JP6903618B2 - Biosensor, target particle detection method and separation method - Google Patents

Description

The present invention relates to biosensors, target particle detection methods and separation methods.

Exosomes are a type of extracellular vesicles, which are minute vesicles contained in a living body having a particle size of about 100 nm. It is known that extracellular vesicles have a lipid bilayer structure containing nucleic acids and the like inside, and have a function of moving between distant organs and transporting substances. Observation results suggesting that proteins, glycoproteins, and the like are contained in the lipid bilayer membrane and that cfDNA is attached around the lipid bilayer membrane have also been reported. They are thought to include information on cells in which extracellular vesicles are produced, information on cells to which they metastasize, and the like.

It has been reported that extracellular vesicles are deeply involved in cancer metastasis. Alternatively, it has been reported that dementia such as Alzheimer's disease, Parkinson's disease, and Levy body dementia, neurodegenerative diseases, and diseases other than cancer such as renal disorder are also involved in the transport of the causative substance. Therefore, extracellular vesicles are attracting attention as biomarkers for use in early detection of diseases, recurrence monitoring, and the like. In addition, since there are many unclear points about its composition and function, 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 specific and high sensitivity, and a biosensor used therein.

A biosensor according to an embodiment is a sensor for detecting target particles 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 has a first insulating layer provided on the first substrate, at least two electrodes embedded in the first insulating layer with a gap from each other, and at least two electrodes and on the gap. A second insulating layer provided on the above and connected to at least two electrodes, a first insulating layer and a second insulating layer provided on the channel and having an opening for exposing the channel corresponding to the gap, and the opening. Also includes 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 showing an example of the biosensor of the embodiment. FIG. 2 is a plan view showing an example of the biosensor of the embodiment. FIG. 3 is a flowchart showing the target particle detection method of the embodiment. FIG. 4 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 5 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 6 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 7 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 8 is a graph showing the current value between the source and drain. FIG. 9 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 10 is a cross-sectional view showing an example of the biosensor of the embodiment. FIG. 11 is a plan view showing an example of the biosensor of the embodiment. FIG. 12 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 13 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 14 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 15 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 16 is a flowchart showing the target particle detection method of the embodiment. FIG. 17 is a cross-sectional view showing an example of the biosensor of the embodiment. FIG. 18 is a flowchart showing a target particle detection method and a separation method of the embodiment. FIG. 19 is a flowchart showing a target particle detection method and a separation method of the embodiment. FIG. 20 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 21 is a diagram showing a state when the biosensor of the embodiment is used. FIG. 22 is a diagram showing a state when the biosensor of the embodiment is used.

Hereinafter, various embodiments will be described with reference to the drawings. Each figure is a schematic view for facilitating the understanding of the embodiment, and the shape, dimensions, ratio, etc. may differ from the actual ones, but these are appropriately redesigned in consideration of the following explanations and known techniques. can do.

A biosensor according to an embodiment is a sensor for detecting target particles 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 has a first insulating layer provided on the first substrate, at least two electrodes embedded in the first insulating layer with a gap from each other, and at least two electrodes and on the gap. And a second insulating layer provided on the first insulating layer and having an opening provided on the channel connected to at least two electrodes and exposing the channel corresponding to the gap, and more than the opening. It is provided with 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, a biosensor is provided that further comprises a probe in a channel exposed from the opening. Further, according to a further embodiment, a target particle detection method and a target particle separation method using a biosensor are provided.

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

(First Embodiment)
-Biosensor FIG. 1 is a cross-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 the first substrate 6. When the plurality of sensor elements 5 are viewed in a plan view, they are arranged in an array, for example, in the matrix direction.

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 the 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 side by side 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.

Channels 12 are provided on the drain electrode 9, the first gap 8a, the first source electrode 10, the second gap 8b, 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 the region and the channel 12 of the first insulating layer 7 that are not covered with the channel 12. The second insulating layer 13 has an opening 15 that exposes a region (channel 12a) of the channel 12 corresponding to the first gap 8a. A shielding electrode 14 is provided on the second insulating layer 13 excluding the opening 15.

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

For example, the first substrate 6 and the second substrate 16 are connected by a wall portion (not shown) located on the outer periphery thereof, whereby the upper electrode 3 is supported. Further, the wall portion forms a sensor array 2, an upper electrode 3, a second substrate 16, and a liquid-tight space 4 surrounded by a wall portion. A sample to be analyzed by the biosensor 1 is housed in the space 4. For example, the second substrate 16 is opened with an injection port (not shown) for injecting a sample and an discharge port (not shown) for discharging the sample. Alternatively, a pillar connecting the first substrate 6 and the second substrate 16 can be formed and reinforced at a place other than the outer peripheral portion as needed.

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

Further, the biosensor 1 has a switch 53 connected to the shielding 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 the switch 53 and can be connected to either the ground line 20 or the DC power supply 57.

Further, each sensor element 5 includes an ammeter 19 and a voltmeter 58, respectively. 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 the sensor elements 5 is not limited to this. For example, 10 to 1,000,000 sensor elements 5, preferably 500 to 10,000, are arranged in one biosensor 1.

FIG. 2 shows a plan view of the biosensor 1 of the first embodiment. Here, for convenience, a sensor array 2 including four sensor elements 5A, 5B, 5C, and 5D is shown. The cross-sectional view of FIG. 1 corresponds to a cross-section obtained by cutting the biosensor of FIG. 2 along BB'. The second insulating layer (not shown) and the shielding electrode 14 have substantially the same planar shape, and openings 15A, 15B, 15C, and 15D are provided above the sensor elements 5A, 5B, 5C, and 5D, respectively. Be prepared. Here, the area of the shielding electrode 14 is larger than the area of each of the channels 12A, 12B, 12C, and 12D exposed from the openings 15A, 15B, 15C, and 15D, respectively. 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 has a rectangular plate shape, for example. Each substrate has, for example, an insulating film on the 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, polymer material, metal, or the like. When a gate electrode is formed by sandwiching an insulating film under the channels 12A, 12B, 12C, 12D, the thickness of the insulating film at the same portion should be as thin as possible without impairing the insulating property, for example. It is preferably about several nm. Such a high quality thin film can be formed by, for example, an ALD (Atomic Layer Deposition) method. Further, the gate voltage can be applied to the channels 12A, 12B, 12C, and 12D from the shielding electrode 14 and, if necessary, the upper electrode 3 via the sample solution. In this case, the thickness of the insulating film under the channel does not need to be reduced as described above.

The size of the first substrate 6 is not limited, but can 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 can 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 made of silicon, for example, a semiconductor circuit can be formed, and the above-mentioned switch circuit, power supply circuit, voltmeter, ammeter, series resistor, and the like can be formed. .. Further, input / output terminals with the semiconductor circuit may be formed on the outer peripheral portion of the first substrate 6.

Further, the second substrate 16 has a sensor element, a second insulating film, a shielding electrode, and the like formed on the first substrate 6, and then a sacrificial layer is formed on the sacrificial layer together with the upper electrode 3. It can be produced by forming a film and then removing the sacrificial layer. When this method is used, the thickness of the sacrificial layer can be formed thinly and uniformly, 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 shielding electrode 14 can be made highly accurate. it can.

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. If you want to use this hole as a sample solution supply port or discharge port, you can leave it as it is, or if you want to form the sample solution supply port and discharge port separately and make the space 4 a closed flow path. , The sacrificial layer removal hole can also be closed. As a method of closing the sacrificial layer removing hole, various methods such as attaching another flat plate or 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. ), Titanium (Ti), Chromium (Cr), Tungsten (W), Ruthenium (Ru), Aluminum (Al) and other metals, or zinc oxide (ZnO), indium tin oxide (ITO), IGZO, conductive It consists of a conductive substance such as a polymer. The size of each electrode can be 100 × 100 nm to 5 × 50 μm, and when the detection target is an extracellular vesicle, it is preferably 100 × 100 nm to 1 × 1 μm. The thickness of each electrode may be, for example, the same as that of the first insulating layer 7, or may be slightly thicker than that of the first insulating layer 7.

In the examples shown in FIGS. 1 and 2, the resistance value of the channel 12a is read by the 3-terminal method, but the resistance value of the channel 12a can also be read by the 4-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 the drain electrode 9 is connected to the drain electrode 9. By installing an ammeter in parallel with the first source electrode 10, the resistance of the channel 12a can be measured. In the case of this form, 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.

The channel 12 is made of, for example, graphene, carbon nanotubes, MoS ₂ , or the like. The channel 12 has a film-like or wire-like shape. The size of the channel 12 is not limited, but can be, for example, 0.1 to 100 μm × 0.1 to 100 μm (width × length). Practically, if it is 20 to 200 nm × 300 to 5,000 nm, it 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 the target particles to be analyzed by the biosensor 1 enter, which will be described in detail later. The opening 15 is preferably sized so that only one target particle can enter. For example, when the target particle is an extracellular vesicle, the opening 15 is preferably 50 × 50 nm to 1 × 1 μm. Further, the size of the channel 12a exposed from the opening 15 can also be 50 × 50 nm to 1 × 1 μm, or the channel width can be narrowed, for example, 30 × 50 nm to 0.5 × 1 μm (channel width × exposed channel). Length) is also acceptable.

The shielding electrode 14 is, 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 toward the shielding electrode 14 side. As a result, the target particles can easily enter the opening 15.

The upper electrode 3 is, 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 produced by, for example, a semiconductor process, a MEMS process, or the like.

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

It is said that about 1% of extracellular vesicles in the blood are derived from cancer. For example, when the sensor array 2 contains 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 are diverse even within a single tumor, and it is said that the extracellular vesicles secreted from the cancer cells are also diverse. When the sensor array 2 contains 10,000 sensor elements, about 100 extracellular vesicles derived from cancer contained at a rate of 1% can be captured, so even if there is diversity, it can be detected without overlooking. can do. In a further embodiment, the biosensor 1 does not have to include the upper electrode 3. In that case, the upper part of the space 4 is open, and the sample is dropped directly 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. The method is a method for detecting target particles in a sample.

The sample is an analytical object that may contain the target particles in it. The sample is, for example, a biological liquid sample. Samples include, for example, blood, serum, plasma, blood cells, urine, stool, sweat, saliva, sputum, lymph, cerebrospinal fluid, tears, 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 during treatment such as surgery or examination. Alternatively, the sample may be an environment-derived sample such as soil, river water, seawater or a mixture thereof.

Alternatively, the sample may be a preparation prepared from the above materials. The preparation may be, for example, one that has undergone 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 membrane having a diameter of 20 nm to 50 μm existing in the living body. For example, the target particle is a particular extracellular vesicle, eg, a particular exosome. Alternatively, the target particle may be a fragment such as a protein, a coagulated product, a cell, a bacterium, a fungus, a virus, or the like.

Here, the dielectric constant of the target particles is larger than that of the liquid (hereinafter, referred to as “sample liquid”) which is the medium of the target particles in the sample. For example, the target particle contains a biological membrane and has a negative charge derived from it. When a sample having a dielectric constant of the target particles smaller than that of the sample liquid is used, alcohol or the like may be mixed with the sample to reduce the dielectric constant of the sample liquid.

FIG. 3 is a diagram showing a schematic flow of the target particle detection method of the first embodiment. The target particle detection method includes the following steps. (S1) Contain the sample solution in the space of the biosensor, (S2) Connect the upper electrode and the shielding electrode to the ground line, connect each sensor element to the AC power supply, and cause dielectric migration to move the target particle to each sensor. Capturing in the opening on the element, (S3) washing the sample solution to expel excess target particles, (S4) adding a trap that specifically binds to the target particles into the space. (S5) Measuring the current value flowing through each sensor element and the potential difference between at least two electrodes (source and 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 the target particles can be detected by performing the above steps will be described with reference to FIGS. 4 and 5. 4 and 5 are views showing a state when the biosensor 1 is used.

Prior to the step (S1), it is preferable to pre-contain the liquid in the space 4 of the biosensor 1. The liquid is, for example, water, physiological water, an ionic liquid, an organic solvent, PBS or the like.

The sample solution is housed in the space 4 in the step (S1).

In the step (S2), the switch 53 is connected to the ground side as shown in FIG. 4, and the shielding 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, but if the resistance of the series resistor 21 is made sufficiently larger than the resistance of the channel 12, a voltage approximately equal to that of the AC power supply is applied to the entire channel 12. Will be 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 to the channel 12a of the opening 15 with the upper electrode 3. When the upper electrode 3 is not used, the electric field directly above the channel 12a of the opening 15 is applied toward infinity. These electric field strength distributions are non-uniform in space 4. That is, since the area of the channel 12a is smaller than that of the upper electrode 3 and the shielding electrode 14, the electric field strength generated in the vicinity of the channel 12a becomes stronger, and the electric lines of force shown by the broken lines in the figure become dense. On the other hand, the electric field strength generated in the vicinity of the upper electrode 3 and the shielding electrode 14 becomes weak, and the electric lines of force become sparse.

Therefore, if particles having different dielectric constants are present in the sample solution, migration due to dielectrophoresis occurs. According to the Clausius-Mossotti equation shown below, when the permittivity of an object is greater than the permittivity of the medium, the object migrates in the direction of strong electric field strength.

In the formula, "F _DEP " is the dielectrophoretic force, "ε _p " is the permittivity of the particle, "ε _m " is the permittivity of the medium, "r" is the permittivity of the particle, 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 dielectric constant larger than that of the sample liquid (for example, the target particles 22) is attracted to the channel 12a where the electric field is concentrated and opens. Enter the part 15. It is left for a few minutes, for example, until the target particles 22 enter the opening 15. Alternatively, from the detection by the following sensor elements, the desired number of sensor elements are left to show the capture of the target particles.

In order to confirm whether or not the target particle 22 has entered the opening 15, the ammeter 19 and the voltmeter 58 of each sensor element 5 are used, and the potential difference and the current between the drain electrode 9 and the first source electrode 10 are used. 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, the current value changes in the sensor element 5a in which the target particles 22 are contained in the opening 15. Since an AC voltage is applied between the electrodes, the current value oscillates in a specific cycle. For example, the change in the current value is a shift in the position where the current value swings. For example, after the target particle 22 enters the opening 15, the position of the amplitude can be shifted to a position having a lower current value (hereinafter, referred to as “lower side”) as shown in FIG. 4 (a). Alternatively, the amplitude may shift to a position where the current value is higher (hereinafter, referred to as "upper side") depending on the configuration of the biosensor 1 and the type of sample.

On the other hand, in the sensor element 5b in which the target particle 22 did not enter the opening 15, the current value does not change as shown in FIG. 4B, and the target particle 22 oscillates at the same position. Although the case where the voltage is constant is described here for convenience, the voltage value fluctuates slightly because the channel resistance actually changes. Therefore, by measuring not only the current value but also the potential difference and measuring it as a change in the channel resistance, it is possible to detect the capture of the target particle 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. Even in that case, the current value changes.

Next, the steps (S4) and subsequent steps will be described with reference to FIG. After washing the sample solution in the step (S3) to wash away the excess target particles, the trap is added into the space 4 in the step (S4). Since the capture body 24 is a substance that specifically binds to the target particle 22, for example, it binds to the target particle 22 that has entered the opening 15. In order to bind the trap 24 to the target particles 22, it is preferable to leave the trap 24 for about 1 hour, for example. On the other hand, the trap 24 does not bind to the non-target particle 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 opening 15.

In the step (S5), the resistance between the source and drain is measured. It is desirable that the measurement is continuously performed after the step (S3) and immediately before the step (S4), but the measurement may be performed intermittently if necessary. For example, if the capture body 24 is labeled with a labeling substance 25 having a negative charge, when the capture body 24 binds to the target particles 22 in the opening 15, the resistance between the source and drain of the sensor element 5a binds to the capture body 24. It changes compared to before. For the sake of convenience, if the voltage is constant, the current value decreases or increases as shown in FIG. 5 (a). On the other hand, in the sensor element 5b in which the non-target particles 23 are contained in 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. 5 (b). There is no change in the current value, and it swings at the same position. Although the change in the current value has been described for convenience, it is the resistance value that actually changes, and the more accurate measurement can be performed by detecting the change in the resistance value is the same as described above.

In the step (S6), the presence or absence or the amount of the target particle 22 is determined from the measurement result of the step (S5). For example, when there is a sensor element 5 whose resistance value has changed, it can be determined that the target particles 22 are present in the sample. On the contrary, 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 having the target particles 22 in the opening 15. On the contrary, the sensor element 5 whose resistance value does not change can be considered that the target particle 22 does not exist in the 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 particles 22 are present in the opening 15 or the target particles 22 are present in the sample. May be good. Such a threshold value can be determined by subjecting a sample in which the target particle 22 is known to be present to the 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 the sensor elements 5 in which the resistance value has changed or the sensor elements 5 in which the amount of change is equal to or greater than the threshold value.

The change in resistance value will be described with reference to an example in which graphene is used as the channel 12. Since graphene has bipolar characteristics, there are a state in which electrons are carriers and a state in which pores are carriers. Therefore, as shown in FIG. 8, _{the relationship between the gate voltage (V g} ) and the source-drain current value ( _ISd ) shows a V shape.

Since the target particles 22 and the trap 24 have a negative charge, electrons are injected from the channel 12a into the channel 12 when they enter the opening 15. 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 drain (indicated by the double-headed arrow in the figure), this shift reduces _ISd. Therefore, when the target particle 22 or the trap 24 enters 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 trapping body 24 do not enter the opening 15, the shift does not occur, so that the particles oscillate at the same position as shown in FIG. 8 (b). In this way, the entry of the target particle 22 or the trap 24 into the opening 15 can be detected as a change in the current value (more accurately, a change in the resistance value).

In a further embodiment, steps (S4)-(S6) may be repeated using different types of traps. That is, after performing the steps (S4) to (S6) by adding the first type of trapping material as described above, a different type of trapping material that specifically binds to a different type of target particle is added. , Steps (S4) to (S6) can be carried out in the same manner. That is, by performing the steps (S4) to (S6) using a plurality of types of traps, it is possible to continuously detect a plurality of types of target particles. If the position of the sensor element 5 whose resistance value has changed is recorded each time the process is repeated, the position information of the sensor element 5 in which the plurality of target particles 22 are present can be obtained.

In the above description, the channel resistance is measured by AC, but DC measurement is also possible. 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 apply a gate voltage for measurement. In that case, as shown in FIG. 7, the switch 53 is connected to the DC power supply 57 side. However, when the DC measurement is performed, the dielectrophoresis is cut off, so that the target particles may go out from the opening 15. In such a case, as shown in FIGS. 6 and C, a probe that captures the target particle may be fixed to the surface of the channel 12a.

To be precise, the capture body recognizes and binds to the biomarker expressed on the surface of the target particle. Therefore, since the number of bindings of the trapping agent changes according to the expression level of the biomarker, the expression level 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 traps, the resistance value changes in each of the traps, so that the particle expresses any biomarker. It is possible to detect that.

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 housed one by one in the opening 15, and the target particles 22 are moved by the change in the resistance value. To detect. Therefore, the target particles in the sample can be digitally counted, and the target particles can be detected with high sensitivity and specifically. Further, since the sample can be detected even if it is used as it is, it is not necessary to perform a step such as centrifugation or extraction in advance, the operation is simple, and contamination and destruction of target particles can be prevented. In addition, 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 traps, information on the type and number of target particles can be obtained.

As the trap 24, for example, an antibody or an antigen-binding fragment, a lectin, or a naturally occurring 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, the trap 24 that binds to a protein, glycoprotein, lipid, sugar chain, cfDNA attached to the surface, or the like expressed on the surface of the extracellular vesicle. Can be used.

As the labeling substance 25 that labels the trap 24, for example, a negatively charged anion, a positively charged cation, a fluorescent dye, a metal bead, a dielectric bead, or the like can be used. Labeling of the trap 24 can be performed using any known method. The trap 24 may be prepared, for example, as a reagent in which the trap 24 is contained 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 feeding unit that sends a sample, a cleaning agent, a reagent, or the like to the space 4, and a data processing unit that stores and processes resistance value information of each sensor element 5. And a control unit that controls the operation of each of these units. The operation of the above steps may be executed by the input of the operator of the apparatus, or may be executed by the program included in the control unit.

By detecting the presence or absence or the 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 particle 22 is an extracellular vesicle, the detection thereof indicates the health condition of the living body from which the sample is derived, for example, the presence / absence, type, course, pharmacological effect or susceptibility to a specific disease. It is possible to obtain the information of. Specific diseases include, but are not limited to, cancer, dementia such as Alzheimer's disease, Parkinson's disease, and Lewy body dementias, neurodegenerative diseases, and acute renal disorders. When a 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 condition, can be obtained.

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

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

In the target particle detection method of the first embodiment, a biosensor 1 having an upper electrode 3 as shown in FIG. 7 and not having a shielding electrode 14 may be used. 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 no voltage is applied to the upper electrode 3 and an AC voltage is applied between the drain electrode 9 and the second source electrode 11 of each sensor element 5, the target particle 22 opens the opening 15. It is possible to 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 are caught in the portion laminated on the channel 12 of the insulating film, so that the size of the channel 12 is large. It is preferable to make the size 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 deleted in order to simplify the configuration. Such a biosensor 1 can also be used in the above-mentioned target particle detection method.

(Second embodiment)
-Biosensor FIG. 10 is a cross-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 this embodiment, the upper electrode 3a is arranged between the openings of the sensor elements arranged in the row direction and between the openings of the sensor elements arranged in the row direction, and has a grid shape. Eggplant. The area of the upper electrode 3a when viewed in a plan view 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 showing how the biosensor of this embodiment is used to capture target particles. The upper electrode 3a is connected to the ground potential via the switch 51 and the switch 53. The shielding electrode 14 is connected to the ground potential via the switch 53. The channel 12 is connected to the AC power supply via the switch 50 and the switch 52. As a result, as in FIG. 4, the distribution is such that the electric field strength becomes stronger toward the inside of the opening 15, so that the target particles penetrate into the opening 15. Similar to FIG. 4, the invasion of the target particles can be electrically detected by the change in the current of the channel 12a (more accurately, the change in resistance).

FIG. 13 is a cross-sectional view showing how the capture body is bound to the target particles using the biosensor of this example. The switches 51, 53, 50, and 52 are as shown in FIG. 12, and the point that the switches 51, 53, 50, and 52 can be electrically detected by the current change (more accurately, the resistance change) of the channel 12a is the same as in FIG.

14 and 15 are cross-sectional views and plan views illustrating a method of recovering or discharging desired target particles using the biosensor of this embodiment. FIG. 14 is a cross-sectional view showing a method of discharging only desired target particles from the opening 15. The upper electrode 3a is connected to the AC power source 18 by the switch 51. The shielding electrode is left grounded by the switch 53. The switch 50 is switched for each individual sensor element 5. For example, the switch 50 connected to the sensor element 5 in which the target particles that are not desired to be collected or discharged are captured remains 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 the target particles to be collected or discharged are captured is connected to the ground line 20 side. As a result, 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 12 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 strength distribution becomes stronger toward the upper electrode 3a side. Therefore, the target particles captured in the channel 12a connected to the ground line 20 are discharged toward the upper electrode 3a by dielectrophoresis. On the other hand, since the channel 12a connected to the AC power supply 18 has the same potential as the upper electrode 3, an electric field distribution with the upper electrode 3a is not formed. Since the electric field distribution with the shielding electrode remains, a local electric field strength distribution is formed around the opening 15. Since this electric field strength distribution is sparse toward the outside of the opening 15, the target particles captured by the channel 12a connected to the AC power source 18 remain inside the opening 15.

FIG. 15 is a plan view showing the state of discharge of the target particles in FIG. Target particles that have entered the opening 15b on the channel connected to the AC power source 18 remain trapped in the opening 15b, whereas the opening on the channel connected to the ground line 20 remains trapped. The target particles contained in 15a are discharged toward the upper electrode 3a. When a flow is applied 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, the target particles discharged along the line of the upper electrode 3a are re-captured. There is no.
-Target particle detection method FIG. 16 is a schematic flow 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) Contain the sample solution in the space of the biosensor, (S2) Connect the upper electrode 3a and the shielding electrode to the ground line, connect each sensor element to the AC power supply, and cause dielectric migration to move the target particles to each. Capturing in the opening on the sensor element, (S3) washing the sample solution to expel excess target particles, and (S4) adding a trap that specifically binds to the target particles into the space. , (S5) to measure the current value flowing through each sensor element and the potential difference between at least two electrodes, (S6) to determine the presence or absence or amount of target particles in the sample according to the result of the measurement, (necessarily). (S3 to 6 are repeated depending on the type of capture body), (S7) the sensor element in which the target particle to be collected or discharged is captured is determined according to the result of the measurement, and (S8) collection or discharge. The sensor element that captures the target particle to be desired and the shielding electrode are grounded, the other sensor element and the upper electrode 3a are connected to the AC power supply, and (S9) the liquid in space 4 is given a flow to collect or discharge the target particle. Is collected or discharged from the space 4 on the sensor array.

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

In the step (S7), from the measurement results up to the step (S6), the target particles showing a desired result for binding of each trap, that is, a positive, negative, or desired response amount (resistance change amount). Read the position of the sensor element in which the subclass is captured. From this result, determine the subclass that you want to collect and send to another analysis, or the subclass that you want to discharge because it is unnecessary.

Next, in the 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, the sensor element containing the subclass to be captured in the opening 15 is connected to the AC power supply. Further, the shielding electrode is grounded to the ground, and the upper electrode 3a is connected to the AC power supply. The exposed portion in the opening 15 of the sensor element grounded to the ground forms an electric field with the upper electrode 3a together with the shielding electrode, and the electric field strength becomes denser on the upper electrode 3a side. The target particles in the part 15 are discharged. On the other hand, the exposed portion in the opening 15 of the sensor element connected to the AC power source forms a local electric field with the shielding electrode, and the electric field strength is sparsely distributed toward the outside of the opening. , The target particle is still retained in the opening 15.

Next, in the step (S9), if some flow is applied to the liquid in the space 4, the target particles discharged from the opening in the 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 showing an example of the biosensor of the third embodiment. The biosensor 1 in this example includes a probe 26 on the surface of the channel 12a exposed to the opening 15. Other than that, the configuration is the same as that 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, a naturally occurring nucleic acid, an artificial nucleic acid, an aptamer, a peptide aptamer, an oligopeptide, a lectin, a protein, or the like.

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, cfDNA attached to the surface, or the like is used. Can be used.

In addition, the probe 26 dissociatively binds the target particle 22 and the channel 12. For example, they are configured to be capable of cleaving the bond between the target particle 22 and the probe 26, within the probe 26 or between the probe 26 and the channel 12, such as by adding appropriate reagents.

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 His-Tag and Ni-NTA. In this case, the addition of imidazole can break the bond between His-Tag and Ni-NTA.

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 the Fc portion of the antibody to the protein A. In this case, the bond between the protein A and the antibody can be cleaved 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 the antibody GST and glutathione. In this case, the addition of reduced glutathione replaces the binding between GST and glutathione with the binding to reduced glutathione, allowing the antibody to cleave the binding to channel 12.

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 bound. In this case, the binding to the probe 26 is replaced by the competing ligand from the target particle 22 by adding a competing ligand that competes with the target particle 22 for binding to the probe 26. As a result, the target particles 22 can be dissociated. The competing ligand is preferably, 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 and the target particle 22 can be dissociated by adding RNase, DNAase, and protease, respectively.

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

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

(A) When a 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 in the channel 12a.

In the step (S13), since the electric field strength gradient opposite to that in the step (S12) is applied, the non-target particles 23 that did not bind to the probe 26 and the contaminants that entered the opening 15 can be removed. On the other hand, since the target particles 22 are captured by the probe 26, they are not released from the opening 15.

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

After that, (S15) the resistance value between the source and drain electrodes of each sensor element 5 may be measured, and (S16) the presence or absence or amount of the target particles 22 in the sample may be determined. If the resistance value was measured during the operation of the step (S12), the target particles 22 remained in the opening 15 after cleaning due to the difference from the resistance value before the target particles 22 entered the opening 15. The sensor element 5 can be specified.

When the presence of the target particle 22 is confirmed by the steps (S15) and (S16), a reagent for dissociating the bond between the channel 12 and the target particle 22 is added in the step (S17) thereafter. In (S18), the target particles 22 are released into the space 4 by applying the same electric field as in the step (S13), and further, by flowing the liquid in the space 4 in the step (S19), the target particles 22 It is also possible to collect. 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 centrifuging or extracting the sample. The method including the steps (S11) to (S19) is also referred to as a "target particle separation method". The separated and recovered target particles 22 can also be further analyzed. For example, when the target particle 22 is an extracellular vesicle, membrane proteins, lipids, sugar chains, inclusions, and the like can be analyzed after recovery. Alternatively, the response to cytokines, ligands, cells and biological tissues can be confirmed in vitro, and the effect on model animals can be confirmed in vivo.

(B) When a probe 26 that selectively captures the target particles 22 is used In this example, by performing the steps (S11) to (S12), for example, a large group of objects including the target particles 22 can be used as the probe 26. Is captured by. In the subsequent step (S13), impurities composed of other objects are discharged from the opening 15 into the space 4 by dielectrophoresis, and these impurities are removed by washing in the step (S20). Can be done. By this step, a large group of objects including the target particles 22 can be once selected from the sample, and the accuracy of the subsequent detection using the trap 24 can be improved.

Next, as a further detection step using the capture body 24, (S21) a capture body that specifically binds to the target particle is added into the space, and (S22) the resistance value between the source and drain electrodes of each sensor element is determined. Measurement, (S23) The presence or absence or amount of target particles in the sample may be determined according to the result of the measurement. The steps (S20) to (S23) can be performed in the same manner as the steps (S3) to (S6) in the above-mentioned 1.5th embodiment, respectively. The step (S21) may be performed by generating dielectrophoresis as in the step (S12). When the catcher 24 has a high dielectric constant or when a label having a high dielectric constant is modified, the trap 24 can be brought closer to the opening 15 by dielectrophoresis to facilitate binding to the target particles 22. Alternatively, if it is considered that the capture body 24 approaches the opening 15 in which the target particle 22 has not entered due to the dielectrophoresis, which adversely affects the detection, the dielectrophoresis may be stopped or the step. Dielectrophoresis of the same reverse gradient as in (S13) may be performed. When the dielectric constant of the trap 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 repeated using the plurality of capture bodies 24 to detect the plurality of target particles 22.

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

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

(Fourth Embodiment)
-Target particle detection method FIG. 21 is a schematic flow showing an example of the target particle detection method of the fourth embodiment. In the method, the steps (S31) to (S33) are the same as the steps (S11) to (S13) of the second embodiment, and the steps (S34) to (S39) are the same as those of the second embodiment. The steps (S14) to (S19) are the same, and the steps (S40) to (S47) are the same as the steps (S20) to (S27) of the second embodiment. The difference between this embodiment and the second embodiment is 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 source 18 by the switch 50 and the switch 52, the shielding electrode is grounded by the switch 53, and the upper electrode is grounded by the switch 51 and the switch 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 strength becomes dense toward the opening 15. Therefore, the target particles are attracted into the opening 15 by dielectrophoresis. Here, if there are non-target particles having a high dielectric constant like the target particles, they are similarly attracted to the opening 15 by dielectrophoresis. In addition, if the properties of the non-target particles are similar to those of the target particles, the electrical signals detected by the sensor element will also be similar, and the difference between the labeled particles and the unlabeled particles cannot be distinguished at this stage. ..

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

FIG. 15 shows a cross-sectional view when the step (S32) is performed again. New particles are attracted to the opening where the non-target particles are 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 remains in the opening is the signal as 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 these detection information. Alternatively, the number of repetitions may be simply determined for processing.

The repeating step is particularly effective when the abundance of the target particles 22 in the sample is expected to be small. For example, cancer-derived extracellular vesicles may be present in only about 1% of the total extracellular vesicles. In this case, when extracellular vesicles are simply collected, even if 1,000 extracellular vesicles are collected, only about 10 extracellular vesicles derived from cancer can be recovered on average. Given the diversity of cancer-derived extracellular vesicles, there is a risk that low-expression subclasses will fail to be recovered. In such cases, for example, if a surface marker characteristically expressed in extracellular vesicles derived from cancer, for example, a probe that binds to CD9, is used, cancer frequently occurs in the captured extracellular vesicles. It will contain the extracellular vesicles of origin.

Therefore, by using this embodiment, the desired target particles can be efficiently recovered, so that the risk of overlooking the subclass contained in the target particles is small, it is possible to detect rare subclasses, and the scale of the sensor array is also large. It can be made smaller.

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 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 ... Current meter 22 ... Target particle 24 ... Capturer 25 ... Labeling substance 26 ... Probe

Claims (28)

A biosensor for detecting target particles in a sample.
A sensor array in which a plurality of sensor elements are arranged on a first substrate is provided.
Each of the plurality of sensor elements
With the first insulating layer provided on the first substrate,
At least two electrodes embedded in the first insulating layer with a gap from each other,
With the at least two electrodes and a channel provided on the gap and connected to the at least two electrodes.
A first insulating layer and a second insulating layer provided on the channel and having an opening corresponding to the gap to expose the channel.
A second electrode provided above the opening is provided.
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 shielding electrode provided on the second insulating layer excluding the opening, or a space is opened above the sensor array so as to face the second insulating layer. The biosensor according to claim 1, which is an upper electrode to be arranged. The second electrode is a shielding electrode provided on the second insulating layer excluding the opening, and is arranged with a space above the sensor array so as to face the second insulating layer. The biosensor according to claim 1, further comprising an upper electrode. The third aspect of claim 3, wherein the area of the upper electrode when the biosensor is viewed in a plan view is smaller than that of the shielding electrode, and the upper electrode is arranged between the opening and the opening of each sensor element. Biosensor. The biosensor according to claim 3 or 4, further comprising a first circuit including a second power source that applies an AC voltage or a DC voltage to the upper electrode. The biosensor according to any one of claims 1 to 5, further comprising a plurality of second circuits including a first power source that applies an AC voltage or a DC voltage to the at least two electrodes of each sensor element. .. Claims 1 to further include a probe immobilized on the channel exposed from the opening and selectively or specifically capturing the target particle, the probe dissociatively binding the channel and the target particle. 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 is bound to Ni-NTA immobilized on the channel. The biosensor according to claim 7, wherein the probe is an antibody, and the antibody is bound to protein A immobilized on the channel. The biosensor according to any one of claims 1 to 9, wherein the opening has a size in which only one target particle can enter. A method for detecting target particles in a sample.
(I) A sensor array in which a plurality of sensor elements are arranged on a first substrate is provided, and the plurality of sensor elements each have a first insulating layer provided on the first substrate and the first insulation. At least two electrodes embedded in the layer with a gap from each other, the at least two electrodes and a channel provided on the gap and connected to the at least two electrodes, the first insulating layer and the channel. To provide a biosensor comprising a second insulating layer provided above and having an opening corresponding to the gap to expose the channel .
(Ii) placing the sample onto the sensor array,
And (iii) to cause the first dielectrophoresis, capturing a target particle in the sample to the opening of each of said sensor element,
( Iv ) Adding a trap that specifically binds to the target particle onto the sensor array,
( V ) Measuring the current value flowing through each sensor element and the potential difference between at least two electrodes provided in the sensor element continuously from immediately before the step ( iv).
( Vi ) A method for detecting target particles, which comprises determining the presence or absence or amount of the target particles in the sample according to the result of the measurement. The prepared biosensor further comprises a second electrode on the second insulating layer, and after the step (iii), the second electrode is subjected to a stronger electric field than the channel exposed in the opening. The method according to claim 11, wherein the second dielectrophoresis for causing the occurrence is caused, and the second dielectrophoresis and the first dielectrophoresis are repeated a plurality of times. Was prepared the biosensor is fixed to the channel that is exposed from the opening, further comprising a probe for capturing the target particles, the probe is detachably connected to said said channel target particles according Item 10. The method according to Item 11. The method according to any one of claims 11 to 13 , further comprising stopping the first dielectrophoresis and cleaning the sensor array after the step ( iii). The method according to any one of claims 11 to 14 , wherein the steps ( iv ) to ( vi ) are repeated using different types of traps. A method for detecting target particles in a sample.
(I) A sensor array in which a plurality of sensor elements are arranged on a first substrate is provided, and the plurality of sensor elements each have a first insulating layer provided on the first substrate and the first insulation. At least two electrodes embedded in the layer with a gap from each other, the at least two electrodes and a channel provided on the gap and connected to the at least two electrodes, the first insulating layer and the channel. To prepare a biosensor including a second insulating layer provided above and having an opening corresponding to the gap to expose the channel and a probe fixed to the surface of the channel exposed from the opening. ,
(Ii) placing the sample onto the sensor array,
( Iii ) Performing the first dielectrophoresis, capturing the target particles in the opening of each sensor element, and specifically capturing the target particles with the probe of the channel exposed from the opening.
(Iv) after the step (ii), continuously from immediately before the step (iii), a value of current flowing to the each sensor element, measuring a potential difference between at least two electrodes of said sensor element,
(V) A method for detecting target particles, which comprises determining the presence or absence or amount of the target particles in the sample according to the result of the measurement. The prepared biosensor further comprises a second electrode on the second insulating layer, and after the step (iii ), the second electrode is subjected to a stronger electric field than the channel exposed in the opening. It Oko a second dielectrophoresis for generating method of claim 16 are repeated a plurality of times and the first dielectrophoretic with the second dielectrophoretic. 16. The method of claim 16, further comprising stopping the first dielectrophoresis and cleaning the sensor array after the step ( iii). The method according to any one of claims 11 to 18 , wherein the opening has a size in which only one target particle can enter. The method according to any one of claims 11 to 19 , wherein the target particle is an extracellular vesicle. A method of separating target particles in a sample.
(I) A sensor array in which a plurality of sensor elements are arranged on a first substrate is provided, and the plurality of sensor elements each have a first insulating layer provided on the first substrate and the first insulation. At least two electrodes embedded in the layer with a gap from each other, the at least two electrodes and a channel provided on the gap and connected to the at least two electrodes, the first insulating layer and the channel. To provide a biosensor comprising a second insulating layer provided above and having an opening corresponding to the gap to expose the channel .
(Ii) placing the sample onto the sensor array,
( Iii ) To cause the first dielectrophoresis and capture the target particles in the sample in the openings of each sensor element.
( Iv ) Adding a trap on the sensor array, which specifically binds to the target particle,
(V) a current value flowing to the each sensor element, measuring the potential difference between the at least two electrodes of said sensor element,
( Vi ) Determining the presence or absence or amount of the target particles in the sample according to the results of the measurement.
( Vii ) A method for separating target particles, which comprises dissociating a channel exposed in the opening from the target particles and recovering the target particles. The prepared biosensor further includes a second electrode on the second insulating layer.
The steps ( iv ) to ( vi ) are repeated using different types of traps, and the steps (iv) to (vi) are repeated.
Wherein step (vii) is at the sensor element in which the target particles are present to be recovered, causing a second dielectrophoresis for generating a strong electric field than the channel exposed to the opening in the second electrode, The method according to claim 21 , wherein the first dielectrophoresis is performed on a plurality of types of the target particles by the other sensor elements, and the plurality of types of the target particles are recovered. A method of separating target particles in a sample.
(I) A sensor array in which a plurality of sensor elements are arranged on a first substrate is provided, and the plurality of sensor elements each have a first insulating layer provided on the first substrate and the first insulation. At least two electrodes embedded in the layer with a gap from each other, the at least two electrodes and a channel provided on the gap and connected to the at least two electrodes, the first insulating layer and the channel. To prepare a biosensor including a second insulating layer provided above and having an opening corresponding to the gap to expose the channel and a probe fixed to the surface of the channel exposed from the opening. ,
(Ii) placing the sample onto the sensor array,
( Iii ) Performing the first dielectrophoresis, capturing the target particles in the opening of each sensor element, and specifically capturing the target particles with the probe of the channel exposed from the opening.
(Iv) after the step (ii), continuously from immediately before the step (iii), a value of current flowing to the each sensor element, measuring a potential difference between at least two electrodes of said sensor element,
(V) Determining the presence or absence or amount of the target particles in the sample according to the result of the measurement (vi) Target particle separation including dissociating the channel and the target particles and recovering the target particles. Method. The biosensor was prepared, the further comprises a second electrode on the second insulating layer, after the step (iii), wherein each sensor element, the second electrode, exposed to the opening the method of claim 23, and caused the second dielectrophoretic for generating a strong electric field than the channel, is repeated a plurality of times and the first dielectrophoretic with the second dielectrophoretic. The prepared biosensor further includes a second electrode on the second insulating layer, and in the step (vi), the opening in the second electrode in the sensor element in which the target particle is present. The method according to claim 23 , wherein the second dielectrophoresis is performed to generate an electric field stronger than the channel exposed in the portion , and the first dielectrophoresis is performed in the other sensor elements. The method according to any one of claims 21 to 25 , further comprising stopping the first dielectrophoresis and cleaning the sensor array after the step ( iii). The method according to any one of claims 21 to 26 , wherein the opening has a size in which only one target particle can enter. The method according to any one of claims 21 to 27 , wherein the target particle is an extracellular vesicle.

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