CN109115846B

CN109115846B - Detection method and sensor of L-cystine based on 3-mercaptopropionic acid modified gate gold electrode

Info

Publication number: CN109115846B
Application number: CN201810984042.1A
Authority: CN
Inventors: 曹忠; 李文锋; 杨佳; 肖忠良; 朱钦; 周立; 冯泽猛; 印遇龙
Original assignee: Changsha University of Science and Technology
Current assignee: Changsha University of Science and Technology
Priority date: 2018-08-28
Filing date: 2018-08-28
Publication date: 2023-09-08
Anticipated expiration: 2038-08-28
Also published as: CN109115846A

Abstract

The invention discloses a detection method and a sensor for L-cystine based on a 3-mercaptopropionic acid modified gate gold electrode, wherein 3-mercaptopropionic acid (MPA) is modified on the surface of a gold film layer of a gold electrode with a gate prolonged, so that a simple MPA self-assembled prolonged Gate Gold Electrode (GGE) is formed, and the L-cystine is sensitively detected by utilizing the amplification effect of an in-situ signal of a prolonged gate field effect transistor. The sensor has good Nernst response relation to L-cystine, and has a linear range of 5.00×10 ^‑8 —1.00×10 ^‑4 mol/L, response sensitivity of 27.20.+ -. 1.5mV/-pc (25 ℃ C.), and detection limit of 1.32X10 ^‑8 The preparation process of the catalyst is simple and convenient, the response time is quick, and the catalyst has potential application prospect.

Description

Detection method and sensor of L-cystine based on 3-mercaptopropionic acid modified gate gold electrode

Technical Field

The invention belongs to the technical field of chemical/biological sensing, and particularly relates to a detection method and a sensor of L-cystine based on a 3-mercaptopropionic acid modified gate gold electrode, namely a selective membrane potential sensor, which are suitable for on-line detection in the aspects of healthy cultivation and life science.

Background

Many sulfur-containing substances are involved in vital activities such as L-cystine, L-cysteine, L-methionine, etc. in human cells. L-cystine and its reduction products play an important role in the life system. L-cystine is involved in the synthesis of insulin and plasma proteins and is the main component of hair and keratin. L-cystine is a structure in which two cysteines are coupled together by an S bond.

Cystine plays a very important role in the human body, and is an autosomal recessive genetic disease, namely cystiuria, caused by the decrease of the reabsorption cystine in the renal tubules and the increase of the cystine content in urine. Cystiuria patients are often associated with cystine stones, which are more particularly shaped, typically hexagonal crystals. Cystine stones are often relatively large and are easily recurrent, and thus patients with cystiuria may need to receive multiple stone removal procedures. The treatment of cystine stones is a very tricky problem because cystine stones are very easy to relapse after operation, and in vitro shock wave lithotripsy (ESWL) can not be crushed and the residual stones after percutaneous nephrolithotomy (PCNL) have high rate. The main goal of cystiuria treatment is to reduce cystine concentration in urine, increase the solubility of cystine in urine, and promote excretion.

Amino acids are important substances constituting animal and human life, and participate in vital activities of human beings, so that it is necessary to detect amino acids. For the detection of amino acids, researchers have constructed a variety of methods such as Kjeldahl method, gas chromatography, capillary electrophoresis, high performance liquid chromatography, etc., long ago. However, these instruments generally have the disadvantages of complicated operation, heavy instruments, etc., and are not suitable for on-line monitoring of amino acids, and are inconvenient to use outdoors, so that the exploration of a rapid and simple amino acid detection method in recent years is an important target for research in the field of amino acids. Compared with other chemical analysis or instrumental analysis methods, electrochemical methods are attracting more and more attention by researchers due to their features of simplicity, sensitivity, no radiation, no pollution, low cost, etc. The electrochemical analysis method can be used for laboratory detection and outdoor use. Various electrochemical techniques such as Differential Pulse Voltammetry (DPV), square Wave Voltammetry (SWV), electrochemical Impedance Spectroscopy (EIS) and ion selective electrode methods (ISEs) have been widely used in various fields. However, a method for electrochemically detecting L-cystine at a potential level has not been reported yet.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a detection method and a sensor of L-cystine based on a 3-mercaptopropionic acid modified gate gold electrode.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

the detection method of the L-cystine based on the 3-mercaptopropionic acid modified gate gold electrode comprises the following steps:

the method comprises the steps of (1) implanting a p-well (2) and an N-type substrate (3) on an Si substrate layer (1) of a field effect transistor, constructing a source electrode (4) and a drain electrode (5) at the p-well (2) by adopting a thermal evaporation and magnetron sputtering technology, constructing a silicon dioxide layer (6) on the Si substrate layer (1) implanted with the p-well (2) and the N-type substrate (3) and constructing the active electrode (4) and the drain electrode (5), plating an aluminum copper alloy layer (8), a chromium palladium alloy layer (9) and a gold film layer (10) on a substrate layer of a polycrystalline silicon gate (7) in sequence by adopting the thermal evaporation and magnetron sputtering technology, and finally constructing a silicon nitride layer (11) on the substrate layer of the polycrystalline silicon gate (7) and the silicon dioxide layer (6); extending the gate portion by a distance of 0.1-500 mm to produce an extended gate field effect transistor having a gate gold electrode;

(2) Preparing an ethanol solution of 3-mercaptopropionic acid, soaking a gate gold electrode of the cleaned extended gate field effect transistor therein, standing at 25 ℃, and then cleaning the soaked gate gold electrode to prepare a 3-mercaptopropionic acid film gate gold electrode modified by a 3-mercaptopropionic acid film (12);

(3) Connecting a reference electrode, a 3-mercaptopropionic acid film grid gold electrode and an electrode interface of an extended grid field effect transistor to form a double high-resistance differential amplification circuit, inserting the reference electrode and the 3-mercaptopropionic acid film grid gold electrode into PBS buffer solution, connecting a power interface of the extended grid field effect transistor with a positive electrode and a negative electrode of a stabilized voltage supply respectively, and connecting a signal output interface of the extended grid field effect transistor with a test port of a universal meter to form a complete sensing detection loop; the potential change of the system can be sensitively detected by utilizing the in-situ signal amplification function of the field effect transistor; the potential of the 3-mercaptopropionic acid membrane gate gold electrode serving as a working electrode gradually tends to be stable along with the increase of time, samples to be detected containing L-cystine (13) with different concentrations are added after the potential is stable, and then corresponding potential response data are obtained, so that the detection of the L-cystine (13) in the samples to be detected is completed.

Preferably, in the step (1), when the aluminum copper alloy layer (8), the chromium palladium alloy layer (9) and the gold film layer (10) are plated on the basal layer of the polycrystalline silicon grid electrode (7) in sequence by using the thermal evaporation and magnetron sputtering technology, si is adopted ₃ N ₄ Passivating; the aluminum copper alloy layer (8) comprises the following components in parts by weight: 40-68 parts of Al, 30-60 parts of Cu, 2-12 parts of Ni, 1-8 parts of Fe, 1-6 parts of Ti and 0.01-0.50 part of Nb; the chromium-palladium alloy layer (9) comprises the following components in parts by weight: cr 40-80, pd 10-40, ni 2-12, fe 1-8, ti 1-6, nb 0.01-0.50; the thickness of the aluminum copper alloy layer (8) is 20-600 nm, the thickness of the chromium palladium alloy layer (9) is 20-600 nm, and the thickness of the gold film layer (10) is 20-1000 nm.

Preferably, step (2) is carried out by preparing an ethanol solution of 1.0 to 20.0mmol/L of 3-mercaptopropionic acid. In the step (2), the gate gold electrode of the extended gate field effect transistor is sequentially cleaned with ultrapure water and absolute ethyl alcohol. And (3) soaking the gate gold electrode in the ethanol solution of 3-mercaptopropionic acid for 1-72 h in the step (2). In the step (2), the soaked grid gold electrode is washed by absolute ethyl alcohol and ultrapure water, dried and stored.

Preferably, the reference electrode in the step (3) is a saturated calomel electrode or an Ag/AgCl electrode with a built-in saturated KCl solution, and the working electrode is a 3-mercaptopropionic acid membrane gate gold electrode. The PBS buffer solution in the step (3) is phosphate buffer solution with pH of 3.0-8.0 and concentration of 0.1mol/L, the pH value is preferably 5.0, and the preparation method is that a certain amount of NaH is added ₂ PO ₄ ·2H ₂ O、Na ₂ HPO ₄ ·12H ₂ O, naCl are mixed and dissolved in water according to a proper proportion, and the pH value is regulated by adopting 0.1mol/L hydrochloric acid.

The invention also provides a sensor for detecting L-cystine, which comprises a field effect transistor, wherein a grid-extended gold electrode, namely a grid gold electrode, is arranged on the field effect transistor, the grid part of the grid-extended gold electrode is extended by a distance of 0.1-500 mm, and a 3-mercaptopropionic acid film (12) is assembled on the surface of a gold film layer (10) of the gold electrode.

The field effect transistor comprises an Si substrate layer (1) and a polysilicon gate (7) arranged on the Si substrate layer (1); the p-well (2) and the N-type substrate (3) are implanted in the Si basal layer (1), a source electrode (4) and a drain electrode (5) are arranged at the p-well (2), the Si basal layer (1) which is implanted in the p-well (2) and the N-type substrate (3) and used for constructing the source electrode (4) and the drain electrode (5) is provided with a silicon dioxide layer (6); an aluminum copper alloy layer (8), a chromium palladium alloy layer (9) and a gold film layer (10) are plated on the basal layer of the polycrystalline silicon grid electrode (7) in sequence; a silicon nitride layer (11) is also arranged on the basal layer of the polysilicon gate (7) and the silicon dioxide layer (6). The thickness of the aluminum copper alloy layer (8) is 20-600 nm, the thickness of the chromium palladium alloy layer (9) is 20-600 nm, and the thickness of the gold film layer (10) is 20-1000 nm.

The sensor has good Nernst response relationship to L-cystine (13), and has a linear range of 5.00×10 ^-8 —1.00×10 ^-4 mol/L, response sensitivity of 27.20.+ -. 1.5mV/-pc (25 ℃ C.), and detection limit of 1.32X10 ^-8 mol/L。

The invention is further described below:

the invention extends the Gate Gold Electrode (GGE) of the Field Effect Transistor (FET) for a certain distance, such as 0.1-500 mm, and self-assembles 3-mercaptopropionic acid (MPA) on the surface of GGE to form a novel sensor capable of detecting L-cystine (L-cysteine). SEM characterization, electrochemical testing and XPS analysis of the sensing interface show that the negatively charged 3-mercaptopropionic acid adsorbs the positively charged target L-cystine by electrostatic action in solution, thereby forming an electric double layer structure to generate a membrane potential for recognizing organic ammonium ions. The electrode has good potential response performance to L-cystine in PBS buffer solution at pH=5.0, and the linear response range of the electrode to L-cystine is 5.00×10 ^-8 —1.00×10 ^-4 mol/L, response sensitivity of 27.20.+ -. 1.5mV/-pC (25 ℃ C.), and detection limit of 1.32X10 ^-8 mol/L. The potential response time of the electrode is fast, about 28.5 seconds. The electrode has better reproducibility and stability. And the electrodeHas good selectivity, and the measurement of L-cystine is not interfered by 13 amino acids such as L-methionine (L-Met), L-aspartic acid (L-Asp), L-proline (L-Pro), L-leucine (L-Leu), L-valine (L-Val), L-lysine (L-Lys), L-tryptophan (L-Trp), L-glutamic acid (L-Glu), L-tyrosine (L-Thr), L-serine (L-Ser), L-glycine (L-Gly), L-arginine (L-Arg), L-histidine (L-His) and the like. In addition, the electrode can be used for measuring the L-cystine in an actual pig serum sample, the recovery rate is 91.0-108.9%, and the novel potential sensor is expected to become a novel means which is simple and convenient in device and can rapidly detect the L-cystine.

In a word, the invention develops a simple selective membrane potential type electrochemical sensor, and provides a novel detection method of L-cystine, namely 3-mercaptopropionic acid (MPA) is modified on the surface of a gold film layer of a gold electrode with a prolonged grid electrode to form a simple MPA self-assembled prolonged Grid Gold Electrode (GGE), and the L-cystine is sensitively detected by utilizing the amplification effect of an in-situ signal of a prolonged grid field effect transistor (EGFET). The experimental test result shows that the concentration of L-cystine of the sensor is 5 multiplied by 10 ^-8 —1.0×10 ^-4 The preparation method has the advantages of good Nernst response in the mol/L range, simple and convenient preparation process, quick response time and potential application prospect.

Drawings

FIG. 1 is a schematic diagram of an extended gate field effect transistor; in the figure: 1. si basal layer, 2, source electrode, 3, drain electrode, 4, p-well, 5, N-type substrate, 6, silicon dioxide layer, 7, polysilicon grid, 8, aluminum copper alloy layer, 9, chromium palladium alloy layer, 10, gold film layer, 11, silicon nitride layer, 12, 3-mercaptopropionic acid film;

FIG. 2 is an SEM characterization diagram of the electrode surface morphology before and after detecting L-cystine by GGE/MPA electrode; FIG. 2a is a scanning electron microscope image of the surface of a bare gold electrode, FIG. 2b is an image after self-assembly of MPA on the electrode surface, and FIG. 2c is a surface image after detection of L-cystine by the electrode;

FIG. 3 is a schematic diagram showing the recognition response principle of the MPA molecule combined with the L-cystine molecule; in the figure: 10. gold film layer, 12, 3-mercaptopropionic acid film, 13, L-cystine;

fig. 4: FIGS. 4A and 4B are respectively electrode solutions in potassium ferricyanide (containing 2.0 mmol/LK) ₃ [Fe(CN) ₆ ]，2.0mmol/L K ₄ [Fe(CN) ₆ ]，0.2mol/LNa ₂ SO ₄ ) An alternating current impedance diagram and a cyclic voltammogram; in fig. 4A and 4B: a. GGE, b, GGE/MPA, c, GGE/MPA/L-cystine;

FIG. 5 shows XPS survey spectra of different electrode surfaces; in the figure: a. GGE, b, GGE/MPA, c, GGE/MPA/L-cystine;

FIG. 6 is a graph of slope of MPA membrane modified GGE electrode as a function of pH;

FIG. 7 is a graph showing the potential response of GGE/MPA electrodes after binding different concentrations of L-cystine in PBS buffer at pH=5.0;

FIG. 8A is a time-potential dynamic plot of GGE/MPA electrode after L-cystine addition to PBS buffer at pH=5.0; FIG. 8B shows the addition of L-cystine (10 ^-6 mol/L) of the dynamic response curve;

FIG. 9 shows the effect of common amino acids on detection of L-cystine by GGE/MPA electrodes.

Detailed Description

1. Experimental procedure

1. Preparation of MPA self-assembled membrane gold electrode

42.46mg of 3-mercaptopropionic acid (MPA) was weighed out and dissolved in 100mL of ethanol to give 4mmol/L of MPA/ethanol solution. The preparation process of the self-assembled film comprises the following steps: sequentially cleaning the gold electrode with ultrapure water and ethanol, soaking in MPA/ethanol solution, standing at 25deg.C for 48 hr, taking out the self-assembled film modified gold electrode, cleaning with ethanol and ultrapure water, drying, and preserving to obtain 3-mercaptopropionic acid (MPA) self-assembled film gold electrode.

2. Design of extended gate field effect transistor and preparation of gate gold electrode

FIG. 1 is a schematic diagram of an extended gate Field Effect Transistor (FET) design, i.e., incorporating the basic structure of a metal-oxide-semiconductor field effect transistor (MOSFET), with a p-well 2 and an N-type substrate 3 implanted in the FET Si base layer 1, with source and drain electrodes 4 and 5 built up at the p-well 2 using thermal evaporation and magnetron sputtering techniques, and then being subjected to a process of forming a semiconductor deviceA silicon dioxide layer 6 is constructed on the Si basal layer 1 implanted with the p-well 2 and the N-type substrate 3 and constructed with the active electrode 4 and the drain electrode 5, then an aluminum copper alloy layer 8, a chromium palladium alloy layer 9 and a gold film layer 10 are plated on the basal layer of the polysilicon gate 7 in sequence by adopting the thermal evaporation and magnetron sputtering technology, and finally a silicon nitride layer 11 is constructed on the basal layer of the polysilicon gate 7 and the silicon dioxide layer 6; the aluminum copper alloy layer 8 comprises the following components in parts by weight: 40-68 parts of Al, 30-60 parts of Cu, 2-12 parts of Ni, 1-8 parts of Fe, 1-6 parts of Ti and 0.01-0.50 part of Nb; the chromium-palladium alloy layer 9 comprises the following components in parts by weight: cr 40-80, pd 10-40, ni 2-12, fe 1-8, ti 1-6, nb 0.01-0.50; the thickness of the aluminum copper alloy layer 8 is 20-600 nm, the thickness of the chromium palladium alloy layer 9 is 20-600 nm, and the thickness of the gold film layer 10 is 20-1000 nm; extending the gold electrode of the gate portion by a distance of 200mm and using SiO ₂ And Si (Si) ₃ N ₄ The field effect transistor wafer is passivated in order to prevent portions of the wafer other than Au from coming into contact with the solution, thereby forming an Extended Gate Field Effect Transistor (EGFET). And carrying out different physical/chemical modification treatments on the surface of an extended Gate Gold Electrode (GGE) film of the EGFET to form a sensitive film so as to realize sensitive detection of the target to be detected. 3-mercaptopropionic acid (MPA) was self-assembled on the surface of the gate gold electrode according to the above-described method to prepare a 3-mercaptopropionic acid film gate gold electrode (GGE/MPA) modified with 3-mercaptopropionic acid film 12.

3. Testing of self-assembled film gate gold electrodes

A buffer system for electrode potential test is phosphate buffer solution (PBS, 0.1 mol/L) with pH 3.0-8.0, and is prepared by mixing NaH with certain amount ₂ PO ₄ ·2H ₂ O、Na ₂ HPO ₄ ·12H ₂ O, naCl are mixed and dissolved in water according to a proper proportion, and the pH value is regulated by adopting 0.1mol/L hydrochloric acid.

Firstly, connecting a saturated calomel electrode and a 3-mercaptopropionic acid membrane grid gold electrode with an electrode interface of an Extended Grid Field Effect Transistor (EGFET) designed in a laboratory to form a double high-resistance differential amplification circuit, inserting the electrodes into PBS buffer solution, connecting a power interface of the EGFET with the positive electrode and the negative electrode of a stabilized voltage supply respectively, and connecting a signal output interface with a test port of a universal meter, thereby forming a complete sensing detection loop. The potential change of the system can be sensitively detected by utilizing the in-situ signal amplification function of the field effect transistor. The potential of the working electrode GGE/MPA in the PBS buffer solution gradually tends to be stable along with the increase of time, and L-cystine with different concentrations of an object to be detected is added after the potential is stable, so that corresponding potential response data are obtained, namely, a standard curve is obtained, and the potential curve of an actual sample is compared with the standard curve when the actual sample is detected, so that the detection can be completed.

2. Experimental results and analysis

1. SEM characterization of GGE/MPA

SEM characterization was performed on the electrode surface morphology features before and after detection of L-cystine by GGE/MPA electrode, as shown in FIG. 2. In fig. 2, (a) is a scanning electron microscope image of the surface of a bare gold electrode, and it can be seen from the image that the electrode surface is smoother; when the electrode was modified (see fig. 2 b), the electron microscopy image showed that an irregular and rough film was formed on the gold electrode surface, indicating successful self-assembly of MPA to the extended gate gold electrode surface. When L-cystine is detected by the electrode, the surface is agglomerated (see FIG. 2 c), and the structural change is probably due to electrostatic adsorption of L-cystine and MPA, so that the morphology of the electrode surface is changed.

2. Electrochemical characterization of GGE/MPA

The 3-mercaptopropionic acid (MPA) is a short-chain mercapto compound containing mercapto groups and carboxyl end groups, sulfur on the mercapto groups can form Au-S bonds with Au to self-assemble on the surface of a gold electrode, after L-cystine is combined, and under acidic conditions, the L-cystine is positively charged, the 3-mercaptopropionic acid is negatively charged in a buffer solution, electrostatic adsorption occurs between the two molecules, so that the potential rising trend is presented, and two 3-mercaptopropionic acid molecules in the 3-mercaptopropionic acid film layer are combined with two-NH of one L-cystine ₃ ⁺ Equivalent to binding a divalent cation, the response scheme is shown in figure 3.

In order to verify the interaction of 3-mercaptopropionic acid with L-cystine, the present invention examined the electrochemical behavior of the different modified electrodes described above using alternating current impedance and cyclic voltammetry, as shown in FIG. 4. FIG. 4 is a graph of electrode at 2.0mM potassium ferricyanideSolution and 0.2M Na ₂ SO ₄ Alternating current impedance diagram (A) and cyclic voltammogram (B) in solution. In fig. 4A, a represents a bare gold electrode, the impedance value is small, and in fig. 4A, b represents an MPA-modified gold electrode, it is obvious that a semicircle appears in the high-frequency portion compared with the bare gold electrode, and the impedance value is large, which indicates that MPA reduces the conductivity of the gold electrode. Binding L-cystine (1.0X10) ^-5 mol/L), the impedance value is further increased (as shown by curve c in FIG. 4A), and the electronic conductivity is reduced due to adsorption of MPA and L-cystine, so that the impedance value of the gold electrode modified with MPA is increased, and the change trend of the impedance can be verified from the corresponding cyclic voltammetry behavior (FIG. 4B).

In fig. 4B, a represents a bare gold electrode, and has obvious oxidation peaks and reduction peaks, which indicate that the conductivity of the pretreated bare gold electrode is relatively good. FIG. 4B shows that B represents MPA modified gold electrode with significantly smaller redox peaks than that of bare electrode, demonstrating that MPA forms a non-conductive single molecule self-assembled film on gold surface via thiol groups, hindering [ Fe (CN) ₆ ] ^3-/4- Electron conduction at the electrode surface. After binding L-cystine (1.0X10) ^-5 mol/L), the oxidation-reduction peak (shown as curve c in FIG. 4B) is significantly reduced, because cystine is adsorbed on the MPA-modified gold electrode, and the electron conductivity is reduced, so that the electrochemical conduction current is reduced, which indicates that adsorption bonding occurs between the MPA gold electrode and L-cystine, and the method is feasible for identifying and detecting the L-cystine.

3. XPS characterization of GGE/MPA electrodes

Further testing and characterization of the interaction between the modified electrode and the target ion were performed by using X-ray photoelectron spectroscopy (XPS), and XPS total spectra of the surfaces of the different electrodes are shown in fig. 5A. As can be seen from FIG. 5A, the gold electrode (curve b) after self-assembling MPA has not only Au but also Au _4f And the characteristic peak of S2p appears at the binding energy of 163.58eV, indicating that-SH in 3-mercaptopropionic acid forms Au-S bond with Au at the electrode surface, i.e., demonstrating that 3-mercaptopropionic acid has successfully self-assembled on the gold electrode surface. Curve c represents GGE/MPA detectionFrom FIG. 5, it can be seen that the peak intensity of sulfur element after the modification of electrode to react L-cystine is higher than that of electrode before reaction. From the data of the binding energies of the different atoms in Table 1, it can be seen that the binding energy of O1s was changed by 0.46eV before and after detecting L-cystine, because 3-mercaptopropionic acid and L-cystine have electrostatic adsorption, and the binding energy of negatively charged 3-mercaptopropionic acid adsorbs positively charged L-cystine, so that the binding energy shifts to low energy. The reduction in binding energy of S2p by 1.35eV further demonstrates that electrostatic adsorption between 3-mercaptopropionic acid and L-cystine occurs.

TABLE 1

4. Selection of optimal pH

Experiments examine the change of the potential of the MPA membrane electrode along with the concentration of L-cystine of a tested object under the conditions that the pH value of the electrode is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, and the Nernst response slope is obtained according to the change, so that a relation diagram of the slope and the pH value is formed, and is shown in figure 6. As can be seen from fig. 6: the slope of the electrode response was greatest at ph=5.0 and its slope value was 27.20±1.5mV/-pC (25 ℃), approaching the theoretical value of the positive divalent nernst response slope (29.25 mV/-pC). The mechanism shown in FIG. 3, i.e., the L-cystine provides two positive charges, is further verified by the slope of the electrode response resulting from pH optimization, and thus the slope is in a positive bivalent Nernst response relationship.

5. GGE/MPA electrode response Performance

The test response performance of the electrode to L-cystine was examined experimentally, and FIG. 7 is a graph showing the potential response of the electrode after binding different concentrations of L-cystine at PBS buffer pH=5.0. As can be seen from FIG. 7, as the L-cystine concentration increases, the electrode potential also gradually increases, indicating that the L-cystine bonded to the surface of the modified electrode increases, and the linear response of the electrode to L-cystine ions in PBS buffer solution with pH=5.0 ranges from 5.0X10 ^-8 mol/L—1.0×10 ^-4 mol/L. Meanwhile, the least square method is adopted for processing, and electricity is obtained after fittingPolar potential linear response plot, linear equation for electrode Δe=214.45+27.20log ₁₀ c, the slope of which is close to the Nernst response slope theory of divalent positive ions, and 2 NH are combined with one MPA molecule deduced before ₃ ⁺ Is identical to the mechanism of the formula (I). The detection limit can be obtained by plotting about 1.32X10 ^- ⁸ mol/L。

6. Determination of response time, stability and reproducibility

The response time and stability of the MPA modified gold electrode for detecting L-cystine are examined experimentally. FIG. 8A is a graph showing the dynamic potential change after adding different concentrations of L-cystine to PBS buffer at pH=5.0, i.e., by adding L-cystine at 1.0X10 ^-8 —1.0×10 ^-4 The continuous measurement is carried out from low concentration to high concentration in the mol/L range and the potential value of the electrode changing along with time is recorded, so that the time for the electrode to reach equilibrium in the whole concentration range is short, the response time is calculated to reach 95% of the maximum value of potential response, namely 28.5s, and the electrode has very high response speed to L-cystine; FIG. 8B is a further addition 10 ^-5 The dynamic response curve after mol/L L-cystine further demonstrates that the response performance of the electrode is stable. The electrode pair was 1.0X10 ^-5 The response slope of the mol/L L-cystine sample is changed to 21.53mV/-pC after 14 days of test, and the slope is reduced by 20.84%, which indicates that the potential sensor can be used for more than 2 weeks and has longer service life.

TABLE 2 GGE/MPA electrode reproducibility

Experiments also examined the reproducibility of the potential response of the prepared MPA modified electrode to different concentrations of L-cystine samples, i.e.to 1X 10 ^-6 mol/L and 1X 10 ^-5 The potential values of the L-cystine samples were measured back and forth 10 times for each of the mol/L-cystine samples (as shown in Table 2). By analysis and processing of the data, the relative standard deviation was found to be 1.96% and 2.18% respectively, which is small, indicating that the electrode was in two different concentrations of L-cystineThe reproducibility of (c) is good.

7. Electrode selectivity

One of the important characteristics of ion selective electrodes is that it is selective and responsive to only a particular substance. Thus, to the PBS buffer solution containing the modified electrode, 10. Mu.M L-cystine, 50 times the concentration of the interfering substance, i.e., 500. Mu.M, was added, and 13 amino acids such as L-methionine (L-Met), L-aspartic acid (L-Asp), L-proline (L-Pro), L-leucine (L-Leu), L-valine (L-Val), L-lysine (L-Lys), L-tryptophan (L-Trp), L-glutamic acid (L-Glu), L-tyrosine (L-Thr), L-serine (L-Ser), L-glycine (L-Gly), L-arginine (L-Arg), L-histidine (L-His) were examined for the interference of the MPA modified gate gold electrode on L-cystine (FIG. 9). The results show that the interference of the 13 interfering substances to the L-cystine to be detected is not obvious, and the sensor can be proved to have good selectivity.

8. Determination and analysis application of recovery rate

Under the optimized experimental condition, the 3-mercaptopropionic acid is utilized to modify the determination of the concentration of L-cystine in the actual sample by the grid gold electrode. In the measurement, pig serum samples C1, C2, C3, C4 and C5 are respectively collected, supernatant is taken, the supernatant is diluted by 10 times by PBS buffer solution with pH of 5.0, L-cystine with known concentration is added into the pig serum samples, and the recovery rate is measured to be 91.0-108.9% by a standard addition method. Experimental results show that the electrode can be used for detecting L-cystine in actual samples.

In summary, the invention provides a selective electrode based on MPA membrane modified gate gold electrode, and experimental results show that the electrode shows a high-sensitivity Nernst response relationship to L-cystine in PBS buffer solution with pH=5.0, and the linear response range is 5.00×10 ^-8 —1.00×10 ^-4 mol/L, detection limit of 1.32X10 ^-8 mol/L. The electrode has the characteristics of short response time (about 28.5 seconds), good selectivity, reproducibility, stability and the like, can be directly applied to measuring the L-cystine in an actual sample, and is expected to become a novel online test means of the L-cystine.

Claims

1. The detection method of the L-cystine based on the 3-mercaptopropionic acid modified gate gold electrode is characterized by comprising the following steps:

(2) Preparing an ethanol solution of 3-mercaptopropionic acid, soaking a gate gold electrode of the cleaned extended gate field effect transistor therein, standing at 25 ℃, and then cleaning the soaked gate gold electrode to prepare a 3-mercaptopropionic acid film gate gold electrode; preparing the 3-mercaptopropionic acid membrane gate gold electrode modified by the 3-mercaptopropionic acid membrane (12)

2. The method according to claim 1, wherein in the step (1), when the aluminum copper alloy layer (8), the chromium palladium alloy layer (9) and the gold film layer (10) are sequentially plated on the substrate layer of the polysilicon gate electrode (7) by using a thermal evaporation and magnetron sputtering technique, si is used ₃ N ₄ Passivating; the aluminum copper alloy layer (8) comprises the following components in parts by weight: 40-68 parts of Al, 30-60 parts of Cu, 2-12 parts of Ni, 1-8 parts of Fe, 1-6 parts of Ti and 0.01-0.50 part of Nb; the chromium-palladium alloy layer (9) comprises the following components in parts by weight: cr 40-80, pd 10-40, ni 2-12, fe 1-8, ti 1-6, nb 0.01-0.50; the thickness of the aluminum copper alloy layer (8) is 20-600 nm, the thickness of the chromium palladium alloy layer (9) is 20-600 nm, and the thickness of the gold film layer (10) is 20-1000 nm.

3. The process according to claim 1, wherein in step (2) an ethanol solution of 1.0 to 20.0mmol/L of 3-mercaptopropionic acid is prepared.

4. The method of claim 1, wherein the gate gold electrode is immersed in the ethanol solution of 3-mercaptopropionic acid in step (2) for a period of time ranging from 1 to 72h.

5. The method of claim 1, wherein the reference electrode in step (3) is a saturated calomel electrode or an Ag/AgCl electrode with a built-in saturated KCl solution, and the working electrode is a 3-mercaptopropionic acid membrane gate gold electrode.

6. The method of claim 1, wherein the PBS buffer in step (3) is phosphate buffer at a concentration of 0.1mol/L at a pH of 3.0-8.0.

7. A sensor for detecting L-cystine, the sensor comprising a field effect transistor, the field effect transistor being provided with a gate-extended gold electrode, characterized in that, in the gate-extended gold electrode, the gate part is extended by a distance of 0.1-500 mm, and a 3-mercaptopropionic acid film (12) is assembled on the surface of a gold film layer (10) of the gold electrode.

8. The sensor according to claim 7, characterized in that the field effect transistor comprises a Si-based layer (1) and a polysilicon gate (7) provided on the Si-based layer (1); the p-well (2) and the N-type substrate (3) are implanted in the Si basal layer (1), a source electrode (4) and a drain electrode (5) are arranged at the p-well (2), the Si basal layer (1) which is implanted in the p-well (2) and the N-type substrate (3) and used for constructing the source electrode (4) and the drain electrode (5) is provided with a silicon dioxide layer (6); an aluminum copper alloy layer (8), a chromium palladium alloy layer (9) and a gold film layer (10) are plated on the basal layer of the polycrystalline silicon grid electrode (7) in sequence; a silicon nitride layer (11) is also arranged on the basal layer of the polysilicon gate (7) and the silicon dioxide layer (6).

9. The sensor according to claim 8, wherein the thickness of the aluminum copper alloy layer (8) is 20-600 nm, the thickness of the chromium palladium alloy layer (9) is 20-600 nm, and the thickness of the gold film layer (10) is 20-1000 nm.

10. The sensor according to any one of claims 7 to 9, wherein the sensor has a good nernst response to L-cystine (13) with a linear range of 5.00 x 10 ^-8 —1.00×10 ^-4 mol/L, response sensitivity of 27.20+ -1.5 mV/-pcThe detection limit was 1.32X10 ^-8 mol/L。