CN104535629A - Preparation method of enzyme biosensor for peroxide value of edible oil - Google Patents
Preparation method of enzyme biosensor for peroxide value of edible oil Download PDFInfo
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- CN104535629A CN104535629A CN201410822047.6A CN201410822047A CN104535629A CN 104535629 A CN104535629 A CN 104535629A CN 201410822047 A CN201410822047 A CN 201410822047A CN 104535629 A CN104535629 A CN 104535629A
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Abstract
The invention provides a preparation method of an enzyme biosensor for a peroxide value of edible oil, and aims at the defects in prior art. According to the preparation method, the industrial production and single use are easily achieved, the treatment and detection time of samples is shortened, and the spot, rapid and accurate detection of the samples is achieved. The preparation method comprises the following steps: (1) printing a layer of chitosan membrane on a working electrode of a detection electrode of the enzyme biosensor by virtue of a silk screen print method, printing mixed slurry containing horse radish peroxidase, and drying at 50 DEG C; (2) reprinting mixed slurry containing an enzyme substrate, a reaction accelerator, namely thiazole, a surfactant and hydroxyethyl cellulose, drying at 50 DEG C, and binding a spacing sheet; (3) mixing a to-be-tested edible oil sample with an extraction solution to form reaction liquid, and dropwise adding the reaction liquid into a sample feeding hole of the detection electrode of the enzyme biosensor; and (4) detecting current between the working electrode and a counter electrode, comparing the current with a standard curve stored in a single chip microcomputer to obtain a result, and translating the result into the concentration of the peroxide value.
Description
Technical field
The present invention relates to field of biological detection, specifically the preparation method of edible oil peroxide value enzyme biologic sensor.
Background technology
Grease, in the processes such as processing, storage, circulation, use, changes because the oxygen in light, heat and air etc. acts on the complexity often becoming sour rotten.Can oxidative rancidity under the effect of the vegetable oil that general grease is especially rich in unsaturated fatty acid oxygen in atmosphere, the intermediate product such as superoxide and hydroperoxides is generated in oxidizing process, they are easy to decompose and produce volatility and non-volatile fatty acid, aldehyde, ketone and alcohol etc., these products that become sour often have special foul smell and pained flavour, greatly affect the organoleptic properties of grease, and cause its nutritive value greatly to decline.The content of the lipid peroxide in grease represents with the milligramequivalent of active oxygen in every kilogram of grease and peroxide value, and peroxide value is the major criterion judging grease freshness and quality grade.
The detection of edible oil peroxide value is the key means guaranteeing oil quality, and domestic and international widely used assay method mainly contains high performance liquid chromatography and chemical titration.High performance liquid chromatography need use large-scale instrument, and real cost is high, is difficult to use as generaI investigation; And the reaction time of chemical titration is long, complex steps, reagent need propose above time preparation the last week, and all manual operations, is subject to the interference from human factor of operating personnel.Iodimetric titration (CB/T5538) is the international analytical approach of oil peroxidation value, simple, just can carry out in common laboratory, but iodometric determination process is subject to the impact of multiple extraneous factor, as the factor such as judgement of the pH value of solution, temperature, amount of water and shake speed, titration end-point, make the undulatory property of its measured value large, poor repeatability, so cause this method sensitivity low, there is sampling amount large simultaneously, be difficult to be adapted to modern society proposes accurately and fast, the food safety detection theory of portable, scene.
Edible oil peroxide value detects all according to the analytical approach (chemical titration) that GB/T5009.37-1996 specifies in the department that respectively prevents epidemic of current China.Domestic and international consecutive publications is detected the invention document patent of oxidation value, as Chinese patent (CN87106131.7) " test paper for determining peroxy-acetic acid and preparation method thereof " relates to the content measuring the Peracetic acid in water-soluble antiseptic liquid with test paper; Jap.P. (53-30383) " in grease, superoxide measures the preparation method of test paper " is using soluble starch, potassium iodide as principal ingredient, all has following defect: to light and oxygen sensitivity, easily lost efficacy; Reaction time was long, more than 5 minutes; Can only measure by quantitative and semi-quantitative, numerical value etc. accurately can not be measured.Have not yet to see about adopting screen printing technique industrialization to make the disposable relevant report detecting enzyme electrode for edible oil peroxide value.
Through finding the retrieval of present technical literature, Chinese Patent Application No. 03132054.6 is called " a kind of method preparing TiO 2 gel film electrochemica biological sensor ", adopt TiO 2 gel film to modify traditional electrode in this patent, then adopt electrochemical method to be detected hydrogen oxide.The process that the method prepares modified electrode is quite complicated, the setup time of electrode more than 2 hours, the modified electrode poor stability prepared, modified electrode can not be preserved for a long time, more than one month when saved, the response sensitivity of electrode decrease beyond 25%, thus can not promote the use of on a large scale.
Summary of the invention
The object of the invention is to for above-mentioned the deficiencies in the prior art, provide a kind of preparation method of enzyme biologic sensor of edible oil peroxide value, it is made to be easy to industrialization making and single use, and substantially reduce sample preparation and detection time, thus realize sample scene, detect fast, accurately.
Principle of the present invention is as follows:
Grease belongs to low conduction or non-conductive, hydrophobic material, and therefore research measures the peroxide ingredient of grease, measures organic peroxide exactly in organic phase, just needs preparation organic phase superoxide enzyme electrode.The polar chemical group of the film forming that the present invention utilizes shitosan good and good cementation power and its inside, horseradish peroxidase (HRP) is fixed on the surface of working electrode in enzyme biologic sensor detecting electrode, make the amperometric biosensor that can carry out detection fast to edible oil peroxide value, redox reaction is there is in the superoxide in edible oil and zymolyte under the catalytic action of HRP, produce electron transfer, form electric current, strength of current is directly proportional to peroxide concentrations, and sensing range is 1meq/kg ~ 60meq/kg.
Be that electrode connecting line is connected with to electrode and form another strip electrode substrate, two strip electrodes are parallel to each other substantially, electrode connecting line applies one deck polycarbonate insulator on the surface, two strip electrodes are substantially disposed on electrode base sheet, connection terminal and exposed conductive material thin film is to electrode, what be parallel to each other, spacer is coated with to electrode and working electrode two ends, spacer with to electrode, working electrode is vertical places, spacer top covers cover plate, and electrode, space between spacer and cover plate form well.The present invention relates to a kind of preparation method applying the enzyme biologic sensor of screen printing electrode detection edible oil peroxide value, comprise the steps:
Step one, the working electrode of enzyme biologic sensor detecting electrode adopt silk screen print method print one deck chitosan film, on the working electrode of electrode, the mixed serum of horseradish peroxidase, buffer solution, swelling agent, enzymatic protective reagent is printed, 50 DEG C of dryings after drying; The concentration of horseradish peroxidase is 0.5 ~ 6.5mg/mL.Use screen process press, make the thickness of enzyme membrane reach 1 ~ 4 μm.
Step 2, the battery lead plate of above-mentioned oven dry is printed again containing zymolyte 3,3,5, the mixed serum of 5-tetramethyl benzidine, reaction promoter thiazole, Surfactant OP-10, swelling agent etc., 50 DEG C of dryings, spacer in stickup, then cover cover plate, under 10-25 DEG C of environment, preserve enzyme bio-sensing detecting electrode; Described surfactant can select lauryl sodium sulfate or OP-10 or polysorbas20.Reaction promoter is the derivant of benzothiazole, and molecular structure is: R
3c
6h
mn:C (R
4) S; Wherein m=3-4; R
3=H, CH
3, C
2h
5; R
4=H, OH, SH, NH
2.Zymolyte is the derivant of biphenylamine, and molecular formula is: [R
1c
6h
mnR
2]
2; Wherein m=2-4, R
1=H, CH
3, C
6h
5, CH
3o, NH
2; R
2=H, CH
3.
Step 3, is mixed to form reactant liquor by edible oil sample to be measured and extract equal-volume, and is added drop-wise to by the reactant liquor of generation in the well of enzyme biologic sensor detecting electrode; Extraction agent is for being selected from one or more the combination in the methanol aqueous solution of 30 ~ 70% (w/w), ethanol water, isopropanol water solution and certain herbaceous plants with big flowers alcohol solution.
Step 4, testing electrode and to the electric current between electrode, if electric current is within the sensing range of current detecting instrument, then automatically process conversion by current detecting instrument, and compare with the typical curve that stores in single-chip microcomputer, be converted into the concentration of peroxide value, directly demonstrate testing result by display, if electric current is below or above the sensing range of current detecting instrument, then the display display minimum value of concentration lower than sensing range of peroxide value or the maximal value higher than sensing range; The sensing range of described current detecting instrument is 1meq/kg-60meq/kg.
Compared with prior art, there is following beneficial effect:
1, the present invention has screen printing electrode structure simply, and simple and convenient processing method, cost are low, are detected oxidation value easy to operate with the sensor that this screen printing electrode is made, sample pre-treatments is simple, and detection time is short, only needs 5 seconds, antijamming capability is strong, and detection sensitivity is high.And in enzyme electrode electrochemical methods modified electrode due to can immobilization and modify electroactive material, effectively improve its selectivity, effectively can eliminate the interference of other materials.
2, the present invention can carry out industrialization production, will have good application prospect in the field quick detection field of food peroxide value.
Embodiment
With embodiment, the present invention is further described below, but the present invention is not limited to these embodiments.
Step one, the working electrode of enzyme biologic sensor detecting electrode adopt silk screen print method print one deck chitosan film, on the working electrode of electrode, the mixed serum of horseradish peroxidase, citric acid solution, hydroxyethyl cellulose, glycocoll, sweet mellow wine is printed, 50 DEG C of dryings after drying;
Step 2, the battery lead plate of above-mentioned oven dry is printed again containing zymolyte 3,3,5, the mixed serum of 5-tetramethyl benzidine, reaction promoter thiazole, Surfactant OP-10, hydroxyethyl cellulose etc., 50 DEG C of dryings, spacer in stickup, then cover cover plate, under 10-25 DEG C of environment, preserve enzyme bio-sensing detecting electrode;
Step 3, is mixed to form reactant liquor by edible oil sample to be measured and extract equal-volume, and is added drop-wise to by the reactant liquor of generation in enzyme biologic sensor detecting electrode well;
Step 4, testing electrode and to the electric current between electrode, if electric current is within the sensing range of current detecting instrument, then automatically process conversion by current detecting instrument, and compare with the typical curve that stores in single-chip microcomputer, be converted into the concentration of peroxide value, directly demonstrate testing result by display, if electric current is below or above the sensing range of current detecting instrument, then the display display minimum value of concentration lower than sensing range of peroxide value or the maximal value higher than sensing range.The sensing range of described current detecting instrument is 1meq/kg-60meq/kg.
In the present embodiment, screen printing technique is adopted conductive carbon paste to be printed on electrode base sheet the matrix of two strip electrodes, their upper ends are exposed connection terminal, the electrode connecting line of their center section applies one deck polycarbonate insulator on the surface, and lower end is the working electrode that is parallel to each other and to electrode.Spacer and working electrode and the justify align to electrode, the hole of the sample holes of formation can not be too large, and sample holes mainly adopts capillarity to allow testing sample automatically enter space, and contact with the responding layer on working electrode and react.Sample preparation:
Sample is mixed by 1: 5 with extract, stirs, leave standstill 5 minutes, as solution to be measured.Be added drop-wise in the well of enzyme bio-sensing detecting electrode by above-mentioned solution to be measured, be detected oxidation value, lowest detectable limit reaches 1meq/kg.
The present embodiment uses enzyme biologic sensor detecting electrode to be detected the principle of work of oxidation value fast for the superoxide generation redox reaction in HRP catalytic reaction liquid on enzyme biologic sensor detecting electrode, ejected electron, electronics again with the zymolyte 3 in reactant liquor, 3, 5, 5-tetramethyl benzidine reacts, generate and pass electron complex, displacement is there is and forms electric current in electronics under the constant voltage excitation of sensor detecting instrument, electric current be converted and after amplifying after analog to digital converter, peroxide value concentration is converted the electrical signal to directly from its reading of liquid crystal display by single-chip microcomputer, also store data in storer simultaneously.
The present embodiment is reproducible, is quick on the draw, and detection time is short, only needs 5 seconds, and sample treatment is simple, and antijamming capability is comparatively strong, will have good application prospect in the field quick detection field of edible oil peroxide value.Concrete test result is in table 1:
Table 1: example 1 test result
Claims (8)
1. the preparation method of edible oil peroxide value enzyme biologic sensor, its step is as follows:
Step one, the working electrode of enzyme biologic sensor detecting electrode adopt reticulated printing method print one deck chitosan film, on the working electrode of electrode, the mixed serum of horseradish peroxidase, buffer solution, swelling agent, enzymatic protective reagent etc. is printed, 50 DEG C of dryings after drying;
Step 2, the battery lead plate of above-mentioned oven dry is printed the mixed serum containing zymolyte, reaction promoter, surfactant, swelling agent etc. again, 50 DEG C of dryings, spacer in stickup, then cover cover plate, under 10-25 DEG C of environment, preserve enzyme biologic sensor detecting electrode;
Step 3, is mixed to form liquid to be measured by edible oil sample to be measured and extract equal-volume, and is added in enzyme bio-sensing detecting electrode well by the drop to be measured generated;
Step 4, testing electrode and to the electric current between electrode, if electric current is within the sensing range of current detecting instrument, then automatically process conversion by current detecting instrument, and compare with the typical curve that stores in single-chip microcomputer, be converted into the concentration of peroxide value, directly demonstrate testing result by display, if electric current is below or above the sensing range of current detecting instrument, then the display display minimum value of concentration lower than sensing range of peroxide value or the maximal value higher than sensing range.
2. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, described step one uses screen process press, makes the thickness of enzyme membrane reach 1 ~ 4 μm.
3. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, selects the concentration of horseradish peroxidase to be 0.5 ~ 6.5mg/mL in described step one.
4. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, in described step 2, zymolyte is the derivant of biphenylamine, and molecular formula is: [R
1c
6h
mnR
2]
2; Wherein m=2-4, R
1=H, CH
3, C
6h
5, CH
30, NH
2; R
2=H, CH
3.
5. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, in described step 2, reaction promoter is the derivant of benzothiazole, and molecular structure is: R
3c
6h
mn:C (R
4) S; Wherein m=3-4; R
3=H, CH
3, C
2h
5; R
4=H, OH, SH, NH
2.
6. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, in described step 2, surfactant is lauryl sodium sulfate, OP-10, polysorbas20.
7. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, in described step 3, extraction agent is for being selected from one or more the combination in the methanol aqueous solution of 30 ~ 70% (w/w), ethanol water, isopropanol water solution and certain herbaceous plants with big flowers alcohol solution.
8. the preparation method of edible oil peroxide value enzyme biologic sensor according to claim 1, described sensing range is 1meq/kg-60meq/kg.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108196015A (en) * | 2018-02-06 | 2018-06-22 | 吉林农业大学 | A kind of food with filling automated detection method |
| CN108802342A (en) * | 2017-05-04 | 2018-11-13 | 陈锡秋 | Oil product detects and application |
| KR20200013860A (en) * | 2018-07-31 | 2020-02-10 | 광운대학교 산학협력단 | Method for Sensing rancidity of oils in situ and the Device thereof |
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| KR20200013860A (en) * | 2018-07-31 | 2020-02-10 | 광운대학교 산학협력단 | Method for Sensing rancidity of oils in situ and the Device thereof |
| KR102147571B1 (en) * | 2018-07-31 | 2020-08-24 | 광운대학교 산학협력단 | Method for Sensing rancidity of oils in situ and the Device thereof |
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