CN108802124B - L-cystine detection method and sensor based on glutathione composite membrane gate gold electrode - Google Patents
L-cystine detection method and sensor based on glutathione composite membrane gate gold electrode Download PDFInfo
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Abstract
The invention discloses a detection method and a sensor for L-cystine based on a glutathione composite film modified gate gold electrode, which are characterized in that dibutyl phthalate/graphene oxide/glutathione (DBP/GO/GSH) composite materials are modified on an extended gate gold film layer of a field effect transistor to form a novel DBP/GO/GSH self-assembled Gate Gold Electrode (GGE), and the L-cystine is sensitively detected by utilizing the in-situ signal amplification effect of the field effect transistor, wherein the glutathione composite film adsorbs and combines a positively charged target substance L-cystine through electrostatic effect to form an electric double layer structure so as to generate a film potential for identifying monovalent organic ammonium ions. The sensor has good Nernst response relation to L-cystine, and has a linear range of 2.5X10 ‑6 —1.0×10 ‑4 mol/L, response sensitivity of 55.72.+ -. 1.5mV/-pC (25 ℃ C.), and detection limit of 1.02X10 ‑ 6 mol/L. The preparation process is simple and convenient, the response time is quick, and the preparation method has potential application prospect.
Description
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 glutathione composite membrane gate gold electrode, namely a selective membrane potential sensor, which are suitable for detection in the aspects of healthy cultivation and life science.
Background
Dibutyl phthalate (DBP) is a colorless oily liquid that gives good flexibility to articles, and is used as a plasticizer for polyvinyl acetate, alkyd resins, nitrocellulose, ethylcellulose, and neoprene and nitrile rubbers because of its excellent stability, flex resistance, adhesion, and water resistance.
Graphene Oxide (GO) is a derivative of Graphene, and has the same structure as Graphene, except that a large number of oxygen-containing groups are connected to a two-dimensional infinitely extending basal plane formed by a layer of carbon atoms, the plane contains-OH and C-O, and the sheet edge contains c=o and-COOH. Compared with graphene, graphene oxide has excellent performances, good wettability and surface activity, and plays a great role in improving the mechanical and electrical properties of materials.
In recent years, many electrochemical researches on graphene oxide composite modified materials are carried out, namely, ya and the like detect carbendazim in food by utilizing a nitrogen-doped graphene oxide modified glassy carbon electrode, the modified materials show excellent electrocatalytic oxidation performance on the carbendazim, the detection range is 5.0-850 mug/L, and the detection lower limit is 1.0 mug/L; the nickel nano material, the attapulgite and the reduced graphene oxide composite material are modified on a glassy carbon electrode by the Shen and the like to detect glucose, and the sensor has the characteristics of high sensitivity, quick response and the like for detecting glucose, and the lower detection limit is 0.37mM; the modified graphene oxide/zinc oxide (GO/ZnO) composite material of Hadi Beitoolahi et al on a screen printing electrode is used for simultaneously detecting levodopa and tyrosine, the electrode has high sensitivity and high selectivity to two detection substances, and the detection lower limit is 4.5 multiplied by 10 respectively -7 M and 3.4X10 -7 M; rao et al studied the electrochemical behavior of two electroactive functional groups on p-nitrophenol on reduced graphene oxide modified glassy carbon electrodes, the sensor had a detection lower limit of 0.55mM for PNP; li Junhua and the like modify the GO/MWCNT nano composite material on the glassy carbon electrode to construct a novel stable stateL-tryptophan sensor with good quality and detection range of 1.0X10 -6 —1.0×10 -4 mol/L, its lower limit of detection is 3.5X10 -7 mol/L; liu Xiaohua and the like prepare a single-walled carbon nanotube-graphene oxide composite modified glassy carbon electrode for measuring catechol, the modified electrode has good electrocatalytic activity to catechol, and the detection limit is 4 multiplied by 10 -7 M。
GSH is formed by glutamic acid, cysteine and glycine through peptide bond, and has molecule of glutamic acid gamma-COOH and cysteine alpha-NH 2 Condensed into specific gamma-peptide bonds. Glutathione has antioxidant and integral detoxification effects and also helps maintain normal immune system function. The glutathione has the functions of antioxidation and integrated detoxification, so that the glutathione has wide application, can be used as a medicine and a base material of functional foods, and can be widely applied to the functional foods such as aging delay, immunity enhancement, tumor resistance and the like.
Glutathione exists in the human body in two forms, one is reduced Glutathione (GSH) and the other is oxidized glutathione (GSSG), so-called glutathione is reduced glutathione. Since glutathione contains thiol groups, glutathione can self-assemble to the gold electrode surface through-SH. There are many reports on self-assembly of glutathione onto gold electrode surfaces for detection of metal ions. Zeng et al self-assembled a mixture of 3-mercaptopropionic acid and glutathione on the surface of gold electrode for Cu detection 2+ Research shows that the sensor is suitable for Cu 2+ Exhibits excellent selectivity, and the detection range is 0.1 mu M-1 mM; chow self-assembles MPA-GSH system on gold electrode surface for detecting Cd 2+ The reproducibility of this electrode was good, with a lower detection limit of 5nM. Glutathione is used as a modification material of the electrode, has wide functions in detecting metal ions and also has important functions in detecting some nitrogen-containing molecules and organic matters. RGO/L-P-GSH composite material is modified on a glassy carbon electrode by Vilia et al, the sensor has excellent electrocatalytic performance on 4-aminophenol, the detection range is 0.4-200 mu M, the detection lower limit reaches 0.03 mu M (S/N=3), and the selectivity is goodThe method can be applied to detection of actual samples; p rez-R afols and the like are modified on screen printing electrode carbon nanofibers by GSH and are used for detecting Cd 2+ And Pb 2+ The lower detection limits are 3.0 mug/L and 3.2 mug/L respectively; han et al utilize Fe 3 O 4 The detection range of the sensor for detecting the estrogen is 0.025-10 mu M, and the detection lower limit is 2.76 mu M.
Cystine (L-cysteine) is an amino acid that contains disulfide bonds (a sulfide bond is a structure where two thiol molecules are joined by an S-S bond). There are many methods for detecting amino acids, such as High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis, and fluorescent probe detection. However, these methods generally have the defects of high cost, expensive precise instruments, complicated sample preparation procedures, skilled operators, inability to carry and use on line or inconvenient, and the like, so the exploration and development of a simple and rapid amino acid analysis method is an important target for research in the field. Compared with other instrumental analysis methods, electrochemical methods are receiving more and more attention due to the features of simplicity, sensitivity, no radiation, no pollution and the like, and can be used for laboratory detection and field analysis. 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 detecting L-cystine by potentiometric electrochemical sensing 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 glutathione composite membrane 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 glutathione composite membrane 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) Immersing a gate gold electrode of the cleaned extended gate field effect transistor in dibutyl phthalate solution, taking out, dripping graphene oxide solution on the gate gold electrode, immersing the gate gold electrode in glutathione solution after the gate gold electrode is dried, and then cleaning the immersed gate gold electrode to prepare a glutathione composite film gate gold electrode modified by a glutathione composite film (12); the glutathione composite membrane is a composite membrane composed of dibutyl phthalate, graphene oxide and glutathione;
(3) Connecting a reference electrode and a glutathione composite film grid gold electrode with 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 glutathione composite 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 power supply respectively, and connecting a signal output interface of the extended grid field effect transistor 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 glutathione composite membrane gate gold electrode serving as a working electrode in PBS buffer solution gradually tends to be stable along with the increase of time, and samples to be detected containing L-cystine (13) with different concentrations are added after the potential is stable, so that corresponding potential response data are obtained, and the detection of the L-cystine (13) in the samples to be detected is completed.
Preferably, in the step (1), aluminum is plated on the basal layer of the polysilicon grid electrode (7) in sequence by using the thermal evaporation and magnetron sputtering technologyIn the case of the copper alloy layer (8), the chromium-palladium alloy layer (9) and the gold film layer (10), 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.
Preferably, the dibutyl phthalate solution in the step (2) is a solution with the concentration of dibutyl phthalate being 0.1-10.0 mmol/L, the graphene oxide solution is a solution with the concentration of graphene oxide being 0.1-10.0 mg/mL, and the ethanol solution of glutathione is an ethanol solution with the concentration of glutathione being 0.1-10.0 mmol/L. 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. In the step (2), the time for immersing the gate gold electrode in the dibutyl phthalate solution is 1.0-300 min, the dosage of the graphene oxide solution is 1.0-10.0 mu L when the graphene oxide solution is dripped on the gate gold electrode, and the time for immersing the gate gold electrode in the ethanol solution of glutathione is 1-72 h. 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 glutathione composite film grid 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 4.0, and the preparation method is to prepare a certain amount of NaH 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 glutathione composite 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 2.5X10 -6 —1.0×10 -4 mol/L, response sensitivity of 55.72.+ -. 1.5mV/-pC (25 ℃ C.), and detection limit of 1.02X10 -6 mol/L。
The invention is further described below:
according to the invention, a Gate Gold Electrode (GGE) of a Field Effect Transistor (FET) is prolonged for a certain distance, for example, the Gate Gold Electrode (GGE) is prolonged for 0.1-500 mm, and a dibutyl phthalate/graphene oxide/glutathione (DBP/GO/GSH) composite material is utilized to modify the Gate Gold Electrode (GGE) of the field effect transistor, so that a novel potential type electrochemical sensor for detecting L-cystine is constructed. Electrochemical tests and XPS analysis of a sensing interface show that two carboxyl groups of the modifier GSH are negatively charged in a solution, and the modifier GSH and L-cystine containing amino groups of a target substance and positively charged are electrostatically adsorbed on the surface of a gold electrode, so that the potential of the surface film of the electrode is changed. The electrode has good potential response to L-cystine in PBS buffer solution with pH=4.0, and the linear response range of the electrode to L-cystine is 5.00×10 -6 —1.00×10 -4 mol/L, response sensitivity of 54.16.+ -. 1.5mV/-pC (25 ℃ C.), and detection limit of 1.02X10 -6 mol/L, response time was 76 seconds. The electrode has betterReproducibility and stability, and the electrode has good selectivity, L-glycine (L-Gly), L-threonine (L-Thy), L-phenylalanine (L-Phe), L-glutamic acid (L-Glu), L-alanine (L-Ala), L-aspartic acid (L-Asp), L-isoleucine (L-Lle), L-histidine (L-His), L-proline (L-Pro), L-lysine (L-Lys), L-methionine (L-Met), L-cysteine (L-Cys) and five metal ions such as Fe 3 + 、Zn 2+ 、Cu 2+ 、Ca 2+ 、Ni 2+ And the like do not interfere with the determination of L-cystine. In addition, the electrode can be used for measuring L-cystine in actual pig serum and pig urine samples, the recovery rate can reach 99.5-105.7%, and the potential sensor is expected to become a simple, convenient and quick new means for L-cystine analysis.
In summary, the invention develops a simple selective membrane potential type electrochemical sensor and provides a novel detection method of L-cystine, namely dibutyl phthalate/graphene oxide/glutathione (DBP/GO/GSH) is modified on a gate gold film layer prolonged by a field effect transistor to form a novel DBP/GO/GSH self-assembled Gate Gold Electrode (GGE), and the in-situ signal amplification effect of the field effect transistor is utilized to realize sensitive detection of L-cystine, wherein the glutathione composite film is combined with a positively charged target L-cystine through electrostatic absorption to form an electric double layer structure so as to generate a membrane potential for identifying monovalent organic ammonium ions. The experimental test result shows that the concentration of L-cystine in the sensor is 5.00 multiplied by 10 -6 —1.00×10 - 4 The method has the advantages of good Nernst response relation 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. the silicon substrate layer, 2, the source electrode, 3, the drain electrode, 4, the p-well, 5, the N-type substrate, 6, the silicon dioxide layer, 7, the polysilicon grid electrode, 8, the aluminum copper alloy layer, 9, the chromium palladium alloy layer, 10, the gold film layer, 11, the silicon nitride layer, 12 and the glutathione composite film;
FIG. 2 is a schematic diagram of recognition response principle of DBP/GO/GSH molecules combined with L-cystine molecules; in the figure: 10. gold film layer, 12, glutathione composite film, 13, L-cystine;
fig. 3: FIGS. 3A and 3B 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. 3A and 3B: a. GGE, b, GGE/DBP/GO/GSH, c, GGE/DBP/GO/GSH/L-cystine;
FIG. 4 shows XPS survey spectra of different electrode surfaces; in the figure: a. GGE, b, GGE/DBP/GO/GSH, c, GGE/DBP/GO/GSH/L-cystine;
FIG. 5 is a graph showing the slope of DBP/GO/GSH glutathione composite membrane modified GGE electrode as a function of pH (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0);
FIG. 6 is a graph showing the potential response of GGE/DBP/GO/GSH electrodes after combining different concentrations of L-cystine in PBS buffer at pH=4.0;
fig. 7: FIG. 7A is a graph showing time-potential dynamics of GGE/DBP/GO/GSH electrodes after adding different concentrations of L-cystine to PBS buffer at pH=4.0; FIG. 7B shows the addition of L-cystine (10 -4 mol/L) of the dynamic response curve;
FIG. 8 is a graph showing the effect of common amino acids on detection of L-cystine by GGE/DBP/GO/GSH electrodes.
Detailed Description
1. Experimental procedure
1. Preparation of DBP/GO/GSH self-assembled membrane gold electrode
The preparation process of the DBP/GO/GSH self-assembled film comprises the following steps: sequentially cleaning an electrode by ultrapure water and ethanol; then soaking GGE in 1mmol/L DBP solution for 1h; after 1h, the electrode was removed, followed by dropping a graphene oxide (1 mg/mL) solution onto the electrode in an amount of 5. Mu.L; after the electrode is dried, soaking the electrode in 1mmol/L GSH/ethanol solution; and finally, taking out the GGE modified by the self-assembled film, washing with ethanol and ultrapure water, drying and preserving to obtain the self-assembled film gold electrode of the composite material.
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, 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 an Si base layer 1 of the field effect transistor, constructing a source electrode 4 and a drain electrode 5 at the p-well 2 by adopting thermal evaporation and magnetron sputtering technology, then constructing a silicon dioxide layer 6 on the Si base 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, then plating an aluminum copper alloy layer 8, a chromium palladium alloy layer 9 and a gold film layer 10 on the base layer of a polysilicon gate 7 in sequence by adopting thermal evaporation and magnetron sputtering technology, and finally constructing a silicon nitride layer 11 on the base 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. And (3) self-assembling the glutathione composite film on the surface of the gate gold electrode according to the method, so as to prepare the glutathione composite film gate gold electrode modified by the glutathione composite film 12.
3. Testing of self-assembled film gate gold electrodes
The buffer system for electrode potential test is phosphate buffer solution (PBS, 0.1 mol/L) with pH of 3.0-8.0, and is prepared by mixing a certain amount of NaCl and NaH 2 PO 4 ·2H 2 O and Na 2 HPO 4 ·12H 2 O is mixed and dissolved in water according to a proper proportion, and the pH value of the mixture is regulated by adopting 0.1mol/L hydrochloric acid. First, calomel electrode, self-assembled Gate Gold Electrode (GGE) and the laboratory designThe Extended Gate Field Effect Transistor (EGFET) is connected to form a double high resistance differential amplifying circuit, electrodes are inserted into PBS buffer solution, the power interface of the EGFET is respectively connected with the positive electrode and the negative electrode of a stabilized voltage power supply, and the signal output interface is connected with the test port of the universal meter, so that a complete sensing detection loop is formed. 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 in the PBS buffer solution gradually tends to be stable along with the increase of time, and L-cystine of an object to be detected is added after the potential is stable, so that corresponding potential response data are obtained.
2. Experimental results and analysis
1. Response mechanism and electrochemical characterization of GGE/DBP/GO/GSH electrode
Glutathione (GSH) is a substance containing a sulfhydryl end group, wherein sulfur in sulfhydryl group can form Au-S bond with Au to self-assemble on the surface of a gold plate electrode, and the carboxyl of the glutathione releases H + And negatively charged with-NH on L-cystine 3 + Electrostatic attraction occurs between the electrodes, thereby causing a change in membrane potential; and glutathione contains two-COOH, while L-cystine contains two amino groups, and one glutathione molecule is combined with one L-cystine molecule, so that the glutathione has the response characteristic of monovalent positive ions, and the response recognition process is shown in figure 2.
In order to verify the interaction of the carrier and L-cystine, the invention adopts alternating current impedance and cyclic voltammetry to examine the electrochemical behaviors of the different modified electrodes, as shown in figure 3. Fig. 3 is an ac impedance plot (a) and cyclic voltammogram (B) of an electrode in a potassium ferricyanide solution. In fig. 3A, a represents a bare gold electrode, the impedance value is very small, and in fig. 3A, b represents a gold electrode modified by a DBP-GO-GSH composite material, compared with the bare gold electrode, the appearance of a small semicircle in the high-frequency part indicates that the impedance value is large, which indicates that the composite material reduces the conductivity of the gold electrode. Binding L-cystine (1.0X10) -5 mol/L), the semicircle of the high frequency part becomes larger, indicating that the resistance value is increased again (as shown by curve c in FIG. 3A), because GSH and L-cystine are adsorbed, and the electron conductivity is reduced, thereby leading to gold electrode resistance modified with GSHThe resistance increases and the trend of the resistance change can be confirmed from the corresponding cyclic voltammetric behavior (fig. 3B).
In fig. 3B, a represents a bare gold electrode, which has obvious oxidation peak and reduction peak, which indicates that the pretreated bare gold electrode has stronger electron transfer capability. FIG. 3B shows that B represents a DBP-GO-GSH composite modified gold electrode with significantly smaller redox peaks than that of a bare gold electrode, indicating that the composite forms a non-conductive single-molecule self-assembled film on the gold surface through thiol groups, impeding [ Fe (CN) 6 ] 3-/4- Electron conduction at the electrode surface. After binding L-cystine (1.0X10) -5 After mol/L), the oxidation-reduction peak (shown as curve c in FIG. 3B) is reduced, because cystine is adsorbed on the gold electrode modified by the composite material, and the electronic conductivity is reduced, so that the electrochemical conduction current is reduced, and the DBP-GO-GSH composite material modified gold electrode has good coordination and combination effect with L-cystine, so that the method is feasible for identifying and detecting the L-cystine.
2. XPS characterization of GGE/DBP/GO/GSH 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. 4. As can be seen from fig. 4, compared with the bare gold electrode (curve a), the gold electrode (curve b) modified with DBP-GO-GSH showed a characteristic peak of S2p at a binding energy of 162.71eV, indicating that the composite material has successfully self-assembled on the gold electrode surface at the electrode surface. Curve c represents the surface XPS spectrum of GGE/DBP/GO/GSH electrode after detecting L-cystine, the peak intensity of carbon element, oxygen element and sulfur element is higher than that of electrode before reaction after modifying electrode to react L-cystine, and the binding energy of O1s is changed by 0.31eV before and after detecting L-cystine from the data of different atom binding energy in table 1, because glutathione and L-cystine have electrostatic adsorption effect, GSH originally charged negatively adsorbs L-cystine charged positively, so the binding energy shifts to low energy. The binding energy of S2p is shifted 0.93eV towards high energy after detection of L-cystine due to the dehydro-COOH on GSHwith-NH 3 + Electrostatic attraction occurs to cause a decrease in electron cloud density of S, resulting in an increase in binding energy thereof. It can also be seen from Table 2 that the change in the highest CPS of C, O, S before and after electrode binding to L-cystine also demonstrates that the electrostatic adsorption of GSH and L-cystine causes a change in the electrode surface.
TABLE 1 binding energy of different atoms
TABLE 2 maximum CPS of different atoms
3. Selection of optimal pH
Experiments respectively research the change relation of the electrode potential along with the concentration of the measured ion solution under the conditions that the pH value is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0, and the Nernst response slope is obtained according to the change relation, and then a relation diagram of the slope and the pH value is made, as shown in figure 5. As can be seen from fig. 5: the slope of the electrode response was greatest at ph=4.0 and the slope value was 55.72±1.5mV/-pC (25 ℃) near the theoretical value of the nernst response slope. This means that at ph=4.0, the electrode response is best, resulting in an optimal pH of 4.0.
4. GGE/DBP/GO/GSH electrode response performance
The test response performance of the electrode to L-cystine was examined experimentally, and fig. 6 is a graph of the potential response of the electrode after combining different concentrations of L-cystine in PBS buffer solution at ph=4.0. As can be seen from the graph, as the concentration of L-cystine increases, the electrode potential also gradually increases, indicating that the surface-bound L-cystine of the modified electrode increases, and the linear response of the electrode to L-cystine ions in PBS buffer solution with pH=4.0 ranges from 2.5X10 -6 mol/L—1.0×10 -4 mol/L. Meanwhile, the least square method is adopted for processing, and an electrode potential linear response diagram is obtained after fitting, wherein the linear equation of the electrode is delta E=324.40+55.72log 10 C. The detection limit can be obtained by plotting, and the value is about 1.02X10 -6 mol/L. The response slope of 55.72mV/-pC is close to the Nernst theoretical response slope value of monovalent positive ions, which further proves the reasonability of one glutathione molecule which we speculate before to bind one L-cystine molecule.
5. Determination of response time, stability and reproducibility
The experiment examines the response time and stability of the DBP/GO/GSH composite material modified gold electrode for detecting L-cystine. FIG. 7A is a graph showing the time-potential dynamic response after adding different concentrations of L-cystine to PBS buffer by adding the same to the buffer at 1.0X10 -6 —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 reaction time for the electrode to reach equilibrium in the whole concentration range is very short, the response time is calculated to reach 95% of the maximum value of potential response, namely 76s, and the electrode can have very high response speed to L-cystine; FIG. 7B is a further addition 10 -4 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 mol/L L-cystine sample is continuously tested for 35 days, the response slope change is 45.36mV/-pC, and the response slope is reduced by 18.59%, which indicates that the potential sensor can be used for more than 1 month and has longer service life.
TABLE 3 reproducibility of GGE/DBP/GO/GSH electrodes
Experiments prove that the potential response reproducibility of the GGE/DBP/GO/GSH composite material modified electrode combined with L-cystine samples with different concentrations, namely the electrode is 1 multiplied by 10 -5 mol/L and 1X 10 -4 The potential values of the L-cystine standards were measured back and forth 10 times for each of the L-cystine standards in mol/L (see Table 3). By analysis and processing of the data, the relative standard deviation was found to be 1.39% and 2.45%, respectively, and was smaller, indicating that the electrode was L-cystine at two different concentrationsThe reproducibility of the (c) is better.
6. Electrode selectivity
One of the important characteristics of ion selective electrodes is that it has good selectivity and responds only to a particular substance. Thus, in a PBS buffer solution with a pH=4.0 containing a modified electrode, 10. Mu.M L-cystine, the concentration of the interfering substance being 10 times the concentration of the substance to be measured, i.e., 100. Mu.M, was added, wherein L-glycine (L-Gly), L-threonine (L-Thy), L-phenylalanine (L-Phe), L-glutamic acid (L-Glu), L-alanine (L-Ala), L-aspartic acid (L-Asp), L-isoleucine (L-Lle), L-histidine (L-His), L-proline (L-Pro), L-lysine (L-Lys), L-methionine (L-Met), L-cysteine (L-Cys) and five metal ions such as iron, zinc, copper, calcium, nickel ions were examined (FIG. 8). The results show that the other 17 interfering substances except L-cysteine have no obvious interference to the L-cystine to be detected, and can show that the sensor has good selectivity.
7. Determination and analysis application of recovery rate
And under the optimized experimental condition, the DB/GO/GSH composite material is used for modifying the grid gold electrode to determine the concentration of the L-cystine in the actual sample. During measurement, the pig serum 1 and 2 and the pig urine 3 and 4 which are actual samples are respectively collected and centrifuged, supernatant is taken, the supernatant is diluted 10 times by PBS buffer solution with pH value of 4.0, L-cystine with known concentration is added into the sample, the recovery rate is measured to be 99.5-105.7% by a standard addition method, and experimental results show that the electrode can be used for detecting the L-cystine in the actual samples.
In summary, the invention provides a selective electrode based on DBP/GO/GSH modified extension gate gold film, and experimental results show that the electrode shows a high-sensitivity Nernst response relationship to L-cystine in PBS buffer solution with pH=4.0, and the linear response range is 2.5X10 -6 —1.0×10 -4 mol/L, detection limit of 1.02X10 -6 mol/L. The electrode has the characteristics of short response time (about 76 seconds), good selectivity, reproducibility, stability and the like. In addition, the electrode can be used for detecting L-cystine in actual samples, and is expected to be a new online detection method for L-cystineA model means.
Claims (10)
1. The method for detecting the L-cystine based on the glutathione composite membrane gate gold electrode is characterized by comprising the following steps of:
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) Immersing a gate gold electrode of the cleaned extended gate field effect transistor in dibutyl phthalate solution, taking out, dripping graphene oxide solution on the gate gold electrode, immersing the gate gold electrode in glutathione solution after the gate gold electrode is dried, and then cleaning the immersed gate gold electrode to prepare a glutathione composite film gate gold electrode modified by a glutathione composite film (12); the glutathione composite membrane is a composite membrane composed of dibutyl phthalate, graphene oxide and glutathione;
(3) Connecting a reference electrode and a glutathione composite film grid gold electrode with 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 glutathione composite 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 power supply respectively, and connecting a signal output interface of the extended grid field effect transistor 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 glutathione composite membrane gate gold electrode serving as a working electrode in PBS buffer solution gradually tends to be stable along with the increase of time, and samples to be detected containing L-cystine (13) with different concentrations are added after the potential is stable, so that corresponding potential response data are obtained, and the detection of the L-cystine (13) in the samples to be detected is completed.
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 method of claim 1, wherein the dibutyl phthalate solution in step (2) is a solution having a dibutyl phthalate concentration of 0.1 to 10.0mmol/L, the graphene oxide solution is a solution having a graphene oxide concentration of 0.1 to 10.0mg/mL, and the ethanol solution of glutathione is an ethanol solution having a glutathione concentration of 0.1 to 10.0 mmol/L.
4. The method of claim 1, wherein the time for immersing the gate gold electrode in the dibutyl phthalate solution in step (2) is 1.0 to 300min, the amount of the graphene oxide solution used when the graphene oxide solution is dropped on the gate gold electrode is 1.0 to 10.0 μl, and the time for immersing the gate gold electrode in the ethanol solution of glutathione is 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 glutathione composite 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, a gate-extended gold electrode being provided on the field effect transistor, characterized in that, in the gate-extended gold electrode, the gate part is extended by a distance of 0.1-500 mm, and a glutathione composite film (12) is assembled on the surface of a gold film layer (10) of the gold electrode; the glutathione composite film is a composite film composed of dibutyl phthalate, graphene oxide and glutathione.
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, characterized in that it has a good nernst response to L-cystine (13), a linear range of 2.5 x 10 -6 —1.0×10 -4 mol/L, response sensitivity at 25℃of 55.72.+ -. 1.5mV/-pC with a detection limit of 1.02X10 -6 mol/L。
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