JPWO2016063858A1 - Electrical measuring device - Google Patents

Abstract

Provided are an electrical measurement chip designed to detect not only a change in steady-state current but also a transient current, so that highly sensitive detection is possible, and an electrical measurement device including the electrical measurement chip. A substrate, a sample moving channel formed on the substrate, and a sample measuring channel, wherein the sample measuring channel is a first measuring channel connected to the sample moving channel and the first measuring channel An electric measurement chip including a second measurement channel connected to the sample movement channel from the opposite side.

Description

  The present invention relates to an electric measurement chip and an electric measurement device, and particularly, when a sample such as a cell, a bacterium, a virus, or a DNA flows through a microchannel, reads not only the change in steady current but also the generation of a transient current. Thus, the present invention relates to an electrical measurement chip designed to perform highly sensitive detection, and an electrical measurement device including the electrical measurement chip.

  Accurate measurement of the size, number, etc. of samples such as cells, fungi, pollen, and PM2.5 contained in a solution is important information for living a healthy life. Improvement is desired. In the field of biochemistry, it is desired to develop an analysis chip that analyzes a DNA fragment as it is.

  FIG. 1 shows a conventional technique for measuring the size and number of samples. The sample is passed through pores (micropores) formed on a substrate such as silicon, and the pores are applied by voltage applied to the pores. The size and hardness of the cell are analyzed from the state in which the steady-state current flowing inside the cell changes (see Non-Patent Document 1). The conventional measurement method shown in FIG. 1 is known to improve the sensitivity as the volume of the pores decreases. In order to reduce the volume of the pores, it is necessary to reduce the diameter and reduce the thickness of the substrate. For this reason, the substrate is used in a vertical orientation as shown in FIG.

  In addition, in order to measure the state of the sample passing through the pores in more detail, the substrate on which the microchannels are formed can be placed sideways so that the pores can be observed with a fluorescence microscope. A method of directly observing an event around a hole is also known (see Non-Patent Document 2). 2 is shown in FIG. 1 is shown.

Wasem A. et al. Lab on a Chip, Vol. 12, pp. 2345-2352 (2012) Naoya. Y et al. , "Tracking single-particle dynamics via combined optical and electrical sensing", SCIENTIFIC REPORTS, Vol. 3, pp. 1-7 (2013)

  However, in the method shown in FIG. 1, the information obtained is only a signal indicating a change in the steady current, and the discrimination of the sample that has passed through the pores can only be inferred from the strength of the current value. Therefore, there is a problem that detailed analysis is difficult (problem 1) when a plurality of samples flow into the pores, or when the shape of the sample to be measured is other than a sphere or is easily changed.

  In the method described in Non-Patent Document 1, the drive circuit for driving the sample and the measurement circuit for measuring the current change when the sample passes through the pore are the same. In general, the measurement sensitivity can be increased by increasing the applied voltage. However, if the drive circuit and the measurement circuit are the same, increasing the applied voltage will overload the ammeter of the measurement circuit and prevent high sensitivity detection. (Problem 2).

  Furthermore, since the time for which the sample passes through the pores is influenced by the surface charge, deformability, etc. of the sample, it is very important information particularly in biomolecule analysis. However, with the sensitivity of the conventional method, only a gradual change in the steady current in the pore can be read, and there is a large error in reading the time for the sample accelerated by the applied voltage to pass through the short pore. In addition, when measuring elongated biomolecules such as nucleic acids, it is necessary to introduce the biomolecules into the pores in an elongated state. To this end, a guide channel for bringing the biomolecules into an elongated state is required. It becomes. However, the provision of the guide channel increases the volume of the pore portion, and there is a problem that a decrease in sensitivity is inevitable (Problem 3).

  On the other hand, as shown in FIG. 2, the above (Problem 1) can be solved by placing the substrate horizontally and observing with a fluorescence microscope. However, the method described in Non-Patent Document 2 is designed such that the sample dispersed in the liquid by the pressure of the pump passes through the pores formed on the substrate. When a sample dispersed in a liquid is flowed by the pressure of the pump, the liquid is less likely to flow through the pores as the pore size is reduced in order to increase measurement sensitivity. Of course, the liquid can be flowed by increasing the pressure of the pump, but if the pressure is increased too much, the pores may be damaged. Further, the method of flowing a sample with the pressure of a pump described in Non-Patent Document 2 has a problem that nucleic acid or protein cannot be flowed. Further, the method described in Non-Patent Document 2 also needs to reduce the volume of the pores in order to increase the sensitivity, similarly to the method described in Non-Patent Document 1, and the above (Problem 3) There is a problem that cannot be solved.

The present invention is an invention made in order to solve the above-mentioned conventional problems.
(1) A sample moving channel capable of flowing a sample is formed, and the first measuring channel connected to the sample moving channel and the sample moving channel are connected from the side opposite to the first measuring channel. By forming the second measurement channel, the sample drive circuit and the measurement circuit can be designed as separate circuits,
(2) By making the sample drive circuit and measurement circuit separate, the detection sensitivity can be increased by setting the voltage of the drive circuit high, and it is possible to measure transient currents that were previously buried in noise,
(3) If a variable resistor is incorporated in the measurement circuit, higher sensitivity can be detected and the transient current can be measured more accurately.
(4) By reading the transient current, it is possible to accurately measure the sample entry / exit timing to the sample movement flow path, and as a result, the surface charge and deformability of the sample are measured by calculating the passage speed of the sample. What you can do,
(5) Since the change in the steady current between the first measurement channel and the second measurement channel can be measured separately from the transient current, the sample moving channel can be designed longer than the conventional pore, It was newly discovered that biomolecules such as nucleic acids and proteins can be measured by creating an extended state of the sample in the movement channel.

  That is, an object of the present invention is to provide an electrical measurement chip designed to detect highly sensitively by reading not only a change in steady current but also a transient current, and an electrical measurement device including the electrical measurement chip. Is to provide.

  The present invention relates to an electrical measurement chip and an electrical measurement apparatus including the electrical measurement chip, which will be described below.

(1) including a substrate, a sample moving channel formed on the substrate, and a sample measuring channel,
The sample measurement flow path includes a first measurement flow path connected to the sample movement flow path and a second measurement flow path connected to the sample movement flow path from the side opposite to the first measurement flow path.
Chip for electrical measurement.
(2) The width of the first measurement channel and the second measurement channel is longer as the distance from the sample movement channel is longer than the length of the portion connected to the sample movement channel (1) The electrical measurement chip according to 1.
(3) The electrical measurement chip according to (1) or (2), wherein the first measurement channel and the second measurement channel are formed at asymmetric positions with the sample movement channel interposed therebetween.
(4) The electrical measurement chip according to any one of (1) to (3), wherein at least one narrow portion is formed in the sample moving flow path.
(5) Any one of the above (1) to (4), including a sample input channel formed at one end of the sample moving channel and a sample recovery channel formed at the other end of the sample moving channel. The electrical measurement chip according to 1.
(6) The electrical measurement according to any one of (1) to (5) above, wherein a first measurement electrode and a second measurement electrode are formed instead of the first measurement channel and the second measurement channel. For chips.
(7) The electrical measurement chip according to any one of (1) to (5) above,
A drive circuit for allowing the sample to move through the sample transfer channel,
A measurement circuit that applies a voltage to the first measurement channel and the second measurement channel and measures a change in current when the sample moves through the sample moving circuit;
Including electrical measuring device.
(8) The electrical measurement chip according to (6) above,
A drive circuit for allowing the sample to move through the sample transfer channel,
A measurement circuit for applying a voltage to the first measurement electrode and the second measurement electrode and measuring a change in current when the sample moves through the sample moving circuit;
Including electrical measuring device.
(9) The measurement circuit includes a variable resistor for making the resistance of the drive circuit and the measurement circuit balanced.
The electrical measurement device according to (7) or (8), wherein the measurement circuit measures a difference in current from a balanced state.
(10) The electrical measurement device according to any one of (7) to (9), wherein the measurement circuit measures changes in transient current and steady current.
(11) The electrical measurement apparatus according to any one of (7) to (10), further including a fluorescence microscope.

The electrical measurement chip of the present invention forms a sample movement channel through which a sample can flow, and the first measurement channel connected to the sample movement channel and the sample from the side opposite to the first measurement channel A second measurement channel connected to the moving channel is formed.
For this reason, the electrical measuring device using the electrical measuring chip of the present invention can design the sample driving circuit and the measuring circuit as separate circuits, so that the voltage of the driving circuit can be set high and the detection sensitivity can be increased. The current can also be read accurately. Furthermore, when a variable resistor is incorporated in the measurement circuit, the difference from the state in which the drive circuit and the measurement circuit are balanced can be read, so that the detection sensitivity can be further increased.
The electrical measurement apparatus of the present invention can accurately measure the sample entry / exit timing to the sample movement channel by reading the transient current, and can measure the surface charge and deformability of the sample from the passing speed. . In addition to the transient current, the change in the steady current between the first measurement channel and the second measurement channel can be measured. Therefore, unlike the conventional pore, the sample movement channel can be designed longer and the sample movement It is possible to create and measure the elongation state of biomolecules such as nucleic acids and proteins in the channel.
Furthermore, since the electrical measuring chip of the present invention can be used in a horizontal orientation, it can be used for a more accurate analysis by using it in combination with fluorescence microscope observation.
In addition, by providing a stenosis portion in the sample movement channel, cells having different deformability can be measured even for the same type of cells.

FIG. 1 shows a conventional technique for measuring the size and number of samples. 2 is shown in FIG. 1 is shown. FIG. 3 is a diagram for explaining the outline of the electrical measurement chip 1 of the present invention. FIG. 4 shows another embodiment of the electric measurement chip 1 of the present invention. FIG. 5 is a diagram for explaining an outline of the electrical measurement chip 1 formed with the first electrode and the second electrode in place of the first measurement channel 6 and the second measurement channel 7. FIG. 6 shows another embodiment of the electric measurement chip 1 of the present invention. FIG. 7 is a cross-sectional view taken along the line A-A ′ of FIG. 4 and shows an example of the manufacturing process of the electrical measuring chip 1. FIG. 8 is a diagram showing another manufacturing process of the electrical measurement chip 1 of the present invention. FIG. 9 is a diagram showing an outline of an electricity measuring device 10 using the electricity measuring chip 1 of the present invention. FIG. 10 is a diagram for explaining the relationship between the position of the sample on the electrical measurement chip 1 and the current value that can be measured when measuring the sample using the electrical measurement apparatus 10 of the present invention. FIG. 11 is a view showing another embodiment of the electricity measuring chip 1. FIG. 12 is a drawing-substituting photograph, FIG. 12 (1) is a photograph of the electrical measurement chip 1 produced in Example 1, and FIG. 12 (2) is the first measurement channel 6 and the second measurement channel 7. This is an enlarged photo of the neighborhood. FIG. 13 is a drawing-substituting photograph, and FIG. 13 (1) is an enlarged photograph of the vicinity of the first measurement channel 6 and the second measurement channel 7 of the electrical measurement chip 1 manufactured in Example 2, and FIG. ) Is an enlarged photograph of the vicinity of the first measurement channel 6 and the second measurement channel 7 of the electrical measurement chip 1 manufactured in Example 3. FIG. FIG. 14 (1) is a graph showing the relationship between the measurement time and the measured steady current value in Example 4, and FIG. 14 (2) shows the relationship between the measurement time and the measured steady current value in Example 5. FIG. 14 (3) is a graph showing the relationship between the measurement time and the measured steady current value in Example 6. FIG. 15 is a diagram for explaining the reason why two peaks were measured when the electrical measurement chip 1 of Example 3 was used. FIG. 16 is a continuous photograph of the position of the sample flowing through the sample moving flow path 3, and a photograph and a graph showing a change in steady-state current value (signal intensity) and a change in fluorescence intensity when the sample flows. FIG. 17 is a graph showing changes (signal intensity) in steady-state current values measured in Example 8. FIG. 18 is a graph showing changes in volume (steady current value) (signal intensity) of a sample prepared based on the results measured in Example 8. FIG. 19 is a diagram showing the relationship between the voltage of the drive circuit and the time for the sample to pass through the sample movement channel. FIG. 20 (1) is a drawing-substituting photograph, an enlarged photograph of the vicinity of the sample movement channel of the electrical measurement chip 1 produced in Example 12, and FIG. 20 (2) is an electrical measurement chip 1 produced in Example 12. It is a figure for demonstrating the dimension of the sample moving flow path vicinity. FIG. 20 (3) is a histogram of steady current values, and is a graph showing the distribution of the number of cells counted at each steady current value. FIG. 21 (1) is a drawing-substituting photograph, which is an enlarged photograph of the vicinity of the narrowed portion 34 of the sample moving channel 3. FIG. 21 (2) is a diagram for explaining the length and width of the sample moving flow path 3 and the narrowed portion 34. FIG. 22 (1) shows the time (in) and time (out) when the HeLa cells flowed from the left to the right of the chip shown in FIG. It is a graph which shows. FIG. 22 (2) is a graph showing the time (in) and time (out) of entering the flow path of each width when the HeLa cells are flowed in the reverse direction, and the steady current value. FIG. 23 (1) is a drawing-substituting photograph, which is an enlarged photograph of the vicinity of the constricted portion 34 of the sample moving channel 3 of the electrical measurement chip 1 produced in Example 14. FIG. 23 (2) is a graph showing the relationship between the steady current value and the passage time in Example 14.

  Hereinafter, the electrical measurement chip and electrical measurement apparatus of the present invention will be described in detail. First, in the present invention, the “steady current” means an ionic current constantly flowing in the measurement circuit. The “transient current” means an ionic current that flows instantaneously in the measurement circuit.

  FIG. 3 is a diagram for explaining the outline of the electrical measurement chip 1 of the present invention. 3 includes a substrate 2, a sample moving channel 3 formed on the substrate 2, a sample input channel 4 connected to one end of the sample moving channel 3, and the sample moving channel 3 A sample recovery channel 5 connected to the end, a first measurement channel 6 connected to the sample transfer channel 3, and a second measurement channel connected to the sample transfer channel 3 from the side opposite to the first measurement channel 6 7 (hereinafter, when the flow paths formed on the substrate are collected, they may be simply referred to as “flow paths”). A sample measurement channel is formed by the first measurement channel 6 and the second measurement channel 7.

  The width and depth of the sample moving flow path 3 are not particularly limited as long as they are larger than the size of the sample. However, in order to increase the measurement sensitivity, it is preferable to appropriately adjust the width and depth so as not to be too large. For example, since the diameter of PM2.5 in the air is about 2.5 μm, the width and depth of the sample moving flow path 3 may be about 3 μm. Moreover, since the diameter of a cedar pollen is said to be about 20-40 micrometers and the diameter of a hinoki pollen is said to be about 28 micrometers-45 micrometers, the width | variety and depth should just be about 50 micrometers. Of course, the above numerical values are only a guide, and when the sample is larger, the width and depth may be increased according to the size of the sample, such as 100 μm, 150 μm, and 200 μm. The lower limit values of the width and depth are about 4 nm in the current microfabrication technology, but may be further reduced as the technology advances.

  The sample input channel 4 and the sample recovery channel 5 are large enough to input the electrodes of the sample drive circuit, and may include a liquid containing a sample (hereinafter, a liquid containing a sample is referred to as a “sample liquid”). ) Is not particularly limited in size and shape, but the depth is preferably the same as that of the sample moving flow path 3. In order to allow the sample to efficiently flow into the sample moving circuit 3, the sample input channel 4 and the sample recovery channel 5 may be tapered so that the width becomes narrower toward the sample moving circuit 3.

  The first measurement channel 6 and the second measurement channel 7 constitute a measurement circuit by inserting electrodes of a sample measurement circuit, which will be described later, and measure changes in steady-state current and transient current (hereinafter, changes in steady-state current). And measuring transient current is sometimes referred to as “measuring current change”). The size and shape of the first measurement channel 6 and the second measurement channel 7 are not particularly limited as long as the electrodes of the sample measurement circuit can be inserted, but in order to increase the measurement sensitivity, resistance is required. It is preferable to reduce it. The resistance value of the flow path filled with the sample liquid is a value obtained by dividing the product of the resistivity of the sample liquid and the length of the flow path by the cross-sectional area of the flow path. Therefore, as the width of the flow path is increased, the area is increased and the resistance can be reduced. Therefore, the widths of the first measurement channel 6 and the second measurement channel 7 may become longer as the distance from the sample movement channel 3 is longer than the length L of the portion connected to the sample movement channel 3. preferable. The shapes of the first measurement channel 6 and the second measurement channel 7 may be the same or different, but if the shapes of the first measurement channel 6 and the second measurement channel 7 are different, the measurement is performed. The resulting signal is also asymmetric. For this reason, it is preferable that the first measurement flow channel 6 and the second measurement flow channel 7 have the same shape when measuring a signal with higher accuracy such as the shape of an object from the measured signal.

  In FIG. 3, the widths of the first measurement channel 6 and the second measurement channel 7 are longer than L by making the first measurement channel 6 and the second measurement channel 7 substantially trapezoidal. However, the shape is not particularly limited as long as the widths of the first measurement channel 6 and the second measurement channel 7 become longer as they move away from the sample moving channel 3. For example, FIG. 4 shows another embodiment of the electrical measurement chip 1 of the present invention. As shown in FIG. 4, the semi-circular shape makes it longer as it moves away from the sample moving channel 3. You may do it.

  The depths of the first measurement channel 6 and the second measurement channel 7 may be the same as the depth of the sample moving channel 3. Further, since the sensitivity is improved as the length L is shorter, it may be shortened to such an extent that it can be manufactured by a microfabrication technique. On the other hand, if the length L is too long, the sample may flow into the first measurement channel 6 or the second measurement channel 7 from the sample movement channel 3, so the length L is larger than the width of the sample movement channel 3. The shorter one is preferable, and it is more preferable to make it shorter than the size of the sample to be measured.

  FIG. 5 is a diagram for explaining the outline of the electrical measurement chip 1 in which the first measurement electrode 61 and the second measurement electrode 71 are formed instead of the first measurement channel 6 and the second measurement channel 7. In the case of forming the first measurement electrode 61 and the second measurement electrode 71, it is not necessary to form the first measurement channel 6 and the second measurement channel 7, and on the substrate 2 on which the sample moving channel 3 is formed. The conductive material may be applied up to a position in contact with the sample moving flow path 3. When the electrical measuring chip 1 is used, the sample moving flow path 3 is filled with the sample liquid because it is covered with a glass plate or the like. Therefore, even if the first measurement electrode 61 and the second measurement electrode 71 are formed on the substrate 2, the sample liquid can be conducted.

  As a material for the first measurement electrode 61 and the second measurement electrode 71, a known conductive metal such as aluminum, copper, platinum, gold, silver, or titanium may be used. The first measurement electrode 61 and the second measurement electrode 71 may be manufactured by masking the substrate 2 and vapor-depositing the material. The resistance can be reduced when the first measurement electrode 61 and the second measurement electrode 71 are formed, as compared with the configuration in which the first measurement channel 6 and the second measurement channel 7 are formed and the electrodes are inserted. Therefore, the voltage applied to the sample moving flow path 3 can be lowered. The length of the connection portion between the sample moving flow path 3 and the electrode may be the same as that of the first measurement flow path 6 and the second measurement flow path 7. In addition, it is desirable that the opposing first measurement electrode 61 and second measurement electrode 71 have the same shape. As described above, in the case of the first measurement electrode 61 and the second measurement electrode 71, the resistance can be reduced. Therefore, as shown in FIGS. 3 and 4, the first measurement electrode 61 and the second measurement electrode are moved away from the sample moving channel 3. The width of the electrode 71 may be increased, but it may be the same width such as a rectangle.

  By the way, it is known that the deformability of cells plays an important role in metastasis of cancer cells. In addition, it is known that the deformability of erythrocytes decreases due to life span and cholesterol in blood, and stem cells before differentiation are easily deformed. FIG. 6 shows another embodiment of the electric measurement chip 1 of the present invention, in which a narrowed portion 34 is formed in the sample moving flow path 3. When the sample passes through the stenosis part 34, if the deformability of the sample is different, the deformation state of the cells when passing through the stenosis part 34 is different. Therefore, even for the same type of sample, the deformability of the sample can be measured by examining the passage time and waveform. 3-5, if the deformability of the sample is high, it flows while deforming the sample moving flow path 3. Therefore, the deformability is measured by examining the passage time and waveform of the sample. However, the electric measuring chip 1 shown in FIG. 6 can measure the deformability of the sample in more detail. The width of the constricted portion 34 is preferably at least smaller than the measurement target sample because it measures the deformability of the measurement target sample, and is preferably 50% to 90% of the size of the measurement target sample, 60% More preferably, it is set to about ˜80%. Further, the depth of the narrowed portion 34 is not particularly limited and may be the same as that of the sample moving flow path 3. In addition, since the narrow part 34 should just make width and / or depth smaller than a sample, for example, the width is made the same as the sample movement flow path 3, and depth is 50%-90% of the magnitude | size of a measurement object sample. %, More preferably about 60% to 80%. Alternatively, both the width and depth may be about 50% to 90%, more preferably about 60% to 80% of the size of the sample to be measured.

  6 shows an example in which one narrow portion 34 is formed, but two or more narrow portions 34 may be formed. When two or more narrow portions 34 are formed, the width of each narrow portion 34 may be the same or different. 6 has the first measurement flow path 6 and the second measurement flow path 7, the first measurement electrode 61 and the second measurement electrode 71 shown in FIG. 5 may be used.

The electrical measuring chip 1 can be manufactured by using a fine processing technique. FIG. 7 is a cross-sectional view taken along the line AA ′ in FIG. 4 and shows an example of the manufacturing process of the electrical measuring chip 1.
(1) An etchable material 8 is applied on the substrate 2 by chemical vapor deposition.
(2) A positive photoresist 9 is applied with a spin coater.
(3) Exposure / development processing is performed using a photomask so that light is irradiated to the portion where the flow path is formed, and the positive photoresist 9 in the portion where the flow path is formed is removed. Note that the shape of the photomask may be changed when the electrical measurement chip 1 shown in FIGS. 3, 5, and 6 is manufactured.
(4) The material 8 at the place where the flow path is formed is etched to form the flow path.
(5) The positive photoresist 9 is removed.

  The substrate 2 is not particularly limited as long as it is a material generally used in the field of semiconductor manufacturing technology. Examples of the material of the substrate 2 include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, and InP.

  The positive photoresist 9 is not particularly limited as long as it is generally used in the semiconductor manufacturing field, such as TSMR V50 and PMER. Further, a negative type photoresist may be used instead of the positive type, and there is no particular limitation as long as it is generally used in the semiconductor manufacturing field, such as SU-8, KMPR. The photoresist removal solution is not particularly limited as long as it is a common removal solution in the semiconductor field, such as dimethylformamide and acetone.

The material 8 deposited on the substrate 2 and forming the flow path and other than the flow path is not particularly limited as long as it is an insulating material, and examples thereof include SiO ₂ , Si ₃ N ₄ , BPSG, and SiON. It is done. In the manufacturing process shown in FIG. 7, the flow path is formed using the etchable material 8, but the material 8 is formed using the photosensitive resin such as the positive photoresist or the negative photoresist described above. Also good. When using a photosensitive resin, the photosensitive resin is applied on the substrate 2, a photomask having a shape capable of forming a flow path is used, and the flow path may be formed of the photosensitive resin by exposure and development.

FIG. 8 is a diagram showing another manufacturing process of the electrical measurement chip 1 of the present invention. In the manufacturing process shown in FIG. 7, the flow path is formed by etching, but in the manufacturing process shown in FIG. 8, the chip 1 for electrical measurement can be manufactured by transferring the mold.
(1) By changing the shape of the photomask, convex portions 8 that form flow paths after transfer are formed on the substrate 2 to produce a mold.
(2) The template is transferred to the transfer material 21.
(3) The mold 1 is peeled to produce the electrical measurement chip 1 in which the flow path is formed.

  Examples of the material 21 for transferring the template include insulating materials such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), and hard polyethylene. The electrical measurement chip 1 produced by transfer may be attached to an auxiliary substrate such as glass or plastic in order to improve the convenience of handling.

  When measuring using the electrical measurement chip 1 produced in the manufacturing process shown in FIGS. 7 and 8, when observing with a fluorescence microscope, the substrate 2, the material 8, the material 21 for transferring the mold, and the auxiliary substrate are: It is desirable to form the light transmissive material.

  Further, the electrical measuring chip 1 may be subjected to a hydrophilic treatment so that the sample liquid can easily flow. Examples of the hydrophilization treatment method include plasma treatment, surfactant treatment, PVP (polyvinylpyrrolidone) treatment, photocatalyst and the like. For example, the surface on which the flow path of the electric measurement chip 1 is formed is plasma for 10 to 30 seconds. By treatment, a hydroxyl group can be introduced on the surface.

  FIG. 9 is a diagram showing an outline of an electricity measuring device 10 using the electricity measuring chip 1 of the present invention. The electricity measuring device 10 includes a drive circuit 30 and a measuring circuit 40 in addition to the electricity measuring chip 1.

  The drive circuit 30 includes a first electrode 31 inserted into the sample input channel 4, a second electrode 32 inserted into the sample recovery channel 5, and a voltage applying unit 33. The first electrode 31 and the second electrode 32 are not particularly limited as long as they are materials that conduct electricity. For example, a known conductive metal such as aluminum, copper, platinum, gold, silver, or titanium may be used. In the example shown in FIG. 9, the first electrode 31 is inserted into the sample input channel 4 and the second electrode is inserted into the sample recovery channel 5, but the first electrode 31 and the second electrode 32 are input into the sample. It may be formed in the flow path 4 and the sample recovery flow path 5 and connected by electric wires. The voltage application means 33 is not particularly limited as long as the sample can be moved by applying a voltage to the drive circuit 30. However, it is preferable that the voltage application means 33 does not easily generate noise, such as a battery box.

  In the embodiment shown in FIG. 9, the electrodes 31 and 32 are loaded into the sample loading channel 4 and the sample collection channel 5 of the electricity measuring chip 1 to move the sample. It may be an embodiment. For example, a hole is formed in a part of the sample recovery flow path 5, one end of the silicon tube is connected to the sample recovery flow path 5, and the other end is connected to an aspirator such as a syringe pump. The sample may be moved by force. This is useful when using large samples such as cells. Further, the sample input channel 4 and the sample recovery channel 5 need not be provided. In that case, holes may be formed at both ends of the sample moving flow path 3, and the first electrode 31 and the second electrode 32 of the drive circuit 30 may be inserted into the sample moving circuit 3. Furthermore, if necessary, a suction device similar to the above is provided in the hole at one end of the sample moving flow path 3, one end of the silicon tube is connected to the hole at the other end, and the other end of the silicon tube is connected to the sample liquid. By connecting to the container, the sample may be moved by suction force in addition to the drive circuit 30.

  The measurement circuit 40 includes at least a third electrode 41 inserted into the first measurement channel 6, a fourth electrode 42 inserted into the second measurement channel 7, and an ammeter 43, and the third electrode 41 and the fourth electrode What is necessary is just to measure the electric current from 42 with the ammeter 43. In the case where the voltages of the drive circuit 30 and the measurement circuit 40 are balanced, and the detection of the difference in current from the balanced state is performed for higher sensitivity detection, the voltage application means 44, the variable voltage is applied to the measurement circuit 40. Only the difference between the currents may be measured by including a resistor 45, a resistor 46, and, if necessary, an amplifying means.

  The third electrode 41 and the fourth electrode 42 may be made of the same material as that of the first electrode 31 and the second electrode 32, and are formed in the first measurement channel 6 and the second measurement channel 7 to form an electric wire. It may be connected with. The voltage application unit 44 may be a battery box or the like, similar to the voltage application unit 33. The ammeter 43 may be a generally used ammeter. Amplifying means may be a commonly used amplifier. When the first measurement electrode 61 and the second measurement electrode 71 are formed, the third electrode 41 and the fourth electrode 42 are not necessary, and the first measurement electrode 61 and the second measurement electrode 71 and the ammeter 43 are connected by an electric wire. Good.

  In the present invention, by using the variable resistor 45 and the resistor 46, the potential difference of the portion sandwiched between the first measurement channel 6 and the second measurement channel 7 in the sample moving channel 3 and the potential difference of the resistor 46 are obtained. Since it is possible to measure the generation of transient current and the change in steady current when the sample enters the sample movement flow path 3 in a balanced state, the deviation from the balanced state can be measured, so that the detection sensitivity can be increased. Commercially available variable resistors 45 and resistors 46 may be used in the present invention.

  FIG. 10 is a diagram for explaining the relationship between the position of the sample on the electrical measurement chip 1 and the current value that can be measured when measuring the sample using the electrical measurement apparatus 10 of the present invention. First, before the measurement, a buffer solution such as PBS, phosphate buffer, TBE buffer or the like is introduced into the channel by capillary action, and then the sample solution is introduced into the sample introduction channel 4. Next, when a voltage is applied to the drive circuit 30, the sample moves toward the sample recovery channel 5. When the sample moves to the vicinity of the boundary between the sample input channel 4 and the sample moving channel 3 (position a in FIG. 10), the measurement circuit 40 first measures the transient current. Next, the change in the steady current is read until the sample moves from the position a to the vicinity of the connection portion of the sample moving flow path 3 and the first measurement flow path 6 (position b in FIG. 10). Then, a larger change in steady current is measured from the position b until the sample exits from the connection portion (position c in FIG. 10) between the sample moving flow path 3 and the second measurement flow path 7. The change in steady current is read until the sample moves from the position c to the vicinity of the boundary between the sample moving flow path 3 and the sample collection flow path 5 (position d in FIG. 10). Upon exiting circuit 5, measurement circuit 40 measures the transient current.

  As shown in FIG. 10, when a sample is measured using the electrical measurement chip 1 of the present invention, the sample is moved by measuring the transient current when the sample enters and leaves the sample moving channel 3. The time for moving 3 (ad in FIG. 10) can be measured accurately. Therefore, the surface charge and deformability of the sample can be measured. In addition, the waveform at the time of providing the constriction part 34 in the sample movement flow path 3 is demonstrated in the Example mentioned later.

  In addition, the particle size and shape of the sample are such that the sample is between the connection portion between the first measurement channel 6 and the sample movement channel 3 and the connection portion between the second measurement channel 7 and the sample movement channel 3 (FIG. 10). It can be measured by the magnitude of the change in the steady current b to c). Therefore, compared with the length of the sample moving flow path 3, since the length which measures the change of the steady current of a sample is short, measurement sensitivity can be maintained. Furthermore, since the sample movement flow path 3 other than between the first measurement flow path 6 and the second measurement flow path 7 can be used as a guide flow path, elongated molecules such as DNA are elongated while maintaining measurement sensitivity. It becomes possible to measure in a state.

  As described above, the electrical measurement device 10 of the present invention measures a change in steady current while the sample passes through the sample moving flow path 3. In particular, the sample measures the first measurement flow path 6 and the second measurement flow path 7. The larger change in steady-state current is measured when moving between. Therefore, the first measurement channel 6 and the second measurement channel 7 may be formed at positions that are asymmetric near both ends of the sample moving channel 3, but in that case, as shown in the examples described later, Since the waveform at the time is linear, it is preferable to reduce the displacement of the positions where the first measurement channel 6 and the second measurement channel 7 are formed. In the present invention, the “displacement” of the position means an intermediate point between the connection portions of the first measurement channel 6 and the sample movement channel 3 and an intermediate point between the connection points of the second measurement channel 7 and the sample movement channel 3. It means a point (⇔ in FIG. 10). On the other hand, as shown in the examples described later, the steady current can be measured even if the first measurement channel 6 and the second measurement channel 7 are formed at symmetrical positions with the sample moving channel 3 interposed therebetween. As described above, since the waveform of the steady current is broken, it is preferably formed at an asymmetric position as described above, and the positional deviation is half the length of the connection portion of the first measurement channel 6 and the sample moving channel 3. It is more preferable that + half the length of the connecting portion between the second measurement channel 7 and the sample moving channel 3 + the sample size.

  FIG. 11 is a view showing another embodiment of the electricity measuring chip 1. The sample input channel 4 and the sample recovery channel 5 of the electrical measurement chip 1 shown in FIGS. 3 to 6 are a single channel, but as shown in FIG. 11, the sample input channel 4 and the sample recovery channel The channel 5 may be formed as a plurality of channels. By making the sample input flow path 4 into a plurality of flow paths, for example, different samples are put in the respective flow paths, and the first electrode 31 and the second electrode 32 of the drive circuit are also put in the respective flow paths, and voltage is applied. By switching the electrodes to be used, different samples can be continuously analyzed and collected in the sample collection channel.

  Note that the plurality of channels may be formed only in one of the sample input channel 4 and the sample recovery channel 5. When only the sample input channel 4 is a plurality of channels, different sample solutions can be continuously analyzed.

  Further, when samples having different surface charges are included in the sample liquid, the moving speed of the sample flowing through the sample moving flow path 3 is different. Therefore, by forming a plurality of flow paths only for the sample recovery flow path 5 and switching the electrodes inserted into the respective flow paths, different samples in the sample liquid can be separated and recovered for further analysis. Can be used.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

[Preparation of chip 1 for electrical measurement]
<Example 1>
The electrical measurement chip 1 was produced by the following procedure.
(1) A silicon substrate 2 (diameter 76 mm, manufactured by Ferrotec Silicon) with a thickness of 600 μm was prepared.
(2) A negative photoresist SU-8 3005 (manufactured by MICRO CHEM) was applied by a spin coater.
(3) It exposed using the photomask so that light might irradiate the part which forms a flow path by photolithography. After exposure, the resist was developed using SU-8 developer (manufactured by MICRO CHEM). After the development, rinsing was performed using ultrapure water, and moisture was removed by a spin dryer and dried to prepare a mold.
(4) Polydimethylsiloxane (SILPOT184, manufactured by Toray Industries, Inc.) was poured into the produced mold and cured.
(5) The cured PDMS was removed from the mold, and then a commercially available cover glass (thickness: 0.17 mm) was brought into close contact with the PDMS to produce a chip 1 for electrical measurement.

  12A is a photograph of the electrical measurement chip 1 produced in Example 1, and FIG. 12B is an enlarged photograph of the vicinity of the first measurement channel 6 and the second measurement channel 7. The sample moving flow path 3 had a length of 150 μm, a width of 4 μm, and a depth of 7.5 μm. The depth of the first measurement channel 6 and the second measurement channel 7 is 7.5 μm, and the length of the connecting portion with the sample movement channel 3 is 10.5 μm. The angle was about 45 °. Further, the positional deviation between the first measurement channel 6 and the second measurement channel 7 across the sample moving channel 3 was 40 μm. The depth of the sample input channel 4 and the sample recovery channel 5 was 7.5 μm.

<Example 2>
The electrical measurement chip 1 is manufactured in the same procedure as in Example 1 except that the shape of the photomask of Example 1 is changed and the positional deviation between the first measurement channel 6 and the second measurement channel 7 is set to 5 μm. did. FIG. 13 (1) is an enlarged photograph of the vicinity of the first measurement channel 6 and the second measurement channel 7 of the electrical measurement chip 1 produced in Example 2.

<Example 3>
The procedure of Example 1 is the same as that of Example 1 except that the shape of the photomask of Example 1 is changed and the first measurement channel 6 and the second measurement channel 7 are formed at symmetrical positions across the sample moving channel 3. An electrical measurement chip 1 was produced. FIG. 13 (2) is an enlarged photograph of the vicinity of the first measurement channel 6 and the second measurement channel 7 of the electrical measurement chip 1 produced in Example 3.

[Fabrication of electrical measuring device 10]
<Example 4>
(1) Production of Drive Circuit 30 The first electrode 31 and the second electrode 32 were produced by peeling a wire (FTAS-408 manufactured by Oyaide Electric Co., Ltd.) and exposing a metal part. As the voltage application means 33, a battery box (manufactured by Seinan Industry Co., Ltd.) was used.
(2) Production of measurement circuit 40 The third electrode 41 and the fourth electrode 42 were produced by peeling the skin of an electric wire (FTVS-408 manufactured by Oyaide Electric Co., Ltd.) to expose the metal part. As the amplification means, Variable Gain Low Noise Current Amplifier manufactured by FEMTO was used. As the voltage applying means 44, a battery box (manufactured by Seinan Industry Co., Ltd.) was used. As the variable resistor 45, a precision potentiometer manufactured by BI Technologies was used. The ammeter 43 converts the signal amplified by the amplifying means into an electrical signal for PC using USB-DAQ (manufactured by National Instruments) and read it with software created using Lab View (manufactured by National Instruments). . The resistor 46 was a metal film resistor (1 kΩ made by Panasonic).
(3) The electrical measurement chip 1 manufactured in Example 1 has the first electrode 31 in the sample input channel 4, the second electrode 32 in the sample recovery channel 5, the third electrode 41 in the first measurement channel 6, By inserting the fourth electrode 42 into the second measurement channel 7, the electrical measurement device 10 of the present invention was produced.

[Measurement using the electrical measuring device 10]
<Example 5>
A sample solution was prepared by dispersing fluorescent microbeads (Fluoresbrite manufactured by Polyscience) as a sample in ultrapure water. Next, 5 × TBE buffer was introduced into the channel by capillary action, 30 μl of the prepared sample solution was introduced into the sample introduction channel 4, and a voltage of 53 V was applied to the drive circuit 30. A voltage of 18 V was applied to the measurement circuit 40. The variable resistor 45 was operated so that the apparent resistances of the drive circuit 30 and the measurement circuit 40 were balanced. Changes in steady-state current and generation of transient current when the sample flowed through the sample moving flow path 3 were measured. FIG. 14A is a graph showing the relationship between the measurement time and the measured steady current value in Example 5.

<Example 6>
Measurement was carried out in the same procedure as in Example 5 except that the electrical measurement chip 1 produced in Example 2 was used. FIG. 14B is a graph showing the relationship between the measurement time and the measured steady current value in Example 6.

<Example 7>
Measurement was performed in the same procedure as in Example 5 except that the electrical measurement chip 1 produced in Example 3 was used. FIG. 14 (3) is a graph showing the relationship between the measurement time and the measured steady current value in Example 7.

  As shown in FIGS. 14 (1) to (3), when any of the electrical measurement chips 1 of Examples 1 to 3 is used, two peaks of transient current are confirmed, and the peak intervals are almost the same. there were. Since Examples 1 to 3 use the same sample, the surface charges are the same. Therefore, regardless of the positional relationship between the first measurement channel 6 and the second measurement channel 7, the time for the sample to move through the sample movement channel 3 can be accurately measured according to the surface charge of the sample.

  Further, when the electrical measurement chip 1 of Example 1 was used, as shown in FIG. 14A, the amount of change in the steady current value was the largest, but the peak waveform was linear. This is because the displacement between the first measurement channel 6 and the second measurement channel 7 is large, so that even if the sample moves between the first measurement channel 6 and the second measurement channel 7, the volume change does not occur. This is probably because the steady state continued.

  On the other hand, as shown in FIG. 14 (2), when the electrical measurement chip 1 of Example 2 is used, the change in steady-state current value is smaller than that of the electrical measurement chip 1 of Example 1, The steady-state current waveform showed a clear peak.

Further, when the electricity measuring chip 1 of Example 3 was used, two peaks were measured as shown in FIG. As shown in FIG.
(1) Since the first measurement flow path 6 and the second measurement flow path 7 are arranged in a symmetrical positional relationship, the current of the measurement circuit 40 is more likely to flow than the chips arranged in the first and second embodiments.
(2) When the sample flows to the ends of the first measurement channel 6 and the second measurement channel 7, the change in the steady current is measured. As described above, the electrical measurement chip 1 of Example 3 is the electrical Therefore, when the sample comes in the middle of the connection part with the sample moving flow path 3, the steady current value returns to a value close to the base value,
(3) Then, when the sample flowed out from the connection part, the change in the steady current value was measured.
It is thought to be for the purpose.

  From the above results, the first measurement channel 6 and the second measurement channel 7 are preferably formed at asymmetric positions with the sample moving channel 3 in between, and the peak value varies depending on the sample size. Arranged so as not to be linear (the position where the end of the first measurement channel 6 and the end of the second measurement channel 7 do not overlap with the sample moving channel 3 and are not too far apart). It is preferable.

[Measurement using the electrical measuring device 10 and a fluorescence microscope]
<Example 8>
Using fluorescent microbeads (Fluoresbrite manufactured by Polyscience) as a sample, a fluorescent microscope (TE300 manufactured by Nikon) is arranged so that the space between the first measurement channel 6 and the second measurement channel 7 of the electrical measurement chip 1 can be observed. Then, the measurement was performed in the same procedure as in Example 5 except that the fluorescence intensity was measured. FIG. 16 shows a photograph of the electrical measurement chip 1, a photograph of fluorescent microbeads flowing between the first measurement channel 6 and the second measurement channel 7, and a change in steady-state current value when the fluorescent microbeads flow ( Signal intensity) and a graph showing changes in fluorescence intensity (the portion surrounded by the line in the graph is the measurement result when the fluorescent microbead flows between the first measurement channel 6 and the second measurement channel 7) is there. As shown in FIG. 16, by using the electrical measuring device 10 of the present invention, a sample flowing through the sample moving channel 3 of the electrical measuring chip 1 is measured with a fluorescence microscope while measuring changes in the transient current and the steady current value. Since it can be observed, the event occurring at the measurement site of the electrical measurement chip 1 can be accurately observed.

<Example 9>
Measurement was performed in the same manner as in Example 8, except that fluorescent microbeads (Fluoresbrite manufactured by Polyscience) having a particle size of about 3.1 μm, 2.08 μm, and 1 μm were used. FIG. 17 is a graph showing changes (signal intensity) in steady-state current values measured in Example 9. Although it was difficult to determine whether substances of the same size overlap each other or substances of different sizes only by measuring changes in the steady-state current value in the past, observe in conjunction with a fluorescence microscope. As a result, the sample could be accurately identified. In addition, since the fluorescence microscope can distinguish different colors, for example, by staining the gram-negative bacteria and the positive bacteria and observing them with a fluorescence microscope, measuring changes in transient currents and steady-state current values, it is possible to distinguish roughly types. Is also possible.

[Relationship between particle size and steady current value]
<Example 10>
Measurement was performed in the same manner as in Example 8 using fluorescent microbeads (Fluoresbrite manufactured by Polyscience) having particle diameters of about 3.1 μm, 2.08 μm, 1.75 μm, 1.1 μm, 1 μm, and 0.75 μm. Went. FIG. 18 is a graph showing the change in the volume of the sample and the steady current value (signal intensity). As shown in FIG. 18, it was confirmed that there was a correlation between the signal intensity and the sample volume.

[Relationship between applied voltage, signal intensity and transit time]
<Example 11>
In Example 5, the measurement was performed in the same procedure as in Example 5 except that the voltage of the drive circuit 30 was measured in place of the three types of 53V, 32V, and 12V. FIG. 19 is a diagram showing the relationship between the voltage of the drive circuit and the time for the sample to pass through the sample movement channel. As shown in FIG. 19, it is clear that the measurement sensitivity can be increased by increasing the voltage of the drive voltage 30, while the passage time is shortened due to the surface charge of the sample. In addition, in the case of 12V, although there was little variation in signal intensity, variation in passage time was large. On the other hand, when the drive voltage was set to 32 V or more, there was almost no variation in the passage time, but there was a variation in the signal intensity. This is presumably because the driving force to the charged sample was reduced under a low voltage, and the moving speed of the sample was affected by the frictional force received from the wall surface.
In the present invention, the length of the sample movement channel 3 and the interval between the first measurement channel 6 and the second measurement channel 7 can be arbitrarily set. Therefore, even if the voltage of the drive circuit 30 is increased, the length of the sample moving flow path 3 and the first measurement flow path 6 and the first measurement flow path 6 are adjusted so that the time required for reading the change in the steady current is the shortest. 2 Since the measurement channel 7 can be set, highly sensitive detection can be performed in a short time.

[Relationship between particle size and steady current when using cells]
<Example 12>
In Example 10 above, fluorescent microbeads having a constant shape were used, but the correlation between the signal intensity and the sample volume in the case of using cells whose shape changed was examined.
First, by changing the shape of the photomask of the first embodiment, the width of the sample movement channel 3 is 20 μm, and the distance between the end of the first measurement channel 6 and the end of the second measurement channel 7 is 20 μm. A measuring chip 1 was produced. 20 (1) is an enlarged photograph of the vicinity of the sample movement channel of the electrical measurement chip 1 produced in Example 12, and FIG. 20 (2) is the sample migration channel of the electrical measurement chip 1 produced in Example 12. It is a figure for demonstrating the dimension of vicinity. Other sizes are the same as those in the first embodiment.
Then, in addition to the drive circuit 30, holes were made in a part of the PDMS sample input channel 4 and sample recovery channel 5 of the manufactured electrical measurement chip 1, and one end of the silicon tube was formed as the sample recovery channel 5. The electrical measuring device 10 was produced in the same procedure as in Example 4 except that the other end was connected to a hole and the other end was connected to a syringe pump (kd Scientific, KDS210).
Next, as a sample
HeLa cell (human cervical cancer-derived cell): about 15 μm (ATCC, CCL-2)
Jurkat cell (human T cell) suspension system: about 10 μm (ATCC, TIB-152),
Was used. HeLa cells were cultured using MEM (Sigma aldrich, M4655) which is a cell culture medium for HeLa. Jurkat was cultured using RPMI1640 (gibco, 11875-093), which is a cell medium for Jurkat.
Then, using the above cells instead of the fluorescent microbeads, the voltage applied to the drive circuit 30 was 3 V, and the experiment was performed in the same procedure as in Example 10 except that the sample solution was sucked at 5 to 10 μL / min with a syringe pump. went.
FIG. 20 (3) is a histogram of steady current values, and is a graph showing the distribution of the number of cells counted at each steady current value. As shown in FIG. 20 (2), it was confirmed that there was a correlation between the intensity of the steady-state current value and the volume of the sample even when using a sample whose shape such as cells easily changed.

[Fabrication of electricity measuring chip 1 having narrowed portion 34]
<Example 13>
By changing the shape of the photomask of Example 1, the electrical measurement chip 1 having the narrowed portion 34 was produced. FIG. 21 (1) is an enlarged photograph of the vicinity of the narrowed portion 34 of the sample moving flow path 3 of the electrical measurement chip 1 manufactured in Example 13. FIG. 21B is a diagram showing the length and width of the sample moving flow path 3 and the narrowed portion 34 of the electrical measurement chip 1 manufactured in Example 13. The sample moving flow path 3 had a width of 25 μm, and a narrowed portion having a width of 15 μm and a narrowed portion having a width of 10 μm were formed at intervals. The stenosis part with a width of 15 μm, the stenosis part with a width of 10 μm, and the length of the sample moving flow path 3 between both the stenosis parts were 30 μm. Further, the narrow portion having a width of 15 μm and the sample moving flow path 3 were connected at an angle of about 45 °, and the length of the connection portion was about 5 μm. The narrow portion having a width of 10 μm and the sample moving flow path 3 were connected at an angle of about 45 °, and the length of the connection portion was about 7.5 μm.
Next, an electrical measuring device 10 was manufactured in the same procedure as in Example 12 using the manufactured electrical measuring chip 1.
Next, HeLa cells described in Example 12 were used as samples, and aspirated at a rate of 5 μl / min with a syringe pump.
FIG. 22 (1) shows the time (in) and the time (in) when the HeLa cells flowed from the left to the right of the chip shown in FIG. 21 (flow path width is 15 μm → 25 μm → 10 μm). It is the graph which shows the change of the last time (out) and the steady-state current value. FIG. 22 (2) shows the time when the HeLa cells flowed in the opposite direction (flow path width is 10 μm → 25 μm → 15 μm) and entered the flow path of each width (in), and the time of exit (out), and It is a graph which shows a steady-state electric current value. As described above, the size of the HeLa cell is about 15 μm. As is clear from FIGS. 22 (1) and (2), even when the flow paths have the same length, the time for the HeLa cells to pass increases as the flow path width decreases. In particular, when a HeLa cell passes through a 10 μm narrow portion that cannot pass through without deformation, it takes a much longer time than when it has a width of 15 μm and 25 μm. From the above results, it was possible to measure the deformability of the sample by forming the narrowed portion 34 in the sample moving flow path 3.

<Example 14>
In Example 13, since the deformability of the sample could be measured by providing the constricted portion 34, in this example, the same type of cells having different deformability were prepared and measured.
First, by changing the shape of the photomask, the electrical measurement chip 1 having one narrow portion 34 having a width of 10 μm and a length of 40 μm was produced. FIG. 23 (1) is an enlarged photograph of the vicinity of the narrowed portion 34 of the sample moving flow path 3 of the electrical measurement chip 1 manufactured in Example 14. Next, an electrical measurement device was produced in the same procedure as in Example 13 using the produced electrical measurement chip 1.
Next, rat trunkrin A (wako, 125-04363), a substance that inhibits the formation of the cytoskeleton by inhibiting actin polymerization, acts on the HeLa cells of Example 12 at a concentration of 0.5 μM. I let you. In addition, since the formation of cytoskeleton is inhibited when latrunculin A is allowed to act on HeLa cells, the cells have different deformability than HeLa cells that do not act on latrunculin A.
Then, using the electrometric apparatus thus prepared, HeLa cells (LatA) that acted on rattrunkin A and HeLa cells that did not act on rattrunkin A (Without LatA) were added in an amount of 10 μl / min using a syringe pump. The experiment was performed in the same procedure as in Example 12 except that the suction was performed.
FIG. 23 (2) is a graph showing the relationship between the steady current value and the passage time. As is clear from FIG. 23 (2), when the same steady-state current value, that is, the cell size is the same, the HeLa cell (Without LatA) not acting on rattrunkin A clearly passes through the stenosis. The time to do was long.
From the above results, it was possible to measure the difference in cell deformability by measuring the time for passing through the stenosis even for the same type of cells. Because cancerous cells have higher deformability than normal cells, for example, by flowing the same cell population solution through an electrical measurement chip with a stenosis, cancer cells can be extracted from the cell population. A device (cell sorter) for discriminating / sorting the transformed cells can be produced.

By using the electrical measurement chip 1 of the present invention, the drive circuit and the measurement circuit can be designed as separate circuits, so that the voltage of the drive circuit can be set high and the detection sensitivity can be increased. Furthermore, since transient currents can be accurately read, the surface charge of the sample can be read, and it is possible to measure the biomolecules such as nucleic acids and proteins by creating an extended state of the sample in the sample movement channel. Become.
Therefore, it is useful for the development of measuring instruments for accurately analyzing samples in companies, research institutions, and the like.

[0001]
Technical field [0001]
The present invention relates to an electric measurement chip and an electric measurement device, and particularly, when a sample such as a cell, a bacterium, a virus, or a DNA flows through a microchannel, reads not only the change in steady current but also the generation of a transient current. Thus, the present invention relates to an electrical measurement chip designed to perform highly sensitive detection, and an electrical measurement device including the electrical measurement chip.
Background art [0002]
Accurate measurement of the size, number, etc. of samples such as cells, fungi, pollen, and PM2.5 contained in a solution is important information for living a healthy life. Improvement is desired. In the field of biochemistry, it is desired to develop an analysis chip that analyzes a DNA fragment as it is.
[0003]
FIG. 1 shows a conventional technique for measuring the size and number of samples. The sample is passed through pores (micropores) formed on a substrate such as silicon, and the pores are applied by voltage applied to the pores. The size and hardness of the cell are analyzed from the state in which the steady-state current flowing inside the cell changes (see Non-Patent Document 1). The conventional measurement method shown in FIG. 1 is known to improve the sensitivity as the volume of the pores decreases. In order to reduce the volume of the pores, it is necessary to reduce the diameter and reduce the thickness of the substrate. For this reason, the substrate is used in a vertical orientation as shown in FIG.
[0004]
In addition, in order to measure the state of the sample passing through the pores in more detail, the substrate on which the microchannels are formed can be placed sideways so that the pores can be observed with a fluorescence microscope. A method of directly observing an event around a hole is also known (see Non-Patent Document 2). 2 is shown in FIG. 1 is shown.
Prior art documents

Claims (11)

Including a substrate, a sample movement channel formed on the substrate, and a sample measurement channel,
The sample measurement flow path includes a first measurement flow path connected to the sample movement flow path and a second measurement flow path connected to the sample movement flow path from the side opposite to the first measurement flow path.
Chip for electrical measurement.   2. The electricity according to claim 1, wherein the width of the first measurement channel and the second measurement channel is longer as the distance from the sample movement channel is longer than the length of the portion connected to the sample movement channel. Measuring chip.   3. The electrical measurement chip according to claim 1, wherein the first measurement channel and the second measurement channel are formed at asymmetric positions with the sample movement channel interposed therebetween.   The electrical measurement chip according to claim 1, wherein at least one narrow portion is formed in the sample moving flow path.   The electrical measurement according to any one of claims 1 to 4, further comprising a sample input channel formed at one end of the sample moving channel and a sample recovery channel formed at the other end of the sample moving channel. For chips.   The electrical measurement chip according to any one of claims 1 to 5, wherein a first measurement electrode and a second measurement electrode are formed instead of the first measurement channel and the second measurement channel. The chip for electrical measurement according to any one of claims 1 to 5,
A drive circuit for allowing the sample to move through the sample transfer channel,
A measurement circuit that applies a voltage to the first measurement channel and the second measurement channel and measures a change in current when the sample moves through the sample moving circuit;
Including electrical measuring device. The electrical measurement chip according to claim 6,
A drive circuit for allowing the sample to move through the sample transfer channel,
A measurement circuit for applying a voltage to the first measurement electrode and the second measurement electrode and measuring a change in current when the sample moves through the sample moving circuit;
Including electrical measuring device. The measuring circuit includes a variable resistor for balancing the resistance of the driving circuit and the measuring circuit;
The electrical measurement device according to claim 7 or 8, wherein the measurement circuit measures a difference in current from a balanced state.   The electrical measurement apparatus according to claim 7, wherein the measurement circuit measures changes in transient current and steady current.   The electrical measurement apparatus according to any one of claims 7 to 10, further comprising a fluorescence microscope.