship between the gate voltage (V _g ) and the source-drain current value (I _Sd ) has a V-shape.

Since the target particles 22 and the capturing body 24 have a negative charge, when they enter the opening 15, electrons are injected from the channel 12 a into the channel 12. As a result, apparently graph shifts to the negative side of the V _g-axis as shown by the broken line in FIG. 8. In the width of the AC voltage applied between the source and the drain (indicated by a double arrow in the figure), _ISd is reduced by this shift. Therefore, when the target particles 22 and the capturing body 24 enter the opening 15, the amplitude of the current value shifts downward as shown in FIG. 8A. On the other hand, when the target particles 22 and the capturing body 24 do not enter the opening 15, no shift occurs, and the amplitude is at the same position as shown in FIG. 8B. In this manner, the entry of the target particles 22 or the capturing body 24 into the opening 15 can be detected as a change in the current value (accurately, a change in the resistance value).

  In a further embodiment, steps (S4) to (S6) may be repeated using different types of capture bodies. That is, after performing the steps (S4) to (S6) by adding the first kind of capturing body as described above, further adding a different kind of capturing body that specifically binds to a different kind of target particle. Steps (S4) to (S6) can be similarly performed. That is, by performing steps (S4) to (S6) using a plurality of types of capturing bodies, it is possible to continuously detect a plurality of types of target particles. By recording the position of the sensor element 5 whose resistance value has changed each time the repetition is performed, it is possible to obtain position information of the sensor element 5 where a plurality of target particles 22 exist.

  In the above description, the case where the channel resistance is measured by AC is described, but DC measurement can also be performed. In that case, as shown in FIG. 6, the switch 52 is connected to the DC power supply 56 side. It is also possible to measure by applying a gate voltage. In that case, as shown in FIG. 7, the switch 53 is connected to the DC power supply 57 side. However, since the dielectrophoresis is cut off when performing the DC measurement, there is a possibility that the target particles may come out of the opening 15. In such a case, a probe that captures the target particles may be fixed to the surface of the channel 12a as shown in FIGS.

  The capturer recognizes and binds to a biomarker expressed on the surface of the target particle to be precise. Therefore, the number of binding of the capturing body changes according to the expression amount of the biomarker, and therefore, the expression amount of the biomarker can be measured from the change amount of the resistance value. Furthermore, even when one particle expresses a biomarker that binds to a plurality of types of capturers, the resistance value changes in any of the capturers, so that the particle expresses any of the biomarkers. Can be detected.

  According to the biosensor and the target particle detection method described above, the target particles 22 in the sample are attracted by dielectrophoresis, the target particles 22 are accommodated one by one in the opening 15, and the target particles 22 are changed by a change in resistance. To detect. Therefore, digital counting of the target particles in the sample is possible, and the target particles can be detected with high sensitivity and specificity. In addition, since detection is possible even when the sample is used as it is, there is no need to perform steps such as centrifugation or extraction in advance, the operation is simple, and contamination and destruction of target particles can be prevented. Further, since the biosensor is small, it is easy to carry and can be used in various places.

  When a plurality of target particles are detected using a plurality of capturing bodies, information on the type and the number of the target particles can be obtained.

  As the capturing body 24, for example, an antibody or an antigen-binding fragment, a lectin, or a naturally-derived nucleic acid, an artificial nucleic acid, an aptamer, a peptide aptamer, an oligopeptide, a protein, or the like can be used. For example, when the target particle 22 is an extracellular vesicle, a capturing body 24 that binds to a protein, a glycoprotein, a lipid, a sugar chain, or cfDNA or the like attached to the surface of the extracellular vesicle. Can be used.

  As the labeling substance 25 for labeling the capturing body 24, for example, a negatively charged anion, a positively charged cation, a fluorescent dye, metal beads, dielectric beads, or the like can be used. Labeling of the capturing body 24 can be performed using any known method. The capturing body 24 may be prepared, for example, as a reagent containing the capturing body 24 in a solvent.

  The target particle detection method may be performed by an apparatus that automatically performs each step. Such a device includes, for example, a biosensor 1, a liquid sending unit that sends a sample, a detergent, a reagent, or the like to the space 4, and a data processing unit that stores and processes information on the resistance value of each sensor element 5. And a control unit that controls operations of these units. The operations in the above steps may be executed by an input of an operator of the apparatus, or may be executed by a program included in the control unit.

  By detecting the presence or absence or amount of the target particles 22, the state of the sample or the state of the living body from which the sample is derived can be known. For example, when the target particles 22 are extracellular vesicles, the detection of the extracellular vesicles allows the detection of the health condition of the living body from which the sample is derived, for example, the presence / absence, type, course, pharmacological effect or susceptibility of a specific disease. Information can be obtained. Examples of the specific disease include, but are not limited to, cancer, dementia such as Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies, neurodegenerative disease, and acute kidney injury. When the specific disease is cancer, information on the site, metastasis destination, or site at risk of metastasis may be obtained. Alternatively, information on properties other than the disease of the living body, such as aging, metabolism, fatigue, stress, pregnancy or fetal status, etc. can also be obtained.

  In addition, by detecting a plurality of types of target particles 22 using a plurality of capturing bodies 24, a health state of a living body may be determined from, for example, a ratio or a combination of the numbers of the plurality of target particles 22. Alternatively, it is also possible to obtain comprehensive information on a plurality of health condition items in a living body.

  For example, from the detection result of extracellular vesicles by the method of the embodiment, it is possible to diagnose a disease or a health condition of a living body from which the sample is derived.

  Note that, in the target particle detection method of the first embodiment, the biosensor 1 having the upper electrode 3 as shown in FIG. In this case, the area of the upper electrode 3 when the biosensor 1 is viewed in a plan view is larger than the area of the channel 12a. According to this configuration, when an AC voltage is applied between the drain electrode 9 and the second source electrode 11 of each sensor element 5 without applying a voltage to the upper electrode 3, the target particles 22 Can cause dielectrophoresis that is attracted to

  However, if the area of the channel 12 when the biosensor 1 is viewed in a plan view is too large than the area of the opening 15, the target particles will be caught on the portion of the insulating film that is stacked on the channel 12. Is preferably as small as possible. Therefore, in the embodiment of FIG. 9, the structure is not the three-terminal method but the two-terminal method. Further, in this embodiment, the voltmeter is omitted to simplify the configuration. Such a biosensor 1 can also be used in the above target particle detection method.

(Second embodiment)
-Biosensor Fig. 10 is a sectional view showing an example of the biosensor of the second embodiment, and Fig. 11 is a plan view of the biosensor 1 of the second embodiment. In the present embodiment, the upper electrodes 3a are arranged between the openings of the sensor elements arranged in the row direction and between the openings of the sensor elements arranged in the column direction, and have a lattice shape. Eggplant The area of the upper electrode 3a when viewed in plan is smaller than the area of the shielding electrode 14. The width w of the upper electrode 3 can be, for example, 50 nm to 5 μm.

  The upper electrode 3a is connected to the switch 51 and can be connected to either the AC power supply 18 or the switch 53. The switch 53 can be connected to either the ground or the DC power supply 57.

  FIG. 12 is a cross-sectional view illustrating a state in which target particles are captured using the biosensor of this example. The upper electrode 3a is connected to a ground potential via a switch 51 and a switch 53. The shield electrode 14 is connected to a ground potential via a switch 53. Channel 12 is connected to an AC power supply via switches 50 and 52. Thereby, similarly to FIG. 4, the distribution is such that the electric field intensity becomes stronger toward the inside of the opening 15, so that the target particles enter the opening 15. FIG. 4 also shows that the intrusion of target particles can be electrically detected by a change in current (more precisely, a change in resistance) in the channel 12a.

  FIG. 13 is a cross-sectional view showing a state where a capturing body is bound to target particles using the biosensor of this example. The switches 51, 53, 50, and 52 are as shown in FIG. 12, and are similar to FIG. 5 in that they can be electrically detected by a current change (correctly, a resistance change) of the channel 12a.

  14 and 15 are a cross-sectional view and a plan view illustrating a method of collecting or discharging desired target particles using the biosensor of the present embodiment. FIG. 14 is a cross-sectional view showing a method for discharging only desired target particles from the opening 15. The upper electrode 3a is connected to the AC power supply 18 by a switch 51. The shield electrode is kept at the ground by the switch 53. The switch 50 switches for each sensor element 5. For example, the switch 50 connected to the sensor element 5 where the target particles not to be collected or discharged are captured is kept connected to the power supply line 54. The power supply line 54 is connected to the AC power supply 18 by a switch 52. On the other hand, the switch 50 connected to the sensor element 5 in which target particles to be collected or discharged are captured is connected to the ground line 20 side. Accordingly, the channel 12 connected to the ground line 20 has the same potential as the shielding electrode, so that the electric field between the channel and the shielding electrode disappears, and an electric field distribution is formed between the channel 12 and the upper electrode 3a. Here, since the upper electrode 3a is smaller than the combined area of the shielding electrode and the channel 12a exposed from the opening of the shielding electrode, the electric field intensity distribution becomes stronger toward the upper electrode 3a. Therefore, the target particles trapped in the channel 12a connected to the ground line 20 are discharged toward the upper electrode 3a by dielectrophoresis. On the other hand, the channel 12a connected to the AC power supply 18 has the same potential as the upper electrode 3, so that no electric field distribution is formed between the channel 12a and the upper electrode 3a. Since the electric field distribution with the shielding electrode remains, a local electric field intensity distribution is formed around the opening 15. Since the electric field intensity distribution becomes sparse toward the outside of the opening 15, the target particles captured in the channel 12 a connected to the AC power supply 18 remain in the opening 15.

FIG. 15 is a plan view showing how the target particles are discharged in FIG. Target particles entering the opening 15b on the channel connected to the AC power supply 18 remain trapped in the opening 15b, whereas the target particles on the channel connected to the ground line 20 remain. The target particles contained in 15a are discharged toward the upper electrode 3a. When a flow is given to the liquid phase filled in the space 4, the target particles discharged from the opening are discharged along the line of the upper electrode 3a. At this time, since the electric field distribution formed by the channel connected to the AC power supply is local around the opening, target particles discharged along the line of the upper electrode 3a may be re-captured. There is no.
-Target particle detection method Fig. 16 is a schematic flow chart showing an example of a target particle detection and separation method using the biosensor of the second embodiment. The target particle detection method includes the following steps. (S1) storing the sample solution in the space of the biosensor; (S2) connecting the upper electrode 3a and the shielding electrode to the ground line, connecting each sensor element to the AC power source, causing dielectrophoresis to cause each target particle to Capturing at the opening on the sensor element, (S3) washing the sample solution and discharging excess target particles, and (S4) adding a capturing body that specifically binds to the target particles into the space. (S5) measuring a current value flowing through each sensor element and a potential difference between at least two electrodes; (S6) determining the presence or absence or amount of target particles in the sample according to the result of the measurement; (S3 to 6 are repeated by changing the type of capturing body), (S7) determining the sensor element capturing the target particles to be collected or discharged according to the result of the measurement, (S8) collecting or Grounding the sensor element capturing the target particles to be discharged and the shielding electrode, connecting the other sensor elements and the upper electrode 3a to an AC power source, (S9) applying a flow to the liquid in the space 4 to collect or discharge the target. Collecting or discharging particles from the space 4 on the sensor array.

  Steps (S1) to (S6) can be performed, for example, in the same manner as steps (S1) to (S6) of the target particle detection method described in the first embodiment.

  In the step (S7), from the measurement results up to the step (S6), the target particles exhibiting a desired result with respect to the binding of each capturer, that is, a positive or negative, or a desired response amount (resistance change amount). Read the position of the sensor element where the subclass is captured. From this result, a subclass that is desired to be collected and sent to another analysis, or a subclass that is unnecessary and should be discharged is determined.

  Next, in step (S8), the sensor element in which the subclass to be collected or discharged is captured is grounded to the ground potential. On the other hand, a sensor element containing a subclass to be captured in the opening 15 is connected to an AC power supply. Further, the shield electrode is grounded, and the upper electrode 3a is connected to an AC power supply. The exposed portion in the opening 15 of the sensor element that is grounded forms an electric field between the upper electrode 3a and the shield electrode together with the shielding electrode, and the electric field strength becomes dense on the upper electrode 3a side. The target particles in the section 15 are discharged. On the other hand, the exposed portion in the opening 15 of the sensor element connected to the AC power supply forms a local electric field with the shielding electrode, and the electric field intensity has a sparse distribution toward the outside of the opening. , The target particles continue to be retained in the openings 15.

  Next, if a certain flow is given to the liquid in the space 4 in the step (S9), the target particles discharged from the opening as described above are collected or discharged from the sensor array. As a method of giving a flow to the liquid in the space 4, a micropump may be used, or electrophoresis may be performed.

(Third embodiment)
-Biosensor Fig. 17 is a cross-sectional view illustrating an example of the biosensor according to the third embodiment. The biosensor 1 in this example includes a probe 26 on the surface of the channel 12a exposed at the opening 15. Other configurations are the same as those of the biosensor of the second embodiment.

  The probe 26 is a molecule that selectively or specifically captures the target particle 22. The probe 26 is, for example, an antibody or an antigen-binding fragment, or a naturally occurring nucleic acid, an artificial nucleic acid, an aptamer, a peptide aptamer, an oligopeptide, a lectin, or a protein.

  For example, when the target particle 22 is an extracellular vesicle, a probe 26 that binds to a protein, glycoprotein, lipid or sugar chain expressed on the surface of the extracellular vesicle, or cfDNA or the like attached to the surface is used. Can be used.

  Further, the probe 26 releasably couples the target particle 22 and the channel 12. For example, they are configured to be able to cleave the bond between the target particle 22 and the probe 26, within the probe 26, or between the probe 26 and the channel 12 by adding an appropriate reagent or the like.

  For example, Ni-NTA is immobilized on the channel 12a, and the probe 26 is modified with His-Tag. Then, the probe 26 is fixed to the channel 12a by binding of His-Tag and Ni-NTA. In this case, the bond between His-Tag and Ni-NTA can be broken by the addition of imidazole.

  Alternatively, when an antibody is used as the probe 26, protein A is immobilized on the channel 12a, and the probe 26 is immobilized on the channel 12a by binding between the Fc portion of the antibody and protein A. In this case, the bond between protein A and the antibody can be broken by setting the sample solution under acidic conditions.

  Alternatively, when a GST-expressing antibody is used as the probe 26, glutathione is immobilized on the channel 12a, and the probe 26 is immobilized on the channel 12a by binding of GST and glutathione of the antibody. In this case, by adding reduced glutathione, the bond between GST and glutathione is replaced by the bond with reduced glutathione, and the bond between the antibody and the channel 12 can be cut off.

  Alternatively, if a probe 26 carrying magnetic beads is used, the channel 12 and the probe 26 can be coupled or dissociated by turning on / off the magnetism of the channel 12.

  Alternatively, the channel 12, the probe 26, and the target particle 22 are directly bonded. In this case, by adding a competitive ligand that competes with the target particle 22 for binding to the probe 26, the binding to the probe 26 is replaced by the competitive ligand from the target particle 22. As a result, the target particles 22 can be dissociated. It is preferable that the competitive ligand is, for example, a ligand having a stronger affinity for the probe 26 than the target particle 22.

  Alternatively, when an RNA aptamer, a DNA aptamer, or a peptide aptamer is used as the probe 26, the probe 26 can be decomposed by adding RNase, DNAase, and protease, respectively, and the target particle 22 can be dissociated.

-Target particle detection method Fig. 18 is a schematic flow chart showing an example of a target particle detection method using the biosensor of the third embodiment. In the method, first, (S11) the sample solution is accommodated in the space of the biosensor, (S12) the upper electrode and the shield electrode are grounded to ground, and an AC power source is connected to the source electrode of each sensor element. The target particles are guided into the opening by dielectrophoresis. Steps (S11) and (S12) can be performed, for example, in the same manner as steps (S1) and (S2) of the target particle detection method described in the third embodiment.

  Next, in step (S13), the sensor element and the shield electrode are grounded to the ground potential, and an AC power supply is connected to the upper electrode. By this operation, particles having no binding ability with the probe 26 described below are discharged from the opening 15 by dielectrophoresis.

(A) In the case where the probe 26 that specifically captures the target particle 22 is used. In this example, when the target particle 22 enters the opening 15 in the step (S12), the target particle 22 binds to the probe 26. As a result, the target particles 22 can be captured by the channel 12a.

  In the step (S13), an electric field strength gradient opposite to that in the step (S12) is applied, so that the non-target particles 23 that have not been bonded to the probe 26 and foreign substances that have entered the opening 15 can be removed. On the other hand, the target particles 22 are not emitted from the openings 15 because they are captured by the probe 26.

  Next, in a step (S14), the non-target particles 23 and foreign substances discharged from the opening 15 and the surplus target particles 22 in the space 4 are washed away.

  After that, (S15) measuring the resistance value between the source and drain electrodes of each sensor element 5 and (S16) determining the presence or absence or amount of the target particles 22 in the sample may be performed. If the measurement of the resistance value is also performed during the operation of the step (S12), the target particles 22 remain in the opening 15 after the cleaning due to the difference from the resistance before the target particles 22 enter the opening 15. The sensor element 5 can be specified.

  When the presence of the target particles 22 is confirmed in the steps (S15) and (S16), a reagent for dissociating the bond between the channel 12 and the target particles 22 is further added in the step (S17). In (S18), the target particles 22 are released into the space 4 by applying the same electric field as in the step (S13), and the liquid in the space 4 is caused to flow in the step (S19). Can also be recovered. As a method of creating a flow in the liquid in the space 4, for example, a micropump or electrophoresis can be performed.

  By this method, the desired target particles 22 can be easily separated from the sample without performing centrifugation, extraction, or the like. The method including the steps (S11) to (S19) is also referred to as “target particle separation method”. The separated and collected target particles 22 can be further analyzed. For example, when the target particles 22 are extracellular vesicles, after collection, membrane proteins, lipids, sugar chains, inclusions, and the like can be analyzed. Alternatively, responses to cytokines, ligands, cells and living tissues can be confirmed in vitro, and effects on model animals can be confirmed in vivo.

(B) In the case of using the probe 26 that selectively captures the target particles 22 In this example, by performing the steps (S11) to (S12), for example, a large classification object group including the target particles 22 Is captured by In the subsequent step (S13), foreign substances consisting of other objects are released into the space 4 through the opening 15 by dielectrophoresis, and the foreign substances are removed by washing in the step (S20). Can be. By this step, a large group of object groups including the target particles 22 can be once selected from the sample, and the accuracy of the subsequent detection using the capturing body 24 can be improved.

  Next, as a further detection step using the capturing body 24, (S21) a capturing body that specifically binds to the target particles is added to the space, and (S22) the resistance value between the source and drain electrodes of each sensor element is determined. Measuring (S23) The presence or absence or amount of target particles in the sample may be determined according to the result of the measurement. Steps (S20) to (S23) can be performed in the same manner as steps (S3) to (S6) in the above-described 1.5th embodiment. The step (S21) may be performed by generating dielectrophoresis as in the step (S12). When the dielectric constant of the capturing body 24 is high or when a label having a high dielectric constant is modified, the capturing body 24 can be brought closer to the opening 15 by dielectrophoresis, and can be easily bound to the target particles 22. Alternatively, if it is conceivable that the capturing body 24 approaches the opening 15 where the target particles 22 do not enter due to dielectrophoresis, adversely affecting the detection, the dielectrophoresis may be stopped or performed. Dielectrophoresis with the same reverse gradient as in (S13) may be performed. When the dielectric constant of the capturing body 24 is low or when a label having a low dielectric constant is modified, the direction of the dielectrophoresis may be reversed. The steps (S20) to (S23) may be repeatedly performed using a plurality of capturing bodies 24 to detect a plurality of target particles 22.

  Furthermore, the space 4 may be washed after the step (S21) and before the step (S22). Thereby, the capturing body 24 that has not bound to the target particle 22 can be removed. For example, the capturing body 24 that has non-specifically entered the opening 15 can also be removed. This allows for more accurate detection.

  The washing step can be performed by, for example, sending a cleaning agent such as water, physiological water, PBS, TBS, or PBS-T or TBS-T into the space 4 and extruding an old liquid.

(Fourth embodiment)
-Target Particle Detection Method FIG. 21 is a schematic flow chart showing an example of the target particle detection method of the fourth embodiment. In this method, steps (S31) to (S33) are the same as steps (S11) to (S13) of the second embodiment, and steps (S34) to (S39) are the same as those of the second embodiment. Steps (S14) to (S19) are the same as steps (S40) to (S47), and are the same as steps (S20) to (S27) of the second embodiment. This embodiment is different from the second embodiment in that steps (S32) and (S33) are repeated as necessary.

  FIG. 22 shows a cross-sectional view in the step (S32). The sensor element is connected to the AC power supply 18 by switches 50 and 52, the shield electrode is grounded to ground by switch 53, and the upper electrode is grounded to ground by switches 51 and 53. At this time, the channel 12a exposed to the opening 15 forms an electric field with respect to the shielding electrode and the upper electrode, and the distribution of the electric field intensity becomes dense toward the opening 15. Thus, the target particles are attracted into the opening 15 by dielectrophoresis. Here, when there is a non-target particle having a high dielectric constant like the target particle, the non-target particle is similarly drawn into the opening 15 by dielectrophoresis. Also, if the properties of the non-target particles are similar to the target particles, the electrical signals detected by the sensor element will also be similar, and at this stage, the difference between the labeled particles and the non-labeled particles cannot be discerned .

  FIG. 14 shows a sectional view in the step (S33). The sensor element is grounded to the ground potential by the switch 50, and the shield electrode is also grounded to the ground potential by the switch 53. The upper electrode is connected by a switch 51 to an AC power supply. An upper electrode forms an electric field with respect to the shield electrode and the channel 12 a exposed in the opening 15. Here, the area of the upper electrode is smaller than the combined area of the shielding electrode and the channel 12a, so that the electric field intensity becomes denser toward the upper electrode, and has a gradient opposite to that of FIG. Accordingly, dielectrophoresis occurs in a direction in which particles are emitted from the opening 15, but the target particles are not emitted from the opening 15 because the target particles are bonded to the probe fixed to the surface of the channel 12 a. On the other hand, since the non-target particles do not bind to the probe, they are released from the opening 15 by dielectrophoresis.

  FIG. 15 shows a cross-sectional view when the step (S32) is performed again. Particles are newly attracted to the opening from which the non-target particles have been released, and the sensor element detects the capture of the new particles accordingly. On the other hand, the signal of the sensor element in which the target particle has remained in the opening is the signal detected in FIG. It is not known whether the newly captured particles are target particles or non-target particles, but it can be predicted that the particles that remain captured are target particles. Steps (S32) and (S33) can be repeated until a desired number of target particles can be captured from the detection information. Alternatively, processing may be performed simply by determining the number of repetitions.

  The repetition step is particularly effective when the amount of the target particles 22 in the sample is expected to be small. For example, extracellular vesicles derived from cancer may be present in only about 1% of the total extracellular vesicles. In this case, when extracellular vesicles are simply collected, only about 10 extracellular vesicles derived from cancer can be collected on average even if 1,000 are collected. Considering the diversity of extracellular vesicles derived from cancer, there is a risk that subclasses with low expression levels may be missed. In such cases, for example, if a surface marker characteristically expressed on cancer-derived extracellular vesicles, such as a probe that binds to CD9, is used, cancer cells frequently appear in the captured extracellular vesicles. Extracellular vesicles of origin will be included.

  Therefore, by using this example, the desired target particles can be efficiently collected, so that the risk of overlooking the subclasses contained in the target particles is small, rare subclasses can be detected, and the size of the sensor array can be reduced. Can be smaller.

DESCRIPTION OF SYMBOLS 1 ... Biosensor 2 ... Sensor array 3,3a ... Upper electrode 4 ... Space 5 ... Sensor element 6 ... First substrate 7 ... First insulating layer 8a ... First gap 8b ... Second gap 9 ... Drain electrode DESCRIPTION OF SYMBOLS 10 ... 1st source electrode 11 ... 2nd source electrode 12 ... Channel 13 ... 2nd insulating layer 14 ... Shielding electrode 15 ... Opening 17 ... 1st AC / DC power supply 18 ... 2nd AC / DC power supply 19 ... Ammeter 22 ... Target particle 24 ... Capturer 25 ... Labeled substance 26 ... Probe

Claims (27)

A biosensor for detecting target particles in a sample,
A sensor array having a plurality of sensor elements arranged on a first substrate;
Each of the plurality of sensor elements is
A first insulating layer provided on the first substrate;
At least two electrodes embedded in the first insulating layer with a gap therebetween;
A channel provided on the at least two electrodes and the gap and connected to the at least two electrodes;
A second insulating layer provided on the first insulating layer and the channel, the second insulating layer having an opening exposing the channel corresponding to the gap;
A second electrode provided above the opening,
A biosensor in which the area of the second electrode is larger than the area of the channel exposed from the opening when the biosensor is viewed in a plan view.   The second electrode is a shield electrode provided on a second insulating layer except for the opening, or a space is provided above the sensor array so as to face the second insulating layer. The biosensor according to claim 1, wherein the biosensor is an upper electrode arranged.   The second electrode is a shielding electrode provided on the second insulating layer excluding the opening, and is arranged above the sensor array with a space therebetween so as to face the second insulating layer. The biosensor according to claim 1, further comprising an upper electrode.   The area of the upper electrode when the biosensor is viewed in a plan view is smaller than the shielding electrode, and the upper electrode is disposed between the openings of the sensor elements. Biosensor.   The biosensor according to claim 3, further comprising a first circuit including a second power supply that applies an AC voltage or a DC voltage to the upper electrode.   The biosensor according to claim 1, further comprising a plurality of second circuits including a first power supply that applies an AC voltage or a DC voltage to the at least two electrodes of each of the sensor elements. .   Further comprising a probe fixed to the channel exposed from the opening and selectively or specifically capturing the target particle, wherein the probe releasably binds the channel and the target particle. 7. The biosensor according to any one of 6.   The biosensor according to claim 7, wherein the probe is modified with His-Tag, and the His-Tag binds to Ni-NTA immobilized on the channel.   The biosensor according to claim 7, wherein the probe is an antibody, and the antibody binds to protein A immobilized on the channel.   The biosensor according to any one of claims 1 to 9, wherein the opening has a size into which only one of the target particles enters. A method for detecting target particles in a sample, comprising:
(I) disposing the sample on a sensor array in which a plurality of sensor elements are disposed on a first substrate;
(Ii) causing first dielectrophoresis to capture target particles in openings on each sensor element;
(Iii) adding a capturer that specifically binds to the target particle onto a sensor array;
(Iv) measuring a current value flowing through each of the sensor elements and a potential difference between at least two electrodes provided in the sensor elements continuously immediately before the step (iii);
(V) A method for detecting target particles, comprising determining the presence or absence or amount of the target particles in the sample according to the result of the measurement.   A probe exposed from the opening and connected to at least two electrodes embedded on the substrate of the sensor element, the probe being configured to selectively or specifically capture the target particles; 12. The method of claim 11, wherein the channel and the target particle are releasably associated.   The method according to claim 11, further comprising stopping the first dielectrophoresis and washing the sensor array after the step (ii).   The method according to any one of claims 11 to 13, wherein the steps (iii) to (v) are repeated using different types of the capturing body. A method for detecting the target particles in a sample,
(I) disposing the sample on a sensor array in which a plurality of sensor elements are disposed on a first substrate;
(Ii) causing first dielectrophoresis, capturing the target particles in the openings on each sensor element, and at least two electrodes embedded on the substrate of the sensor element exposing the target particles from the openings; Specifically capturing the target particles with a probe fixed to a connected channel; (iv) after the step (i), immediately after the step (ii), immediately before the step (ii); Measuring a flowing current value and a potential difference between at least two electrodes of the sensor element;
(V) A method for detecting target particles, comprising determining the presence or absence or amount of the target particles in the sample according to the result of the measurement.   After the step (ii), causing a second dielectrophoresis to generate a stronger electric field in a second electrode provided above the opening than a channel exposed in the opening; and The method according to claim 11 or 15, further comprising causing a first dielectrophoresis, wherein the second dielectrophoresis and the first dielectrophoresis are repeated a plurality of times.   16. The method according to claim 11 or 15, further comprising, after step (ii), stopping the first dielectrophoresis and washing on the sensor array.   The method according to any one of claims 11 to 17, wherein the opening is sized to allow only one of the target particles to enter.   19. The method according to any one of claims 11 to 18, wherein the target particles are extracellular vesicles. A method for separating target particles in a sample, comprising:
(I) disposing the sample on a sensor array in which a plurality of sensor elements are disposed on a first substrate;
(Ii) causing first dielectrophoresis to capture target particles in openings on each sensor element;
(Iii) adding a capturing body, which specifically binds to the target particle, onto the sensor array;
(Iv) measuring a current value flowing through each of the sensor elements and a potential difference between at least two electrodes of the sensor elements;
(V) determining the presence or absence or amount of the target particles in the sample according to the result of the measurement;
(Vi) A method for separating target particles, comprising dissociating the channel exposed in the opening from the target particles and collecting the target particles. The steps (iii) to (v) are repeated using different types of the capturing bodies,
In the step (vi), a stronger electric field is generated in the second electrode provided above the opening in the sensor element in which the target particles to be collected are present than in the channel exposed in the opening. Causing a second dielectrophoresis to cause the first dielectrophoresis to occur in the other sensor elements for a plurality of types of the target particles, and collecting a plurality of types of the target particles,
The method according to claim 20. A method for separating target particles in a sample, comprising:
(I) disposing the sample on a sensor array in which a plurality of sensor elements are disposed on a first substrate;
(Ii) causing first dielectrophoresis, capturing the target particles in the openings on each sensor element, and at least two electrodes embedded on the substrate of the sensor element exposing the target particles from the openings; Specifically capturing the target particles with a probe connected and fixed to a channel, (iv) after the step (i), continuously flowing to each of the sensor elements immediately before the step (ii). Measuring a current value and a potential difference between at least two electrodes of the sensor element;
(V) determining the presence or absence or amount of the target particles in the sample according to the result of the measurement; and (vi) separating target channels including dissociating the channel and the target particles and collecting the target particles. Method.   After the step (ii), in each of the sensor elements, a second dielectric for generating a stronger electric field in the second electrode provided above the opening than in the channel exposed in the opening. 23. The method of claim 22, further comprising causing electrophoresis and causing the first dielectrophoresis, wherein the second dielectrophoresis and the first dielectrophoresis are repeated a plurality of times.   The step (vi) is for generating a stronger electric field in the second electrode provided above the opening in the sensor element in which the target particles are present than in the channel exposed in the opening. 24. The method according to any one of claims 20 to 23, wherein the method is performed by causing the second dielectrophoresis and causing the first dielectrophoresis with the other sensor elements.   25. The method according to any one of claims 20 to 24, further comprising stopping the first dielectrophoresis and washing the sensor array after the step (ii).   The method according to any one of claims 20 to 25, wherein the opening is sized to allow only one of the target particles to enter.   The method according to any one of claims 20 to 26, wherein the target particles are extracellular vesicles.

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