阅读说明：本技术 一种检测肌苷酸的酶生物传感器、其制备方法和应用 (A kind of enzyme biologic sensor, preparation method and application detecting inosinicacid ) 是由 刘源 刘静思 王广现 姜水 于 2019-09-05 设计创作，主要内容包括：本发明属于生物传感器技术领域,公开了一种检测肌苷酸的酶生物传感器、其制备方法和应用,其线性检测范围为0.313～210μg/L,检出限为0.238μg/L,由参比电极、对电极和由工作电极表面固化对肌苷酸敏感的物质识别膜得到的修饰电极组成,物质识别膜由复合溶液a、由5’-核苷酸酶溶液和黄嘌呤氧化酶溶液按体积比1：1组成的双酶复合溶液b和牛血清白蛋白溶液按体积比1:1:1组成,复合溶液a由MXene-Ti<Sub>3</Sub>C<Sub>2</Sub>Tx溶液(T选自-OH、-O或-F)、壳聚糖溶液、氯金酸溶液和氯铂酸溶液按体积比12：1.2：1：1组成。本发明酶生物传感器简单、快速、准确,灵敏度高,可用于食品中肌苷酸定量检测。(The invention belongs to biosensor technology fields, disclose a kind of enzyme biologic sensor for detecting inosinicacid, preparation method and application, its linear detection range is 0.313~210 μ g/L, detection is limited to 0.238 μ g/L, by reference electrode, to electrode and the modified electrode obtained to the material identification film of inosine acid-sensitive is solidified by working electrode surface forms, material identification film is by composite solution a, by 5'-NT solution and xanthine oxidase solution double enzyme composite solution b that 1:1 is formed by volume and bovine serum albumin solution, 1:1:1 is formed by volume, composite solution a is by MXene-Ti 3 C 2 12:1.2:1:1 is formed by volume for Tx solution (T is selected from-OH ,-O or-F), chitosan solution, chlorauric acid solution and platinum acid chloride solution.Enzyme biologic sensor of the present invention is simple, quick, accurate, and high sensitivity can be used for inosinicacid quantitative detection in food.)

1. detecting the enzyme biologic sensor of inosinicacid, which is characterized in that its linear detection range is 0.313~210 μ g/L, by joining It is formed than electrode, to electrode and modified electrode, and the modified electrode is solidified by working electrode surface to inosine acid-sensitive Material identification film obtains；Wherein:

The material identification film by composite solution a, double enzyme composite solution b and 10mg/mL bovine serum albumin solution according to body Product is than being that 1:1:1 is formed；

The composite solution a by 0.5~1.25mg/mL MXene-Ti₃C₂Tx solution, the chitosan solution of 5mg/mL, 5mmol/ 12:1.2:1:1 is formed the platinum acid chloride solution of the chlorauric acid solution of L and 3mmol/L by volume；

Double enzyme composite solution b press body by the 5'-NT solution of 2mg/mL and the xanthine oxidase solution of 2mg/mL Product is formed than 1:1；

The MXene-Ti₃C₂T is selected from-OH ,-O or-F in Tx.

2. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that

The 5'-NT solution is dissolved in pH 6.0 by 5'-NT, the PBS solution of 0.01M obtains；

The xanthine oxidase solution is dissolved in pH 6.0 by xanthine oxidase, the PBS solution of 0.01M obtains.

3. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the MXene-Ti₃C₂Tx's The preparation method comprises the following steps: 1.6gLiF to be slowly dissolved in the HCL aqueous solution of 20mL9mol/L, 5min is stirred, 1.0gTi is then added₃AlC₂ Powder, under room temperature for 24 hours with 400rpm magnetic agitation；Then 5min is centrifuged with 3500rpm, by gained sediment ultrapure water Washing；Ultrasound and washing operation 5~8 times are repeated, when measuring pH value of solution is 6, sediment is collected, is dissolved in 100mL water, In Under argon gas protection, the ultrasound 3h in 4 DEG C of ice baths makes the Ti generated₃C₂T_xThin slice delamination；It is centrifuged 1h with 8000rpm, collects supernatant Liquid, 60 DEG C of vacuum drying obtain black powder, as MXene-Ti₃C₂T_x。

4. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the working electrode is glass carbon Electrode, the reference electrode are Ag/AgCl electrode, and described is platinum electrode to electrode.

5. the enzyme biologic sensor of detection inosinicacid as described in claim 1, which is characterized in that the enzyme biologic sensor Detection is limited to 0.238 μ g/L.

6. the preparation method of any one of Claims 1 to 5 enzyme biologic sensor, which comprises the following steps:

(1) surface preparation is carried out to working electrode；

(2) the composite solution a is added drop-wise to the working electrode surface after step (1) surface preparation, room temperature is dried；

(3) double enzyme composite solution b are added drop-wise to step (2) treated electrode surface, room temperature is dried；

(4) bovine serum albumin solution is added drop-wise to step (3) treated electrode surface, room temperature is dried, and electricity must be modified Pole；

(5) by modified electrode obtained by step (4) and the reference electrode and it is described to electrode composition three-electrode system to get；Its In: the dripping quantity volume ratio of the composite solution a, double enzyme composite solution b and bovine serum albumin solution are 1:1:1.

7. the preparation method of enzyme biologic sensor as claimed in claim 6, which is characterized in that in step (1), the working electrode The step of surface preparation are as follows: working electrode successively uses to 0.3 μm and 0.05 μm of alumina powder polishes on polishing cloth At mirror surface, then with ultrapure water, it is then ultrasonically treated 1min in ultrapure water, is subsequently placed in potassium ferricyanide solution at activation Reason is taken out, with ultrapure water, is dried with nitrogen；Wherein, the potassium ferricyanide solution is by K₃[Fe(CN)₆]、K₄[Fe(CN)₆] It is in molar ratio the mixed solution of 1:1:100 composition with KCl.

8. application of any one of Claims 1 to 5 biosensor in inosinicacid quantitative detection, which is characterized in that institute The linear detection range for stating inosinicacid is 0.313~210 μ g/L.

9. application as claimed in claim 8, which is characterized in that the detection of the inosinicacid is limited to 0.238 μ g/L.

Technical field

The present invention relates to biosensor technology field more particularly to it is a kind of detect inosinicacid enzyme biologic sensor, its Preparation method and application, the detection for inosinicacid (IMP) in food.

Background technique

Inosinicacid (IMP) is a kind of mononucleotide, is the Metabolic Intermediate of ATP in organism.Inosinicacid and its esters energy Delicate flavour is enough generated, is one of flavor substance important in meat, it can also act synergistically with sodium glutamate, make delicate flavour at multiplication Add, is a kind of common flavoring agent.In addition to this, inosinicacid is also the internationally recognized important finger for measuring meat freshness Mark.Currently, the method for quantitative detection inosinicacid includes spectrophotometry, high performance liquid chromatography etc., but these methods there is The problems such as testing cost is high, consuming time is long, complicated for operation, in contrast, electrochemical enzymatic biosensor is with higher quick Perception and selectivity, can effectively amplified signal, and amount of samples is less, and response is rapidly, easy to operate, is more applicable for eating The detection of inosinicacid in product.

Committed step in enzyme electrode preparation process is enzyme immobilizatio, and in recent decades, researcher is always continuous Find suitable zymophore material and process for fixation.MXene-Ti₃C₂Tx (T=OH, O or F) material is a kind of magnesium-yttrium-transition metal Carbide, the two-dimensional structure with class graphene, specific surface area is high, conductivity is high, and stability, good biocompatibility can promote Into the electronics transfer between enzyme active center and electrode interface, there is good application prospect in terms of biosensor zymophore, Nanometer Au, Pt particle can reduce the overpotential of enzymolysis product hydrogen peroxide, so that the detection performance of enzyme biologic sensor is improved, Chitosan has good filming, is commonly used for enzyme immobilizatio.

Summary of the invention

In order to overcome the problems, such as that the method testing cost of existing detection inosinicacid is high, consuming time is long, complicated for operation, this hair Bright primary and foremost purpose is to provide a kind of enzyme bio-sensing of detection inosinicacid with good selectivity, sensitivity and stability Device.

Another object of the present invention is to provide the preparation methods of above-mentioned enzyme biologic sensor, pass through suitable zymophore material Material and process for fixation obtain the stable enzyme biologic sensor of performance.

A further object of the present invention is to provide application of the above-mentioned enzyme biologic sensor in inosinicacid quantitative detection.

Above-mentioned purpose of the invention is realized by following technical method:

In a first aspect, the enzyme biologic sensor of detection inosinicacid of the invention, by reference electrode, to electrode and modified electrode Composition, the modified electrode is solidified by working electrode surface obtains the material identification film of inosine acid-sensitive；Wherein:

The material identification film is pressed by the bovine serum albumin solution of composite solution a, double enzyme composite solution b and 10mg/mL It is 1:1:1 composition according to volume ratio；Wherein, the composite solution a by 0.5~1.25mg/mL MXene-Ti₃C₂Tx solution, 5mg/ The platinum acid chloride solution of the chitosan solution of mL, the chlorauric acid solution of 5mmol/L and 3mmol/L 12:1.2:1:1 group by volume At double enzyme composite solution b press volume by the 5'-NT solution of 2mg/mL and the xanthine oxidase solution of 2mg/mL It is formed than 1:1；

The MXene-Ti₃C₂T is selected from-OH ,-O or-F in Tx；

The 5'-NT solution is dissolved in pH 6.0 by 5'-NT, the PBS solution of 0.01M obtains, the Huang Purine oxidase solution is dissolved in pH 6.0 by xanthine oxidase, the PBS solution of 0.01M obtains.

Preferably, the MXene-Ti₃C₂Tx's the preparation method comprises the following steps: 1.6gLiF to be slowly dissolved in the HCl of 20mL9mol/L Aqueous solution stirs 5min, 1.0gTi is then added₃AlC₂Powder, under room temperature for 24 hours with 400rpm magnetic agitation；Then with 3500rpm is centrifuged 5min, by gained sediment milli-Q water；Ultrasound and washing operation 5~8 times are repeated, when measuring solution When pH is 6, sediment is collected, is dissolved in 100mL water, under protection of argon gas, the ultrasound 3h in 4 DEG C of ice baths makes gained Ti₃C₂T_x Thin slice delamination；1h is finally centrifuged with 8000rpm, collects supernatant, 60 DEG C of vacuum drying obtain black powder, as MXene- Ti₃C₂T_x。

Preferably, the working electrode is glass-carbon electrode, and the reference electrode is Ag/AgCl electrode, described to be to electrode Platinum electrode.

Preferably, the linear detection range of the enzyme biologic sensor is 0.313~210 μ g/L.

Preferably, the detection of the enzyme biologic sensor is limited to 0.238 μ g/L.

Second aspect, the preparation method of the enzyme biologic sensor, comprising the following steps:

(1) surface preparation is carried out to working electrode；

(2) by MXene-Ti₃C₂Tx solution, chitosan solution, chlorauric acid solution, platinum acid chloride solution are uniformly mixed according to a ratio, Composite solution a is obtained, and is added dropwise to the working electrode surface after step (1) surface preparation, room temperature is dried；

(3) 5'-NT solution and xanthine oxidase solution are uniformly mixed according to a ratio, obtain double enzyme composite solution b, And it is added dropwise to step (2) treated electrode surface, room temperature is dried；

(4) bovine serum albumin solution is added drop-wise to step (3) treated electrode surface, room temperature is dried, and electricity must be modified Pole；

(5) by modified electrode obtained by step (4) and the reference electrode and it is described three-electrode system is formed to electrode, i.e., ；

Wherein, the dripping quantity volume ratio of the composite solution a, double enzyme composite solution b and bovine serum albumin solution are 1: 1:1。

Preferably, in step (1), the pretreated step of working electrode surface are as follows: working electrode is successively used 0.3 μm and 0.05 μm of alumina powder be polished to mirror surface on polishing cloth, it is then ultrasonic in ultrapure water then with ultrapure water 1min is handled, is subsequently placed in potassium ferricyanide solution and is activated, takes out, with ultrapure water, is dried with nitrogen；The iron Potassium cyanide solution is by K₃[Fe(CN)₆]、K₄[Fe(CN)₆] and KCl be in molar ratio 1:1:100 composition mixed solution.

The third aspect, application of the above-mentioned biosensor in inosinicacid quantitative detection, linear detection range be 0.313~ 210 μ g/L, detection are limited to 0.238 μ g/L.

The present invention selects New Two Dimensional nano material MXene-Ti₃C₂Tx (T=-OH ,-O or-F), in conjunction with nanometer Au, Pt grain Son and using the film forming of chitosan and inclusion, constructs the zymophore of electrochemical enzymatic biosensor, increases enzyme catalyst and exist The fixed amount and stability of electrode surface, facilitate the catalysis to substrate.It is added dropwise by 5'-NT and xanthine oxidase group At double enzyme composite solutions after use bovine serum albumin solution close film forming, obtain modified electrode, then cooperate reference electrode and right Electrode forms three-electrode system, and the enzyme biologic sensor for detecting inosinicacid is made, and linear detection range is 0.313~210 μ G/L, detection are limited to 0.238 μ g/L.

Compared with prior art, the beneficial effects of the present invention are:

(1) biosensor of the invention has good electron transmission, and the electronics that can generate reaction is in enzymatic activity Good transfer is carried out between center and electrode surface, improves the reaction speed of biosensor.

(2) biosensor of the invention has good selectivity, and can accurately be detected to inosinicacid, anti-interference energy Power is strong, responds to the chaff interferents no current such as cysteine, methionine, inosine diphosphate and inosine triphosphate.

(3) biosensor of the invention is with good stability and reproducibility, is continuously measured by square wave voltammetry It 10 times, as a result only shows 3.4% relative standard deviation, remains to reach original response electric current after storing 2 weeks at 4 DEG C 95%.

(4) biosensor of the invention can be used for the detection of inosinicacid in food, and preparation process is simple, safety, can be It is detected under room temperature environment testing conditions, there is wider detection range, lower detection limits, and application prospect is good.

Detailed description of the invention

Fig. 1 is the overall structure diagram of enzyme biologic sensor in the present invention.

Fig. 2 is the preparation flow figure of enzyme biologic sensor working electrode in the present invention.

Fig. 3 is circulation volt of the enzyme biologic sensor working electrode in PBS solution (0.01M, pH=6.0) in embodiment 2 Pacify curve graph；Wherein, a indicates that sweep speed is 160mV/s, and b indicates that sweep speed is 120mV/s, and c indicates that sweep speed is 80mV/s, d indicate that sweep speed is 40mV/s.

Fig. 4 be in embodiment 3 enzyme biologic sensor there are 3 μm of ol/L inosinicacids and there is no the PBS solutions of inosinicacid Cyclic voltammetry curve figure in (0.01M, pH=6.0)；Wherein, a indicates that there are the inosines that concentration is 3 μm of ol/L in PBS solution Acid, b indicate that inosinicacid is not present in PBS solution.

Fig. 5 be in embodiment 3 enzyme biologic sensor there are 3 μm of ol/L inosinicacids and there is no the PBS solutions of inosinicacid In (0.01M, pH=6.0) differential pulse voltammetry curve graph；Wherein, a indicates that inosinicacid is not present in PBS solution, and b is indicated There are the inosinicacids of 3 μm of ol/L in PBS solution.

Fig. 6 be in embodiment 4 enzyme biologic sensor in PBS solution with 100mV/s sweep speed, -0.15V potential condition Under, to the current-vs-time response curve of various concentration inosinicacid.

Fig. 7 be in embodiment 4 enzyme biologic sensor to the standard curve of the response current of various concentration inosinicacid.

Fig. 8 is that enzyme biologic sensor rings the current-vs-time of disturbance object and inosinicacid in PBS solution in embodiment 5 Answer curve graph.

Specific embodiment

Below with reference to example and attached drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to This.

MXene-Ti of the embodiment of the present invention₃C₂Tx material can be commercial product or is prepared using following methods: will 1.6gLiF is slowly dissolved in the HCL aqueous solution of 20mL9mol/L, stirs 5min, 1.0gTi is then added₃AlC₂Powder, room temperature condition Under with 400rpm magnetic agitation for 24 hours；Then 5min is centrifuged with 3500rpm, by gained sediment milli-Q water；Repeat ultrasound And washing operation 5~8 times, when measuring pH value of solution is 6, sediment is collected, is dissolved in 100mL water, under protection of argon gas, in 4 Ultrasound 3h in DEG C ice bath makes gained Ti₃C₂T_xThin slice delamination is finally centrifuged 1h with 8000rpm, collects supernatant, and 60 DEG C of vacuum are dry It is dry to obtain black powder, as MXene-Ti₃C₂T_x。