CN208860792U - A kind of sensor detecting l-cysteine - Google Patents

Abstract

The utility model discloses a kind of sensors for detecting l-cysteine, the sensor includes field effect transistor, the field effect transistor is equipped with the extended gold electrode of grid, in the extended gold electrode of grid, grid part extends the distance of 0.1-500mm, and gold electrode surfaces are assembled with polydithio-dipropyl sulfonate film.Polydithio-dipropyl sulfonate film is assembled in the golden film layer surface of gold electrode by the utility model, the grid to extend out as field effect transistor, the Sensitive Detection to l-cysteine is realized using field effect transistor signals in situ amplification, wherein, the polyanion of polydithio-dipropyl sulfonate film is adsorbed by electrostatic interaction and combines positively charged object l-cysteine, is formed double electrical layers and is generated the film potential for identifying monovalence organoammonium ions.The sensor has this special response relation of good energy to l-cysteine, and the range of linearity is 5.0 × 10^‑6—1.0×10^‑3Mol/L, response sensitivity are 58.25 ± 1.5mV/-pc (25 DEG C), and detection is limited to 2.69 × 10^‑6mol/L。

Description

A kind of sensor detecting l-cysteine

Technical field

The utility model belongs to chemical/biological field of sensing technologies, and in particular to a kind of ion selective membrane electric potential type The sensor for detecting l-cysteine, suitable for the detection in terms of healthy aquaculture and life science.

Background technique

L-cysteine (L-cystine) is a kind of sulfur-containing amino acid, is present in the keratin of hair, paw etc. more, removes It constitutes outside body protein, is also widely present in organism living cells and tissue, there are a variety of important physiological functions.L- Guang Propylhomoserin, which has, promotes body cell oxidation and reduction function, keeps liver function vigorous, and can neutralize a toxin, leucocyte is promoted to increase Raw, prevention pathogen develops, and has the effects of Protein requirement configuration.This kind of sulfur-containing amino acid of l-cysteine can be used as adding Agent is added to be added to the nutrition for increasing food in food, and in sides such as animal digestion absorption, Nutrition and Metabolism and immune functions Face all plays critically important effect.

Common amino acid detection method has high performance liquid chromatography (HPLC), tablets by HPLC-MS (HPLC-MS), gas chromatography-mass spectrography (GC-MS), capillary electrophoresis, fluorescence probe detection method etc..At present both at home and abroad to L- The research report of cystine detection is few, and the method mainly used is chromatographic isolation analysis method, such as Xaisur's victory using before column Derivative HPLC method measures l-cysteine, and the range of linearity is 0.02-1.00mg/mL；Zhang etc. detects l-cysteine using HPLC, The range of linearity is 2-15 μM；Alwael etc. detects l-cysteine, the range of linearity using reversed-phase liquid chromatography and mass spectrometry combination method It is 0.25-250 μM.However, these methods in the prevalence of at high cost, need expensive precision instrument, complicated sample preparation Process, and cannot online or inconvenient the disadvantages of using outdoors, so exploring and developing a kind of simple, fast Amino acid score Analysis method is the important trend of the area research.Compared with Other Instruments analysis method, electrochemical method is simple with its, it is sensitive, Quickly the features such as, has been to be concerned by more and more people, such as cyclic voltammetry (CV), Differential Pulse Voltammetry (DPV), square wave volt-ampere Method (SWV) etc. has been used for the detection of some amino acid.

Currently, being mainly amperometric sensor using the report of electrochemical method detection cystine, Salimi etc. is prepared for Nickel doped carbon ceramic electrode detects l-cysteine using CV method, and the range of linearity is 1.0-450 μM, and detection is limited to 0.64 μM；Zor Deng using reduction-oxidation graphite it is dilute/beta-cyclodextrin compound film electrode, be 1.0-100 μM to the range of linearity of l-cysteine, detection 1.0 μM are limited to, the binding constant that can get D type and L-type cystine is calculated in combination with DPV detection and model.Existing electric current The generally existing instrument of type method is inconvenient to carry, is difficult to the problems such as on-line checking, and both at home and abroad for cystine detection method It inquires into still seldom, seeks a kind of simpler, quickly and the electrochemical method of sensitive determination l-cysteine seems particularly important.

Relative to the amperometric sensor modified based on composite material, electric potential type electrochemical sensor have instrument it is light and handy, The advantages that preparation is simple, response quickly, is conducive to realize online, Real Time Monitoring to amino acid in actual sample, at present It is successfully applied to the measurement of different aminoacids, such as L-Histidine, L-cysteine, Pidolidone, L-lysine.Moreover, In electric potential type electrochemical sensing method, there is the ion selective membrane of electrochemical properties and be based on metal-dielectric-semiconductor device The combination of the field effect transistor (FET) of part is an important developing direction.Generally the FET device with ion selection function Part is known as ion sensing fet (ISFET), and ISFET device has at low cost, high sensitivity, response fast, easily minimize, The advantages that integrated and easy to use, easy realization is monitored on-line is gradually applied to DNA, inosine, table in recent years The detection of the biomolecule such as skin staphylococcus, prion.Currently, the method for electric potential type Electrochemical Detection l-cysteine is shown in not yet Report.

Utility model content

The utility model overcomes the shortcomings of the prior art, and provides a kind of sensor for detecting l-cysteine.

In order to achieve the above object, technical solution provided by the utility model are as follows:

The sensor of the detection l-cysteine includes field effect transistor, and the field effect transistor prolongs equipped with grid Long gold electrode, that is, grid gold electrode；In the extended gold electrode of grid, the grid part extends 0.1-500mm's Golden membranous layer (10) surface-assembled of distance, gold electrode has polydithio-dipropyl sulfonate film (12).

Wherein, the field effect transistor includes Si basal layer (1) and the polysilicon gate on Si basal layer (1) (7)；It is implanted into p trap (2) and N-type substrate (3) on the Si basal layer (1), is equipped with source electrode (4) and drain electrode at the p trap (2) (5), p trap (2) and N-type substrate (3) are implanted and the Si basal layer (1) for constructing active electrode (4) and drain electrode (5) is equipped with two Silicon oxide layer (6)；Successively plated on the basal layer of the polysilicon gate (7) aluminum-copper alloy layer (8), chromium palladium alloy layer (9) and Golden membranous layer (10)；Silicon nitride layer (11) are additionally provided on the basal layer of polysilicon gate (7) and silicon dioxide layer (6).The aluminium Copper alloy layer (8) with a thickness of 20-600nm, chromium palladium alloy layer (9) with a thickness of 20-600nm, the thickness of golden membranous layer (10) For 20-1000nm.

The sensor has this special response relation of good energy to l-cysteine (13), and the range of linearity is 5.0 × 10^-6— 1.0×10^-3Mol/L, response sensitivity are 58.25 ± 1.5mV/-pc (25 DEG C), and detection is limited to 2.69 × 10^-6mol/L。

The detection method of l-cysteine based on the sensor includes the following steps:

(1) p trap (2) and N-type substrate (3) are implanted on the Si basal layer (1) of scene effect transistor, using thermal evaporation and magnetic Control sputtering technology constructs source electrode (4) and drain electrode (5) at p trap (2), is then being implanted p trap (2) and N-type substrate (3) simultaneously It constructs and constructs silicon dioxide layer (6) on the Si basal layer (1) of active electrode (4) and drain electrode (5), then use thermal evaporation and magnetic control Sputtering technology successively plates aluminum-copper alloy layer (8), chromium palladium alloy layer (9) and golden membranous layer on the basal layer of polysilicon gate (7) (10), silicon nitride layer (11) finally are constructed on the basal layer of polysilicon gate (7) and silicon dioxide layer (6)；By grid part Extend the distance of 0.1-500mm, the polydithio-dipropyl sulfonate film grid gold of polydithio-dipropyl sulfonate film (12) modification is made Electrode；

(2) ethanol solution of sodium polydithio-dipropyl sulfonate is prepared, and by the extension grid field effect transistor after cleaning Grid gold electrode is soaked in wherein, is stood under the conditions of 25 DEG C, then cleans the grid gold electrode after impregnating, poly- two sulphur two is made Propane sulfonic acid (SPS) film grid gold electrode；

(3) by reference electrode, polydithio-dipropyl sulfonate film grid gold electrode and the electrode for extending grid field effect transistor Interface is connected to form double high resistant differential amplifier circuits, and reference electrode, polydithio-dipropyl sulfonate film grid gold electrode are inserted into PBS In buffer solution, the power interface for extending grid field effect transistor is connected with the positive and negative anodes of regulated power supply respectively, grid will be extended The signal output interface of field effect transistor is connected with the test port of universal meter, thus constitutes a complete sensing detection Circuit；Using the signals in situ amplification of field effect transistor, can Sensitive Detection system potential change；As working electrode Polydithio-dipropyl sulfonate film grid gold electrode can be intended to as time increases and gradually in the current potential of PBS buffer solution Stablize, the sample to be tested for containing various concentration l-cysteine (13) is added after current potential is stablized, and then obtains corresponding current potential and ring Data are answered, the detection of l-cysteine (13) in sample to be tested is completed.

Preferably, in step (1), with thermal evaporation and magnetron sputtering technique on the basal layer of polysilicon gate (7) successively When plating aluminum-copper alloy layer (8), chromium palladium alloy layer (9) and golden membranous layer (10), using Si₃N₄It is passivated；Aluminum-copper alloy layer (8) Component including following parts by weight: Al 40-68, Cu 30-60, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01- 0.50；Chromium palladium alloy layer (9) includes the component of following parts by weight: Cr 40-80, Pd 10-40, Ni 2-12, Fe 1-8, Ti 1—6,Nb 0.01—0.50；Aluminum-copper alloy layer (8) with a thickness of 20-600nm, chromium palladium alloy layer (9) with a thickness of 20-600nm, golden membranous layer (10) with a thickness of 20-1000nm.

It preferably, is the ethanol solution for preparing the sodium polydithio-dipropyl sulfonate of 1.0-10.0mmol/L in step (2). It is the grid gold electrode for successively extending grid field effect transistor with ultrapure water and washes of absolute alcohol in step (2).In step (2) Grid gold electrode is soaked in the time in the ethanol solution of sodium polydithio-dipropyl sulfonate as 1-72h.Step is to use nothing in (2) Water-ethanol and ultrapure water clean the grid gold electrode after impregnating, and are dried, save.

Preferably, the reference electrode in step (3) is the Ag/AgCl electricity of saturated calomel electrode or built-in saturation KCl solution Pole, working electrode are polydithio-dipropyl sulfonate film grid gold electrode.PBS buffer solution in step (3) be pH3.0-8.0, Concentration is the phosphate buffer solution of 0.1mol/L, and pH value is preferably 5.0, and preparation method is by a certain amount of NaH₂PO₄· 2H₂O、Na₂HPO₄·12H₂O, NaCl is dissolved in water by proper proportion mixing, adjusts its pH value using the hydrochloric acid of 0.1mol/L.

The utility model is described in further detail below:

The grid gold electrode (GGE) of field effect transistor (FET) is extended certain distance by the utility model, is such as extended 0.1-500mm, polydithio-dipropyl sulfonic acid (SPS) on GGE surface self-organization form a kind of detectable l-cysteine (L- Cystine novel sensor (GGE/SPS electrode)).The SEM characterization of sensing interface, electro-chemical test, XPS analysis show Negatively charged SPS polyanionic membrane is adsorbed by electrostatic interaction and combines positively charged object l-cysteine, shape in solution The film potential of identification monovalence organoammonium ions is generated at double electrical layers.The electrode is at phosphate buffer solution (pH=5.0) In the potential response performance that has had to l-cysteine, the range of linearity is 5.0 × 10^-6—1.0×10^-3Mol/L, response sensitivity For 58.25 ± 1.5mV/-pc (25 DEG C), detection is limited to 2.69 × 10^-6mol/L.The electrode response is fast (30 seconds), reproducibility, steady It is qualitative good.Common amino acid such as L- glycine, l-Alanine, Valine, L-Aspartic acid, L-PROLINE, L-threonine, L- Histidine, L-Leu, L-Trp, l-methionine etc. do not interfere measurement of the electrode to l-cysteine.And the electrode can For the measurement of l-cysteine in practical pig anteserum sample, the rate of recovery is 91.2-107.8%, illustrates the method for the utility model It can a kind of easy as device, energy Accurate Determining l-cysteine new tool.

In short, the utility model develops a kind of selective membrane electric potential type electrochemical sensor of simplicity, one kind is provided Polydithio-dipropyl sulfonic acid (SPS) anionic membrane is assembled in the golden membranous layer of gold electrode by the detection method of new l-cysteine It is realized using field effect transistor signals in situ amplification to L- as the grid that field effect transistor extends out on surface The Sensitive Detection of cystine, the sensor have this special response relation of good energy, can be used for l-cysteine in pig anteserum sample Rapid sensitive detection, in the fields such as healthy aquaculture and life science with potential application prospect.

Detailed description of the invention

Fig. 1 is the design diagram for extending gate field effect transistor；In figure: 1, Si basal layer, 2, source electrode, 3, electric leakage Pole, 4, p trap, 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, polydithio-dipropyl sulfonate film；

Fig. 2 is the electrode surface pattern SEM phenogram before and after GGE/SPS electrode detection l-cysteine；Fig. 2 a is naked gold electricity The scanning electron microscope (SEM) photograph of pole surface, Fig. 2 b are the figures after electrode surface self assembly SPS, and Fig. 2 c is after electrode detection l-cysteine Exterior view；

Fig. 3 is the identification response theory schematic diagram of SPS molecule combination l-cysteine molecule；In figure: 10, golden membranous layer, 12, poly- Two sulphur, two propane sulfonic acid film, 13, l-cysteine；

Fig. 4: Fig. 4 A and Fig. 4 B be respectively electrode in potassium ferricyanide solution (containing 2.0mmol/LK₃[Fe(CN)₆], 2.0mmol/LK₄[Fe(CN)₆], 0.2mol/LNa₂SO₄) in AC impedance figure and cyclic voltammogram；In Fig. 4 A and 4B: a, GGE, b, GGE/SPS, c, GGE/SPS/L-cystine；

Fig. 5 is the full spectrogram of XPS on Different electrodes surface；In figure: a, GGE, b, GGE/SPS, c, GGE/SPS/L- cystine；

Fig. 6 is that the slope of the film modified GGE electrode of SPS changes (pH 3.0,4.0,4.5,5.0,6.0,7.0,8.0) with pH Relational graph；

Fig. 7 is that potential ion responds recognition mechanism schematic diagram；

Fig. 8 is GGE/SPS electrode in the PBS buffer solution of pH=5.0, in conjunction with what is obtained after various concentration l-cysteine Potential response curve graph；

Fig. 9 is response slope histogram of the extended naked GGE and GGE/SPS electrode to l-cysteine；

Figure 10 is that the when m- potential response figure after various concentration l-cysteine is added in electrode in PBS buffer solution；

Figure 11 is influence of the common amino acid to GGE/SPS electrode detection l-cysteine.

Specific embodiment

One, experimentation

1, the preparation of SPS self-assembled film gold electrode

It weighs 14.16mg sodium polydithio-dipropyl sulfonate to be dissolved in 100mL ethyl alcohol, obtains poly- two sulphur two of 4.0mmol/L Propane sulfonic acid sodium/ethanol solution.Gold electrode is successively cleaned with ultrapure water and ethyl alcohol, then is soaked in polydithio-dipropyl sulfonic acid In sodium/ethanol solution, 48h is stood at 25 DEG C, is then taken out the gold electrode of modification, with ethyl alcohol and ultrapure water cleaning, drying It saves, obtains polydithio-dipropyl sulfonic acid (SPS) self-assembled film gold electrode.

2, extend the design of gate field effect transistor and the preparation of grid gold electrode

Fig. 1 is the design diagram for extending gate field effect transistor, that is, combines Metal-oxide-semicondutor field-effect The basic structure of transistor (MOSFET) is implanted into p trap 2 and N-type substrate 3 on the Si basal layer 1 of scene effect transistor, using heat Evaporation and magnetron sputtering technique construct source electrode 4 and drain electrode 5 at p trap 2, are then being implanted p trap 2 and N-type substrate 3 and structure Building silicon dioxide layer 6 on the Si basal layer 1 of source electrode 4 and drain electrode 5 is had, then is existed using thermal evaporation and magnetron sputtering technique Aluminum-copper alloy layer 8, chromium palladium alloy layer 9 and golden membranous layer 10 are successively plated on the basal layer of polysilicon gate 7, finally in polysilicon gate Silicon nitride layer 11 is constructed on the basal layer and silicon dioxide layer 6 of pole 7；Aluminum-copper alloy layer 8 includes the component of following parts by weight: Al 40—68,Cu 30—60,Ni 2—12,Fe 1—8,Ti 1—6,Nb 0.01—0.50；Chromium palladium alloy layer 9 includes following weight Measure the component of part: Cr 40-80, Pd 10-40, Ni 2-12, Fe 1-8, Ti 1-6, Nb 0.01-0.50；Aluminum bronze closes Layer gold 8 with a thickness of 20-600nm, chromium palladium alloy layer 9 with a thickness of 20-600nm, golden membranous layer 10 with a thickness of 20- 1000nm；The gold electrode of grid part is extended to the distance of 200mm, and utilizes SiO₂And Si₃N₄It is brilliant to be passivated field effect transistor Piece, it is therefore an objective to prevent part of the chip in addition to Au from contacting with solution, extend gate field effect transistor (EGFET) to be formed. Different physical/chemical moditied processings is carried out to extension grid gold electrode (GGE) film surface of EGFET, forms sensitive membrane with reality Now to the Sensitive Detection of target to be measured.By preceding method, by the self assembly of SPS polyanion in the surface of grid gold electrode, system Obtain the polydithio-dipropyl sulfonate film grid gold electrode (GGE/SPS) that polydithio-dipropyl sulfonate film 12 is modified.

3, the test of self-assembled film grid gold electrode

The electrode potential test phosphate buffer solution (PBS, 0.1mol/L) that buffer system is pH3.0-8.0, matches Method processed is by a certain amount of NaH₂PO₄·2H₂O、Na₂HPO₄·12H₂O, NaCl is dissolved in water by proper proportion mixing, uses The hydrochloric acid of 0.1mol/L adjusts its pH value.

Firstly, by saturated calomel electrode, the extension of polydithio-dipropyl sulfonate film grid gold electrode and this lab design The electrode interface of gate field effect transistor (EGFET) is connected to form double high resistant differential amplifier circuits, inserts electrodes into PBS buffering In solution, the power interface of EGFET is connected with the positive and negative anodes of regulated power supply respectively, the survey of signal output interface and universal meter It tries port to be connected, thus constitutes a complete sensing detection circuit.Using the signals in situ amplification of field effect transistor, Can Sensitive Detection system potential change.Working electrode GGE/SPS PBS buffer solution current potential can as time increases and Gradually it is intended to stablize, various concentration determinand l-cysteine is added after current potential is stablized, and then obtain corresponding potential response Data, that is, obtain standard curve, when detecting actual sample, the potential curve of actual sample and standard curve are compared Complete detection.

Two, experimental result and analysis

1, the SEM characterization of GGE/SPS

SEM characterization (as shown in Figure 2) has been carried out to the electrode surface pattern before and after GGE/SPS electrode detection l-cysteine, (a) is the scanning electron microscope (SEM) photograph on naked gold electrode surface in Fig. 2, it is seen that electrode surface is relatively flat smooth.When electrode surface self assembly After SPS (Fig. 2 b), show electrode surface forms finer and close film, some are coarse, illustrate that SPS forms self assembly on the surface GGE Film.After electrode detection l-cysteine, there are many granular substances (Fig. 2 c) in surface, and the variation of this structure may be It is reacted since certain combination such as Electrostatic Absorption has occurred with SPS in l-cysteine, stronger complex aggregation is formed, to make Electrode surface form is obtained to change.

2, the Response Mechanism and electrochemical Characterization of GGE/SPS

Polydithio-dipropyl sulfonic acid (SPS) is a kind of containing disulfide bond and disulfide bond both sides are a kind of object of symmetrical structure Matter, the sulphur in disulfide bond can form Au-S key with Au and self assembly in gold electrode surfaces.As disulfide (RSSR), for SPS in acidic electrolyte bath (the PBS buffer solution of 0.1mol/L, pH=5.0), disulfide bond is easily reduced fracture, Two identical structures with sulfydryl are formed, reaction equation can derive as follows:

Moreover, in acid condition, l-cysteine is positively charged, and it is negatively charged containing sulfonic one end in SPS, it will it inhales Draw the positively charged amino of l-cysteine.Due to steric hindrance structure, two sulfonic groups of SPS molecule respectively with l-cysteine two Electrostatic Absorption occurs between positively charged amino to combine, i.e., a SPS molecule can be in conjunction with a l-cysteine molecule, identification Response theory schematic diagram is as shown in Figure 3.

In order to verify the interaction of polydithio-dipropyl sulfonic acid carrier and l-cysteine, the present invention using AC impedance and Cyclic voltammetric method has investigated the electrochemical behavior of above-mentioned different modifying membrane electrode, as shown in Figure 4.Fig. 4 A and B are respectively electrode AC impedance figure and cyclic voltammogram in potassium ferricyanide solution.A represents naked gold electrode, impedance value very little, Fig. 4 A in Fig. 4 A Middle b represents the gold electrode of SPS modification, compared with naked gold electrode, it is evident that semicircle occurs in high frequency section, and impedance value is bigger, says Bright SPS forms finer and close self-assembled monolayer layer by disulfide bond on a gold surface, hinders [Fe (CN)₆]^3-/4-? The electronics of electrode surface conducts.In conjunction with l-cysteine (1.0 × 10^-5Mol/L after), impedance value significantly enhances (such as Fig. 4 A again Shown in middle curve c), this is because very strong absorption combination occurs for SPS and l-cysteine, so that the film of gold electrode surfaces Layer thickens and greatly reduces electronic conductivity, so that electrode impedance value be made to continue to increase very much, the variation of impedance value can be from (as shown in Figure 4 B) is confirmed in corresponding electrochemistry cycle voltammetry behavior.

A represents naked gold electrode in Fig. 4 B, there is apparent oxidation peak and reduction peak, illustrates pretreated naked gold electrode transmitting Electronic capability is very strong.B represents SPS modified gold electrode in Fig. 4 B, and redox peaks are obvious small much compared with naked gold electrode, says Bright SPS forms nonconducting self-assembled monolayer layer by disulfide bond on a gold surface, hinders [Fe (CN)₆]^3-/4-? The electronics of electrode surface conducts, and peak current is caused to decline.When electrode combination l-cysteine (1.0 × 10^-5Mol/L after), oxidation is also Parent peak electric current significantly reduces very much (as shown in curve c in Fig. 4 B), this is because the gold electrode of SPS modification has adsorbed L- Guang ammonia Acid reduces electronic conductivity, to reduce electrochemical transduction electric current, illustrates that SPS and l-cysteine have occurred strong absorption and tie It closes.Therefore, the electrochemical behavior of electrode shows that the sensing interface can be used for the recognition detection of l-cysteine.

3, the XPS characterization of GGE/SPS electrode

Modification membrane electrode has been investigated to the recognition reaction feelings of object l-cysteine by x-ray photoelectron spectroscopy (XPS) Condition, Fig. 5 are the full spectrogram of XPS on the surface above-mentioned Different electrodes (GGE, GGE/SPS, GGE/SPS/L-cystine).As shown in Figure 5, Compared to naked gold electrode, ((curve b) goes out the Au of cash to the gold electrode after curve a), self assembly SPS_4f、Au_4dAnd Au_4pCharacteristic peak, And combination can be 161.71eV at there is the S of sulphur_2pCharacteristic peak shows to form Au-S key in electrode surface, i.e. proof SPS Successfully it is assembled in gold electrode surfaces.Curve c represents GGE/SPS identification and combines the surface XPS map after l-cysteine.By curve A, b, c are it can be found that the intensity of 3 characteristic peaks of gold successively subtracts according to GGE > GGE/SPS > GGE/SPS/L-cystine order It is weak very much, it may be possible to due to electrode surface successively in conjunction with SPS, l-cysteine and cause surface to thicken very much, further prove GGE/SPS is firmly adsorbed and is combined l-cysteine molecule.Different atomic binding energy data can be found that O from table 1_1sCombination 0.33eV can be increased after combining SPS, and further combined with correspondingly reducing 0.46eV after l-cysteine, this is because In acid medium, the electronegative sulfonic group of SPS and Na⁺Weak (the Na of binding force⁺Separate out) so that oxygen shows strong combination Energy；When touching l-cysteine, strong Electrostatic Absorption occurs between the positively charged amino of l-cysteine and SPS sulfonic group and makees With causing the combination of O atom that can reduce；Moreover, N in curve c_1sPeak in conjunction with energy is slightly wider and big than curve b, also indirect proof SPS electrode interface has adsorbed l-cysteine, to show that the sensing interface has recognition reaction to l-cysteine.

The not homoatomic combination energy of table 1

4, the selection of Optimal pH

It is film modified that the SPS at condition of different pH (pH 3.0,4.0,4.5,5.0,6.0,7.0,8.0) has been investigated in experiment The case where current potential of GGE electrode changes with measured object l-cysteine concentration, and finds out response slope according to this, makes slope and pH Relational graph, as shown in Figure 6.As can be seen from Figure 6: electrode response maximum slope when pH=5.0, electrode response is best, and its Slope value is 58.25 ± 1.5mV/-pc (25 DEG C), and close to this special response slope theoretical value of the energy of positive monovalent ion, this explanation exists The charge overall contribution of l-cysteine is equivalent to positive monovalent ion when pH=5.0.

Furthermore, it is also possible to further derive, in acid medium (pH=5.0), the sulfonic group of SPS is negatively charged, can shape At the negative electrical charge band of polyanion, and l-cysteine amino in acid medium is positively charged, the very weak negative electricity of carboxyl band, due to Sulfonic strong electrostatic adsorption and molecularly oriented movement occurs, the positive charge band an of ammonium ion is formed, to constitute double Electric layer structure and generate identification combine monovalence organoammonium ions interphase potential it is poor, potential ion respond recognition mechanism schematic diagram As shown in Figure 7.

5, GGE/SPS Responsibility of the electrode

Experiment exam test response performance of the extended naked GGE and GGE/SPS electrode to l-cysteine.Fig. 8 is GGE/ SPS electrode is in the PBS buffer solution of pH=5.0, in conjunction with the potential response curve graph obtained after various concentration l-cysteine.By Fig. 8 it is found that with l-cysteine concentration increase, electrode potential is also gradually increased, and illustrates the L- of GGE/SPS electrode surface bonding Cystine increases, and the electrode is 5.0 × 10 to the linear response range of l-cysteine in the PBS buffer solution of pH=5.0^-6—1.0×10^-3mol/L.Meanwhile using Least Square in Processing, electrode potential linear response curve, line are obtained after being fitted Property equation be Δ E=328.6+58.25lgc；Known according to graphing method, electrode detection is limited to 2.69 × 10^-6Mol/L (Fig. 8).

Fig. 9 is extended naked GGE and GGE/SPS electrode to the response slope histogram of l-cysteine, can from Fig. 9 Out, naked GGE to the potential response very little of l-cysteine or there is no response, therefore SPS plays the role of sensitive membrane, by SPS It is feasible that modification, which is used to detect l-cysteine on extended GGE,.

It (is shown in Table in addition, GGE/SPS electrode is compared with the reported l-cysteine electrochemical sensor of other documents 2), discovery GGE/SPS Copper diethlydithiocarbamate is relative to amperometric sensor, and the range of linearity is wider, selectivity is preferable, and makes Simply, portable, easy realization miniaturization and on-line monitoring are operated.

The performance of 2 Different electrodes of table compares

Note: Ni-CCE: nickel doped carbon ceramic electrode；β-CD: beta-cyclodextrin；GCE: glass-carbon electrode；GGE: grid gold electrode； RGO: redox graphene；SPS: polydithio-dipropyl sulfonic acid

6, the measurement of response time, stability and reproducibility

Response time and the stability of extended GGE/SPS electrode detection l-cysteine have been investigated in experiment.Figure 10 is electrode When m- potential response figure after various concentration l-cysteine is added in PBS buffer solution, by 1.0 × 10^-5—5.0× 10^-4Follow-on test is carried out within the scope of mol/L from low concentration to high concentration and records its potential value changed over time, can be seen Out, in entire concentration range electrode reach balance reaction time it is very short, to reach potential response maximum value 95% in terms of Calculate response time, about 30s.Meanwhile by the electrode to l-cysteine sample follow-on test 14 days, response slope, which changes, is 41.55mV/-pc has dropped 28.67%, illustrates that the membrane electrode can be used 2 weeks or more, has preferable stability and longer Service life.

The potential response that prepared GGE/SPS electrode combination various concentration l-cysteine sample has been investigated in experiment reappears Property, i.e., 1.000 × 10^-5Mol/L and 1.000 × 10^-4Potential response is measured back and forth in the l-cysteine sample solution of mol/L Value, it is each to survey 10 times.By the statistical disposition of data, the opposite of potential response value of the electrode in two various concentration solution is found Standard deviation is respectively 2.43% and 0.85%, and relative standard deviation is smaller, shows the favorable reproducibility of the SPS membrane electrode.

7, the selectivity of electrode

In the PBS buffer solution of pH=5.0, common amino acid has been investigated to GGE/SPS electrode detection l-cysteine It influences (Figure 11).Using mixed solution method, the concentration of fixed l-cysteine is 1.000 × 10^-550 times of concentration are added in mol/L Interfering component such as common amino acid substance, wherein the concentration of L-cysteine (L-Cys) is 1.000 × 10^-5mol/L.As a result table It is bright, L- glycine (L-Gly), l-Alanine (L-Ala), Valine (L-Val), L-Aspartic acid (L-Asp), L- dried meat is added Propylhomoserin (L-Pro), L-threonine (L-Thr), L-Histidine (L-His), L-Leu (L-Leu), L-Trp (L-Trp), After l-methionine (L-Met), the potential value of SPS membrane electrode response l-cysteine has almost no change (Figure 11), that is, generates Interfere very little；And L-cysteine (L-Cys) then has a certain impact.

8, the measurement of the rate of recovery and analysis are applied

In the case where optimizing experiment condition, using extended GGE/SPS electrode to the l-cysteine in practical pig anteserum sample into Row measurement.The pig anteserum sample handled well is taken (to be derived from 6 miscellaneous piggys of living body ternary, weight is 7-15Kg, by Chinese section respectively Subtropical zone Agro-ecology research institute, institute provides), 10 times are diluted with the PBS buffer solution of pH5.0, the L- Guang ammonia of known concentration is added Acid, using Standard Addition Method for Determination, measure the rate of recovery of l-cysteine in pig anteserum sample between 91.2-107.8% (see Table 3), illustrate that the accuracy of the GGE/SPS electrode is good, can be used for the rapid sensitive inspection of l-cysteine in practical pig anteserum sample It surveys, there is potential using value in the fields such as life medicine and animal-breeding.

The content and its rate of recovery of l-cysteine in 3 GGE/SPS determination of electrode pig anteserum sample of table

In short, GGE/SPS electrode of the present invention shows sensitively l-cysteine in PBS (pH=5.0) solution This spy's response of energy, linear response range is 5.00 × 10^-6—1.00×10^-3Mol/L, detection are limited to 2.69 × 10^-6mol/L。 The electrode response time is short (30 seconds), has good selectivity, reproducibility and the features such as stability.And the electrode can be answered directly For measuring the l-cysteine in practical pig anteserum sample, it is expected to the on-line testing means new as one kind of l-cysteine.

Claims (3)

1. a kind of sensor for detecting l-cysteine, the sensor includes field effect transistor, on the field effect transistor Equipped with the extended gold electrode of grid, which is characterized in that in the extended gold electrode of grid, grid part extends 0.1-500mm Distance, golden membranous layer (10) surface-assembled of gold electrode has polydithio-dipropyl sulfonate film (12).

2. sensor as described in claim 1, which is characterized in that the field effect transistor includes Si basal layer (1) and sets In the polysilicon gate (7) on Si basal layer (1)；P trap (2) and N-type substrate (3), the p are implanted on the Si basal layer (1) It is equipped with source electrode (4) and drain electrode (5) at trap (2), be implanted p trap (2) and N-type substrate (3) and constructs active electrode (4) and leakage The Si basal layer (1) of electrode (5) is equipped with silicon dioxide layer (6)；Successively plated aluminum on the basal layer of the polysilicon gate (7) Copper alloy layer (8), chromium palladium alloy layer (9) and golden membranous layer (10)；In the basal layer and silicon dioxide layer (6) of polysilicon gate (7) On be additionally provided with silicon nitride layer (11).

3. sensor as claimed in claim 2, which is characterized in that the aluminum-copper alloy layer (8) with a thickness of 20-600nm, Chromium palladium alloy layer (9) with a thickness of 20-600nm, golden membranous layer (10) with a thickness of 20-1000nm.