A detection method and sensor for L-cystine

Abstract

The present invention discloses a method and sensor for detecting L-cystine. A poly(disulfide-dipropanesulfonic acid) film is assembled on the surface of a gold film layer of a gold electrode as a gate extending from a field effect transistor. The field effect transistor in-situ signal amplification function is used to realize sensitive detection of L-cystine. The polyanion of the poly(disulfide-dipropanesulfonic acid) film adsorbs and binds the positively charged target L-cystine through electrostatic action to form a double electric layer structure to generate a membrane potential for identifying monovalent organic ammonium ions. The sensor has a good Nernst response relationship for L-cystine, and the linear range is 5.0×10 ‑6 —1.0×10 ‑3 mol / L, the response sensitivity was 58.25±1.5mV / ‑pc (25°C), and the detection limit was 2.69×10 ‑6 mol / L, which can be used for the rapid and sensitive detection of L-cystine in pig serum samples, and has potential application prospects in the fields of healthy breeding and life sciences.

Description

Technical Field

[0001] The invention belongs to the field of chemical / biological sensor technology, and specifically relates to an ion-selective membrane potential type L-cystine detection method and sensor, which is suitable for online detection in healthy breeding and life science. Background Art

[0002] L-cystine is a sulfur-containing amino acid, mostly found in keratin in hair, nails, etc. In addition to constituting body proteins, it is also widely present in living cells and tissues of organisms and has many important physiological functions. L-cystine can promote the oxidation and reduction functions of body cells, make the liver function vigorous, neutralize toxins, promote leukocyte proliferation, prevent the development of pathogens, and maintain protein configuration. Sulfur-containing amino acids such as L-cystine can not only be added to food as additives to increase the nutrition of food, but also play an important role in animal digestion and absorption, nutrient metabolism and immune function.

[0003] Commonly used amino acid detection methods include high performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis, fluorescent probe detection, etc. At present, there are few reports on the detection of L-cystine at home and abroad. The main method used is the chromatographic separation and analysis method. For example, Xia Sujie et al. used pre-column derivatization HPLC to determine L-cystine, with a linear range of 0.02-1.00 mg / mL; Zhang et al. used HPLC to detect L-cystine, with a linear range of 2-15 μM; Alwael et al. used reversed phase liquid chromatography coupled to mass spectrometry to detect L-cystine, with a linear range of 0.25-250 μM. However, these methods generally have the disadvantages of high cost, expensive precision instruments, complex sample preparation processes, and cannot be used online or inconveniently outdoors. Therefore, exploring and developing a simple and fast amino acid analysis method is an important trend in this field. Compared with other instrumental analysis methods, electrochemical methods are gaining more and more attention due to their simplicity, sensitivity and rapidity. For example, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) have been used for the detection of some amino acids.

[0004] At present, the reports on the detection of cystine by electrochemical methods are mainly amperometric sensors. Salimi et al. prepared nickel-doped carbon ceramic electrodes and used CV method to detect L-cystine, with a linear range of 1.0-450μM and a detection limit of 0.64μM; Zor et al. used reduced graphene oxide / β-cyclodextrin composite membrane electrodes, with a linear range of 1.0-100μM and a detection limit of 1.0μM for L-cystine. At the same time, combined with DPV detection and model calculation, the binding constants of D-type and L-type cystine can be obtained. The existing amperometric methods generally have problems such as inconvenient instruments to carry and difficulty in online detection. There are still few discussions on cystine detection methods at home and abroad. It is extremely important to explore a simpler, faster and more sensitive electrochemical method for determining L-cystine.

[0005] Compared with the current-type sensor modified by composite materials, the potential-type electrochemical sensor has the advantages of light instrument, simple preparation, and fast response, which is conducive to the online and real-time monitoring and analysis of amino acids in actual samples. It has been successfully applied to the determination of different amino acids, such as L-histidine, L-cysteine, L-glutamic acid, L-lysine, etc. Moreover, in the potential-type electrochemical sensing method, the combination of ion-selective membranes with electrochemical properties and field-effect transistors (FETs) based on metal-dielectric-semiconductor devices is an important development direction. Generally, FET devices with ion selection functions are called ion-sensitive field-effect transistors (ISFETs). ISFET devices have the advantages of low cost, high sensitivity, fast response, easy miniaturization, integration, and easy use, easy to achieve online monitoring, etc. In recent years, they have gradually been applied to the detection of biological molecules such as DNA, inosine, Staphylococcus epidermidis, and prions. At present, there is no report on the method of potential-type electrochemical detection of L-cystine. Summary of the invention

[0006] The present invention aims to overcome the deficiencies of the prior art and provides a method and a sensor for detecting L-cystine.

[0007] In order to achieve the above object, the technical solution provided by the present invention is:

[0008] The detection method of L-cystine comprises the steps:

[0009] (1) implanting a p-well (2) and an N-type substrate (3) on a 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 thermal evaporation and magnetron sputtering technology, then 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 having the source electrode (4) and the drain electrode (5), then sequentially plating an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9) and a gold film layer (10) on the substrate layer of a polycrystalline silicon gate (7) by 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 obtain a polydisulfide-dipropanesulfonic acid film gate gold electrode modified by a polydisulfide-dipropanesulfonic acid film (12);

[0010] (2) preparing an ethanol solution of sodium poly(disulfide-bispropanesulfonate), and immersing the cleaned gate gold electrode of the extended gate field effect transistor in the ethanol solution, allowing the solution to stand at 25° C., and then washing the immersed gate gold electrode to obtain a poly(disulfide-bispropanesulfonate) (SPS) film gate gold electrode;

[0011] (3) The reference electrode, the poly(disulfide-propane sulfonic acid) film gate gold electrode and the electrode interface of the extended gate field effect transistor are connected to form a dual high-resistance differential amplifier circuit, the reference electrode and the poly(disulfide-propane sulfonic acid) film gate gold electrode are inserted into a PBS buffer solution, the power interface of the extended gate field effect transistor is connected to the positive and negative electrodes of the voltage-stabilized power supply respectively, and the signal output interface of the extended gate field effect transistor is connected to the test port of the multimeter, thereby forming a complete sensing detection circuit; utilizing the in-situ signal amplification effect of the field effect transistor, the potential change of the system can be sensitively detected; the potential of the poly(disulfide-propane sulfonic acid) film gate gold electrode as the working electrode in the PBS buffer solution will gradually tend to be stable with the increase of time, and after the potential is stable, the test sample containing different concentrations of L-cystine (13) is added to obtain the corresponding potential response data, and the detection of L-cystine (13) in the test sample is completed.

[0012] Preferably, in 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 base layer of the polysilicon gate (7) in sequence by thermal evaporation and magnetron sputtering technology, Si3N4 is used for passivation; the aluminum-copper alloy layer (8) comprises the following components in parts by weight: Al 40-68, Cu 30-60, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01-0.50; 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.

[0013] Preferably, in step (2), a 1.0-10.0 mmol / L ethanol solution of sodium polydisulfide propane sulfonate is prepared. In step (2), the gate gold electrode of the extended gate field effect transistor is cleaned with ultrapure water and anhydrous ethanol in sequence. In step (2), the gate gold electrode is immersed in the ethanol solution of sodium polydisulfide propane sulfonate for 1-72 hours. In step (2), the immersed gate gold electrode is cleaned with anhydrous ethanol and ultrapure water, and then dried and stored.

[0014] Preferably, 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 polydisulfide dipropane sulfonic acid film gate gold electrode. The PBS buffer solution in step (3) is a phosphate buffer solution with a pH of 3.0-8.0 and a concentration of 0.1 mol / L, and the pH value is preferably 5.0. The preparation method is to mix a certain amount of NaH2PO4·2H2O, Na2HPO4·12H2O, and NaCl in appropriate proportions and dissolve them in water, and use 0.1 mol / L hydrochloric acid to adjust its pH value.

[0015] The present invention also provides a sensor for detecting L-cystine, the sensor comprising a field effect transistor, the field effect transistor being provided with a gold electrode with an extended gate, i.e., a gate gold electrode; in the gate-extended gold electrode, the gate portion is extended by a distance of 0.1-500 mm, and a poly(disulfide-bispropanesulfonic acid) film (12) is assembled on the surface of a gold film layer (10) of the gold electrode.

[0016] The field effect transistor comprises a Si substrate layer (1) and a polysilicon gate (7) arranged on the Si substrate layer (1); a p-well (2) and an N-type substrate (3) are implanted on the Si substrate layer (1); a source electrode (4) and a drain electrode (5) are arranged at the p-well (2); a silicon dioxide layer (6) is arranged on the Si substrate layer (1) implanted with the p-well (2) and the N-type substrate (3) and having the source electrode (4) and the drain electrode (5); an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9) and a gold film layer (10) are plated on the substrate layer of the polysilicon gate (7) in sequence; and a silicon nitride layer (11) is also arranged on the substrate 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.

[0017] The sensor has a good Nernst response relationship to L-cystine (13) with a linear range of 5.0×10 -6 —1.0×10 -3 mol / L, the response sensitivity was 58.25±1.5mV / -pc (25℃), and the detection limit was 2.69×10 -6 mol / L.

[0018] The present invention will be further described below:

[0019] The present invention extends the gate gold electrode (GGE) of the field effect transistor (FET) by a certain distance, such as 0.1-500 mm, and self-assembles polydisulfide propane sulfonic acid (SPS) on the surface of the GGE to form a new sensor (GGE / SPS electrode) that can detect L-cystine. SEM characterization, electrochemical testing, and XPS analysis of the sensing interface show that the negatively charged SPS polyanion membrane in the solution adsorbs and binds the positively charged target L-cystine through electrostatic action to form a double electric layer structure to generate a membrane potential that recognizes monovalent organic ammonium ions. The electrode has good potential response performance to L-cystine in a phosphate buffer solution (pH=5.0), and the linear range is 5.0×10 -6 —1.0×10 -3 mol / L, the response sensitivity was 58.25±1.5mV / -pc (25℃), and the detection limit was 2.69×10 -6mol / L. The electrode responds quickly (30 seconds) and has good reproducibility and stability. Common amino acids such as L-glycine, L-alanine, L-valine, L-aspartic acid, L-proline, L-threonine, L-histidine, L-leucine, L-tryptophan, L-methionine, etc. do not interfere with the electrode's determination of L-cystine. The electrode can be used for the determination of L-cystine in actual pig serum samples, with a recovery rate of 91.2-107.8%, indicating that the method of the present invention can become a new means of simple device and accurate determination of L-cystine.

[0020] In summary, the present invention develops a simple selective membrane potential electrochemical sensor and provides a new method for detecting L-cystine, that is, a poly (disulfide propane sulfonic acid) (SPS) anion membrane is assembled on the gold film surface of the gold electrode as a gate extending from a field effect transistor, and the in-situ signal amplification effect of the field effect transistor is utilized to realize sensitive detection of L-cystine. The sensor has a good Nernst response relationship and can be used for rapid and sensitive detection of L-cystine in pig serum samples, and has potential application prospects in the fields of healthy breeding and life sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the design of an extended gate field effect transistor; in the figure: 1, Si substrate layer, 2, p-well, 3, N-type substrate, 4, source electrode, 5, drain electrode, 6, silicon dioxide layer, 7, polysilicon gate, 8, aluminum-copper alloy layer, 9, chromium-palladium alloy layer, 10, gold film layer, 11, silicon nitride layer, 12, polydisulfide dipropane sulfonic acid film;

[0022] Figure 2 This is the SEM characterization of the electrode surface morphology before and after the GGE / SPS electrode detects L-cystine; Figure 2 a is a scanning electron microscope image of the bare gold electrode surface. Figure 2 b is the image of the electrode surface after self-assembly SPS, Figure 2 c is the surface image when the electrode detects L-cystine;

[0023] Figure 3 Schematic diagram of the recognition response principle of SPS molecules binding to L-cystine molecules; in the figure: 10, gold film layer, 12, polydisulfide propane sulfonic acid film, 13, L-cystine;

[0024] Figure 4 : Figure 4 A and Figure 4 B are the AC impedance diagram and cyclic voltammogram of the electrode in potassium ferricyanide solution (containing 2.0mmol / L K3[Fe(CN)6], 2.0mmol / LK4[Fe(CN)6], 0.2mol / L Na2SO4); Figure 4In A and 4B: a, GGE, b, GGE / SPS, c, GGE / SPS / L-cystine;

[0025] Figure 5 The XPS spectra of different electrode surfaces are shown in the figure: a, GGE, b, GGE / SPS, c, GGE / SPS / L-cystine;

[0026] Figure 6 This is the relationship diagram of the slope of the SPS membrane modified GGE electrode with pH changes (pH is 3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0);

[0027] Figure 7 Schematic diagram of the mechanism of potential ion response recognition;

[0028] Figure 8 The potential response curve of the GGE / SPS electrode in a PBS buffer solution of pH = 5.0 combined with different concentrations of L-cystine;

[0029] Fig. 9 The bar graphs are the response slopes of the extended bare GGE and GGE / SPS electrodes to L-cystine;

[0030] Fig.10 The time-potential response diagram of the electrode after adding different concentrations of L-cystine in PBS buffer solution;

[0031] Fig.11 The effect of common amino acids on the detection of L-cystine by GGE / SPS electrode. DETAILED DESCRIPTION

[0032] 1. Experimental process

[0033] 1. Preparation of SPS self-assembled film gold electrode

[0034] Weigh 14.16 mg of sodium polydisulfide propane sulfonate and dissolve it in 100 mL of ethanol to obtain a 4.0 mmol / L sodium polydisulfide propane sulfonate / ethanol solution. Wash the gold electrode with ultrapure water and ethanol in turn, then soak it in the sodium polydisulfide propane sulfonate / ethanol solution, and let it stand at 25°C for 48 hours. Then take out the modified gold electrode, wash it with ethanol and ultrapure water, and dry it to obtain a polydisulfide propane sulfonic acid (SPS) self-assembled film gold electrode.

[0035] 2. Design of extended gate field effect transistor and preparation of gate gold electrode

[0036] Figure 1The invention is a schematic diagram of a design of an extended gate field effect transistor, that is, combining the basic structure of a metal-oxide-semiconductor field effect transistor (MOSFET), implanting a p-well 2 and an N-type substrate 3 on a Si substrate layer 1 of the field effect transistor, constructing a source electrode 4 and a drain electrode 5 at the p-well 2 by using thermal evaporation and magnetron sputtering technology, and then 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 having the source electrode 4 and the drain electrode 5, and then plating an aluminum-copper alloy layer 8, a chromium-palladium alloy layer 9 and a gold film layer 10 on the substrate layer of a polycrystalline silicon gate 7 by thermal evaporation and magnetron sputtering technology in sequence, and finally constructing a silicon nitride layer 11 on the substrate layer of the polycrystalline silicon gate 7 and the silicon dioxide layer 6; the aluminum-copper alloy layer 8 includes the following components in parts by weight: Al40-68, Cu 30-60, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01-0.50; the chromium-palladium alloy layer 9 includes 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 20-600nm, the thickness of the chromium-palladium alloy layer 9 is 20-600nm, and the thickness of the gold film layer 10 is 20-1000nm; the gold electrode of the gate part is extended by 200mm, and SiO2 and Si3N4 are used to passivate the field effect transistor wafer to prevent the wafer from contacting the solution except Au, thereby forming an extended gate field effect transistor (EGFET). The surface of the extended gate gold electrode (GGE) film of the EGFET is subjected to different physical / chemical modification treatments to form a sensitive film to achieve sensitive detection of the target to be measured. According to the above method, the SPS polyanion is self-assembled on the surface of the gate gold electrode to obtain a polydisulfide propane sulfonic acid film gate gold electrode (GGE / SPS) modified with a polydisulfide propane sulfonic acid film 12.

[0037] 3. Testing of self-assembled film gate gold electrode

[0038] The buffer system used for electrode potential testing is a phosphate buffer solution (PBS, 0.1 mol / L) with a pH of 3.0-8.0. The preparation method is to mix a certain amount of NaH2PO4·2H2O, Na2HPO4·12H2O, and NaCl in appropriate proportions and dissolve them in water, and use 0.1 mol / L hydrochloric acid to adjust the pH value.

[0039] First, the saturated calomel electrode, the polydisulfide-dipropane sulfonic acid film gate gold electrode and the electrode interface of the extended gate field effect transistor (EGFET) designed by this laboratory are connected to form a dual high-resistance differential amplifier circuit, the electrodes are inserted into the PBS buffer solution, the power interface of the EGFET is connected to the positive and negative poles of the voltage-stabilized power supply respectively, and the signal output interface is connected to the test port of the multimeter, thus forming a complete sensing detection circuit. By utilizing the in-situ signal amplification effect of the field effect transistor, the potential change of the system can be sensitively detected. The potential of the working electrode GGE / SPS in the PBS buffer solution will gradually tend to stabilize with the increase of time. After the potential stabilizes, different concentrations of the test substance L-cystine are added to obtain the corresponding potential response data, that is, to obtain the standard curve. When detecting the actual sample, the potential curve of the actual sample is compared with the standard curve to complete the detection.

[0040] 2. Experimental Results and Analysis

[0041] 1. SEM characterization of GGE / SPS

[0042] The surface morphology of the GGE / SPS electrode before and after detecting L-cystine was characterized by SEM (e.g. Figure 2 shown), Figure 2 (a) is a SEM image of the bare gold electrode surface, showing that the electrode surface is relatively flat and smooth. Figure 2 b), showing that a dense film is formed on the electrode surface, which is somewhat rough, indicating that SPS forms a self-assembled film on the GGE surface. When the electrode detects L-cystine, many granular substances appear on the surface ( Figure 2 c) This structural change may be due to some kind of binding reaction between L-cystine and SPS, such as electrostatic adsorption reaction, forming a strong complex aggregation, thereby changing the electrode surface morphology.

[0043] 2. Response mechanism and electrochemical characterization of GGE / SPS

[0044] Poly(disulfide propane sulfonic acid) (SPS) is a substance containing a disulfide bond with a symmetrical structure on both sides of the disulfide bond. The sulfur in the disulfide bond can form an Au-S bond with Au and self-assemble on the surface of the gold electrode. As a disulfide compound (RSSR), the disulfide bond of SPS is easily reduced and broken in an acidic electrolyte (0.1 mol / L PBS buffer solution, pH = 5.0), forming two identical structures with thiol groups. The reaction formula can be derived as follows:

[0045]

[0046] Moreover, under acidic conditions, L-cystine is positively charged, while the end of SPS containing the sulfonic acid group is negatively charged, which will attract the positively charged amino group of L-cystine. Due to the steric hindrance structure, the two sulfonic acid groups of the SPS molecule are electrostatically adsorbed and bound to the two positively charged amino groups of L-cystine, that is, one SPS molecule can bind to one L-cystine molecule. The schematic diagram of its recognition response principle is shown in the figure below. Figure 3 shown.

[0047] In order to verify the interaction between the poly(disulfide-bispropane sulfonic acid) carrier and L-cystine, the present invention used the electrochemical behavior of the above-mentioned different modified membrane electrodes by using the electrochemical impedance spectroscopy and cyclic voltammetry methods. Figure 4 shown. Figure 4 A and B are the AC impedance diagram and cyclic voltammogram of the electrode in potassium ferrocyanide solution, respectively. Figure 4 The a in A represents a bare gold electrode, which has a very small impedance value. Figure 4 b in A represents the SPS-modified gold electrode. Compared with the bare gold electrode, a semicircle appears in the high-frequency part, and the impedance value is relatively large, indicating that SPS forms a dense monomolecular self-assembled film layer on the gold surface through disulfide bonds, which hinders the [Fe(CN)6] 3- / 4- Electron conduction on the electrode surface. Combined with L-cystine (1.0×10 -5 mol / L), the impedance value increases greatly (e.g. Figure 4 This is because SPS and L-cystine have a very strong adsorption and binding effect, which makes the film on the surface of the gold electrode thicker and greatly reduces the electronic conductivity, thereby causing the electrode impedance value to continue to increase a lot. The change in the impedance value can be confirmed from the corresponding electrochemical cyclic voltammetric behavior (such as Figure 4 B).

[0048] Figure 4 In B, a represents a bare gold electrode, which has obvious oxidation peaks and reduction peaks, indicating that the bare gold electrode after pretreatment has a strong ability to transfer electrons. Figure 4 b in B represents the SPS-modified gold electrode, whose redox peak is significantly smaller than that of the bare gold electrode, indicating that SPS forms a non-conductive monomolecular self-assembled film layer on the gold surface through disulfide bonds, which hinders the [Fe(CN)6] 3- / 4- The electron conduction on the electrode surface leads to a decrease in the peak current. When the electrode is combined with L-cystine (1.0×10 -5 mol / L), the redox peak current decreased significantly (e.g. Figure 4 (shown as curve c in B), this is because the SPS-modified gold electrode adsorbs L-cystine, reducing the electronic conductivity and thus reducing the electrochemical conduction current, indicating that SPS and L-cystine are strongly adsorbed and bound. Therefore, the electrochemical behavior of the electrode indicates that the sensing interface can be used for the recognition and detection of L-cystine.

[0049] 3. XPS characterization of GGE / SPS electrode

[0050] The recognition of the target substance L-cystine by the modified membrane electrode was investigated by X-ray photoelectron spectroscopy (XPS). Figure 5 The XPS spectra of the above different electrodes (GGE, GGE / SPS, GGE / SPS / L-cystine) are shown in Figure 2. Figure 5 It can be seen that compared with the bare gold electrode (curve a), the gold electrode after self-assembled SPS (curve b) has Au 4f 、Au 4d and Au 4p The characteristic peak of sulfur S appears at a binding energy of 161.71 eV. 2p The characteristic peaks indicate that Au-S bonds are formed on the electrode surface, which proves that SPS is successfully assembled on the gold electrode surface. Curve c represents the surface XPS spectrum after GGE / SPS recognizes and binds to L-cystine. From curves a, b, and c, it can be found that the intensity of the three characteristic peaks of gold is greatly weakened in the order of GGE>GGE / SPS>GGE / SPS / L-cystine. This may be due to the fact that the electrode surface is successively combined with SPS and L-cystine, resulting in a lot of surface thickening, which further proves that GGE / SPS firmly adsorbs and binds to L-cystine molecules. From the data of different atomic binding energies in Table 1, it can be found that O 1s The binding energy of SPS increased by 0.33 eV, and further decreased by 0.46 eV after binding to L-cystine. This is because in acidic medium, the negatively charged sulfonic acid groups of SPS react with Na + The binding force is weak (Na + When it comes into contact with L-cystine, a strong electrostatic adsorption occurs between the positively charged amino group of L-cystine and the sulfonic acid group of SPS, resulting in a decrease in the binding energy of the O atom. Moreover, N 1s The peak of the binding energy is slightly wider and larger than that of curve b, which also indirectly proves that L-cystine is adsorbed on the SPS electrode interface, indicating that the sensing interface has a recognition effect on L-cystine.

[0051] Table 1 Binding energy of different atoms

[0052]

[0053] 4. Selection of optimal pH

[0054] The experiment investigated the change of the potential of the SPS membrane modified GGE electrode with the concentration of the measured substance L-cystine under different pH conditions (pH 3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0), and calculated the response slope accordingly, and made a relationship diagram between the slope and pH, as shown in the figure. Figure 6 As shown. Figure 6 It can be seen that when pH = 5.0, the electrode response slope is the largest and the electrode response is the best, and its slope value is 58.25±1.5mV / -pc (25°C), which is close to the theoretical value of the Nernst response slope of a positive monovalent ion. This shows that at pH = 5.0, the overall charge contribution of L-cystine is equivalent to that of a positive monovalent ion.

[0055] Moreover, it can be further deduced that in an acidic medium (pH = 5.0), the sulfonic acid group of SPS carries a negative charge, which will form a negative charge band of the polyanion, while the amino group of L-cysteine ​​carries a positive charge in the acidic medium, and the carboxyl group carries a very weak negative charge. Due to the strong electrostatic adsorption with the sulfonic acid group, the molecular orientation movement occurs, forming a positive charge band of ammonium ions, thereby forming a double-layer structure and generating a phase potential difference for recognizing and binding monovalent organic ammonium ions. The schematic diagram of the potential ion response recognition mechanism is shown in Figure 7 shown.

[0056] 5. GGE / SPS electrode response performance

[0057] The experiment examined the test response performance of the extended bare GGE and GGE / SPS electrodes to L-cystine. Figure 8 This is the potential response curve of the GGE / SPS electrode in a PBS buffer solution with pH = 5.0 and combined with different concentrations of L-cystine. Figure 8 It can be seen that with the increase of L-cystine concentration, the electrode potential also gradually increases, indicating that the L-cystine bonded to the surface of the GGE / SPS electrode increases, and the linear response range of the electrode to L-cystine in PBS buffer solution with pH = 5.0 is 5.0×10 -6 —1.0×10 -3 mol / L. At the same time, the least square method was used to fit the electrode potential linear response curve, and the linear equation was ΔE=328.6+58.25lgc. According to the drawing method, the electrode detection limit was 2.69×10 -6 mol / L( Figure 8 ).

[0058] Fig. 9 is the extended slope bar graph of the bare GGE and GGE / SPS electrodes’ responses to L-cystine. Fig. 9As can be seen in the figure, the potential response of naked GGE to L-cystine is small or almost non-responsive, so SPS acts as a sensitive membrane, and it is feasible to modify SPS on the extended GGE to detect L-cystine.

[0059] In addition, the GGE / SPS electrode was compared with other L-cystine electrochemical sensors reported in the literature (see Table 2). It was found that the GGE / SPS potentiometric sensor had a wider linear range and better selectivity than the amperometric sensor. It was also simple to make, portable, and easy to miniaturize and monitor online.

[0060] Table 2 Performance comparison of different electrodes

[0061]

[0062] Note: Ni-CCE: nickel-doped carbon ceramic electrode; β-CD: β-cyclodextrin; GCE: glassy carbon electrode; GGE: gate gold electrode; rGO: reduced graphene oxide; SPS: poly (disulfide-bis(propylene glycol) sulfonate).

[0063] 6. Determination of response time, stability and reproducibility

[0064] The extended response time and stability of the GGE / SPS electrode in detecting L-cystine were investigated. Fig.10 The time-potential response diagram of the electrode after adding different concentrations of L-cystine in PBS buffer solution was obtained by -5 —5.0×10 -4 mol / L range from low concentration to high concentration, and record the potential value over time. It can be seen that the electrode reaches equilibrium in a short time in the entire concentration range. The response time is calculated to reach 95% of the maximum potential response, which is about 30s. At the same time, the electrode was tested for L-cystine samples for 14 consecutive days, and its response slope changed to 41.55mV / -pc, a decrease of 28.67%, indicating that the membrane electrode can be used for more than 2 weeks, with good stability and long service life.

[0065] The experiment investigated the reproducibility of the potential response of the prepared GGE / SPS electrode combined with L-cystine samples of different concentrations. -5 mol / L and 1.000×10 -4 The potential response value was measured back and forth in a mol / L L-cystine sample solution, and each measurement was performed 10 times. Through statistical processing of the data, it was found that the relative standard deviations of the potential response values ​​of the electrode in two different concentration solutions were 2.43% and 0.85% respectively, and the relative standard deviations were small, indicating that the SPS membrane electrode has good reproducibility.

[0066] 7. Electrode selectivity

[0067] The effects of common amino acids on the detection of L-cystine by GGE / SPS electrode were investigated in PBS buffer solution at pH 5.0. Fig.11 ). Using the mixed solution method, the concentration of L-cystine was fixed at 1.000×10 -5 mol / L, add 50 times the concentration of interfering components such as common amino acids, among which the concentration of L-cysteine ​​(L-Cys) is 1.000×10 -5 mol / L. The results show that after adding L-glycine (L-Gly), L-alanine (L-Ala), L-valine (L-Val), L-aspartic acid (L-Asp), L-proline (L-Pro), L-threonine (L-Thr), L-histidine (L-His), L-leucine (L-Leu), L-tryptophan (L-Trp), and L-methionine (L-Met), the potential value of the SPS membrane electrode in response to L-cystine has almost no change ( Fig.11 ), that is, the interference is very small; while L-cysteine ​​(L-Cys) has a certain effect.

[0068] 8. Determination and analytical application of recovery rate

[0069] Under optimized experimental conditions, the extended GGE / SPS electrode was used to determine L-cystine in actual porcine serum samples. The treated porcine serum samples (taken from 6 live three-way hybrid piglets, weighing 7-15 kg, provided by the Institute of Subtropical Agriculture Ecology, Chinese Academy of Sciences) were diluted 10 times with pH 5.0 PBS buffer, and a known concentration of L-cystine was added. The standard addition method was used for determination, and the recovery rate of L-cystine in the porcine serum samples was between 91.2% and 107.8% (see Table 3), indicating that the GGE / SPS electrode has good accuracy and can be used for rapid and sensitive detection of L-cystine in actual porcine serum samples, and has potential application value in the fields of life medicine and animal breeding.

[0070] Table 3 Determination of the content and recovery rate of L-cystine in pig serum samples by GGE / SPS electrode

[0071]

[0072] In summary, the GGE / SPS electrode of the present invention exhibits a sensitive Nernst response to L-cystine in PBS (pH = 5.0) solution, and its linear response range is 5.00 × 10 -6 —1.00×10 -3 mol / L, the detection limit is 2.69×10 -6mol / L. The electrode has a short response time (30 seconds) and has good selectivity, reproducibility and stability. Moreover, the electrode can be directly applied to the determination of L-cystine in actual pig serum samples, and is expected to become a new online test method for L-cystine.

Claims

1. A method for detecting L-cystine, characterized in that, The method comprises the following steps: (1) implanting a p-well (2) and an N-type substrate (3) on a 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 using thermal evaporation and magnetron sputtering technology, then 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 having the source electrode (4) and the drain electrode (5), then using thermal evaporation and magnetron sputtering technology to sequentially plate an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9) and a gold film layer (10) on the substrate layer of a polycrystalline silicon gate (7), 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 obtain an extended gate field effect transistor having a gate gold electrode; (2) preparing an ethanol solution of sodium poly(disulfide-bispropane sulfonate), and immersing the cleaned gate gold electrode of the extended gate field effect transistor in the ethanol solution, allowing the solution to stand at 25° C., and then washing the immersed gate gold electrode to obtain a poly(disulfide-bispropane sulfonate) film gate gold electrode modified with a poly(disulfide-bispropane sulfonate) film (12); (3) The reference electrode, the poly(disulfide-propane sulfonic acid) film gate gold electrode and the electrode interface of the extended gate field effect transistor are connected to form a dual high-resistance differential amplifier circuit, the reference electrode and the poly(disulfide-propane sulfonic acid) film gate gold electrode are inserted into a PBS buffer solution, the power interface of the extended gate field effect transistor is connected to the positive and negative electrodes of the voltage regulator, respectively, and the signal output interface of the extended gate field effect transistor is connected to the test port of the multimeter, thereby forming a complete sensing detection circuit; utilizing the in-situ signal amplification effect of the field effect transistor, the potential change of the system can be sensitively detected; the potential of the poly(disulfide-propane sulfonic acid) film gate gold electrode as the working electrode in the PBS buffer solution will gradually tend to stabilize with the increase of time, and after the potential stabilizes, the test sample containing different concentrations of L-cystine (13) is added to obtain the corresponding potential response data, thereby completing the detection of L-cystine (13) in the test sample.

2. The method according to claim 1, characterized in that In step (1), when an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9) and a gold film layer (10) are plated on a base layer of a polysilicon gate (7) in sequence by thermal evaporation and magnetron sputtering technology, Si3N4 is used for passivation; the aluminum-copper alloy layer (8) comprises the following components in parts by weight: Al 40-68, Cu 30-60, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01-0.50; 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 method according to claim 1, characterized in that Step (2) is to prepare a 1.0-10.0 mmol / L ethanol solution of sodium polydisulfide propane sulfonate.

4. The method according to claim 1, characterized in that In step (2), the gate gold electrode is immersed in the ethanol solution of sodium polydisulfide propane sulfonate for 1-72 h.

5. The method according to claim 1, characterized in that 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 poly(disulfide-bis(propylene disulfide)) film gate gold electrode.

6. The method according to claim 1, characterized in that The PBS buffer solution in step (3) is a phosphate buffer solution with a pH of 3.0-8.0 and a concentration of 0.1 mol / L.

7. A sensor for detecting L-cystine, the sensor comprising a field effect transistor, the field effect transistor being provided with a gold electrode with an extended gate, characterized in that: In the gold electrode with extended gate, the gate portion is extended by a distance of 0.1-500 mm, and a poly(disulfide-bispropanesulfonic acid) film (12) is assembled on the surface of the 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 substrate layer (1) and a polysilicon gate (7) arranged on the Si substrate layer (1); a p-well (2) and an N-type substrate (3) are implanted on the Si substrate layer (1); a source electrode (4) and a drain electrode (5) are provided at the p-well (2); a silicon dioxide layer (6) is provided on the Si substrate layer (1) implanted with the p-well (2) and the N-type substrate (3) and having the source electrode (4) and the drain electrode (5); an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9) and a gold film layer (10) are plated on the substrate layer of the polysilicon gate (7) in sequence; and a silicon nitride layer (11) is also provided on the substrate layer of the polysilicon gate (7) and the silicon dioxide layer (6).

9. The sensor according to claim 8, characterized in that 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 20-600 nm.

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