WO2020043026A1 - 一种l-胱氨酸的检测方法及传感器 - Google Patents

一种l-胱氨酸的检测方法及传感器 Download PDF

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WO2020043026A1
WO2020043026A1 PCT/CN2019/102372 CN2019102372W WO2020043026A1 WO 2020043026 A1 WO2020043026 A1 WO 2020043026A1 CN 2019102372 W CN2019102372 W CN 2019102372W WO 2020043026 A1 WO2020043026 A1 WO 2020043026A1
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electrode
cystine
layer
gate
gold
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French (fr)
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曹忠
杨佳
刘陈
肖忠良
李丹
张玲
张煜杨
李佳欣
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长沙理工大学
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Priority claimed from CN201810984084.5A external-priority patent/CN109115856A/zh
Priority claimed from CN201821387669.0U external-priority patent/CN208860792U/zh
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Priority to US17/258,932 priority Critical patent/US11939625B2/en
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Definitions

  • the invention belongs to the technical field of chemical / biological sensing, and particularly relates to a method and a sensor for detecting ion-selective membrane potential type L-cystine, which are suitable for on-line detection in aspects of health breeding and life sciences.
  • L-cystine is a sulfur-containing amino acid, which is mostly found in keratin such as hair and finger claws. In addition to constituting body proteins, it is also widely present in living cells and tissues of organisms. An important physiological function. L-cystine has the functions of promoting cell oxidation and reduction, making the liver function strong, and neutralizing toxins, promoting leukocyte proliferation, preventing the development of pathogenic bacteria, and maintaining protein configuration. S-sulfur amino acids such as L-cystine can not only be added to food as additives to increase food nutrition, but also play a very important role in animal digestion and absorption, nutrient metabolism and immune function.
  • 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. .
  • HPLC high performance liquid chromatography
  • HPLC-MS high performance liquid chromatography-mass spectrometry
  • GC-MS gas chromatography-mass spectrometry
  • capillary electrophoresis fluorescent probe detection, etc.
  • fluorescent probe detection etc.
  • the main method used is chromatographic separation and analysis. For example, Xia Sujie et al. Used pre-column derivatization HPLC to determine L-cystine. The linear range is 0.02-1.00. mg / mL; Zhang et al.
  • potential electrochemical sensors Compared to current sensors based on composite materials modification, potential electrochemical sensors have the advantages of light weight, simple preparation, and fast response, which are conducive to online and real-time monitoring and analysis of amino acids in actual samples. They have been successfully applied to different amino acids. Assay, such as L-histidine, L-cysteine, L-glutamic acid, L-lysine, etc.
  • Assay such as L-histidine, L-cysteine, L-glutamic acid, L-lysine, etc.
  • FET field effect transistor
  • ISFETs ion-sensitive field effect transistors
  • ISFET devices have the advantages of low cost, high sensitivity, fast response, easy miniaturization and integration, easy use, and easy online monitoring. In recent years, it has been gradually applied to the detection of DNA, hypoxanthine nucleosides, Staphylococcus epidermidis, prions and other biomolecules. At present, no potential electrochemical detection method for L-cystine has been reported.
  • the present invention aims to overcome the shortcomings of the prior art, and provides a method and a sensor for detecting L-cystine.
  • the method for detecting L-cystine includes the following steps:
  • a p-well (2) and an N-type substrate (3) are implanted on the Si base layer (1) of the field effect transistor, and a source electrode is constructed at the p-well (2) by using thermal evaporation and magnetron sputtering techniques ( 4) and drain electrode (5), and then build on the Si base layer (1) that is implanted with p-well (2) and N-type substrate (3) and builds active electrode (4) and drain electrode (5)
  • the silicon dioxide layer (6) is then plated with an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9), and a gold film in order on the base layer of the polysilicon gate (7) by thermal evaporation and magnetron sputtering techniques.
  • a silicon nitride layer (11) is formed on the base layer of the polysilicon gate (7) and the silicon dioxide layer (6); the gate portion is extended by a distance of 0.1-500 mm to obtain polydisulfide Dipropanesulfonic acid film (12) modified polydithiodipropanesulfonic acid film gate gold electrode;
  • a complete sensing and detection circuit is formed; the in-situ signal amplification of the field effect transistor can be used to sensitively detect the potential change of the system; the polydisulfidepropanesulfonic acid film gate gold electrode as a working electrode is buffered in PBS The potential of the solution will gradually stabilize with the increase of time. After the potential is stabilized, test samples containing different concentrations of L-cystine (13) are added to obtain the corresponding potential response data to complete the L in the test sample. -Detection of cystine (13).
  • an aluminum-copper alloy layer (8), a chromium-palladium alloy layer (9), and a gold film are sequentially plated on the base layer of the polysilicon gate (7) by using thermal evaporation and magnetron sputtering techniques.
  • the aluminum-copper alloy layer (8) includes the following components 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) includes the following components 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
  • the thickness of the gold film layer (10) is 20-1000 nm.
  • step (2) an ethanol solution of sodium polydithiopropane sulfonate (1.0-10.0 mmol / L) is prepared.
  • step (2) the gate gold electrode of the extended gate field effect transistor is sequentially washed with ultrapure water and anhydrous ethanol. The time for immersing the gold electrode in the step (2) in the ethanol solution of sodium polydisulfidepropane sulfonate is 1-72 hours.
  • step (2) the soaked grid gold electrode is washed with absolute ethanol and ultrapure water, and dried and stored.
  • the reference electrode in step (3) is a saturated calomel electrode or an Ag / AgCl electrode with a saturated KCl solution built in, and the working electrode is a polydithiodipropanesulfonic 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 NaH 2 PO 4 ⁇ 2H 2 O , Na 2 HPO 4 ⁇ 12H 2 O, NaCl are mixed and dissolved in water at an appropriate ratio, and the pH value is adjusted with 0.1 mol / L hydrochloric acid.
  • the invention also provides a sensor for detecting L-cystine, the sensor includes a field effect transistor, and the field effect transistor is provided with a gold electrode with extended gate, that is, a gate gold electrode; the gate In the extended gold electrode, the gate portion is extended by a distance of 0.1-500 mm, and the surface of the gold film layer (10) of the gold electrode is assembled with a polydithiodipropanesulfonic acid film (12).
  • the field effect transistor includes a Si base layer (1) and a polysilicon gate (7) provided on the Si base layer (1); a p-well (2) and N are implanted on the Si base layer (1).
  • Type substrate (3), the p-well (2) is provided with a source electrode (4) and a drain electrode (5), is implanted into the p-well (2) and the N-type substrate (3) and an active electrode is constructed (4) and the Si base layer (1) of the drain electrode (5) are provided with a silicon dioxide layer (6);
  • the base layer of the polysilicon gate (7) is sequentially plated with an aluminum-copper alloy layer (8), A chromium-palladium alloy layer (9) and a gold film layer (10); a silicon nitride layer (11) is further provided on the base 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
  • 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 and a response sensitivity of 58.25 ⁇ 1.5mV / -pc (25 °C), the detection limit was 2.69 ⁇ 10 -6 mol / L.
  • the invention extends the gate gold electrode (GGE) of a field effect transistor (FET) by a certain distance, for example, by 0.1-500 mm, and self-assembles polydithiodipropanesulfonic acid (SPS) on the surface of the GGE to form a detectable A novel sensor for L-cystine (GGE / SPS electrode).
  • GGE gate gold electrode
  • FET field effect transistor
  • SPS polydithiodipropanesulfonic acid
  • the electrode has fast response (30 seconds), good reproducibility and stability.
  • the present invention has developed a simple, selective membrane potential-type electrochemical sensor, and provided a new method for detecting L-cystine. That is, a polydithiodipropanesulfonic acid (SPS) anion membrane is assembled in gold. The surface of the gold film layer of the electrode is used as the extended gate of the field effect transistor. The in-situ signal amplification of the field effect transistor is used to realize the sensitive detection of L-cystine.
  • the sensor has a good Nernst response relationship and can be used for The rapid and sensitive detection of L-cystine in swine serum samples has potential application prospects in the fields of healthy farming and life sciences.
  • Figure 1 is a schematic diagram of the design of an extended gate field effect transistor; in the figure: 1, Si base layer, 2, source electrode, 3, drain electrode, 4, p-well, 5, N-type substrate, 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, polydithiodipropanesulfonic acid film;
  • Figure 2 shows the SEM characterization of the electrode surface morphology before and after the detection of L-cystine by the GGE / SPS electrode;
  • Figure 2a is a scanning electron microscope image of the bare gold electrode surface;
  • Figure 2b is a diagram of the electrode surface after self-assembly SPS, and
  • Figure 2c is Surface image after the electrode detects L-cystine;
  • Figure 3 is a schematic diagram of the recognition response principle of SPS molecules bound to L-cystine molecules; in the figure: 10, gold film layer, 12, polydithiodipropanesulfonic acid film, 13, L-cystine;
  • Figure 4 Figures 4A and 4B are electrodes in potassium ferricyanide solution (containing 2.0mmol / L K 3 [Fe (CN) 6 ], 2.0mmol / L K 4 [Fe (CN) 6 ], 0.2mol / L Na 2 SO 4 ) AC impedance and cyclic voltammograms; in Figures 4A and 4B: a, GGE, b, GGE / SPS, c, GGE / SPS / L-cystine;
  • Figure 5 is the XPS full spectrum of different electrode surfaces; in the figure: a, GGE, b, GGE / SPS, c, GGE / SPS / L-cystine;
  • FIG. 6 is a relationship diagram of the slope of the SPS film-modified GGE electrode as a function of pH (pH 3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0);
  • FIG. 7 is a schematic diagram of the recognition mechanism of the potential ion response
  • Figure 9 is a histogram of the slope of the response of extended naked GGE and GGE / SPS electrodes to L-cystine;
  • FIG. 10 is a time-potential response diagram of an electrode after adding different concentrations of L-cystine to a PBS buffer solution
  • Figure 11 shows the effects of common amino acids on the detection of L-cystine by GGE / SPS electrodes.
  • Figure 1 is a schematic diagram of the design of an extended gate field effect transistor, that is, combining the basic structure of a metal-oxide-semiconductor field effect transistor (MOSFET), p-well 2 and an N-type substrate are implanted on the Si base layer 1 of the field effect transistor. 3.
  • MOSFET metal-oxide-semiconductor field effect transistor
  • p-well 2 and N-type substrate are implanted on the Si base layer 1 of the field effect transistor.
  • a silicon dioxide layer 6 is formed on the Si base layer 1, and then an aluminum-copper alloy layer 8, a chromium-palladium alloy layer 9 and a gold film layer 10 are sequentially plated on the base layer of the polysilicon gate 7 by using thermal evaporation and magnetron sputtering techniques. Finally, a silicon nitride layer 11 is constructed on the base layer of the polysilicon gate 7 and the silicon dioxide layer 6; the aluminum-copper alloy layer 8 includes the following components by weight: Al 40—68, Cu 30—60, Ni 2— 12.
  • the chromium-palladium alloy layer 9 includes the following components by weight: Cr 40-80, Pd 10-40, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01-0.50; thickness of aluminum-copper alloy layer 8 is 20-600 nm, thickness of chromium-palladium alloy layer 9 is 20-600 nm, and thickness of gold film layer 10 is 20-1000 nm;
  • the divided gold electrode is extended by a distance of 200 mm, and the field effect transistor wafer is passivated with SiO 2 and Si 3 N 4 to prevent the portion of the wafer other than Au from contacting the solution, thereby forming an extended gate field effect transistor (EGFET).
  • EGFET extended gate field effect transistor
  • GGE extended gate gold electrode
  • the buffer system for electrode potential test is a phosphate buffer solution (PBS, 0.1mol / L) with a pH of 3.0-8.0.
  • the preparation method is to mix a certain amount of NaH 2 PO 4 ⁇ 2H 2 O, Na 2 HPO 4 ⁇ 12H 2 O and NaCl are mixed and dissolved in water at an appropriate ratio, and the pH value is adjusted with 0.1 mol / L hydrochloric acid.
  • FIG. 2 The morphology of the electrode surface before and after the detection of L-cystine by the GGE / SPS electrode was characterized by SEM (as shown in Figure 2).
  • Figure 2 (a) is a scanning electron microscope image of the bare gold electrode surface. It can be seen that the electrode surface is relatively flat. smooth. After the electrode surface self-assembled SPS ( Figure 2b), it was shown that a denser film was formed on the electrode surface, which was somewhat rough, indicating that the SPS formed a self-assembled film on the GGE surface.
  • Figure 2c When the electrode detected L-cystine, a lot of particulate matter appeared on the surface ( Figure 2c). This structural change may be due to a certain binding effect between L-cystine and SPS, such as electrostatic adsorption reaction, to form The stronger complexes aggregate, which changes the surface morphology of the electrode.
  • SPS Polydithiodipropanesulfonic acid
  • RSSR disulfide compound
  • its disulfide bond is easily reduced and broken, forming two identical structures with sulfhydryl groups, the reaction formula It can be derived as follows:
  • L-cystine under acidic conditions, L-cystine is positively charged, and the end containing SPS in the SPS is negatively charged, which will attract L-cystine to positively charged amino groups. Due to the steric hindrance structure, the two sulfonic acid groups of the SPS molecule and the two positively charged amino groups of the L-cystine are electrostatically bound to each other, that is, one SPS molecule can bind one L-cystine molecule, which The schematic diagram of the identification response principle is shown in Figure 3.
  • FIG. 4A and 4B are an AC impedance diagram and a cyclic voltammogram of an electrode in a potassium ferricyanide solution, respectively.
  • a represents a bare gold electrode, and the impedance value is very small.
  • b represents a SPS-modified gold electrode. Compared with the bare gold electrode, it is obvious that a semicircle appears in the high-frequency part, and the impedance value is relatively large.
  • the bond formed a dense single-molecule self-assembled film layer on the gold surface, which prevented [Fe (CN) 6 ] 3- / 4- electron conduction on the electrode surface.
  • L-cystine 1.0 ⁇ 10 -5 mol / L
  • the impedance value is greatly increased (as shown by curve c in FIG. 4A), which is due to the very strong adsorption of SPS and L-cystine
  • the combined effect makes the film layer on the surface of the gold electrode thicker and greatly reduces the electronic conductivity, so that the electrode impedance value continues to increase a lot, and the change in its impedance value can be confirmed by the corresponding electrochemical cyclic voltammetry behavior (such as Figure 4B).
  • a represents a bare gold electrode, and there are obvious oxidation peaks and reduction peaks, which indicates that the bare gold electrode after pretreatment has a strong ability to transfer electrons.
  • b represents the SPS-modified gold electrode, and its redox peak is significantly smaller than that of the bare gold electrode, indicating that SPS forms a non-conductive single-molecule self-assembled film layer on the gold surface through disulfide bonds, which prevents [Fe (CN) 6 ] 3- / 4- Electron conduction on the electrode surface causes the peak current to decrease.
  • X-ray photoelectron spectroscopy was used to investigate the recognition effect of the modified membrane electrode on the target L-cystine.
  • Figure 5 shows the surface of the different electrodes (GGE, GGE / SPS, GGE / SPS / L-cystine). XPS full spectrum. It can be seen from FIG.
  • the Au electrode 4f , Au 4d, and Au 4p characteristic peaks of gold appear in the gold electrode (curve b) after self-assembly SPS, and appear at a binding energy of 161.71eV
  • the characteristic S 2p peak of sulfur indicates that the Au-S bond was formed on the electrode surface, which proves that the SPS was successfully assembled on the surface of the gold electrode.
  • Curve c represents the surface XPS pattern of GGE / SPS recognition after binding to L-cystine.
  • the linear response range for L-cystine in the medium is 5.0 ⁇ 10 -6 —1.0 ⁇ 10 -3 mol / L.
  • the least square method was used to obtain the linear response curve of the electrode potential after fitting.
  • the electrode detection limit was 2.69 ⁇ 10 -6 mol / L ( Figure 8).
  • Figure 9 is a histogram of the slope of the response of extended naked GGE and GGE / SPS electrodes to L-cystine. As can be seen from Figure 9, the potential response of naked GGE to L-cystine is little or no response Therefore, SPS acts as a sensitive membrane, and it is feasible to modify SPS to detect L-cystine on extended GGE.
  • Ni-CCE nickel-doped carbon ceramic electrode
  • ⁇ -CD ⁇ -cyclodextrin
  • GCE glassy carbon electrode
  • GGE grid gold electrode
  • rGO reduced graphene oxide
  • SPS polydithiodipropane Sulfonic acid.
  • Fig. 10 is the time-potential response diagram of the electrode after adding different concentrations of L-cystine to the PBS buffer solution, which was performed from low concentration to high concentration in the range of 1.0 ⁇ 10 -5 -5.0 ⁇ 10 -4 mol / L. Continuously test and record the time-varying potential value. It can be seen that the response time of the electrode to reach equilibrium in the entire concentration range is very short. The response time is calculated at 95% of the maximum value of the potential response, which is about 30s.
  • the electrode pair L-cystine sample was continuously tested for 14 days, and the 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, and has good stability and Longer service life.
  • the extended GGE / SPS electrode was used to determine L-cystine in actual pig serum samples. Take processed pig serum samples (from 6 live ternary hybrid piglets, weighing 7-15Kg, provided by the Institute of Subtropical Agriculture and Ecology, Chinese Academy of Sciences), dilute 10 times with PBS buffer pH 5.0, add The known concentration of L-cystine was determined by standard addition method. The recovery of L-cystine in pig serum samples was between 91.2 and 107.8% (see Table 3), indicating that the GGE / SPS electrode Good accuracy, can be used for rapid and sensitive detection of L-cystine in actual pig serum samples, and has potential application value in the fields of life medicine and animal breeding.
  • the electrode has a short response time (30 seconds) and has good selectivity, reproducibility and stability.
  • the electrode can be directly applied to the determination of L-cystine in actual pig serum samples, and it is expected to become a new online test method for L-cystine.

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Abstract

一种L-胱氨酸的检测方法及传感器，检测方法包括：将聚二硫二丙烷磺酸膜（12）组装在金电极的金膜层（10）表面，作为场效应晶体管延伸出来的栅极，利用场效应晶体管原位信号放大作用实现对L-胱氨酸的灵敏检测，其中，聚二硫二丙烷磺酸膜（12）的聚阴离子通过静电作用吸附结合带正电荷的目标物L-胱氨酸，形成双电层结构而产生识别一价有机铵离子的膜电位。传感器对L-胱氨酸具有良好的能斯特响应关系，线性范围为5.0×10 -6—1.0×10 -3mol/L，响应灵敏度为58.25±1.5mV/-pc，检出限为2.69×10 -6mol/L，可用于猪血清样品中L-胱氨酸的快速灵敏检测，在健康养殖和生命科学等领域具有潜在的应用前景。

Description

一种L-胱氨酸的检测方法及传感器 技术领域
本发明属于化学/生物传感技术领域，具体涉及一种离子选择性膜电位型的L-胱氨酸的检测方法及传感器，适用于健康养殖与生命科学方面的在线检测。
背景技术
L-胱氨酸(L-cystine)是一种含硫氨基酸，多存在于头发、指爪等的角蛋白中，除构成机体蛋白质外，还广泛存在于生物体活细胞与组织中，具有多种重要的生理功能。L-胱氨酸具有促进机体细胞氧化和还原机能，使肝脏功能旺盛，并能中和毒素、促进白细胞增生、阻止病原菌发育，并有维持蛋白质构型等作用。L-胱氨酸这类含硫氨基酸不仅可作为添加剂加入到食品中以增加食物的营养，而且在动物消化吸收、营养代谢以及免疫功能等方面都起到了很重要的作用。
常用的氨基酸检测方法有高效液相色谱法(HPLC)、高效液相色谱-质谱联用法(HPLC-MS)、气相色谱-质谱法(GC-MS)、毛细管电泳法、荧光探针检测法等。目前国内外对L-胱氨酸检测的研究报道不多，主要采用的方法是色谱分离分析方法，如夏苏捷等利用柱前衍生HPLC法测定L-胱氨酸，线性范围为0.02—1.00mg/mL；Zhang等采用HPLC检测L-胱氨酸，线性范围为2—15μM；Alwael等利用反相液相色谱与质谱联用方法检测L-胱氨酸，线性范围是0.25—250μM。然而，这些方法普遍存在着成本高，需要昂贵的精密仪器、复杂的样品制备流程，且不能在线或不方便在户外使用等缺点，所以探索并发展一种简便、快捷的氨基酸分析方法是该领域研究的重要趋势。与其它仪器分析方法相比，电化学方法以其简单、灵敏、快速等特点越来越受到人们的关注，如循环伏安法(CV)、微分脉冲伏安法(DPV)、方波伏安法(SWV)等已用于一些氨基酸的检测。
目前，利用电化学方法检测胱氨酸的报道主要是电流型传感器，Salimi等制备了镍掺杂碳陶瓷电极，采用CV法检测L-胱氨酸，线性范围为1.0—450μM，检出限为0.64μM；Zor等采用还原氧化石墨稀/β-环糊精复合膜电极，对L-胱氨酸的线性范围为1.0—100μM，检出限为1.0μM，同时结合DPV检测和模型计算可获得D型和L型胱氨酸的结合常数。现存的电流型方法普遍存在仪器不方便携带、难以在线检测等问题，而国内外对于胱氨酸检测方法的探讨依然很少，探寻一种更加简单、快速和灵敏测定L-胱氨酸的电化学方法显得极为重要。
相对于基于复合材料修饰的电流型传感器，电位型电化学传感器具有仪器轻巧、制 备简易、响应快速等优点，有利于对实际样品中氨基酸实现在线、实时监测分析，目前已成功应用于不同氨基酸的测定，如L-组氨酸、L-半胱氨酸、L-谷氨酸、L-赖氨酸等。而且，在电位型电化学传感方法中，具有电化学特性的离子选择性膜和基于金属-电介质-半导体器件的场效应晶体管(FET)的结合是一个重要的发展方向。一般把具有离子选择功能的FET器件称为离子敏场效应晶体管(ISFET)，ISFET器件具有成本低、灵敏度高、响应快，易小型化、集成化，且使用方便、易实现在线监测等优点，近年来已逐渐应用于DNA、次黄嘌呤核苷、表皮葡萄球菌、朊病毒等生物分子的检测。目前，电位型电化学检测L-胱氨酸的方法还没有见报道。
发明内容
本发明旨在克服现有技术的不足，提供一种L-胱氨酸的检测方法及传感器。
为了达到上述目的，本发明提供的技术方案为：
所述L-胱氨酸的检测方法包括如下步骤：
(1)在场效应晶体管的Si基底层(1)上植入p阱(2)和N型衬底(3)，采用热蒸发和磁控溅射技术在p阱(2)处构建源电极(4)和漏电极(5)，然后在被植入p阱(2)和N型衬底(3)并构建有源电极(4)和漏电极(5)的Si基底层(1)上构建二氧化硅层(6)，再采用热蒸发和磁控溅射技术在多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)，最后在多晶硅栅极(7)的基底层和二氧化硅层(6)上构建氮化硅层(11)；将栅极部分延长0.1—500mm的距离，制得聚二硫二丙烷磺酸膜(12)修饰的聚二硫二丙烷磺酸膜栅极金电极；
(2)制备聚二硫二丙烷磺酸钠的乙醇溶液，并将清洗后的延长栅场效应晶体管的栅极金电极浸泡于其中，于25℃条件下静置，然后洗净浸泡后的栅极金电极，制得聚二硫二丙烷磺酸(SPS)膜栅极金电极；
(3)将参比电极、聚二硫二丙烷磺酸膜栅极金电极与延长栅场效应晶体管的电极接口相连形成双高阻差分放大电路，将参比电极、聚二硫二丙烷磺酸膜栅极金电极插入PBS缓冲溶液中，将延长栅场效应晶体管的电源接口分别与稳压电源的正负极相连，将延长栅场效应晶体管的信号输出接口与万用电表的测试端口相连，由此构成一个完整的传感检测回路；利用场效应晶体管的原位信号放大作用，可灵敏检测体系的电位变化；作为工作电极的聚二硫二丙烷磺酸膜栅极金电极在PBS缓冲溶液的电位会随着时间的增加而逐渐趋向于稳定，待电位稳定后加入含有不同浓度L-胱氨酸(13)的待测样品，进而获得相应的电位响应数据，完成待测样品中L-胱氨酸(13)的检测。
优选地，步骤(1)中，用热蒸发和磁控溅射技术在多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)时，采用Si 3N 4进行钝化；铝铜合金层(8)包括如下重量份的组分：Al 40—68、Cu 30—60、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铬钯合金层(9)包括如下重量份的组分：Cr 40—80、Pd 10—40、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铝铜合金层(8)的厚度为20—600nm，铬钯合金层(9)的厚度为20—600nm，金膜层(10)的厚度为20—1000nm。
优选地，步骤(2)中是制备1.0—10.0mmol/L的聚二硫二丙烷磺酸钠的乙醇溶液。步骤(2)中是依次用超纯水和无水乙醇清洗延长栅场效应晶体管的栅极金电极。步骤(2)中栅极金电极浸泡于聚二硫二丙烷磺酸钠的乙醇溶液中的时间为1—72h。步骤(2)中是用无水乙醇和超纯水洗净浸泡后的栅极金电极，并进行干燥、保存。
优选地，步骤(3)中的参比电极为饱和甘汞电极或内置饱和KCl溶液的Ag/AgCl电极，工作电极为聚二硫二丙烷磺酸膜栅极金电极。步骤(3)中的PBS缓冲溶液为pH3.0—8.0、浓度为0.1mol/L的磷酸盐缓冲溶液，pH值优选为5.0，其配制方法是将一定量的NaH 2PO 4·2H 2O、Na 2HPO 4·12H 2O、NaCl按适当比例混合溶于水，采用0.1mol/L的盐酸调节其pH值。
本发明还提供了一种检测L-胱氨酸的传感器，所述传感器包括场效应晶体管，所述场效应晶体管上设有栅极延长的金电极，即，栅极金电极；所述栅极延长的金电极中，所述栅极部分延长0.1—500mm的距离，金电极的金膜层(10)表面组装有聚二硫二丙烷磺酸膜(12)。
其中，所述场效应晶体管包括Si基底层(1)和设于Si基底层(1)上的多晶硅栅极(7)；所述Si基底层(1)上植入p阱(2)和N型衬底(3)，所述p阱(2)处设有源电极(4)和漏电极(5)，被植入p阱(2)和N型衬底(3)并构建有源电极(4)和漏电极(5)的Si基底层(1)上设有二氧化硅层(6)；所述多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)；在多晶硅栅极(7)的基底层和二氧化硅层(6)上还设有氮化硅层(11)。所述铝铜合金层(8)的厚度为20—600nm，铬钯合金层(9)的厚度为20—600nm，金膜层(10)的厚度为20—1000nm。
该传感器对L-胱氨酸(13)具有良好的能斯特响应关系，线性范围为5.0×10 -6—1.0×10 -3mol/L，响应灵敏度为58.25±1.5mV/-pc(25℃)，检出限为2.69×10 -6mol/L。
下面对本发明作进一步说明：
本发明将场效应晶体管(FET)的栅极金电极(GGE)延长一定距离，如延伸0.1—500 mm，在GGE表面自组装上聚二硫二丙烷磺酸(SPS)，形成一种可检测L-胱氨酸(L-cystine)的新型传感器(GGE/SPS电极)。传感界面的SEM表征、电化学测试、XPS分析表明，在溶液中带负电荷的SPS聚阴离子膜通过静电作用吸附结合带正电荷的目标物L-胱氨酸，形成双电层结构而产生识别一价有机铵离子的膜电位。该电极在磷酸盐缓冲溶液(pH＝5.0)中对L-胱氨酸有好的电位响应性能，线性范围为5.0×10 -6—1.0×10 -3mol/L，响应灵敏度为58.25±1.5mV/-pc(25℃)，检出限为2.69×10 -6mol/L。该电极响应快(30秒)，重现性、稳定性好。常见氨基酸如L-甘氨酸、L-丙氨酸、L-缬氨酸、L-天冬氨酸、L-脯氨酸、L-苏氨酸、L-组氨酸、L-亮氨酸、L-色氨酸、L-甲硫氨酸等均不干扰电极对L-胱氨酸的测定。且该电极可用于实际猪血清样品中L-胱氨酸的测定，回收率为91.2—107.8％，说明本发明的方法可成为一种装置简便、能准确测定L-胱氨酸的新手段。
总之，本发明开发了一种简便的选择性膜电位型电化学传感器，提供了一种新的L-胱氨酸的检测方法，即将聚二硫二丙烷磺酸(SPS)阴离子膜组装在金电极的金膜层表面，作为场效应晶体管延伸出来的栅极，利用场效应晶体管原位信号放大作用实现对L-胱氨酸的灵敏检测，该传感器具有良好的能斯特响应关系，可用于猪血清样品中L-胱氨酸的快速灵敏检测，在健康养殖与生命科学等领域具有潜在的应用前景。
附图说明
图1为延长栅极场效应晶体管的设计示意图；图中：1、Si基底层，2、源电极，3、漏电极，4、p阱，5、N型衬底，6、二氧化硅层，7、多晶硅栅极，8、铝铜合金层，9、铬钯合金层，10、金膜层，11、氮化硅层，12、聚二硫二丙烷磺酸膜；
图2为GGE/SPS电极检测L-胱氨酸前后的电极表面形貌SEM表征图；图2a是裸金电极表面的扫描电镜图，图2b是电极表面自组装SPS后的图，图2c是当电极检测L-胱氨酸后的表面图；
图3为SPS分子结合L-胱氨酸分子的识别响应原理示意图；图中：10、金膜层，12、聚二硫二丙烷磺酸膜，13、L-胱氨酸；
图4：图4A和图4B分别为电极在铁氰化钾溶液(含有2.0mmol/L K 3[Fe(CN) 6]，2.0mmol/L K 4[Fe(CN) 6]，0.2mol/L Na 2SO 4)中的交流阻抗图和循环伏安图；图4A和4B中：a、GGE，b、GGE/SPS，c、GGE/SPS/L-cystine；
图5为不同电极表面的XPS全谱图；图中：a、GGE，b、GGE/SPS，c、GGE/SPS/L-cystine；
图6为SPS膜修饰GGE电极的斜率随pH变化(pH为3.0、4.0、4.5、5.0、6.0、7.0、8.0)的关系图；
图7为电位离子响应识别机理示意图；
图8为GGE/SPS电极在pH＝5.0的PBS缓冲溶液中，结合不同浓度L-胱氨酸后得到的电位响应曲线图；
图9为延长的裸GGE和GGE/SPS电极对L-胱氨酸的响应斜率柱状图；
图10为电极在PBS缓冲溶液中加入不同浓度L-胱氨酸后的时间-电位响应图；
图11为常见氨基酸对GGE/SPS电极检测L-胱氨酸的影响。
具体实施方式
一、实验过程
1、SPS自组装膜金电极的制备
称取14.16mg聚二硫二丙烷磺酸钠溶于100mL乙醇中，得到4.0mmol/L的聚二硫二丙烷磺酸钠/乙醇溶液。将金电极依次用超纯水和乙醇清洗，再浸泡于聚二硫二丙烷磺酸钠/乙醇溶液中，于25℃下静置48h，然后将修饰的金电极取出，用乙醇和超纯水洗净，干燥保存，得到聚二硫二丙烷磺酸(SPS)自组装膜金电极。
2、延长栅极场效应晶体管的设计及栅极金电极的制备
图1是延长栅极场效应晶体管的设计示意图，即结合金属-氧化物-半导体场效应晶体管(MOSFET)的基本结构，在场效应晶体管的Si基底层1上植入p阱2和N型衬底3，采用热蒸发和磁控溅射技术在p阱2处构建源电极4和漏电极5，然后在被植入p阱2和N型衬底3并构建有源电极4和漏电极5的Si基底层1上构建二氧化硅层6，再采用热蒸发和磁控溅射技术在多晶硅栅极7的基底层上依次镀上铝铜合金层8、铬钯合金层9和金膜层10，最后在多晶硅栅极7的基底层和二氧化硅层6上构建氮化硅层11；铝铜合金层8包括如下重量份的组分：Al 40—68、Cu 30—60、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铬钯合金层9包括如下重量份的组分：Cr 40—80、Pd 10—40、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铝铜合金层8的厚度为20—600nm，铬钯合金层9的厚度为20—600nm，金膜层10的厚度为20—1000nm；将栅极部分的金电极延长200mm的距离，并且利用SiO 2和Si 3N 4钝化场效应晶体管晶片，目的是防止晶片除Au外的部分与溶液接触，从而形成延长栅极场效应晶体管(EGFET)。对EGFET的延长栅极金电极(GGE)膜表面进行不同的物理/化学修饰处理，形成敏感膜以实现对待测目标物的灵敏检测。按前述方法，将SPS聚阴离子自组装在栅极金电极的表面，制得聚二硫二丙烷磺酸膜12修饰的聚二硫二丙烷磺酸膜栅极金电极(GGE/SPS)。
3、自组装膜栅极金电极的测试
电极电位测试用缓冲体系为pH3.0—8.0的磷酸盐缓冲溶液(PBS，0.1mol/L)，其配制方法是将一定量的NaH 2PO 4·2H 2O、Na 2HPO 4·12H 2O、NaCl按适当比例混合溶于水，采用0.1mol/L的盐酸调节其pH值。
首先，将饱和甘汞电极、聚二硫二丙烷磺酸膜栅极金电极与本实验室设计的延长栅极场效应晶体管(EGFET)的电极接口相连形成双高阻差分放大电路，将电极插入PBS缓冲溶液中，将EGFET的电源接口分别与稳压电源的正负极相连，信号输出接口与万用电表的测试端口相连，由此构成一个完整的传感检测回路。利用场效应晶体管的原位信号放大作用，可灵敏检测体系的电位变化。工作电极GGE/SPS在PBS缓冲溶液的电位会随着时间的增加而逐渐趋向于稳定，待电位稳定后加入不同浓度待测物L-胱氨酸，进而获得相应的电位响应数据，即，获得标准曲线，在检测实际样本时，将实际样本的电位曲线与标准曲线比对即可完成检测。
二、实验结果与分析
1、GGE/SPS的SEM表征
对GGE/SPS电极检测L-胱氨酸前后的电极表面形貌进行了SEM表征(如图2所示)，图2中(a)是裸金电极表面的扫描电镜图，可见电极表面比较平整光滑。当电极表面自组装SPS后(图2b)，显示电极表面形成较致密的膜，有些粗糙，说明SPS在GGE表面形成了自组装膜。当电极检测L-胱氨酸后，表面出现了很多颗粒状的物质(图2c)，这种结构的变化可能是由于L-胱氨酸与SPS发生了某种结合作用如静电吸附反应，形成较强的配合物聚集，从而使得电极表面形态发生改变。
2、GGE/SPS的响应机理及电化学表征
聚二硫二丙烷磺酸(SPS)是一种含有二硫键且二硫键两边为对称结构的一种物质，其二硫键中的硫可以与Au形成Au-S键而自组装在金电极表面上。作为二硫化合物(RSSR)，SPS在酸性电解质(0.1mol/L的PBS缓冲溶液，pH＝5.0)中，其二硫键易被还原断裂，形成两个相同的带巯基的结构，其反应式可推导如下：
Figure PCTCN2019102372-appb-000001
而且，在酸性条件下，L-胱氨酸带正电，而SPS中含有磺酸基的一端带负电，将会吸引L-胱氨酸带正电的氨基。由于空间位阻结构，SPS分子的两个磺酸基分别与L-胱氨酸两个带正电的氨基之间发生静电吸附结合，即一个SPS分子可以结合一个L-胱氨酸分子，其识别响应原理示意图如图3所示。
为了验证聚二硫二丙烷磺酸载体与L-胱氨酸的相互作用，本发明采用交流阻抗和循环伏安方法考察了上述不同修饰膜电极的电化学行为，如图4所示。图4A和B分别为电极在铁氰化钾溶液中的交流阻抗图和循环伏安图。图4A中a代表裸金电极，阻抗值很小，图4A中b代表SPS修饰的金电极，与裸金电极相比，很明显高频部分出现半圆，阻抗值比较大，说明SPS通过二硫键在金表面上形成了较致密的单分子自组装膜层，阻碍了[Fe(CN) 6] 3-/4-在电极表面的电子传导。结合L-胱氨酸(1.0×10 -5mol/L)后，阻抗值又非常大地增强(如图4A中曲线c所示)，这是由于SPS与L-胱氨酸发生非常强的吸附结合作用，使得金电极表面的膜层增厚而大大降低了电子传导性，从而使电极阻抗值继续增大很多，其阻抗值的变化可从相应的电化学循环伏安行为中得到印证(如图4B所示)。
图4B中a代表裸金电极，有明显的氧化峰与还原峰，说明预处理后的裸金电极传递电子能力很强。图4B中b代表SPS修饰金电极，其氧化还原峰较裸金电极的明显小了很多，说明SPS通过二硫键在金表面上形成了不导电的单分子自组装膜层，阻碍了[Fe(CN) 6] 3-/4-在电极表面的电子传导，导致峰电流下降。当电极结合L-胱氨酸(1.0×10 -5mol/L)后，氧化还原峰电流明显降低了很多(如图4B中曲线c所示)，这是由于SPS修饰的金电极吸附了L-胱氨酸，降低了电子传导性，从而降低了电化学传导电流，说明SPS与L-胱氨酸发生了强吸附结合。因此，电极的电化学行为表明该传感界面可用于L-胱氨酸的识别检测。
3、GGE/SPS电极的XPS表征
借助X-射线光电子能谱(XPS)考察了修饰膜电极对目标物L-胱氨酸的识别作用情况，图5为上述不同电极(GGE，GGE/SPS，GGE/SPS/L-cystine)表面的XPS全谱图。由图5可知，相比于裸金电极(曲线a)，自组装SPS后的金电极(曲线b)出现金的Au 4f、Au 4d和Au 4p特征峰，而且在结合能为161.71eV处出现硫的S 2p特征峰，表明在电极表面形成了Au-S键，即证明SPS成功组装在金电极表面上。曲线c代表GGE/SPS识别结合L-胱氨酸后的表面XPS图谱。由曲线a、b、c可以发现，金的3个特征峰的强度按照GGE>GGE/SPS>GGE/SPS/L-cystine次序依次减弱很多，可能是由于电极表面依次结合SPS、L-胱氨酸而导致表面增厚很多，进一步证明GGE/SPS牢牢地吸附结合了L-胱氨酸分子。从表1中不同原子结合能数据可以发现O 1s的结合能在结合SPS后增加了0.33eV，而进一步结合L-胱氨酸后又相应降低了0.46eV，这是因为在酸性介质中，SPS带负电的磺酸基与Na +的结合力弱(Na +游离出来)，使得氧表现出强的结合能；当接触到L-胱氨酸时，L-胱氨酸带正电的氨基与SPS磺酸基之间发生强的静电吸附作用，导致O原子的结合能降低；而且，曲线c中N 1s结合能的峰比曲线b的略宽且大，也间接证明SPS电极界面吸附 了L-胱氨酸，从而表明该传感界面对L-胱氨酸有识别作用。
表1不同原子的结合能
Figure PCTCN2019102372-appb-000002
4、最佳pH的选择
实验考察了在不同pH条件(pH为3.0、4.0、4.5、5.0、6.0、7.0、8.0)下SPS膜修饰的GGE电极的电位随被测物L-胱氨酸浓度变化的情况，并依此求出响应斜率，做出斜率与pH的关系图，如图6所示。从图6中可以看出：pH＝5.0时电极响应斜率最大，电极响应最好，且其斜率值为58.25±1.5mV/-pc(25℃)，接近正一价离子的能斯特响应斜率理论值，这说明在pH＝5.0时L-胱氨酸的电荷总体贡献相当于正一价离子。
而且，也可以进一步推导，在酸性介质(pH＝5.0)中，SPS的磺酸基带负电荷，会形成聚阴离子的负电荷带，而L-胱氨酸在酸性介质中氨基带正电，羧基带很弱的负电，由于与磺酸基的强静电吸附作用而发生分子取向运动，形成一个铵离子的正电荷带，从而构成双电层结构而产生识别结合一价有机铵离子的相间电位差，其电位离子响应识别机理示意图如图7所示。
5、GGE/SPS电极响应性能
实验考查了延长的裸GGE和GGE/SPS电极对L-胱氨酸的测试响应性能。图8是GGE/SPS电极在pH＝5.0的PBS缓冲溶液中，结合不同浓度L-胱氨酸后得到的电位响应曲线图。由图8可知，随着L-胱氨酸浓度的增加，电极电位也逐渐增大，说明GGE/SPS电极表面键合的L-胱氨酸增加，且该电极在pH＝5.0的PBS缓冲溶液中对L-胱氨酸的线性响应范围为5.0×10 -6—1.0×10 -3mol/L。同时，采用最小二乘法处理，经拟合后得到电极电位线性响应曲线，线性方程为ΔE＝328.6+58.25lgc；根据作图法知，电极检出限为2.69×10 -6mol/L(图8)。
图9是延长的裸GGE和GGE/SPS电极对L-胱氨酸的响应斜率柱状图，从图9中可看出，裸GGE对L-胱氨酸的电位响应很小或基本上没有响应，因此SPS起到敏感膜作用，将SPS修饰在延长的GGE上用来检测L-胱氨酸是可行的。
此外，将GGE/SPS电极与其它文献已报道的L-胱氨酸电化学传感器进行比较(见表2)，发现GGE/SPS电位型传感器相对于电流型传感器，其线性范围较宽、选择性较好， 且制作简单、操作便携，易实现小型化和在线监测。
表2不同电极的性能比较
Figure PCTCN2019102372-appb-000003
注:Ni-CCE:镍掺杂碳陶瓷电极；β-CD:β-环糊精；GCE:玻碳电极；GGE:栅极金电极；rGO:还原氧化石墨烯；SPS:聚二硫二丙烷磺酸.
6、响应时间、稳定性和重现性的测定
实验考察了延长的GGE/SPS电极检测L-胱氨酸的响应时间和稳定性。图10是电极在PBS缓冲溶液中加入不同浓度L-胱氨酸后的时间-电位响应图，通过在1.0×10 -5—5.0×10 -4mol/L范围内从低浓度到高浓度进行连续测试并记录其随时间变化的电位值，可以看出，在整个浓度范围内电极达到平衡的反应时间很短，以达到电位响应最大值的95％来计算响应时间，约为30s。同时，将该电极对L-胱氨酸样品连续测试14天，其响应斜率变化为41.55mV/-pc，下降了28.67％，说明该膜电极可以使用2周以上，具有较好的稳定性和较长的使用寿命。
实验考察了所制备的GGE/SPS电极结合不同浓度L-胱氨酸样品的电位响应重现性，即在1.000×10 -5mol/L和1.000×10 -4mol/L的L-胱氨酸样品溶液中来回测定电位响应值，各测10次。通过数据的统计处理，发现电极在两个不同浓度溶液中的电位响应值的相对标准偏差分别为2.43％和0.85％，且相对标准偏差较小，表明该SPS膜电极的重现性好。
7、电极的选择性
在pH＝5.0的PBS缓冲溶液中，考察了常见氨基酸对GGE/SPS电极检测L-胱氨酸的影响(图11)。采用混合溶液法，固定L-胱氨酸的浓度为1.000×10 -5mol/L，加入50倍浓度的干扰组分如常见氨基酸物质，其中L-半胱氨酸(L-Cys)的浓度为1.000×10 -5mol/L。结果表明，加入L-甘氨酸(L-Gly)、L-丙氨酸(L-Ala)、L-缬氨酸(L-Val)、L-天冬氨酸(L-Asp)、L-脯氨酸(L-Pro)、L-苏氨酸(L-Thr)、L-组氨酸(L-His)、L-亮氨酸(L-Leu)、L-色氨酸(L-Trp)、L-甲硫氨酸(L-Met)后，SPS膜电极响应L-胱氨酸的电位值几乎没有变化(图 11)，即产生的干扰很小；而L-半胱氨酸(L-Cys)则有一定的影响。
8、回收率的测定与分析应用
在优化实验条件下，利用延长的GGE/SPS电极对实际猪血清样品中的L-胱氨酸进行测定。分别取处理好的猪血清样品(取自6头活体三元杂小猪，体重为7—15Kg，由中国科学院亚热带农业生态研究所提供)，用pH5.0的PBS缓冲液稀释10倍，加入已知浓度的L-胱氨酸，采用标准加入法测定，测得猪血清样品中L-胱氨酸的回收率在91.2—107.8％之间(见表3)，说明该GGE/SPS电极的准确性好，可以用于实际猪血清样品中L-胱氨酸的快速灵敏检测，在生命医学和动物养殖等领域具有潜在应用价值。
表3 GGE/SPS电极测定猪血清样品中L-胱氨酸的含量及其回收率
Figure PCTCN2019102372-appb-000004
总之，本发明所述GGE/SPS电极在PBS(pH＝5.0)溶液中，对L-胱氨酸表现出灵敏的能斯特响应，其线性响应范围为5.00×10 -6—1.00×10 -3mol/L，检出限为2.69×10 -6mol/L。该电极响应时间短(30秒)，具有良好的选择性、重现性和稳定性等特点。且该电极能直接应用于测定实际猪血清样品中的L-胱氨酸，有望成为L-胱氨酸的一种新的在线测试手段。

Claims (10)

  1. 一种L-胱氨酸的检测方法，其特征在于，所述方法包括如下步骤：
    (1)在场效应晶体管的Si基底层(1)上植入p阱(2)和N型衬底(3)，采用热蒸发和磁控溅射技术在p阱(2)处构建源电极(4)和漏电极(5)，然后在被植入p阱(2)和N型衬底(3)并构建有源电极(4)和漏电极(5)的Si基底层(1)上构建二氧化硅层(6)，再采用热蒸发和磁控溅射技术在多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)，最后在多晶硅栅极(7)的基底层和二氧化硅层(6)上构建氮化硅层(11)；将栅极部分延长0.1—500mm的距离，制得具有栅极金电极的延长栅场效应晶体管；
    (2)制备聚二硫二丙烷磺酸钠的乙醇溶液，并将清洗后的延长栅场效应晶体管的栅极金电极浸泡于其中，于25℃条件下静置，然后洗净浸泡后的栅极金电极，制得聚二硫二丙烷磺酸膜(12)修饰的聚二硫二丙烷磺酸膜栅极金电极；
    (3)将参比电极、聚二硫二丙烷磺酸膜栅极金电极与延长栅场效应晶体管的电极接口相连形成双高阻差分放大电路，将参比电极、聚二硫二丙烷磺酸膜栅极金电极插入PBS缓冲溶液中，将延长栅场效应晶体管的电源接口分别与稳压电源的正负极相连，将延长栅场效应晶体管的信号输出接口与万用电表的测试端口相连，由此构成一个完整的传感检测回路；利用场效应晶体管的原位信号放大作用，可灵敏检测体系的电位变化；作为工作电极的聚二硫二丙烷磺酸膜栅极金电极在PBS缓冲溶液的电位会随着时间的增加而逐渐趋向于稳定，待电位稳定后加入含有不同浓度L-胱氨酸(13)的待测样品，进而获得相应的电位响应数据，完成待测样品中L-胱氨酸(13)的检测。
  2. 如权利要求1所述的方法，其特征在于，步骤(1)中，用热蒸发和磁控溅射技术在多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)时，采用Si 3N 4进行钝化；铝铜合金层(8)包括如下重量份的组分：Al 40—68、Cu 30—60、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铬钯合金层(9)包括如下重量份的组分：Cr 40—80、Pd 10—40、Ni 2—12、Fe 1—8、Ti 1—6、Nb 0.01—0.50；铝铜合金层(8)的厚度为20—600nm，铬钯合金层(9)的厚度为20—600nm，金膜层(10)的厚度为20—1000nm。
  3. 如权利要求1所述的方法，其特征在于，步骤(2)中是制备1.0—10.0mmol/L的聚二硫二丙烷磺酸钠的乙醇溶液。
  4. 如权利要求1所述的方法，其特征在于，步骤(2)中栅极金电极浸泡于聚二硫二丙烷磺酸钠的乙醇溶液中的时间为1—72h。
  5. 如权利要求1所述的方法，其特征在于，步骤(3)中的参比电极为饱和甘汞电极或内置饱和KCl溶液的Ag/AgCl电极，工作电极为聚二硫二丙烷磺酸膜栅极金电极。
  6. 如权利要求1所述的方法，其特征在于，步骤(3)中的PBS缓冲溶液为pH3.0—8.0、浓度为0.1mol/L的磷酸盐缓冲溶液。
  7. 一种检测L-胱氨酸的传感器，所述传感器包括场效应晶体管，所述场效应晶体管上设有栅极延长的金电极，其特征在于，所述栅极延长的金电极中，栅极部分延长0.1—500mm的距离，金电极的金膜层(10)表面组装有聚二硫二丙烷磺酸膜(12)。
  8. 如权利要求7所述的传感器，其特征在于，所述场效应晶体管包括Si基底层(1)和设于Si基底层(1)上的多晶硅栅极(7)；所述Si基底层(1)上植入p阱(2)和N型衬底(3)，所述p阱(2)处设有源电极(4)和漏电极(5)，被植入p阱(2)和N型衬底(3)并构建有源电极(4)和漏电极(5)的Si基底层(1)上设有二氧化硅层(6)；所述多晶硅栅极(7)的基底层上依次镀上铝铜合金层(8)、铬钯合金层(9)和金膜层(10)；在多晶硅栅极(7)的基底层和二氧化硅层(6)上还设有氮化硅层(11)。
  9. 如权利要求8所述的传感器，其特征在于，所述铝铜合金层(8)的厚度为20—600nm，铬钯合金层(9)的厚度为20—600nm，金膜层(10)的厚度为20—1000nm。
  10. 如权利要求7至9任一项所述的传感器，其特征在于，所述传感器对L-胱氨酸(13)具有良好的能斯特响应关系，线性范围为5.0×10 -6—1.0×10 -3mol/L，响应灵敏度为58.25±1.5mV/-pc(25℃)，检出限为2.69×10 -6mol/L。
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