JP2004020238A - Bio-reaction-of measuring chip, and method and apparatus for measuring bio-reaction using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for easily and reliably detecting a weak physical or chemical signal generated by a functional protein and its change, and to provide a method and an apparatus for measuring the physical or chemical signal of the functional protein by using the device. <P>SOLUTION: The chip for detecting a change in the physical or chemical characteristics of the functional protein comprises a board having at least one electrode and a conductive wire connected to the electrode; and a separation section having at least one chamber for accommodating the functional protein or a constituent containing the functional protein. In the chip, the functional protein or the constituent containing the functional protein is arranged on the electrode, and the separation section has a reference electrode and can derive the physical or chemical signal generated by the functional protein. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biological reaction measurement chip, that is, a chip for measuring a physical or chemical signal of a functional protein and its change, and a biological reaction measurement method and apparatus using the same. The present invention particularly relates to a chip that can be used for measuring a physical change or a chemical change of an antibody, an enzyme, and a receptor, and a measurement method and a measurement apparatus using the chip.
[0002]
[Prior art]
Conventionally, a physical or chemical signal generated by a biological component accompanying the activity of the biological component is measured as a digital signal based on an electrical change of the biological component or a fluorescence intensity emitted by an indicator incorporated in the biological component. It is measured by taking in. For example, when measuring channel activity of a cell at a single channel level, a digital signal (electrical quantity passing through a channel) obtained by an electrophysiological measuring device using a microelectrode probe such as a patch clamp and a dedicated control device is used. , The channel opening / closing time, timing, the number of times, etc. are calculated, and the channel activity is measured (intracellular recording method). For the whole cell, the amount of ions flowing into the cell is measured as a digital signal by a fluorescence measurement method, and this is used as the cell channel activity.
[0003]
The patch clamp method is a method in which ion transport through a single channel protein molecule is electrically recorded by a microelectrode probe using a microportion (patch) of a cell membrane attached to a tip portion of a micropipette. Patch clamping is one of the few techniques in cell biology that allows one to study the function of a single protein molecule in real time (eg, Molecular Biology of Cells, Third Edition, Garland Publishing). , Inc., New York, 1994, Japanese edition, translated by Keiko Nakamura et al., Pp. 181-182, 1995, Kyoikusha.
[0004]
The fluorescent antibody method combines a luminescence indicator or a fluorescent dye that emits light in response to a change in the concentration of a specific ion with the latest image processing methods (for example, by taking a fluorescent image of a cell with a CCD camera, etc. Monitor the movement of ions) and measure the electrical activity of the whole cell.
[0005]
However, the patch clamp method requires special techniques for micropipette fabrication and manipulation, and requires a lot of time to measure one sample, making it suitable for high-speed screening of a large number of drug candidate compounds. Not. In addition, the fluorescence measurement method can screen a large number of drug candidate compounds at high speed, but requires a step of staining cells. In the measurement, the background is high due to the effect of the dye, and the fluorescence intensity decreases with time. Therefore, there is a disadvantage that the S / N ratio is poor.
[0006]
In addition, methods for observing electrochemical changes in functional proteins include those described in Japanese Patent No. 2949845, US Pat. No. 5,810,725, US Pat. No. 5,56,067, JP-A-9-823318, WO 01/25769 A2, and US Pat. No. 5,187,696. , WO 98/54294, WO 99/66329, WO 99/31503, and the like using a substrate on which a multi-electrode is arranged.
[0007]
Japanese Patent No. 2949845, US Pat. No. 5,810,725, US Pat. No. 5,56,067, and Japanese Patent Application Laid-Open No. 9-823318 disclose microelectrodes formed on a glass substrate using photolithographic technology, and the electrical changes of cells are multi-pointed. An integrated composite electrode characterized in that it can be measured extracellularly and a measurement system using the same.
[0008]
WO 01/25769 A2 has a through-hole on an insulating substrate, and a functional protein such as a cell including an ion channel is arranged in the through-hole to form a gigashield on the surface of the insulating substrate with the cell or the like. A substrate capable of measuring a current generated when ions pass through an ion channel by using a reference electrode and a measurement electrode provided in two regions separated by is described.
[0009]
U.S. Patent No. 5,187,696 describes a device that can monitor cell growth by culturing cells on electrodes and measuring impedance changes.
[0010]
WO 98/54294 describes a device for adhering cells on a plate electrode and measuring its electrical signal.
[0011]
WO 99/66329 describes a device for observing the activity of cells on a porous material by means of resistance or impedance changes, and an assay using the same.
[0012]
WO 99/31503 describes a method of forming a patch clamp by trapping cells in a through-hole using a substrate having a through-hole and measuring a change in current.
[0013]
The above-mentioned prior art uses a flat plate electrode to carry out electrical activity of cells, and forms a small through hole on an insulating substrate to form a patch clamp between the cell and the substrate, which is generated by ions passing through an ion channel. It is characterized by monitoring the current flowing. However, in the case of such a plate electrode, it is possible to detect a potential change of the cell membrane, but it is not possible to measure with high sensitivity due to leakage of a signal into a liquid. In addition, the method of forming a small through hole on an insulating substrate to form a patch clamp with a cell has a low probability of forming a patch clamp, and also destroys the cell membrane to cause physical damage. The condition cannot be measured. Furthermore, when forming a patch clamp system, it is difficult to adjust the pressure for performing suction, and it is necessary to provide an adjusting mechanism in order to perform the operation with multiple channels, which is not practical. From these viewpoints, the conventional plate electrode is suitable for high-speed drug screening, but has poor sensitivity. Further, an insulating substrate provided with minute through holes is not suitable for high-speed drug screening. From a similar point of view, automated patch clamp robots take time to process samples and are also unsuitable for rapid drug screening.
[0014]
[Problems to be solved by the invention]
SUMMARY OF THE INVENTION The present invention has been made to solve the above problems of the related art, and an object of the present invention is to improve a conventional electrophysiological measuring device to eliminate the above problems. The present invention is a device capable of easily and reliably detecting a minute physical signal or a chemical signal emitted by a biological component and a change thereof, which cannot be detected by the conventional extracellular recording method, And a method and apparatus for measuring a physical or chemical signal of a biological component using the same.
[0015]
[Means for Solving the Problems]
The present invention relates to a chip for detecting a change in a physical property or a chemical property of a functional protein, the chip having at least one electrode and a conductive line connected to the electrode; and a chip disposed on the substrate; An isolator having at least one chamber for containing a functional protein or a component containing the functional protein, wherein the functional protein or the component containing the functional protein is disposed on the at least one electrode, The isolation unit has a reference electrode, and can derive a physical signal or a chemical signal emitted by the functional protein.
[0016]
As used herein, the term "physical signal or chemical signal" generated by a functional protein refers to a signal generated by a component of a living body including a functional protein represented by a cell membrane or the like. , A signal originating from a specific site of a measurement target, or a signal that changes due to a specific event in a measurement target, such as activation of an ion channel of a cell in response to a drug. As used herein, the term "significant signal" or "significant signal" is used primarily to refer to the latter meaning, that is, a signal that has changed due to a particular event in a measurement object.
[0017]
Preferably, the substrate and the isolation unit are integrated.
[0018]
Preferably, the substrate is formed of an insulating material.
[0019]
Preferably, the insulating material is glass, plastic, or SOI.
[0020]
Preferably, the electrode is formed from a material including metal, conductive plastic, or conductive rubber.
[0021]
Preferably, said metal is selected from the group consisting of platinum, platinum-platinum black, gold, silver, nickel, titanium, silver-silver chloride, and copper.
[0022]
Preferably, the surface of the electrode is plasma-treated.
[0023]
Preferably, a self-assembled monolayer is formed on the surface of the electrode. .
[0024]
Preferably, a biocompatible substance or a functional protein is arranged on the self-assembled monolayer.
[0025]
Preferably, a mixture of a lipid and a functional protein is disposed on the self-assembled monolayer.
[0026]
Preferably, the biocompatible substance is selected from the group consisting of collagen, fibronectin, vitronectin, poly-L-lysine, polyornithine, and polyethyleneimine.
[0027]
The tip may include a plurality of electrodes.
[0028]
Preferably, each of the plurality of electrodes and the conductive line connected thereto are insulated from other electrodes and the conductive lines connected thereto.
[0029]
Preferably, the substrate has at least one through hole, and the electrode is disposed on at least a part of a periphery of the through hole or on the through film.
[0030]
Preferably, the substrate has at least one through hole, and the electrode is arranged in the through hole.
[0031]
In the chip, a voltage change at a constant frequency is given through the electrode, and a frequency that changes according to the reactivity of the functional protein can be detected.
[0032]
In the chip, a current change at a constant frequency is given through the electrode, and a frequency that changes according to the reactivity of the functional protein can be detected.
[0033]
In the chip, a physical or chemical signal generated when a substance that specifically reacts with the functional protein acts on the functional protein can be derived.
[0034]
Preferably, the functional protein is selected from the group consisting of an antibody, an enzyme, and a receptor, and the substance that specifically reacts with the functional protein is selected from the group consisting of an antigen, a substrate, and a ligand.
[0035]
Typically, specific combinations of a functional protein and a substance that specifically reacts with the functional protein include acetylcholine receptor and acetylcholine, NMDA receptor and glutamate, Ca channel and calcium, dopamine receptor and dopamine, and VEGF receptor ( Vascular growth factor receptor) and VEGF (vascular growth factor), EGF receptor and EGF, T cell and CD4 antibody, cytochrome P450 and lansopropazolo, G protein and hormone, and the like.
[0036]
Preferably, a conductive line electrically connected to the through hole is arranged on a front surface or a back surface of the substrate.
[0037]
Preferably, the functional protein or a component containing the functional protein is disposed in the opening of the through-hole, and the interaction between the functional protein and a substance that specifically reacts with the functional protein is electrochemically detected. .
[0038]
Preferably, the depth of the through hole is 1 to 10000 micrometers.
[0039]
Preferably, the portion of the substrate that does not include the through-hole has a thickness in the range of 10 to 10000 micrometers.
[0040]
Preferably, the surface area of the electrode is smaller than the surface area of the reference electrode.
[0041]
Preferably, the chip further includes a heating unit disposed on the substrate.
[0042]
Preferably, the heating means is a Peltier element or an IH heater.
[0043]
The present invention also relates to a method for measuring a physical signal or a chemical signal emitted by at least one kind of functional protein. This method comprises the steps of: (A) generating a plurality of time-series signals derived from the device on which a functional protein is arranged; (B) acquiring a plurality of extracted data groups from the data group, wherein each of the extracted data groups is a fixed number selected from the data group. (C) calculating a standard deviation of the plurality of extracted data groups to obtain a standard deviation group, and (D) referring to each of the standard deviations constituting the standard deviation group. Selecting a physical or chemical signal.
[0044]
Preferably, the reference in the step (D) is to calculate an average value of each standard deviation constituting the standard deviation group.
[0045]
Preferably, the reference in the step (D) classifies each of the standard deviations into a plurality of classes each having a unit of a standard deviation of a fixed width, and each of the standard deviations exists in the class and each of the classes. Plotting on the coordinate with the number of the standard deviation as an axis, approximating the obtained graph to a normal distribution, and calculating the average value, half width, and variance or standard deviation of the obtained normal distribution It is to be.
[0046]
Preferably, the method further includes performing the steps (B) to (C) a plurality of times by changing the number of data constituting the extracted data group.
[0047]
Preferably, the method further includes performing the steps (B) to (D) a plurality of times by changing the number of data constituting the extracted data group and the width of the class. The physical signal or the chemical signal is selected from a plurality of average values, half-value widths, and variances or standard deviations of the normal distribution.
[0048]
Preferably, the method further includes, after the step (A), adding a data group obtained for each of the functional proteins.
[0049]
Preferably, the method further comprises, after the step (A), a step of subtracting a data group obtained for each of the functional proteins.
[0050]
Preferably, the method further comprises, prior to the step (A), simultaneously stimulating each of the functional proteins.
[0051]
Preferably, the reference in the step (D) classifies each of the standard deviations into a plurality of classes each having a unit of a standard deviation of a fixed width, and the plurality of standard deviations are present in the class and each of the classes. Plotting on the coordinate with the number of the standard deviation as an axis, the obtained graph, the exponential decrease, exponential increase, Gaussian, Lorentz, sigma, by a curve approximation analysis selected from the group consisting of multiple peaks and nonlinear Approximation and obtaining the slope before and after the vertex of the obtained approximation curve.
[0052]
Preferably, the selection in the step (B) is performed in chronological order.
[0053]
Preferably, in the method, the steps (B) to (D) are repeatedly performed, the selection in the step (B) is performed in time series, and the first data of the extracted data group in each repetition is constant. And the method further comprises the step of comparing the average value of each of the standard deviation groups obtained at each iteration.
[0054]
Preferably, in the method, the steps (B) to (D) are repeatedly performed, the selection in the step (B) is performed in time series, and the first data of the extracted data group in each repetition is constant. And the method further comprises comparing the mean of the normal distribution of each of the standard deviations obtained at each iteration.
[0055]
Preferably, in the method, the steps (B) to (D) are repeatedly performed, the selection in the step (B) is performed in time series, and the first data of the extracted data group in each repetition is constant. Selected at time intervals and the method further comprises comparing the half-width, variance or standard deviation of the normal distribution of each of the standard deviation groups obtained at each iteration.
[0056]
Preferably, in the above method, the reference in the step (D) includes obtaining a plurality of extracted standard deviation groups from the standard deviation group, wherein each of the extracted standard deviation groups is different from the standard deviation group. The method comprising calculating a mean value of the fixed number of standard deviations and a time series increase rate of the mean value, wherein the extracted standard deviations are obtained. Comparing the average value of each of the groups with a preset set value, and generating time (a) of a time series signal corresponding to a standard deviation group that gives an average value that is equal to or greater than the set value. A standard deviation group that gives a time-series increase rate of the average value of the extracted standard deviation group after (a) equal to or smaller than a preset value. Corresponding to Step of identifying occurrence time of (b) of the time series signal, further comprising the step of obtaining a significant signal based on the occurrence time (a) and (b).
[0057]
Preferably, in the method, the physical or chemical signal comprises binding of an antigen to an antibody, binding of a substrate to an enzyme, or binding of a ligand to a receptor, or activation of an ion channel, receptor, or intracellular. This is a significant signal accompanying the operation of the signal transmission system.
[0058]
Preferably, in the method, the physical signal or the chemical signal is a significant signal generated by a cell responding to the test chemical, and the step (A) is performed when the action on the cell is known. The method is performed in the presence of a certain standard chemical substance and in the presence of the test chemical substance, and the method is performed in the presence of the test chemical substance and the average value obtained in the presence of the standard chemical substance. The method further includes a step of comparing the obtained average value.
[0059]
Preferably, in the method, the physical signal or the chemical signal is a significant signal generated by a cell responding to the test chemical, and the step (A) is performed when the action on the cell is known. The method is carried out in the presence of a certain standard chemical substance and in the coexistence of the standard chemical substance and the test chemical substance, wherein the method comprises: obtaining the average value obtained in the presence of the standard chemical substance; The method further includes comparing the average value obtained in the presence of a substance and the test chemical substance.
[0060]
The present invention also relates to an apparatus for measuring an action of a test chemical substance on a functional protein, the apparatus comprising: the device, a time-series signal generated from the functional protein, a means for acquiring a data group including a plurality of data; Means for obtaining a plurality of extracted data groups from a group, wherein each of the extracted data groups is constituted by a fixed number of data selected from the data group; and a standard for the plurality of extracted data groups. Means for calculating a deviation to obtain a standard deviation group, and means for selecting a physical signal or a chemical signal with reference to each of the standard deviations constituting the standard deviation group.
[0061]
The method of measuring a physical signal or a chemical signal of a functional protein and its change utilizing the chip according to the present invention records a signal emitted from a signal source containing a functional protein as time-series signal data, From the data, a standard deviation value for each predetermined sample time is calculated, and a physical or chemical signal derived from a functional protein is indirectly calculated from the average value of the standard deviation values.
[0062]
Alternatively, the method of measuring a physical signal or a chemical signal of a functional protein and its change utilizing the chip of the present invention is a method in which a signal emitted from a signal source containing a functional protein is recorded as time-series signal data and set in advance. The standard deviation value for each predetermined sample time is calculated, the standard deviation value is approximated to a normal distribution, and a two-dimensional feature amount is extracted from the average value and the half width of the normal distribution.
[0063]
Alternatively, the method of measuring a physical signal or a chemical signal of a functional protein and its change utilizing the chip of the present invention is a method in which a signal emitted from a signal source containing a functional protein is recorded as time-series signal data and set in advance. The standard deviation value for each of two or more different sample times is calculated, the standard deviation value is approximated to a normal distribution for each of the different sample times, and the average of the normal distribution obtained for each of the different sample times is calculated. From the value and the half width, a two-dimensional feature amount is extracted for each different sample time.
[0064]
If desired, the time-series signal data can be from two or more signal sources and added. Preferably, the time-series signal data from the two or more signal sources may be tuned.
[0065]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described with reference to the accompanying drawings.
[0066]
(Embodiment 1)
FIG. 1 is a schematic sectional view ((a) of FIG. 1) and a plan view ((b) of FIG. 1) showing a chip for detecting a physicochemical change of a functional protein according to one embodiment of the present invention. . In the present embodiment, a glass substrate (Corning 1737F) having a thickness of 1.1 mm is used as the substrate 5. An electrode 4 as a measuring electrode and a conductive wire 3 for deriving an electric signal from the electrode 4 are patterned on a glass substrate 5 by gold sputtering, and the other parts than the electrode are covered with an insulating layer 7. The surface of the electrode 4 is substantially flat or planar. The conductive line 3 is led out to the edge of the substrate, forming a terminal 6 at the edge of the substrate. A method for sputtering a conductive material typified by gold is known to those skilled in the art. For example, using gold, in the presence of an inert gas such as argon, a plasma is generated by high frequency between electrodes under low vacuum conditions, and the gold on the cathode is repelled by the energy of ions, resulting in a porous layer on the opposite anode. Form on film. Alternatively, the electrodes and the conductive wires may be formed using a method such as a vacuum evaporation method. Further, it can be formed by printing. Methods for forming the insulating layer 7 are also known to those skilled in the art.
[0067]
A plastic substrate 2 having a through-hole 1 for holding a solution containing a functional protein is arranged and adhered on a glass substrate provided with the electrode 4, the insulating layer 7, and the conductive wire 3 thus formed. The plastic substrate 2 serves as a functional protein or a biological component-containing portion containing a functional protein for isolating a plurality of measurement samples, and the through-hole 1 forms a chamber for accommodating the functional protein to be measured together with the electrode 4. . This plastic substrate 2 is adhered to the substrate with a silicon-based adhesive.
[0068]
(Embodiment 2)
2 and 3 are diagrams showing another embodiment of the present invention. FIG. 2 is a schematic cross-sectional view (FIG. 2A) and a plan view (FIG. 2B) of the chip of the present embodiment, and FIG. 3 further includes a reference electrode 10 on the chip shown in FIG. It is a perspective view of the arrange | positioned chip.
[0069]
As shown in FIG. 2, the chip according to the present embodiment uses an insulating SOI (Silicon on insulator) as the substrate 8, and this substrate 8 is compatible with the through hole 1 of the plastic substrate 2 and communicates therewith. Are provided. On the through hole 1 'on the substrate 8, a flat electrode 4 is formed as a measurement electrode so as to cover the opening. The conductive wire 3 is formed on the side wall of the through hole 1 'so as to be in contact with the lower surface of the electrode. Other configurations are the same as those of the chip of the first embodiment.
[0070]
The chip shown in FIG. 3 is provided with reference electrodes 10 on the side surfaces of the through-holes 1 formed in the plastic substrate 2, and independently from each reference electrode 10, a conductive line connected to a signal amplifier is drawn out and a terminal 9 connected thereto is connected. It has.
[0071]
(Embodiment 3)
FIG. 4 is a schematic sectional view (FIG. 4 (a)) and a plan view (FIG. 4 (b)) showing a chip according to still another embodiment of the present invention. In this embodiment, the measurement electrode 4 is formed so as to penetrate the insulating substrate 8, and one end of the measurement electrode 4 is formed so as to form a contact 11 with a connection portion connected to a signal amplifier. We are excited to type 4 '. The conductive wire 3 is connected to the other end of the measurement electrode. Other configurations are the same as those of the chip described in the second embodiment. A circle surrounded by a dotted line on the right side of FIG. 4A is a partial cross-sectional view showing an enlarged mushroom-shaped head of the measurement electrode 4.
[0072]
(Embodiment 4)
FIG. 5 is a perspective view of a sensor-measuring instrument integrated chip according to still another embodiment of the present invention. The sensor-measuring device integrated chip shown in FIG. 5 includes a sensor unit 13 and a measuring device 17. The sensor unit 13 includes an insulating substrate 5, a plastic substrate 2 having a through-hole 1 forming a chamber for accommodating a functional protein or a component containing a functional protein, and a bottom opening of the through-hole 1 or a peripheral edge thereof. The apparatus includes a measurement electrode 4 provided at least in part, a conductive wire 3, and a heater 12 for adjusting conditions in a chamber, that is, a measurement environment atmosphere, and the above-described components are integrated. The heater 12 is arranged on a heater support member 12 'as required, and the measurement electrode 4 and the conductive wire 3 can also be arranged on the heater support member 12'. As the heater support member, a metal material, a resin material, ceramic, or the like can be used. In addition, as the heater 12, specifically, for example, an IH heater shown in 15 of FIG. 6 or a Peltier element shown in 16 of FIG. 7 can be used. The heater 12 is led out by the conductive wire 3 'to form a terminal 14, and is connected to an external power supply (not shown).
[0073]
The terminals 9 and 14 of the conductive wires of the sensor section 13 protrude from the ends of the substrate 2 and the plastic substrate 5 and can be connected to the measuring instrument 17. The measuring device 17 comprises a receiving part 18 (only a part of which is shown in FIG. 5) which is compatible with the terminals 9 and 14, and the receiving part for receiving the terminal 14 further comprises a heater for operating the heater. It can be configured to be connected to an external power supply. The measuring device 17 displays the data of physical or chemical signals derived from the functional protein in each chamber, adjusts the temperature, and applies disturbance (that is, the obtained data reduces the influence of noise, deviates from the average). "Applying a" disturbance component "for correction). The chip according to the present embodiment is configured to detect a physical change or a chemical change of a functional protein and to display data.
[0074]
6 and 7 are exploded views of a modified example of the sensor unit 13 of the sensor-measuring instrument integrated chip. The reference numeral 15 in FIG. 6 denotes an IH heater, which spirals along the wall of the through hole 1 ′ provided in the substrate 15 as shown in a partially enlarged view below FIG. Are located in In this embodiment and the embodiment shown in FIG. 7, the measurement electrode 4 is formed on the heater support member 12 '. In FIG. 7, reference numeral 16 denotes a Peltier element. As is well known, a Beltier element is a heat exchange device that operates as a heat pump by passing a direct current, and performs cooling, heating, and temperature control.
[0075]
(Example 1)
FIG. 8 is a diagram schematically showing an outline of an apparatus provided with a chip for detecting a physical or chemical change of a functional protein used in an example of the present invention. The configuration of the device shown in FIG. 8 is as follows. A: A warming device that houses a chip for detecting a physical or chemical change of a functional protein. An environment suitable for the functional protein is maintained depending on the functional protein to be tested. B: Means (robot arm) for moving the cell potential measurement chip from the warming device. C: Signal amplification device (amplifier). D: Signal leading cable. E: Signal processing device (computer). Note that the connection between the above-described components is performed by using a means known in the art, and thus the details are not particularly described.
[0076]
FIG. 9 is a diagram schematically illustrating an apparatus used for measuring the ion channel activity in each embodiment of the present invention for each process. In this example, a crude vascular smooth muscle cell membrane fraction prepared from rat vascular smooth muscle cells was used as a component containing a functional protein. The apparatus shown in FIG. 9 includes a signal source 101 having therein the chip shown in FIG. 4 on which a crude membrane fraction of rat vascular smooth muscle cells is arranged, and a standard deviation calculating unit 102 for calculating a standard deviation at regular time intervals. An average calculation unit 105 that calculates an average value of standard deviation values output every fixed time seconds, and an activity calculation unit 108 that calculates ion channel activity from the average value output by the average calculation unit 105. And a data display section 110 for displaying the activity. The standard deviation calculation unit 102, the average value calculation unit 105, and the activity calculation unit 108 are hard disks in which a program for executing these steps is recorded and which is built in a computer. The data display unit is a CRT of a computer.
[0077]
In FIG. 9, the communication of each part is indicated by a dotted line or a solid line. In FIG. 9, reference numerals 103 and 106 denote a normal distribution approximation unit and an average / half-value width calculation unit, respectively, which will be described below. In addition, the sample number distribution unit 104 instructs the standard deviation calculation unit 102 on a method of sampling the signal from the signal source 101 as necessary (for an outline of the configuration of the apparatus including the sample number distribution unit 104, see FIG. 11). Shown). The signal adding unit 107 includes a unit for adding signals from a plurality of signal sources 101 as necessary (an outline of a device including the signal adding unit 107 is shown in FIG. 12). The activity classifying unit 109 compares the average value and the half-value width of the normal distribution obtained by the average / half-value width calculating unit 106 with an existing database, if necessary, to determine the signal change pattern of the functional protein. Including means for classification.
[0078]
The rat vascular smooth muscle cell crude membrane fraction was prepared as follows.
[0079]
Four 75 cm with DMEM medium containing 10% FBS and antibiotics ² Using a volumetric flask, smooth muscle cells were cultured in a thermostat adjusted to 37 ° C. in the presence of 5% carbon dioxide. When the cultured smooth muscle cells reached 80% confluency, the medium was replaced with DMEM medium without FBS, and the cells were further cultured overnight in a thermostat at 37 ° C. Next, the cells were washed twice with PBS at 4 ° C., ice-cooled in advance, and homogenized using a Dounce homogenizer.
[0080]
The obtained homogenized solution was transferred to a 15-ml polypropylene tube, and centrifuged at 1,000 × g for 10 minutes to obtain a supernatant. The supernatant was further centrifuged at 4 ° C. and 100,000 g for 45 minutes to obtain a precipitate. The obtained precipitate was washed with Tris buffer (75 mM Tris (pH 7.5), 15 mM MgCl 2) containing 2 × protease inhibitor. ₂ ) To obtain a solution containing a crude membrane fraction of rat vascular smooth muscle cells.
[0081]
The surface of the electrode of the chip shown in FIG. 4 was processed by the following method.
[0082]
The chip shown in FIG. 4 was immersed in a 1 mM n-octanethiolate (hereinafter referred to as OT) solution dissolved in ethanol and allowed to stand for 4 hours to form a self-assembled monolayer (SAM) composed of OT molecules. . Further, the membrane was immersed in 10 mM 1-methyoxy-5-methylphenazinium (hereinafter referred to as MMP) for 1 hour to conjugate MMP to the self-assembled monolayer. Thereafter, the chip was washed with ethanol and water.
[0083]
10 mM β-oleoyl-γ-palmitoyyl (hereinafter referred to as PCOP) and the vascular smooth muscle cell crude membrane fraction obtained as described above were mixed using ultrasonic waves. The obtained mixture was dropped on the surface of the electrode 4 of the chip treated as described above, and left overnight. Then, a solution containing 0, 0.1, 0.3, 1, 3, 10, 30, and 100 μM of norepinephrine is introduced into the chamber with respect to the crude membrane fraction of vascular smooth muscle cells, and is emitted in response to norepinephrine. The electrical signal was measured. FIG. 13 shows the result of calculating the standard deviation of the time-series data for 10 seconds for each time-series data every 100 milliseconds, plotting the average value and displaying the average value on the data display unit 110. As described above, in the ion channel activity extraction method according to the present embodiment, the distribution depending on the norepinephrine concentration, that is, as the norepinephrine concentration increases, the distribution of the standard deviation value every 100 milliseconds is on the right side, that is, the standard value every 100 milliseconds. It was confirmed that the deviation value was increasing.
[0084]
(Example 2)
FIG. 10 is a diagram schematically illustrating a modified example of the apparatus used for measuring the ion channel activity in the embodiment of the present invention for each process. In this example, a rat hippocampus-derived crude membrane fraction was used as the functional protein. The device shown in FIG. 10 includes the chip shown in FIG. 4 in the signal source 101. This device is different from the device shown in FIG. 103, an average / half-width calculator 106, and an activity classifier 109. The communication of each part is shown by a dotted line or a solid line in FIG.
[0085]
The normal distribution approximation unit 103 classifies the values of the plurality of standard deviations obtained by the unit deviation value calculation unit into a plurality of classes each having a unit of a standard deviation of a fixed width. Is plotted on a coordinate having the number of standard deviations existing in the Y axis as the Y axis, and the obtained graph is approximated to a normal distribution. The average value / half width calculation unit 106 calculates the average value and the half width of the obtained normal distribution. The activity classifying unit 109 for classifying the activity classifies the ion channel activity from the obtained average value and half width. Note that, similarly to the apparatus shown in FIG. 9, the unit standard deviation calculation unit 102, the normal distribution approximation unit 103, the average / half-width calculation unit 106, and the activity classifying unit 109 perform these calculations. It is a computer with a built-in hard disk on which programs are recorded. The data display unit 110 is a CRT.
[0086]
The crude membrane fraction derived from rat hippocampus was prepared as follows. That is, the whole brain is excised from the rat under ice-cooling, and the whole brain is further subjected to a sherbet-like Eagle's balanced salt solution ( E arle's b alternated s alts s solution (abbreviated as EBSS) The component composition is as follows: CaCl ₂ 0.02, KCl 0.40, MgSO ₄ ・ 7H ₂ O 0.20, NaCl 6.68, NaHCO ₃ 2.20, NaH ₂ PO ・ 4H ₂ O 0.14, D-glucose 1.00, phenol red 0.01 pH 7.2: each of the above numbers represents the number of grams of each component contained in 1 L of the solution) for 3 minutes. The EBSS used at this time was 95% O for 5 minutes before forming the sherbet. ₂ + 5% CO ₂ Ventilated. After standing for 3 minutes, only the hippocampus was excised, crushed with scissors, and treated with EBSS containing 0.02% trypsin-0.53 mM EDTA at 37 ° C for 5 minutes. Thereafter, pipetting was performed with a glass pipette whose tip was smoothed by a fire to prepare a cell suspension. Immediately after preparation, centrifuge at 600 g for 10 minutes. Qing Was discarded, and the pelleted cells were washed twice by centrifugation using 10 ml EBSS. Next, the pelleted cells were suspended in 0.6 ml of EBSS, mixed with 0.1 ml of 90% (w / v) glycerol, and allowed to stand at room temperature for 5 minutes. Further, 0.1 ml of 90% (w / v) glycerol was added to the mixture, and the mixture was allowed to stand for 5 minutes. This operation was repeated twice at room temperature. Thereafter, the cells were ice-cooled (0 to 4 ° C.) for 10 minutes, and washed by centrifugation at 600 g for 10 minutes at 4 ° C. Next, cells were added to Lysing buffer (HEPES; 10 mM, CaCl 2). ₂ 1 mM, MgSO ₄ ; 1 mM) and allowed to stand for 5 minutes to explode the cells.
[0087]
Then, the mixture was centrifuged at 600 g for 15 minutes at 4 ° C., and the obtained supernatant was collected in another tube (the pellet obtained by centrifugation contains nuclei, fragments of cell walls, etc. The cell membrane is contained in the supernatant. The supernatant is gently placed on 0.1 ml of 40% (w / w) sucrose, centrifuged at 20,000 G for 90 minutes at 4 ° C., and the precipitate (the cell membrane is present at the interface between water and sucrose) is removed. The precipitate was resuspended in EBSS to prepare a cell membrane solution.
[0088]
Subsequently, the surface of the chip shown in FIG. 4 was processed as follows.
[0089]
First, the chip shown in FIG. 4 was immersed in acetone for 20 minutes. Subsequently, the chip was washed with a UV ozone cleaner for 10 minutes, and the chip was dried under a nitrogen atmosphere. Next, the chip was immersed in an ethanol solution containing 10 μM of dithiobis (succinimidyl undecanoate) for 12 hours to form a SAM composed of Dithiobis molecules on the electrode. Thereafter, the electrode surface was washed with ethanol and pure water in this order, and then dried under a nitrogen atmosphere. The rat hippocampus-derived crude membrane fraction prepared as described above was dropped on the surface of the electrode 4 of the chip treated in this manner, and allowed to stand at 37 ° C. for 30 minutes, whereby the cell membrane fraction containing the receptor was placed on the electrode. Was placed. After arranging the cell membrane fraction, using the apparatus shown in FIG. 10, 0.1, 0.3, 1, 3, and 10 μM concentrations of carbachol solutions were added to the chambers in the chip, respectively. The signals were measured, and the standard deviation calculation unit 102 calculated the standard deviation of the time-series data for 10 seconds for each time-series data every 100 milliseconds. Next, in the normal distribution approximation unit, the values of the plurality of standard deviations obtained by the unit deviation value calculation unit are classified into a plurality of classes each having a unit of a standard deviation of a fixed width, and the class is defined as an X axis. The calculated standard deviation value was plotted on a coordinate having the number of standard deviations present in the class as the Y axis, and the obtained graph was displayed on the data display unit 110 by approximating a normal distribution. FIG. 14 shows the results. As shown in FIG. 14, different normal distributions of potential change were obtained depending on the concentration of added carbachol.
[0090]
【The invention's effect】
A chip capable of detecting a functional protein-functional protein interaction, a functional protein-substance-specific substance-reactive substance interaction, and a significant signal associated with a functional protein-substance-substance interaction, a device equipped therewith, There is provided a method of detecting the significant signal using the same. According to the present invention, the above-mentioned significant signal can be detected by electrochemical recording which has been difficult to detect conventionally. By using the chip of the present invention, an apparatus for performing high-speed drug screening, cell diagnosis, and measurement of environmental pollutants can be provided. Furthermore, the calculation of the binding-dissociation constant of a functional protein and a substance specifically reacting with the functional protein and the interaction between the functional protein and the ligand specifically reacting with the functional protein and the functional protein and the chemical It also makes it possible to provide a means for electrochemically measuring the affinity test.
[0091]
ADVANTAGE OF THE INVENTION According to this invention, the device which can measure channel activity easily from the fluctuation | variation of the signal which arises by opening and closing a channel without forming a giga shield between a functional protein and an insulating substrate is provided.
[0092]
According to the present invention, when performing a measurement, a chip having a member that does not require a skill and time, or a dedicated control device for its production and operation, such as a flat electrode, is used, and thus the work in its production and operation is performed. Since the efficiency is good and no chemical substance is used, there is no need to consider the side effect and the change in fluorescence sensitivity with time, and an apparatus which is inexpensive and has high productivity is provided.
[0093]
According to the present invention, it is also possible to provide a high-speed drug screening and cytodiagnosis device, particularly a cytodiagnosis method and apparatus capable of performing on-site cytodiagnosis at an operation site simply and quickly without performing immunostaining. .
[0094]
By utilizing the chip of the present invention and arranging functional proteins such as a cell membrane fraction, an enzyme, an antibody, and various receptors as functional proteins on an electrode, the functional proteins and various chemical substances are interposed between the functional proteins. It is also possible to detect a physical change or a chemical change due to the interaction between and.
[Brief description of the drawings]
FIG. 1 is a schematic sectional view and a schematic plan view showing a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 2 is a schematic sectional view and a schematic plan view showing a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 3 is a perspective view showing a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 4 is a schematic cross-sectional view and a schematic plan view showing a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 5 is a perspective view showing a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 6 is an exploded view of a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 7 is an exploded view of a modified example of a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 8 is a diagram schematically showing an outline of an apparatus provided with a chip for detecting a physical change or a chemical change of a functional protein of the present invention.
FIG. 9 is a diagram schematically showing an apparatus provided with a chip for detecting a physical change or a chemical change of a functional protein of the present invention for each step.
FIG. 10 is a diagram schematically illustrating, by process, a modified example of an apparatus provided with a chip for detecting a physical or chemical change of a functional protein of the present invention.
FIG. 11 is a diagram schematically illustrating, by process, a modified example of an apparatus provided with a chip for detecting a physical or chemical change of a functional protein of the present invention.
FIG. 12 is a diagram schematically illustrating a modification example of a device provided with a chip for detecting a physical change or a chemical change of a functional protein of the present invention for each step.
FIG. 13 is a view showing the display of the results of measuring the response to norepinephrine in the nerve cell membrane fraction prepared from the rat hippocampus according to the method of the present invention.
FIG. 14 is a view showing the display of the results of measuring the response of the membrane fraction derived from rat hippocampus to carbachol according to the method of the present invention.
[Explanation of symbols]
1, 1 'through hole
2 Plastic substrate
3, 3 'conductive wire
4 electrodes
4 'mushroom-shaped electrode end
5,8 substrate
6, 9, 14 Terminal
7 Insulation layer
10 Reference electrode
11 contacts
12 heater
12 'heater support
13 Sensor part
15 IH heater
16 Peltier element
17 Measuring instrument
18 Terminal receiving section
101 signal source
102 Standard deviation calculator
103 Normal distribution approximation unit
104 Sample number distribution section
105 Average value calculation unit
106 Average / Half Width Calculator
107 signal adder
108 Activity calculator
109 Activity Classification Unit
110 Data display section

Claims (45)

A chip for detecting a change in a physical property or a chemical property of a functional protein,
A substrate having at least one electrode and a conductive line connected to the electrode; and an isolator disposed on the substrate and having at least one chamber for containing a functional protein or a component containing the functional protein. Prepare,
Here, the functional protein or a component containing the functional protein is disposed on the at least one electrode, the isolation unit has a reference electrode, and can derive a physical signal or a chemical signal emitted by the functional protein. , Chips. The chip according to claim 1, wherein the substrate and the isolation unit are integrated. The chip according to claim 1, wherein the substrate is formed of an insulating material. The chip according to claim 3, wherein the insulating material is glass, plastic, or SOI. The chip according to claim 1, wherein the electrode is formed from a material including metal, conductive plastic, or conductive rubber. The chip of claim 5, wherein the metal is selected from the group consisting of platinum, platinum-platinum black, gold, silver, nickel, titanium, silver-silver chloride, and copper. The chip according to claim 1, wherein a surface of the electrode is plasma-treated. The chip according to claim 1, wherein a self-assembled monolayer is formed on a surface of the electrode. The chip according to claim 8, wherein a biocompatible substance or a functional protein is arranged on the self-assembled monolayer. The chip according to claim 8, wherein a mixture of a lipid and a functional protein is arranged on the self-assembled monolayer. The chip according to claim 9, wherein the biocompatible substance is selected from the group consisting of collagen, fibronectin, vitronectin, poly-L-lysine, polyornithine, and polyethyleneimine. 2. The chip according to claim 1, comprising a plurality of electrodes. 13. The chip according to claim 12, wherein each of the plurality of electrodes and a conductive line connected thereto are insulated from other electrodes and conductive lines connected thereto. The chip according to claim 1, wherein the substrate has at least one through hole, and the electrode is disposed on at least a part of a periphery of the through hole or on the through film. The chip according to claim 1, wherein the substrate has at least one through hole, and the electrode is disposed in the through hole. The chip according to claim 1, wherein a voltage change at a constant frequency is given through the electrode, and a frequency that changes according to the reactivity of the functional protein is detected. 2. The chip according to claim 1, wherein a current change at a constant frequency is given through the electrode, and a frequency that changes according to the reactivity of the functional protein is detected. The chip according to claim 1, wherein the substance that specifically reacts with the functional protein derives a physical or chemical signal generated when the substance acts on the functional protein. 19. The method according to claim 18, wherein the functional protein is selected from the group consisting of an antibody, an enzyme, and a receptor, and the substance that specifically reacts with the functional protein is selected from the group consisting of an antigen, a substrate, and a ligand. Chips. The chip according to claim 14, wherein a conductive line that is connected to the through hole is arranged on a front surface or a back surface of the substrate. The functional protein or a component containing the functional protein is arranged in an opening of the through-hole, and an interaction between the functional protein and a substance that specifically reacts with the functional protein is electrochemically detected. A chip according to claim 1. The chip according to claim 14, wherein the depth of the through hole is 1 to 10000 micrometers. 15. The chip of claim 14, wherein the portion of the substrate that does not include the through hole has a thickness in the range of 10 to 10000 micrometers. The tip according to claim 1, wherein the surface area of the electrode is smaller than the surface area of the reference electrode. The chip according to claim 1, further comprising a heating unit disposed on the substrate. The chip according to claim 25, wherein the heating means is a Peltier element or an IH heater. A method for measuring a physical signal or a chemical signal emitted by at least one kind of functional protein,
(A) a step of acquiring a time-series signal derived from the device according to claim 1, wherein a functional protein is arranged, as a data group including a plurality of data;
(B) a step of acquiring a plurality of extracted data groups from the data group, wherein each of the extracted data groups is composed of a fixed number of data selected from the data group; Calculating a standard deviation of the plurality of extracted data groups to obtain a standard deviation group, and (D) selecting a physical signal or a chemical signal with reference to each of the standard deviations constituting the standard deviation group A method comprising: The method according to claim 27, wherein a physical signal or a chemical signal emitted by a plurality of types of functional proteins is measured. 28. The method of claim 27, wherein the reference in step (D) is to calculate the average of each standard deviation that makes up the group of standard deviations. The reference in the step (D) classifies each of the standard deviations into a plurality of classes each having a unit of a standard deviation of a fixed width, and each of the standard deviations is present in the class and each of the classes. Plotting on the coordinate with the number of the standard deviation as an axis, approximating the obtained graph to a normal distribution, and calculating the average value, half width, and variance or standard deviation of the obtained normal distribution 28. The method of claim 27, wherein 28. The method according to claim 27, further comprising: performing the steps (B) to (C) a plurality of times by changing the number of data constituting the extracted data group. 31. The method according to claim 30, further comprising performing the steps (B) to (D) a plurality of times by changing the number of data constituting the extracted data group and the width of the class. Selecting said physical or chemical signal from the mean, half-width, and variance or standard deviation of a plurality of said normal distributions obtained. 28. The method according to claim 27, wherein a physical signal or a chemical signal emitted by the functional protein is measured, and after the step (A), a step of adding a data group obtained for each of the functional proteins is included. The method further encompasses. 28. The method according to claim 27, wherein a physical signal or a chemical signal emitted by the functional protein is measured, and after the step (A), a step of subtracting a data group obtained for each of the functional proteins is included. The method further encompasses. 28. The method according to claim 27, wherein a physical signal or a chemical signal emitted by the functional protein is measured, further comprising a step of simultaneously stimulating each of the functional proteins before the step (A). . The reference in the step (D) classifies each of the standard deviations into a plurality of classes each having a unit of a standard deviation of a certain width, and divides the plurality of standard deviations into the classes and the classes present in each of the classes. Plotting on the coordinate with the number of standard deviation as an axis, and approximating the obtained graph by curve approximation analysis selected from the group consisting of exponential decrease, exponential increase, Gauss, Lorentz, sigma, multiple peaks and nonlinear 28. The method of claim 27, comprising: and obtaining slopes around vertices of the resulting fitted curve. 28. The method of claim 27, wherein the selections in step (B) are performed in chronological order. 30. The method according to claim 29, wherein the steps (B) to (D) are repeatedly performed, wherein the selection in the step (B) is performed in chronological order, and the first data of the extracted data group in each repetition. Is selected at regular time intervals, and further comprising comparing the average value of each of the standard deviation groups obtained at each iteration. 31. The method according to claim 30, wherein the steps (B) to (D) are repeatedly performed, wherein the selection in the step (B) is performed in chronological order, and the first data of the extracted data group in each repetition. Is selected at regular time intervals and further comprising comparing the mean of the normal distribution of each of the standard deviations obtained at each iteration. 31. The method according to claim 30, wherein (B) to (D) are repeatedly performed, wherein the selection in the step (B) is performed in chronological order, and the first data of the extracted data group in each repetition is determined. A method further comprising comparing the half-width, variance or standard deviation of the normal distribution of each of the standard deviation groups selected at regular time intervals and obtained at each iteration. The reference in step (D) includes obtaining a plurality of extracted standard deviation groups from the standard deviation group, wherein each of the extracted standard deviation groups is selected in time series from the standard deviation group. 28. The method of claim 27, comprising the standard deviation of the number of
Calculating the average value of the certain number of standard deviations and a time-series increase rate of the average value,
A step of comparing the average value of each of the obtained extracted standard deviation groups with a preset set value,
Identifying an occurrence time (a) of a time-series signal corresponding to a group of standard deviations giving an average value equal to or greater than the set value;
The time series increase rate of the average value of the extracted standard deviation group after the above (a) is set to a time series corresponding to a standard deviation group that provides an increase rate equal to or smaller than a preset value. Identifying the time of occurrence (b) of the signal;
Obtaining a significant signal based on said occurrence times (a) and (b). The physical or chemical signal is used for binding of an antigen to an antibody, binding of a substrate to an enzyme, or binding of a ligand to a receptor, or activation of an ion channel, a receptor, or activation of an intracellular signaling system. 28. The method of claim 27, wherein the signal is a significant signal. 28. The method of claim 27, wherein the physical or chemical signal is a significant signal generated by a cell responding to the test chemical, wherein the step (A) comprises: Performed in the presence of a known standard chemical, and in the presence of the test chemical, respectively, the average value obtained in the presence of the standard chemical, and the average obtained in the presence of the test chemical The method further comprising the step of comparing with said average value. 28. The method of claim 27, wherein the physical or chemical signal is a significant signal generated by a cell responding to the test chemical, wherein the step (A) comprises: In the presence of a known standard chemical, and in the presence of the standard chemical and the test chemical, respectively, the average value obtained in the presence of the standard chemical, and the standard chemical, A method further comprising comparing the average value obtained in the coexistence with the test chemical. An apparatus for measuring an effect of a test chemical substance on a functional protein,
The device of claim 1,
Means for acquiring a time-series signal generated from the functional protein as a data group including a plurality of data,
Means for acquiring a plurality of extracted data groups from the data group, wherein each of the extracted data groups comprises a fixed number of data selected from the data group; An apparatus comprising: means for calculating a standard deviation of the standard deviation to obtain a group of standard deviations; and means for referring to each of the standard deviations constituting the group of standard deviations and selecting a physical signal or a chemical signal.

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