CN114894875A - Device for determining isoelectric point of protein by using indium tin oxide field effect transistor and using method - Google Patents
Device for determining isoelectric point of protein by using indium tin oxide field effect transistor and using method Download PDFInfo
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- CN114894875A CN114894875A CN202210567980.8A CN202210567980A CN114894875A CN 114894875 A CN114894875 A CN 114894875A CN 202210567980 A CN202210567980 A CN 202210567980A CN 114894875 A CN114894875 A CN 114894875A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/403—Cells and electrode assemblies
- G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
- G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
Abstract
The invention discloses a device for detecting isoelectric points of protein by using an indium tin oxide field effect transistor and a using method thereof. The device comprises an indium tin oxide field effect transistor, wherein the indium tin oxide field effect transistor is sequentially provided with a substrate, a high-kappa dielectric layer and an ITO channel layer from bottom to top, source and drain electrodes are arranged at two ends of the ITO channel layer, gold nanoparticles for adsorbing protein are deposited on the surface of the middle part of the ITO channel layer, and an insulating layer is arranged on the source and drain electrodes; the upper part of the indium tin oxide field effect transistor is provided with a sample chamber, and the top of the sample chamber is provided with a plurality of sample inlets and a single sample outlet. The present invention exploits this "limitation" in reverse to determine the isoelectric point (pI) of proteins: by continuously varying the pH of the test solution (pH), the channel current (I) of the field effect transistor is measured when the pH of the solution is equal to the pI of the protein ds ) The signal exhibits a knee.
Description
Technical Field
The invention belongs to the technical field of biosensors. In particular to a device for measuring the isoelectric point of protein by using an indium tin oxide field effect transistor and a using method thereof.
Background
Protein is an ampholyte, a colloidal macromolecular substance with positive and negative charges, and its dissociation state and dissociation degree are influenced by the pH of the solution. When the pH of the solution reaches a certain value, the positive and negative charges on the protein are equal, and the protein is electrically neutral, at which time the pH of the solution is called the isoelectric point (pI) of the protein. At isoelectric point, the conductivity, osmotic pressure, solubility and viscosity of protein are all the smallest, the stability of colloidal solution is the worst, and protein is easy to precipitate. The isoelectric precipitation of the protein is utilized, and the method plays an important role in the separation and purification of the protein.
The current methods for determining the isoelectric point of proteins are mainly two types: (1) precipitation method. Taking casein as an example, a certain concentration of casein is added into acetic acid/sodium acetate buffers with different pH values, when the pH value reaches a certain value, the casein is precipitated and the precipitation amount is maximum, and the pH value is the isoelectric point of the protein. Although the method is simple and easy, the consumed protein has large mass, and the method is not suitable for measuring the isoelectric point of the protein with extremely low concentration, and the measurement precision of the isoelectric point is low. (2) Gel isoelectric focusing electrophoresis. In the electrophoresis system, a continuous and stable pH gradient environment with the pH from low to high is formed from an anode to a cathode, the protein moves in the environment under the action of an electric field, and when a certain pH value is reached, the protein stops moving and focuses at the pH value to generate a concentrated band, wherein the pH value is the isoelectric point of the protein. The gel isoelectric focusing electrophoresis method has high precision, and can distinguish protein with isoelectric point difference of 0.01-0.02pH unit. However, this method has a disadvantage in that the protein solution is required to contain no salt. Secondly, the protein is required to be stable at the isoelectric point and cannot be denatured or insoluble. Thirdly, expensive reagents and equipment are needed, such as ampholyte Ampholine, staining solution, destaining solution, focusing indicator and the like for constructing a continuous and stable pH gradient environment, and equipment such as an electrophoresis apparatus, a gel imager and the like. In addition, the complicated processes of glue preparation, sample application, electrophoresis, glue stripping, dyeing, decoloring and the like are also needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention providesAn apparatus for measuring isoelectric point of protein by using indium tin oxide field effect transistor (ITO FET) and its application method are provided. The field effect transistor as a biosensor has a limitation: it cannot be used to directly detect uncharged species. The present invention exploits this "limitation" in reverse to determine the isoelectric point (pI) of a protein: by continuously varying the pH of the test solution, the signal I of the field effect transistor is obtained when the pH of the solution is equal to the pI of the protein ds An inflection point will appear. Because the ITO film growth process is compatible with the mainstream film growth processes such as magnetron sputtering and the like, and the ITO semiconductor performance is excellent, the ITO is adopted as the channel material of the field effect transistor. The pI of the protein is determined by adopting the ITO FET, the concentration of the protein can be as low as pg/mL, the volume of the protein sample only needs 10-50uL, and the measurement precision can be accurate to 0.1pH unit.
In order to achieve the purpose, the invention adopts the following technical scheme:
a device for detecting protein isoelectric points by using an indium tin oxide field effect transistor comprises an indium tin oxide field effect transistor, wherein the indium tin oxide field effect transistor is sequentially provided with a substrate, a high-kappa dielectric layer and an ITO channel layer from bottom to top, two ends of the ITO channel layer are provided with source and drain electrodes, gold nanoparticles for adsorbing protein are deposited on the surface of the middle part of the ITO channel layer, and an insulating layer is arranged on the source and drain electrodes; the upper part of the indium tin oxide field effect transistor is provided with a sample chamber, and the top of the sample chamber is provided with a plurality of sample inlets and a single sample outlet for introducing buffers with different pH values.
Preferably, the substrate is a heavily doped silicon sheet which is used as a grid electrode of the indium tin oxide field effect transistor.
Preferably, the high-kappa dielectric layer is a gate dielectric, specifically HfO 2 、HfLaO、 Al 2 O3 or SiO 2 The high-k dielectric layer is prepared by an atomic layer deposition method, magnetron sputtering or evaporation, and the thickness of the high-k dielectric layer is 3-10 nm.
Preferably, the ITO channel layer grows by magnetron sputtering, atomic layer deposition or electron beam evaporation film-making process, and the thickness of the ITO channel layer is 5-10 nm.
Preferably, the source and drain electrodes are Ni and Au composite metal layers, the thickness of Ni is 1-10nm, and the thickness of Au is 10-50 nm.
Preferably, the insulating layer is SiO 2 、SiN x PMMA or SU 8.
Preferably, the sample chamber is made of PDMS and is fixed on the indium tin oxide field effect transistor by adopting a thermal bonding process; the sample chamber has dimensions of an internal length of 1-10mm, an internal width of 1-10mm and an internal height of 1-10 mm.
Preferably, the sample inlet of the sample chamber is connected to an external microsyringe via a conduit.
The use method of the device for detecting the isoelectric point of the protein by using the indium tin oxide field effect transistor comprises the following steps:
(1) dripping 1pg/mL-1ng/mL protein solution on the surface of an ITO channel layer of an indium tin oxide field effect transistor, wherein the solvent for dissolving the protein is 1 XPBS phosphate buffer solution with pH =7.4, the dripping volume is 50-100 mu L, then standing and incubating for 12-24h at 4 ℃, and after the incubation is finished, washing away the unfixed protein by adopting 1 XPBS phosphate buffer solution with pH = 7.4;
(2) setting electrical test parameters: the substrate is used as a gate electrode, fixed gate voltage and source-drain voltage are set, and channel current I is monitored in real time ds A time-dependent profile;
(3) test I by injecting a buffer solution of pH1=7.4 into the sample chamber through the injection port at a rate of 10-50 μ L/s using a microsyringe ds As a baseline;
(4) when the base line is stable, stopping injecting the buffer solution with the pH of 1=7.4, sequentially introducing the buffer solutions with the pH from high to low into the microsyringe at the speed of 10-50 mu L/s, and detecting and monitoring the channel current I in real time ds The variation curve of (d); when the introduced buffer solution makes the channel current I ds When an inflection point appears, the pH of the buffer solution is the isoelectric point of the protein.
Preferably, the pH interval of the buffer solution with the pH being changed from high to low in step (4) is 0.1.
The invention has the beneficial effects that: (1) simple operation, and complicated processes of glue preparation, sample application, electrophoresis, glue stripping, dyeing, decoloration and the like. (2) Expensive reagents and equipment are not required. (3) The amount of protein sample required is extremely small, with a protein concentration of pg/mL and a volume of about 50-100. mu.L. (4) The test precision is high, and the accuracy can be up to 0.02pH unit (5) without pretreatment such as desalting of protein.
Drawings
FIG. 1 is a schematic diagram of an ITO field effect transistor;
FIG. 2 is a schematic side view of an apparatus for measuring isoelectric points of proteins;
FIG. 3 is a schematic top view of an apparatus for measuring isoelectric points of proteins;
FIG. 4 is a schematic diagram of the principle for determining the isoelectric point of protein, when the pH of the buffer solution is less than the pI of the protein, the protein is positively charged, and ITO is induced to be negatively charged, so that I ds Increase and further decrease the buffer solution pH, I ds Further increase, when the pH of the buffer solution is equal to the pI of the protein, I ds A sharp decrease (signal inflection point) when the buffer pH continues to decrease, at a pH less than pI, I ds But also exhibits an increased phenomenon; however, when the pH value of the buffer solution is larger than the pI of the protein, the protein is negatively charged, ITO is induced to be positively charged, and I is enabled to be ds Decrease;
FIG. 5 shows the determination of the isoelectric point of human prostate antigen PSA using ITO FET; as the pH of the continuous solution decreases in sequence, the more negatively charged the PSA, I ds The larger; when the solution pH =6.8 is equal to the isoelectric point of PSA (pI = 6.8), PSA is uncharged, resulting in I ds Sharply decreases and an inflection point appears; when the solution pH =6.7 is further lowered, PSA is negatively charged, I ds Rising; PSA is positively charged when solution pH =7.4, I ds Reducing to the vicinity of a reference value;
in the figure, 1 is a silicon substrate, 2 is a high-kappa dielectric layer, 3 is an ITO channel layer, 4 is a source drain electrode, 5 is an insulating layer, 6 is a sample chamber, 7 is a sample inlet, and 8 is a sample outlet.
Detailed Description
In order that the objects, features and advantages of the invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings, which are illustrated in detail in order to provide a thorough understanding of the invention, but which may be carried out in other ways than those described. Accordingly, the invention is not limited by the specific implementations disclosed below.
Example 1
A device for detecting protein isoelectric points by using an indium tin oxide field effect transistor comprises an indium tin oxide field effect transistor, wherein the indium tin oxide field effect transistor is sequentially provided with a substrate 1, a high-kappa dielectric layer 2 and an ITO channel layer 3 from bottom to top, two ends of the ITO channel layer 3 are provided with source and drain electrodes 4, gold nanoparticles for adsorbing protein are deposited on the surface of the middle part of the ITO channel layer 3, and an insulating layer 5 is arranged on the source and drain electrodes 4; a sample chamber 6 is arranged at the upper part of the indium tin oxide field effect transistor, and a plurality of sample inlets 7 and a single sample outlet 8 for introducing buffer solutions with different pH values are arranged at the top of the sample chamber 6; the preparation method comprises the following steps:
preparation of PDMS sample cavity
(1) Firstly, designing a template by using AutoCAD, and then manufacturing the template on glass by using SU8 by using a micro-nano processing technology.
(2) PDMS (Dow coming SYLGARD 184) master and coagulant were mixed well at a ratio of 5:1 and left to stand in a vacuum chamber for about 30 min to remove air bubbles. After removing air bubbles, pouring the mixture on an SU8 template, wherein the liquid level of PDMS is 1mm, and placing the mixture in an oven at 80 ℃ for baking for 2 h. The PDMS sample chamber has an internal length of 5mm, an internal width of 3mm, an internal height of 1mm and a wall thickness of 1 mm.
(3) After drying, the PDMS was peeled off from the SU8 template. And punching holes at the tip ends of two sides of the elliptic cavity by using a puncher (phi = 0.5 mm) to serve as a liquid inlet and a liquid outlet.
Preparation of (II) ITO FET
(1) Heavily doped p-type silicon (100) is cleaned using a standard RCA clean process.
(2) An atomic layer deposition process is used for depositing HfLaO with the thickness of 10nm at the temperature of 200-300 ℃ to be used as a gate dielectric layer.
(3) ITO with the thickness of 5nm is deposited by a magnetron sputtering process to be used as a channel. The deposition temperature was 300 ℃ and the ratio of argon to oxygen was 9: 1.
(4) Forming a source electrode and a drain electrode at two ends of the stone ITO through photoetching and evaporation processes: by adopting the photoetching process, the electron beam evaporation process, the stripping process and other processes, the FET with the ITO as the channel is prepared, the source electrode and the drain electrode are composed of 5nm Ni and 50nm Au, the length of the conductive channel is 10 micrometers, and the width of the conductive channel is 20 micrometers.
(5) And (3) manufacturing SU8 insulating layers on the source electrode and the drain electrode by the process steps of glue homogenizing, baking, exposing, developing, fixing, removing glue and the like so as to isolate the source electrode and the drain electrode from contacting with the test sample. (1) And spin-coating SU-8 at 800 rpm/3 s and 3000 rpm/30 s, and baking at 110 ℃ for 3 min. (2) Exposure is carried out for 6s, and baking is carried out for 2min at 110 ℃. (3) PGMEA developed for 60s and IPA developed for 30 s. And cleaning with deionized water, and blowing with nitrogen. The ITO channel is exposed by photolithography and development processes.
(6) And depositing gold nanoparticles on the surface of the ITO. Gold nanoparticles are deposited on the surface of the ITO channel by a magnetron sputtering process to adsorb protein. The deposition conditions were: the temperature was 200 ℃ and the deposition rate was 0.5A/s for 10 s.
Bonding of PDMS sample chamber and ITO FET hydrogen peroxide sensor
Treating the PDMS sample chamber with oxygen plasma, treatment conditions: the ratio of argon to oxygen is 5:1, the treatment time is 5min, and the power is 30W. And after the oxygen plasma treatment is finished, aligning and attaching the PDMS sample chamber and the ITO FET, and standing for 2 hours in a vacuum oven.
The use method of the device for detecting the isoelectric point of the protein by using the indium tin oxide field effect transistor comprises the following steps: in the case of human prostate antigen (PSA), the isoelectric point of PSA is reported in several publications, with an isoelectric point of 6.8. PSA is purchased from American biosciences (United States Biological Inc.).
(1) The outlet of the sample chamber was closed, and the microsyringe injected the PSA solution into the sample chamber from the inlet of the sample chamber at a rate of 10 μ L/s, with the protein-dissolving solvent being 1 x PBS phosphate buffer pH = 7.4. After 10s, the injection was stopped and the cells were incubated at 4 ℃ for 12 h. After the incubation was completed, the outlet was opened, and a 1 × PBS phosphate buffer solution with pH =7.4 was injected from the inlet at a rate of 50 μ L/s by a microsyringe, and the unfixed PSA was washed off for 60 s.
(2) The microsyringe measures the pH at a rate of 50. mu.L/s 1 1 XPBS phosphate buffer solution of =7.4 was injected into the sample chamber, test I ds As a baseline, the test parameter is the gate voltage V gs = 0.1V, source-drain voltage V ds = 0.05V。
(3) After the baseline had stabilized, the microsyringe was used to measure the pH at a rate of 50. mu.L/s 2 1 XPBS phosphate buffer solution of =7 was injected into the sample chamber, test I ds To be stable.
(4) The microsyringe measures the pH at a rate of 50. mu.L/s 3 1 XPBS phosphate buffer solution of =6.9 was injected into the sample chamber, test I ds To be stable.
(5) The microsyringe measures the pH at a rate of 50. mu.L/s 4 1 XPBS phosphate buffer solution of =6.8 was injected into the sample chamber, test I ds To be stable.
(6) The microsyringe measures the pH at a rate of 50. mu.L/s 5 1 XPBS phosphate buffer solution of =6.7 was injected into the sample chamber, test I ds To be stable.
(7) The microsyringe measures the pH at a rate of 50. mu.L/s 6 1 XPBS phosphate buffer solution of =7.4 was injected into the sample chamber, test I ds To be stable.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed composition method, i.e. it is not meant that the present invention must rely on the above detailed composition and method to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. An apparatus for detecting isoelectric point of protein by using indium tin oxide field effect transistor, characterized in that: the device comprises an indium tin oxide field effect transistor, wherein the indium tin oxide field effect transistor is sequentially provided with a substrate, a high-kappa dielectric layer and an ITO channel layer from bottom to top, source and drain electrodes are arranged at two ends of the ITO channel layer, gold nanoparticles for adsorbing protein are deposited on the surface of the middle part of the ITO channel layer, and an insulating layer is arranged on the source and drain electrodes; the upper part of the indium tin oxide field effect transistor is provided with a sample chamber, and the top of the sample chamber is provided with a plurality of sample inlets and a single sample outlet.
2. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the substrate is a heavily doped silicon sheet which is used as a grid electrode of the indium tin oxide field effect transistor.
3. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the high-k dielectric layer is a gate dielectric, specifically HfO 2 、HfLaO、 Al 2 O 3 Or SiO 2 The high-k dielectric layer is prepared by an atomic layer deposition method, magnetron sputtering or evaporation, and the thickness of the high-k dielectric layer is 3-10 nm.
4. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the ITO channel layer is grown by magnetron sputtering, atomic layer deposition or electron beam evaporation film-making process, and the thickness of the ITO channel layer is 5-10 nm.
5. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the source electrode and the drain electrode are Ni and Au composite metal layers, the thickness of Ni is 1-10nm, and the thickness of Au is 10-50 nm.
6. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the insulating layer is SiO 2 、SiN x PMMA or SU 8.
7. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the sample chamber is made of PDMS and is fixed on the indium tin oxide field effect transistor by adopting a thermal bonding process; the sample chamber has dimensions of an internal length of 1-10mm, an internal width of 1-10mm and an internal height of 1-10 mm.
8. The apparatus for detecting isoelectric point of protein according to claim 1 comprising: the sample inlet of the sample chamber is connected with an external microsyringe through a conduit.
9. A method of using the device for detecting isoelectric point of protein with indium tin oxide field effect transistor according to any one of claims 1 to 8, comprising the steps of:
(1) dripping 1pg/mL-1ng/mL protein solution on the surface of an ITO channel layer of an indium tin oxide field effect transistor, wherein the solvent for dissolving the protein is 1 XPBS phosphate buffer solution with pH =7.4, the dripping volume is 50-100 mu L, then standing and incubating for 12-24h at 4 ℃, and after the incubation is finished, washing away the unfixed protein by adopting 1 XPBS phosphate buffer solution with pH = 7.4;
(2) setting electrical test parameters: the substrate is used as a gate electrode, fixed gate voltage and source-drain voltage are set, and channel current I is monitored in real time ds A time-dependent profile;
(3) the pH is measured by microsyringe through an inlet at a rate of 10-50. mu.L/s 1 Injection of buffer solution of =7.4 into the sample chamber, test I ds As a baseline;
(4) when the baseline stabilized, pH 1 Stopping injecting the buffer solution with the concentration of 7.4, sequentially introducing the buffer solution with the pH value from high to low into the microsyringe at the speed of 10-50 mu L/s, and detecting and monitoring the channel current I in real time ds The variation curve of (d); when the introduced buffer solution makes the channel current I ds When an inflection point appears, the pH of the buffer solution is the isoelectric point of the protein.
10. The method of using the device for detecting isoelectric point of protein according to claim 9 wherein the pH interval of the buffer solution from high to low pH in step (4) is 0.1.
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