JP2010243301A - Measuring system, measuring instrument, transistor, and measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein quantification system capable of detecting target protein with high sensitivity. <P>SOLUTION: The protein quantification system 20 is equipped with the source region 4 and drain region 5 formed on the surface of a substrate, the gate part 7 formed on the surface of the substrate between the source region 4 and the drain region 5, and the reference electrode 1 applying voltage to the gate part 7 through the electrolyte solution 12 present on the side opposite to the side facing the surface of the substrate of the gate part 7 and includes a transistor 10 wherein the ligand 13, which specifically interacts with the target protein 14, is bonded to the surface on the side opposed to the side facing the surface of the substrate of the gate part 7 and an ammeter 9 measuring the current flowing through the channel formed between the source region 4 and the drain region 5 by bonding a charge imparting reagent 15 to the target protein 14 specifically interacting with the ligand 13. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a measurement system, a measurement device, a transistor, and a measurement method for measuring a protein concentration in a solution.

  Conventionally, various methods have been used to quantify proteins with unknown concentrations. One of the most commonly used protein quantification methods is Western blotting. Western blotting is a method of separating proteins in a sample by electrophoresis such as SDS-PAGE, then transferring the proteins to a membrane electrically, and using antibodies specific to the target protein in the sample on the membrane. In this method, the target protein is detected by reaction.

  The Western blot method has been widely used because the equipment and reagents to be used are inexpensive, but it takes a long time for a series of experimental operations and is complicated, so it relates to protein quantification using a transistor. There is a lot of research.

  A field effect transistor designed to detect an antigen that reacts with a specific antibody has been proposed as a transistor for detecting a biological substance using an electrical signal (see Patent Document 1). A field effect transistor designed to detect purines, pyrimidines, and nucleotides has also been proposed (see Patent Document 2).

  Currently known transistors that detect biological substances are predominantly those that detect DNA (see Non-Patent Document 1), and few transistors still detect proteins.

U.S. Pat. No. 4,238,757 (publication date: 1980, 12, 9) U.S. Pat. No. 4,777,019 (publication date: 1988, 10, 11)

Japan Journal of Applied Physics, Vol. 44, no. 4B, 2005, pp2854-2859

  A conventional transistor for quantifying proteins has the following problems.

  First, when DNA is quantified with a transistor, the negative charge of the phosphate group of the nucleotide matches the electrical signal of the DNA, but each protein has a complex three-dimensional structure. The calculated charge amount does not match the surface charge. As a result, the surface charge is not proportional to the protein amount, and it is difficult to accurately quantify the protein amount from the surface charge.

  Second, proteins are macromolecules, and it is difficult to fit the entire target protein bound and adsorbed to the ligand within the Debye length. For this reason, the protein must be detected with high sensitivity at a part of the electric signal potential (tailing portion) within the Debye length. Therefore, there arises a problem that it is difficult to detect the protein.

  Therefore, a measuring apparatus that can be used for protein quantification and can detect a small amount of protein charge with high sensitivity has been strongly desired.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a measurement device (transistor) and a measurement system that detect proteins with higher sensitivity than in the past.

  In order to solve the above problems, the measurement system according to the present invention is a measurement system that measures the concentration of a target protein that is a measurement target, the source region and the drain region formed on the substrate surface, the source region, A reference for applying a voltage to the gate part through an electrolyte solution existing on the gate part formed on the substrate surface between the drain region and the side of the gate part facing the substrate surface A transistor having a ligand that specifically interacts with the target protein on a surface opposite to the surface of the gate portion facing the substrate surface, and a specific electrode for the ligand. When a charge-carrying molecule binds to the target protein that interacted, the current flowing through the channel between the source region and the drain region is measured. It is characterized in that it comprises a measuring device for.

  According to said structure, the transistor which comprises a measurement system is provided with the source region, the drain region, the gate part, and the reference electrode. The reference electrode applies a voltage to the gate portion via an electrolyte solution that is present on the opposite side of the gate portion facing the substrate surface. A ligand that specifically interacts with the target protein is bound to the gate portion on the side opposite to the side facing the substrate surface. With this configuration, it is possible to keep only the target protein on the surface of the gate portion.

  Then, the measuring apparatus measures a drain current (current flowing between the source region and the drain region) flowing in the channel generated by the binding of the charge-providing molecule to the target protein that specifically interacts with the ligand.

  By binding the charge-providing reagent to the target protein, even if the amount of the target protein is small, the drain current corresponding to the target protein concentration can be measured by increasing the charge. Therefore, the target protein can be detected with higher sensitivity than before.

  The charge possessed by the charge-providing reagent may be a positive charge or a negative charge.

  The charge-providing molecule is preferably selected from the group consisting of CBB G-250, sodium dodecyl sulfate, and a cationic polymer.

  With the above configuration, the charge of the target protein can be increased, and the target protein can be detected with higher sensitivity than before.

  The gate portion preferably includes a gate insulating layer formed on the substrate surface and an immobilizing layer laminated on the gate insulating layer for immobilizing the ligand.

  With the above configuration, it becomes easy to fix the ligand to the gate portion.

  The transistor used in the measurement system includes the source region, the drain region, the gate portion, and the reference electrode, and is opposite to the side of the gate portion facing the substrate surface. A transistor having a ligand bonded to the surface is also included in the technical scope of the present invention.

  The measurement method according to the present invention is a measurement method using the measurement system, and includes an interaction step in which the ligand and the target protein interact with each other, and a protein that has not interacted with the ligand in the interaction step. A washing step for removing the charge, a charge giving step for binding the charge-providing molecule to a protein interacting with the ligand after the washing step, and a source region and the drain region after the charge giving step. And a measuring step for measuring a current flowing between them.

  According to the above method, the target protein can be specifically retained on the gate insulating layer, and the charge of the protein is increased by binding the target protein to the target protein, resulting in higher sensitivity than before. It becomes possible to detect the target protein.

  The measuring apparatus according to the present invention is a measuring apparatus for measuring the concentration of a target protein, and is formed on a substrate surface between a source region and a drain region formed on the substrate surface and between the source and the drain. A gate part and a reference electrode for applying a voltage to the gate part via an electrolyte solution present on the opposite side of the gate part facing the substrate surface; and on the substrate surface of the gate part A transistor to which a ligand that specifically interacts with the target protein is bound to a surface opposite to the facing side, and a charge-imparting molecule binds to the target protein that specifically interacts with the ligand. And a measuring unit for measuring a change in current flowing through the channel generated between the source region and the drain region.

  According to said structure, the measuring apparatus is provided with the transistor provided with a source region, a drain region, a gate part, and a reference electrode. The reference electrode provided in the transistor applies a voltage to the gate portion via an electrolyte solution that is present on the opposite side of the gate portion facing the substrate surface. A ligand that specifically interacts with the target protein is bound to the gate portion on the side opposite to the side facing the substrate surface. With this configuration, the target protein can be specifically retained on the surface of the gate portion.

  Then, the measurement device measures the drain current flowing in the channel that is generated when the charge-providing molecule binds to the target protein that specifically interacts with the ligand.

  By binding the charge-providing reagent to the target protein, even if the amount of the target protein is small, the drain current corresponding to the target protein concentration can be measured by increasing the charge. Therefore, the target protein can be detected with higher sensitivity than before.

  The charge possessed by the charge-providing reagent may be a positive charge or a negative charge.

  As described above, the protein quantification system according to the present invention includes a source region and a drain region formed on the substrate surface, a gate portion formed on the substrate surface between the source region and the drain region, and A reference electrode for applying a voltage to the gate part via an electrolyte solution present on the opposite side of the gate part from the side facing the substrate surface, and a side of the gate part facing the substrate surface On the opposite side, a transistor in which a ligand that specifically interacts with the target protein is bound, and a charge-providing molecule binds to the target protein that specifically interacts with the ligand. And a measuring device for measuring a current flowing through a channel generated between the drain region and the drain region.

  Therefore, there is an effect that the target protein can be detected with higher sensitivity than before.

It is a figure which shows the structure of the protein quantification system which concerns on one Embodiment of this invention. It is a figure which shows reaction in the gate part of target protein, a ligand, and a charge provision reagent. (A) is a figure which shows the structure of the protein quantification system based on one Example of this invention, (b) is a graph for demonstrating the principle of protein quantification.

  An embodiment of the present invention will be described below with reference to FIGS. The protein quantification system 20 of this embodiment is a quantification system that quantifies a target protein (target protein, target protein) 14 that is a detection and measurement target. As will be described later, in the protein quantification system 20, the target protein 14 specifically bound to the ligand 13 bound to the gate portion 7 of the transistor 10 is treated with the charge imparting reagent 15, and the charge imparting reagent 15 is bound to the target protein 14. The target protein 14 is detected and quantified with high sensitivity by measuring the drain current that increases or decreases as a result.

(1) Configuration of Protein Quantification System 20 The configuration of the protein quantification system 20 of this embodiment is shown in FIG. As shown in FIG. 1, the protein quantification system 20 includes a transistor 10 as a protein sensor (biosensor), a fixed power source 8 that supplies a constant voltage, an ammeter (current measuring device) 9, and a variable that can change the supplied voltage. A power supply 16 is included.

  The transistor 10 is basically a field-effect transistor (FET), and includes a reference electrode 1, a source region 4 and a drain region 5 formed on the surface of the substrate 6, a source region 4 and a drain region 5. A gate portion 7 and a liquid tank 11 are formed on the surface of the substrate.

  The reference electrode 1 applies a voltage to the gate portion 7 via the electrolyte solution 12 present on the opposite side of the gate portion 7 from the side facing the substrate surface. The reference electrode 1 is connected to the anode of the variable power source 16, and the tip of the reference electrode 1 is inserted into the electrolyte solution 12 stored in the liquid tank 11. The cathode of the variable power source 16 is grounded.

  The source region 4 and the drain region 5 are regions that are heavily doped with N-type impurities, and the substrate 6 is a silicon substrate that is heavily doped with P-type impurities. The source region 4 and the drain region 5 are separated by the gate portion 7. In other words, the transistor 10 has an N-channel depletion type ion-sensitive field-effect transistor (ISFET) as its basic structure. A source electrode and a drain electrode are connected to the source region 4 and the drain region 5, respectively, but these electrodes are not shown in FIG. Note that the transistor 10 may have a P-channel MOS transistor as its basic structure.

  The gate part 7 has a gate insulating layer 3 formed on the substrate surface. The gate insulating layer 3 is, for example, a silicon oxide film.

The immobilization layer 2 is a layer for immobilizing the ligand 13 that specifically interacts (bonds or adsorbs) with the target protein 14, and is a layer of a silicon nitride film (Si ₃ N ₄ ). Since the silicon nitride film has a strong protective action against the solution, it has an object of protecting the silicon oxide film from the solution. The immobilization layer 2 may contain Au, Pt, or a polymer so that the ligand 13 can be easily immobilized.

  The ligand 13 is bonded to the surface (ligand binding surface) of the fixed layer 2 opposite to the side in contact with the gate insulating layer 3. In other words, the ligand 13 is bonded to the surface of the gate portion 7 opposite to the surface facing the substrate surface. The ligand binding surface of the immobilization layer 2 is in direct contact with the electrolyte solution 12.

  The ligand 13 captures the target protein 14 by specifically interacting with the target protein 14. As the ligand 13, an antibody, an artificial antibody, a peptide, a protein, an aptamer, or a combination thereof can be used.

  As a method of immobilizing the ligand 13 on the immobilization layer 2, a physical adsorption method in which the ligand 13 is directly adsorbed on the immobilization layer 2, and a chemistry in which Au or Pt or the like is applied on the immobilization layer 2 and chemically bonded to the ligand 13. A binding method, a comprehensive method including the ligand 13 using a polymer, or the like can be used. The concentration of the solution containing the ligand 13 used when the ligand 13 is immobilized on the immobilization layer 2 is, for example, 5 μg / mL. The concentration range of the ligand solution is preferably 1 to 20 μg / mL, and more preferably 5 μg / mL.

  The ammeter 9 measures the drain current flowing from the source region 4 to the drain region 5 when a voltage is applied from the reference electrode 1. When a voltage is applied from the reference electrode 1, a drain current corresponding to the applied voltage flows from the source region 4 to the drain region 5, and the drain current is detected by the ammeter 9. In particular, the ammeter 9 measures the drain current generated by the binding of the charge-providing reagent 15 to the target protein 14 that specifically interacts with the ligand 13.

(2) Binding of target protein to ligand and treatment with charge imparting reagent Next, a method of specifically binding the target protein 14 to the ligand 13 and treating the bound target protein 14 with the charge imparting reagent 15 will be described. FIG. 2 is a diagram showing a reaction of the target protein 14, the ligand 13, and the charge-providing reagent (charge-providing molecule) 15 at the gate portion 7.

  As shown in FIG. 2, first, ligand 13 is immobilized on immobilization layer 2 in order to prevent nonspecific adsorption of target protein 14 and / or substances other than target protein 14 to immobilization layer 2. The surface of the immobilization layer 2 is blocked with a blocking agent. Examples of the blocking agent include 10% bovine serum albumin (BSA) solution. Only the target protein 14 specifically bound to the ligand 13 can be detected by the blocking treatment.

  Next, the ligand 13 and the target protein 14 are bound. That is, a solution containing the target protein 14 is dropped on the ligand 13 to bind to the ligand 13. The target protein 14 may be collected from a biological tissue or may be purified using a purification method such as chromatography, and the method for preparing the target protein 14 is not particularly limited.

  The reaction time between the ligand 13 and the target protein 14 may be determined by the concentration of the ligand 13 and the target protein 14 within a range in which the ligand 13 and the target protein 14 are not degraded and / or denatured. It is. The antigen-antibody reaction may take several minutes to 3 hours, and more preferably 5 minutes to 1 hour. In this example, it was performed in 10 minutes.

  Thereafter, the target protein 14 not bound to the ligand 13 is washed away using a suitable washing buffer such as PBS (Phosphate buffered saline).

  Next, the charge-imparting reagent 15 is dropped onto the immobilization layer 2 and allowed to stand for a predetermined time, whereby the charge-imparting reagent 15 is bound to the target protein 14 specifically bound to the ligand 13. The reaction time between the charge-providing reagent 15 and the target protein 14 may be appropriately set according to the type of the charge-providing reagent 15. When CBB G-250 is used as the charge-providing reagent 15, the reaction time is, for example, 5 minutes.

  As the charge-providing reagent 15, in addition to CBB G-250, sodium dodecyl sulfate (SDS), a cationic polymer (a polymer having a positive charge), and the like can be used. Of these, CBB G-250 is particularly preferred.

  When CBB G-250 is used as the charge-providing reagent 15, a negative charge can be imparted to the entire target protein 14 while maintaining the higher-order structure or complex structure of the target protein 14. Therefore, the target protein 14 may be a polymer that is difficult to separate by a conventional method, a membrane protein, or the like.

  When SDS is used as the charge-imparting reagent 15, since SDS binding involves denaturation of the target protein 14, the influence of denaturation on the antigen-antibody reaction is minimized as much as possible by shortening the reaction time between SDS and the target protein 14. It is preferable to do.

  When a cationic polymer is used as the charge-providing reagent 15, the cationic polymer is chemically bonded to the surface of the target protein 14 in a limited manner. An example of a protein modification method that can be used in this case is disclosed in JP-A-6-184190.

  Thereafter, the charge-providing reagent 15 released without being bound to the target protein 14 is removed by washing with 0.1 × PBS or the like.

  Finally, an electrolyte solution 12 (for example, 0.1 × PBS) as a measurement solution is injected into the liquid tank 11.

(3) Principle of Quantifying Target Protein Concentration In the transistor 10, a drain current flows from the source region 4 to the drain region 5 due to a voltage load by the reference electrode 1. At this time, a drain current flowing from the source region 4 to the drain region 5 increases due to the binding of the charged charge-providing reagent 15 to the target protein 14 specifically bound to the ligand 13. Since the charge-providing reagent 15 binds nonspecifically to the target protein 14, the drain current increases as the amount of the target protein 14 specifically bound to the ligand 13 increases. As described above, the protein quantification system 20 detects the amount of the target protein 14 specifically bound to the ligand 13 as an increase in drain current. The change in the drain current may be measured by measuring the Vg-Id characteristic.

  In order to measure the concentration of the target protein 14 contained in the sample to be measured, first, while changing the voltage applied to the reference electrode 1 (that is, the voltage of the variable power source 16), at least three different known concentrations are obtained. Using the protein solution, the Vg value (gate voltage) at which a predetermined Id value (drain current) is obtained is measured for each protein solution having a known concentration. The voltage change of the reference electrode 1 may be inserted / extracted at −3 to + 3V, for example. Then, a calibration curve is created by plotting the protein concentration against the Vg value.

  Thereafter, the Vg value in the Id value of the sample containing the target protein 14 is detected using the ammeter 9, and the concentration of the target protein 14 in the sample is calculated by applying the Vg value to the calibration curve.

  By measuring the Id value (drain current) when three different protein solutions having different concentrations were used with the voltage of the variable power supply 16 kept constant, and plotting the protein concentration against the Id value. A calibration curve may be created.

  In any case, it is only necessary to measure an increase in drain current due to the binding of the charge-providing reagent 15 to the target protein 14 that specifically interacts with the ligand 13.

  As described above, the protein quantification method in the protein quantification system 20 removes the interaction process in which the ligand 13 and the target protein 14 interact specifically, and the protein that did not interact with the ligand 13 in the interaction process. A washing step, a charge giving step for binding the charge-providing reagent 15 to the target protein 14 interacting with the ligand 13 after the washing step, and a measurement step for measuring the drain current after the charge giving step. Yes.

(4) Control The CBB G-250 also binds to the ligand 13 and the blocking agent. Since the ligand 13 used for preparing the calibration curve and the ligand 13 used for measuring the protein concentration of the protein solution of unknown concentration are basically the same, it is necessary to take control for removing the background. Not necessarily.

  When control is performed to remove the influence of drift and noise of the transistor 10, two transistors 10 are used to measure the concentration of one target protein 14. One transistor 10 performs ligand 13 fixation, blocking, antigen-antibody reaction, and processing with the charge-providing reagent 15, and the other transistor 10 performs blocking, antigen-antibody reaction, and processing with the charge-providing reagent 15. Since the latter is a measurement value including noise, the latter measurement value is subtracted from the former measurement value and used as a measurement value for the amount of protein.

(5) Effect of protein quantification system 20 In the present invention, CBB G-250 used in BN-PAGE, which is a kind of electrophoresis, is used as an example of the charge-imparting reagent 15 in order to increase the charge amount of the target protein 14. We are using. In general, SDS-PAGE or Native-PAGE is used for protein electrophoresis.

  In SDS-PAGE, a negative charge of SDS is imparted to a protein having a small charge, thereby facilitating migration in a gel and separation. However, since this SDS binds while destroying the higher-order structure and complex structure of the protein, separation based on the original shape of the protein cannot be performed.

  Native-PAGE gives charge to the protein itself by examining the electrophoresis buffer conditions, and separates the protein in its original form. However, a protein having a basic isoelectric point or a protein having a small surface charge cannot have a sufficient charge for electrophoresis, and makes accurate separation difficult.

  On the other hand, BN-PAGE uses and separates a dye called CBB G-250 instead of SDS to migrate and separate proteins in an overall negatively charged state. Since this CBB G-250 can give a negative charge to the whole protein while maintaining the higher-order structure and complex structure of the protein, migration is easy and separation based on the original form of the protein can be performed. For this reason, application to a polymer or membrane protein which is difficult to separate becomes possible.

  Moreover, the detection of the protein by electrophoresis is to separate the protein for each size. For this reason, in order to obtain confirmation that the protein is the target protein, such as when there is a possibility that the same size protein is contained in the sample solution, Western blotting is performed after electrophoresis, and a specific reaction by the antibody is performed. It needs to be detected. These operations are hours consuming and time consuming.

  When the protein quantification system 20 is used, the charge amount of a small amount of protein can be detected with high sensitivity without requiring a complicated operation that is faster in operation than the conventional electrophoresis method.

  Further, since the transistor 10 can be highly integrated and can be easily manufactured, it can be easily used industrially.

[Example of experimental results]
An example of the experimental result of creating a calibration curve for measuring protein concentration using the protein quantification system 20 will be described. FIG. 3A is a diagram showing the configuration of the protein quantification system 20 according to one embodiment of the present invention, and FIG. 3B is a graph for explaining the principle of protein quantification in the protein quantification system 20.

In this example, an anti-adiponectin antibody (Anti Adiponectin [Acrp30], Human, (Mouse), RSD) was used as the ligand 13, and adiponectin was used as the target protein 14. Three protein solutions were prepared by diluting commercially available adiponectin (1 μg / mL) (Acrp30 / Adiponectin, CF, Human, RSD) to 0, 25, 62.5 μg / mL with 2.5 mM KH ₂ PO _4. It was adjusted.

Further, in this embodiment, a transistor 10 having a proton-sensitive film _(Si 3 _{N 4)} as a fixing layer 2. As shown in FIG. 3A, the transistor 10 has an N-channel depletion type CMOS-ISFET as a basic structure, and an N-type region that bridges the source region 4 and the drain region 5 has a substrate surface. Is formed. The N-type region is a region doped with N-type impurities at a high concentration, and a gate portion 7 is formed on the surface thereof. The substrate 6 is N-type silicon, and a P well is formed around the source region 4 and the drain region 5.

First, three transistors 10 were prepared, and an anti-adiponectin antibody was immobilized on the immobilization layer 2 by a physical adsorption method as shown in FIG. Antibody solution used to dissolve the antibody 500 [mu] g to _{2.5mM KH 2 PO 4 /2.5mM Na 2} HPO 4 1mL, the antibody solution 500 [mu] g / mL, was used which was further diluted to 5 [mu] g / mL. An anti-adiponectin antibody solution was dropped on the immobilization layer 2 and allowed to stand for 1 hour.

  After immobilization, the surface of the immobilization layer 2 was washed with 0.1 × PBS to remove antibodies that were not bound to the immobilization layer 2.

  Next, 0.1 × PBS containing 10% BSA was dropped onto the immobilization layer 2 and allowed to stand for 1 hour for blocking. After blocking, the plate was washed with 0.1 × PBS to remove free BSA.

<Addition of charge-imparting reagent to target protein>
Next, each diluted antibody solution was dropped onto the immobilized layer 2 of the three transistors 10 on which the ligand 13 was immobilized, and allowed to stand for 10 minutes to carry out an antigen-antibody reaction. After washing with 0.1 × PBS to remove free antibodies, CBB G-250 solution was added dropwise as a charge-imparting reagent and allowed to stand for 5 minutes. By this treatment, CBB G-250 was bound to adiponectin. CBB G-250 solution was used without diluting PROTEIN ASSAY Bradford, BIO-RAD.

  Next, it was washed with 0.1 × PBS, free CBB G-250 was removed, and the measurement solution (0.1 × PBS) was filled in the liquid tank 11.

  Vg-Id characteristics of each transistor 10 thus set were measured using a semiconductor parameter analyzer (4155A SEMICONDUCTOR PARAMETER ANALYZER, hp, and 16442A TEST FIXTURE, hp) as the ammeter 9. Each measurement value (the gate electrode Vg is inserted at -3 to +3 V, the source and well contact are grounded, the drain voltage is +50 mV, the substrate potential is +1 V) is input to the semiconductor parameter analyzer, and as shown in FIG. Vg when Id = 3 μA was recorded as a measured value.

  As a result, Vg was 970, 960, and 940 mV in the order of anti-adiponectin antibody concentrations of 0, 25, and 62.5 μg / mL, respectively. Using this result, a calibration curve was created by the method of least squares. The three points corresponding to each anti-adiponectin antibody concentration are almost in a straight line, indicating that the antibody was quantified with high accuracy.

(Example of change)
The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and embodiments obtained by appropriately combining the technical means disclosed in the embodiments. Is also included in the technical scope of the present invention.

  For example, the transistor 10 and the ammeter 9 may be integrally formed and realized as a protein quantification apparatus. Further, the variable power source 16 and the fixed power source 8 may be incorporated in the protein quantification apparatus.

  That is, the present invention is a measuring apparatus for measuring the concentration of a target protein to be measured, which is formed on a substrate surface between a source region and a drain region formed on the substrate surface and between the source and the drain. And a reference electrode for applying a voltage to the gate portion via an electrolyte solution present on the opposite side of the gate portion facing the substrate surface, and the substrate of the gate portion. A charge-binding molecule binds to a transistor in which a ligand that specifically interacts with the target protein is bound to a surface opposite to the surface facing the surface, and to the target protein that specifically interacts with the ligand Therefore, it can also be expressed as a measurement device including a measurement unit that measures a change in current flowing between the source region and the drain region.

  The present invention can also be expressed as follows.

  That is, the present invention is an FET type sensor having a source, a gate and a drain electrode, wherein a ligand that specifically reacts or adsorbs to a protein to be detected (target protein) is introduced on the gate surface. is there.

  The sensor is a biosensor that detects the channel drain current changed by the charge of the CBB G-250 by binding the CBB G-250 after specifically reacting or adsorbing the ligand and the target protein. Is preferred.

  If the protein quantification system of the present invention is used, the protein can be quantified easily and with high sensitivity. Therefore, it can be suitably used for protein quantification in research and testing in the life science field, medical field, and the like.

DESCRIPTION OF SYMBOLS 1 Gate electrode 2 Immobilization layer 3 Gate insulating layer 4 Source region 5 Drain region 7 Gate part 6 Substrate 9 Ammeter 12 Electrolyte solution 13 Ligand 14 Target protein (target protein)
15 Charge-providing reagents (charge-providing molecules)
20 Protein quantification system

Claims (6)

A measurement system for measuring the concentration of a target protein to be measured,
A source region and a drain region formed on the substrate surface;
A gate portion formed on the substrate surface between the source region and the drain region;
A reference electrode for applying a voltage to the gate portion via an electrolyte solution present on the opposite side of the gate portion from the side facing the substrate surface, and a side of the gate portion facing the substrate surface; Is a transistor to which a ligand that specifically interacts with the target protein is bonded to the opposite surface;
And a measuring device that measures a current flowing through a channel generated between the source region and the drain region by binding a charge-providing molecule to a target protein that specifically interacts with the ligand. Measuring system.   The measurement system according to claim 1, wherein the charge-imparting molecule is selected from the group consisting of CBB G-250, sodium dodecyl sulfate, and a cationic polymer.   2. The gate portion includes a gate insulating layer formed on the surface of the substrate and an immobilization layer that is laminated on the gate insulating layer and immobilizes the ligand. Or the measuring system of 2. A transistor used in the measurement system according to claim 1,
The device includes the source region, the drain region, the gate portion, and the reference electrode, and a ligand is bonded to a surface of the gate portion opposite to the side facing the substrate surface. Transistor. A measurement method using the measurement system according to any one of claims 1 to 3,
An interaction step for specifically interacting the ligand and the target protein;
A washing step for removing proteins that did not interact with the ligand in the interaction step;
A charge imparting step of binding the charge imparting molecule to the protein interacting with the ligand after the washing step;
And a measuring step of measuring a current flowing between the source region and the drain region after the charge applying step. A measuring device for measuring the concentration of a target protein,
A source region and a drain region formed on the substrate surface; and a gate portion formed on the substrate surface between the source region and the drain region;
A reference electrode for applying a voltage to the gate portion via an electrolyte solution present on the opposite side of the gate portion from the side facing the substrate surface, and a side of the gate portion facing the substrate surface; Is a transistor to which a ligand that specifically interacts with the target protein is bonded to the opposite surface;
And a measurement unit that measures a change in a current flowing through a channel generated between the source region and the drain region by binding a charge-providing molecule to a target protein that specifically interacts with the ligand. A measuring device.

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