CN102297886A - Glucose electrode based on electron mediator of selena ferrocene, and preparation method thereof - Google Patents
Glucose electrode based on electron mediator of selena ferrocene, and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a glucose electrode based on an electron mediator of selena ferrocene, and a preparation method thereof, and belongs to the technical field of electroanalytical chemistry detection. The preparation method for the glucose electrode comprises that: the selena ferrocene is modified on the surface of a glassy carbon electrode; glucose oxidase and bovine serum albumin are cross linked to glutaral, then the resulting cross linked material is modified on the surface of the selena ferrocene; a drying treatment is performed for 18-24 hours at a temperature of 0-4 DEG C; a uniform composite film is formed on the surface of the electrode to prepare the modified glassy carbon electrode. According to the present invention, the selena ferrocene is mainly adopted as the electron mediator to promote the electron transfer between the electrode and the enzyme reaction layer; the prepared glucose electrode can be directly applicable for rapid detection of the glucose concentration, and has characteristics of good catalytic performance, high stability and high detection sensitivity on the glucose.
Description
Technical field
The present invention relates to a kind of is glucose electrode of crucial trim and its production and application with assorted ferrocene derivatives of selenium and glucose oxidase, belongs to Electroanalytical Chemistry detection technique field.
Background technology
Analysis tool or system that biology sensor is made up of bio-identification original paper and two parts of transducer, wherein biological identification element comprises bioactivators such as enzyme, antibody, microorganism.These active substances are fixed in the certain substrate, and contact closely with suitable converter.When measured object by diffusing in the bio-sensing rete, through molecular recognition, biochemical reaction takes place after, the information that is produced is converted into quantitative physical signalling such as light, electricity etc. by corresponding transducer, thereby realizes the fast detecting to specific substrates.Electrochemica biological sensor is with the biology sensor of electrochemical sensor as basic electrode and bioactive materials composition, is called for short bioelectrode.Electrochemica biological sensor mainly comprises enzyme electrode, microorganism electrode, immunoelectrode, tissue electrode, organelle electrode and DNA electrode etc., and wherein the development of enzyme electrode is the most representative in field of biosensors.It is that first generation biology sensor is the electro-catalysis of relaying body with oxygen that Study on Biosensor has experienced three developing stage, and second generation biology sensor is based on the electro-catalysis of artificial mediator, the direct electro-catalysis of third generation biology sensor.Utilize the artificial electron mediator with electrocatalysis that adds, transmitting electronics between enzyme and electrode is big characteristics of second generation biology sensor, and wherein the character of electron mediator is one of deciding factor of sensor performance.But, existing glucose electrode poor stability, sensitivity is not high, and it is good to need a kind of catalytic performance badly, and stability is high, the glucose electrode that the detection sensitivity of glucose is significantly improved.
Summary of the invention
The purpose of this invention is to provide a kind of glucose electrode, specifically utilize selenium to mix ferrocene, the common modified glassy carbon of glucose oxidase as glucose sensor based on the assorted ferrocene of electron mediator selenium.The present invention significantly improves the response sensitivity of sensor to glucose by the operating voltage of the good redoxomorphism reduction sensor of the assorted ferrocene of selenium.
Another object of the present invention provides the preparation method of this glucose electrode.
The objective of the invention is to realize in the following manner:
A kind of glucose electrode based on the assorted ferrocene of electron mediator selenium, this glucose electrode comprises mediator layer and enzyme reaction layer, and described mediator layer is the assorted ferrocene mediator layer of selenium, and wherein, the assorted ferrocene of mediator selenium is selected FcSeSeFc, FcSeCH for use
2SeFc and FcSe
3In a kind of, the assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator.
Mediator is for composite modified dose to prepare with the assorted ferrocene acetonitrile solution of selenium, water phase surfactant mixture and Nafion aqueous solution.Described surfactant is preferably a kind of among polyvinyl pyrrolidone, department class 60, polysorbas20 or the OP, and the quality percentage composition is 2.7%～12.5% of composite modified dose an of mediator.Nafion quality percentage composition is 1.35%～8.33% of composite modified dose an of mediator in the described Nafion aqueous solution.
Enzyme reaction layer of the present invention is prepared by glucose oxidase (glucose oxidase), bovine serum albumin(BSA) and glutaraldehyde cross-linking.Enzyme reaction layer is 1～3: 2～3 by volume ratio preferably: 1～2 glucose oxidase solution, mass percentage concentration are that 1% bovine serum albumin solution and mass percentage concentration are that 1% glutaraldehyde solution is mixed and made into.Glucose oxidase solution is a glucose oxidase solution of making 2000～2500 unit of activity/mL with the phosphate buffered solution of pH 6.0～7.0; Mass percent concentration is that 1% bovine serum albumin solution and mass percent concentration are that 1% glutaraldehyde solution is respectively with the phosphate buffered solution preparation of pH 6.0～7.0.
A kind of preparation method of the glucose electrode based on the assorted ferrocene of electron mediator selenium comprises the pre-service of electrode, and this method may further comprise the steps:
A. drip composite modified dose of the assorted ferrocene electron mediator of selenium on pretreated glass-carbon electrode surface, make it cover glass-carbon electrode fully, dry, obtain the glass-carbon electrode of the assorted ferrocene layer of surface coverage electron mediator selenium; The assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator.
B. will be modified at behind glucose oxidase and bovine serum albumin(BSA) and the glutaraldehyde cross-linking on the assorted ferrocene layer of selenium,, form the layer of even composite membrane, promptly make modified glassy carbon electrode at electrode surface at 0～4 ℃ of down dry 18～24h.
The preparation method of above-mentioned glucose electrode based on the assorted ferrocene of electron mediator selenium specifically may further comprise the steps:
A. on pretreated glass-carbon electrode, drip composite modified dose of the assorted ferrocene electron mediator of selenium, make it cover glass-carbon electrode fully, dry, obtain the glass-carbon electrode of surface coverage electron mediator layer;
The assorted ferrocene electron mediator of this selenium prepares for composite modified dose by the following method: select FcSeSeFc, FcSeCH for use
2SeFc, FcSe
3In a kind ofly be made into the assorted ferrocene acetonitrile solution of selenium with acetonitrile; With selenium assorted ferrocene acetonitrile solution, water phase surfactant mixture and Nafion aqueous solution; Wherein, the assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator;
B. glucose oxidase is dissolved in the glucose oxidase solution that is made into 2000～2500 unit of activity/mL in the phosphate buffer of pH 6.0～7.0; Being mixed with mass percent concentration respectively with the phosphate buffered solution of pH 6.0～7.0 is that 1% bovine serum albumin solution and mass percent concentration are 1% glutaraldehyde solution;
Volume ratio is 1～3: 2～3: after 1～2 glucose oxidase solution, 1% bovine serum albumin solution and 1% glutaraldehyde solution are crosslinked, dropping is on the glass-carbon electrode of overlay electronic mediator layer and it is covered fully, with electrode at 0～4 ℃ of down dry 18～24h.
Surfactant described in this method is a kind of among polyvinyl pyrrolidone, department class 60, polysorbas20, the OP, and the quality percentage composition is 2.7%～12.5% of composite modified dose an of mediator.Nafion quality percentage composition is 1.35%～8.33% of composite modified dose an of mediator in the described Nafion aqueous solution.
The pre-service of the glass-carbon electrode described in this method is to pass through following steps: with the glass-carbon electrode polishing, be the Al of 1.0 μ m, 0.3 μ m and 0.05 μ m more successively with particle diameter at first
2O
3Water slurry is polished to minute surface, is at last 40～60% HNO successively in volumetric concentration
3Ultrasonic cleaning in aqueous solution, absolute ethyl alcohol and the distilled water.
Composite modified dose of preparation method of described mediator preferably is that the assorted ferrocene acetonitrile solution of 0.1% selenium, mass percentage concentration are that 1% water phase surfactant mixture and mass concentration are that 5% Nafion aqueous solution obtains with mass concentration.
Raw material of the present invention is all commercially available to be got.
The present invention utilizes and adds the assorted ferrocene derivatives of selenium as electron mediator between electrode and enzyme reaction layer.Ferrocene is a kind of metallorganics with sandwich structure, and two folded ferric ions of pentadiene ring are positive divalence.As the redox center, repeatedly reversible one-electron oxidation reduction reaction can take place in ferrocene in most solution, is a kind of desirable redox modification group.Found through experiments, selenium has bigger electronegativity than oxygen atom, and the assorted ferrocene derivatives of selenium has lower oxidation-reduction potential than ferrocene and transmits the ability of electronics.The standard speed constant k
0Can simply be interpreted as the tolerance of oxidation-reduction pair to the dynamics complexity.One has big k
0The system of value will reach balance within a short period of time, and k
0It is very slow that the less system of value reaches balance.The AC impedance experiment can obtain Fc and FcSeSeFc, FcSeCH by experiment
2SeFc, FcSe
3Standard speed constant as mediator is respectively 4.88 * 10
-5S/cm, 9.47 * 10
-5S/cm, 7.24 * 10
-5S/cm, 6.38 * 10
-5S/cm, the assorted ferrocene derivatives of selenium has bigger standard speed constant, easier generation redox reaction.
The glucose electrode that the present invention is based on the assorted ferrocene of electron mediator selenium can be directly used in the electrochemical gaging of concentration of glucose.Using method is that assay method is as follows: as working electrode, saturated calomel electrode is as contrast electrode with the assorted ferrocene modified glassy carbon of selenium, and platinum electrode is formed three-electrode system as auxiliary electrode; During mensuration, described modified electrode is placed the phosphate buffer of the pH 7.0 that constant rate of speed stirs, on working electrode, apply certain current potential, record current-time curve, after background current reaches stable state, add the glucose solution sample with microsyringe, and the record current response.The electrode that the inventive method prepares under different concentration of glucose determination sensor to the current-responsive (see figure 1) of glucose, response time 3-5 second, and in concentration 0.02～10.0mmol/L scope, obtain the linear relationship curve (see figure 2) of electric current and concentration of glucose, utilize this linear relationship curve and corresponding linear equation, can get the concentration of glucose solution sample.
Beneficial effect of the present invention compared with the prior art: modified electrode of the present invention has used a kind of new electron mediator, its catalytic performance is good, stability is high, detection sensitivity height to glucose, obtained modified electrode is the time spent not, can preserve in phosphate buffer or in 4 ℃ of refrigerators, inner sensor still can keep 93% initial current response in one month.
Description of drawings
Fig. 1 for modified electrode of the present invention to the electric current of the glucose solution of 0.02～10.0mmol/L-time response curve map
Fig. 2 is the canonical plotting of modified electrode among the present invention to glucose responding
Fig. 3 is the structural formula of the assorted ferrocene derivatives of used selenium among the present invention
Embodiment
Below by specific embodiment the present invention is further elaborated.
Embodiment 1
Preparation process and step in the present embodiment are as follows:
(1) pre-service of glass-carbon electrode: the glass-carbon electrode that at first with diameter is 3mm polishes with abrasive paper for metallograph, is the Al of 1.0 μ m, 0.3 μ m and 0.05 μ m more successively with particle diameter
2O
3Water slurry drops on the chamois leather, to minute surface, is at last 50% HNO with the glass-carbon electrode surface finish successively with volumetric concentration
3Aqueous solution, absolute ethyl alcohol and each 2min of distilled water ultrasonic cleaning, standby;
(2) preparation FcSeSeFc electron mediator modified glassy carbon electrode: on the good glass-carbon electrode of pre-service, drip composite modified dose of 30uLFcSeSeFc electron mediator, make electron mediator cover glass-carbon electrode for composite modified dose, naturally dry, obtain the glass-carbon electrode of surface coverage electron mediator layer;
This compound method of composite modified dose is: FcSeSeFc is dissolved in the acetonitrile, be made into the assorted ferrocene acetonitrile solution of selenium of mass concentration 0.1%, the Nafion aqueous solution that in the assorted ferrocene acetonitrile solution of above-mentioned selenium, adds 1% polyvinyl pyrrolidone aqueous solution and 5%, three's volume ratio is: the 0.1% selenium ferrocene acetonitrile solution of mixing: 1% polyvinyl pyrrolidone solution: 5%Nafion solution=60: 2.5: 1, mix, get composite modified dose of electron mediator.Wherein, the assorted ferrocene quality percentage composition of selenium is 9.4% of composite modified dose an of mediator, surfactant polyvinyl pyrrolidone quality percentage composition is 3.9% of composite modified dose an of mediator, and Nafion quality percentage composition is 1.57% of composite modified dose an of mediator.
(3) preparation FcSeSeFc electron mediator and the common modified glassy carbon electrode of glucose oxidase: glucose oxidase is dissolved in the glucose oxidase solution that is made into 2000～2500 unit of activity/mL in the phosphate buffer of pH 6.0～7.0; Preparing mass percent concentration respectively with the phosphate buffered solution of pH 6.0～7.0 is that 1% bovine serum albumin solution and mass percent concentration are 1% glutaraldehyde solution.
Again with glucose oxidase solution, 1% bovine serum albumin solution and after 1% glutaraldehyde solution mixes, drip on the glass-carbon electrode of overlay electronic mediator layer and it is covered fully, dripping quantity is 30uL; Dry 24h under 4 ℃, electrode surface forms the layer of even composite membrane, promptly makes modified glassy carbon electrode.The volume ratio of used each dressing agent is: glucose oxidase solution: 1% bovine serum albumin solution: 1% glutaraldehyde solution=2: 2: 1.
Modified glassy carbon electrode is as follows to the assay method and the process of glucose solution concentration as glucose sensor:
As working electrode, saturated calomel electrode is as contrast electrode with the assorted ferrocene modified glassy carbon of selenium, and platinum electrode is formed three-electrode system as auxiliary electrode.During mensuration, described modified electrode is placed the phosphate buffer of the pH 7.0 that constant rate of speed stirs, on working electrode, apply certain current potential, record current-time curve, after background current reaches stable state, add the glucose solution sample with microsyringe, and the record current response.The electrode for preparing according to this method under different concentration of glucose determination sensor to the current-responsive (see figure 1) of glucose, response time 3-5 second.And in concentration 0.02～10.0mmol/L scope, obtain the linear relationship curve (see figure 2) of electric current and concentration of glucose, and utilize this linear relationship curve and corresponding linear equation, obtain the concentration of glucose solution sample.
Obtained modified electrode is the time spent not, preserves in phosphate buffer or in 4 ℃ of refrigerators, and inner sensor still can keep 93% initial current response in one month.
Embodiment 2
Preparation process and step in the present embodiment are as follows:
(1) pre-service of glass-carbon electrode: the glass-carbon electrode that at first with diameter is 3mm polishes with abrasive paper for metallograph, is the Al of 1.0 μ m, 0.3 μ m and 0.05 μ m more successively with particle diameter
2O
3Water slurry drops on the chamois leather, to minute surface, is at last 50% HNO with the glass-carbon electrode surface finish successively with volumetric concentration
3Aqueous solution, absolute ethyl alcohol and each 2min of distilled water ultrasonic cleaning, standby;
(2) preparation FcSeCH
2SeFc electron mediator modified glassy carbon electrode: on the good glass-carbon electrode of pre-service, drip 30uLFcSeCH
2Composite modified dose of SeFc electron mediator makes electron mediator cover glass-carbon electrode for composite modified dose, dries naturally, obtains the glass-carbon electrode of surface coverage electron mediator layer;
This compound method of composite modified dose is: with FcSeCH
2SeFc is dissolved in the acetonitrile, be made into the assorted ferrocene acetonitrile solution of selenium of mass concentration 0.1%, the Nafion aqueous solution that in the assorted ferrocene acetonitrile solution of above-mentioned selenium, adds 1% department's class's 60 aqueous solution and 5%, three's volume ratio is: the 0.1% selenium ferrocene acetonitrile solution of mixing: 1% class of department, 60 solution: 5%Nafion solution=50: 3: 1, mix, get composite modified dose of electron mediator.Wherein, the assorted ferrocene quality percentage composition of selenium is 9.25% of composite modified dose an of mediator, and class of surfactant department 60 quality percentage compositions are 5.5% of composite modified dose of mediator, and Nafion quality percentage composition is 1.88% of composite modified dose an of mediator.
(3) preparation FcSeCH
2The modified glassy carbon electrode that SeFc electron mediator and glucose oxidase are common: glucose oxidase is dissolved in the glucose oxidase solution of joining 2000～2500 unit of activity/mL in the phosphate buffer of pH 6.0～7.0; Preparing mass percent concentration respectively with the phosphate buffered solution of pH 6.0～7.0 is that 1% bovine serum albumin solution and mass percent concentration are 1% glutaraldehyde solution;
Again with glucose oxidase solution, 1% bovine serum albumin solution and after 1% glutaraldehyde solution mixes, drip on the glass-carbon electrode of overlay electronic mediator layer and it is covered fully, dripping quantity is 30uL; Dry 24h under 4 ℃ forms the layer of even composite membrane at electrode surface, promptly makes modified glassy carbon electrode.The volume ratio of used each dressing agent is: glucose oxidase solution: 1% bovine serum albumin solution: 1% glutaraldehyde solution=1: 3: 1.
Modified glassy carbon electrode is as follows to the assay method and the process of glucose solution concentration as glucose sensor:
As working electrode, saturated calomel electrode is as contrast electrode with the assorted ferrocene modified glassy carbon of selenium, and platinum electrode is formed three-electrode system as auxiliary electrode; During mensuration, described modified electrode is placed the phosphate buffer of the pH 7.0 that constant rate of speed stirs, on working electrode, apply certain current potential, record current-time curve, after background current reaches stable state, add the glucose solution sample with microsyringe, and the record current response.
Obtained modified electrode is the time spent not, preserves in phosphate buffer or in 4 ℃ of refrigerators, and inner sensor still can keep 93% initial current response in one month.
Embodiment 3
Preparation process and step in the present embodiment are as follows:
(1) pre-service of glass-carbon electrode: the glass-carbon electrode that at first with diameter is 3mm polishes with abrasive paper for metallograph, is the Al of 1.0 μ m, 0.3 μ m and 0.05 μ m more successively with particle diameter
2O
3Water slurry drops on the chamois leather, to minute surface, is at last 50% HNO with the glass-carbon electrode surface finish successively with volumetric concentration
3Aqueous solution, absolute ethyl alcohol and each 2min of redistilled water ultrasonic cleaning, standby;
(2) preparation FcSe
3Electron mediator modified glassy carbon electrode: on the good glass-carbon electrode of pre-service, drip FcSe
3Composite modified dose of electron mediator makes electron mediator cover glass-carbon electrode for composite modified dose, dries naturally, obtains the glass-carbon electrode of surface coverage electron mediator layer;
The compound method of this dressing agent is: with a certain amount of FcSe
3Be dissolved in the acetonitrile, be made into the assorted ferrocene acetonitrile solution of selenium of mass concentration 0.1%, the Nafion aqueous solution that in the assorted ferrocene acetonitrile solution of above-mentioned selenium, adds 1% polysorbas20 aqueous solution and 5%, three's volume ratio is: the selenium ferrocene acetonitrile solution of mixing: 1% polysorbas20 solution: 5%Nafion solution==55: 2: 1, mix, get composite modified dose of electron mediator.Wherein, the assorted ferrocene quality percentage composition of selenium is 9.48% of composite modified dose an of mediator, and surfactant polysorbas20 quality percentage composition is 3.4% of composite modified dose an of mediator, and Nafion quality percentage composition is 1.7% of composite modified dose an of mediator.
(3) preparation FcSe
3The modified glassy carbon electrode that electron mediator and glucose oxidase are common: glucose oxidase is dissolved in the glucose oxidase solution that is made into 2000～2500 unit of activity/mL in the phosphate buffer of pH 6.0～7.0; Being mixed with mass percent concentration respectively with the phosphate buffered solution of pH6.0～7.0 is that 1% bovine serum albumin solution and mass percent concentration are 1% glutaraldehyde solution;
Again with glucose oxidase solution, 1% bovine serum albumin solution and after 1% glutaraldehyde solution mixes, drip on the glass-carbon electrode of overlay electronic mediator layer and it is covered fully, dripping quantity is 30uL; Dry 24h under 4 ℃ forms the layer of even composite membrane at electrode surface, promptly makes modified glassy carbon electrode.The volume ratio of used each dressing agent is: glucose oxidase solution: 1% bovine serum albumin solution: 1% glutaraldehyde solution=3: 1: 2.
Modified glassy carbon electrode is as follows to the assay method and the process of glucose solution concentration as glucose sensor:
As working electrode, saturated calomel electrode is as contrast electrode with the assorted ferrocene modified glassy carbon of selenium, and platinum electrode is formed three-electrode system as auxiliary electrode; During mensuration, described modified electrode is placed the phosphate buffer of the pH 7.0 that constant rate of speed stirs, on working electrode, apply certain current potential, record current-time curve, after background current reaches stable state, add the glucose solution sample with microsyringe, and the record current response.3～5 seconds response times.
Obtained modified electrode is the time spent not, preserves in phosphate buffer or in 4 ℃ of refrigerators, and inner sensor still can keep 93% initial current response in one month.
In the above-mentioned embodiment, bovine serum albumin(BSA) (BR) is available from Fluka, USA; Nafion (5wt.%) is available from the intelligent space Energy Science Co., Ltd in Nanjing; OP is available from Shanghai Ling Feng chemical reagent company limited.
FcSe
3Can prepare by the following method: with the there-necked flask vacuum nitrogen filling gas of a 250mL; three times repeatedly; add ferrocene (3.7272g; 20mmol); vacuum nitrogen filling gas repeats twice again, then tetrahydrofuran (150mL) is injected flask under nitrogen protection, and ice bath stirs; the n-BuLi 25mL (40mmol) that under nitrogen protection, adds (1.6M in hexane) with syringe; ice bath reacted 30 minutes down, reacted 2 hours under the room temperature afterwards, and solution colour is by the orange red peony that becomes; slowly add selenium powder (3.2006g again; 40mmol), react half an hour, add selenium powder (1.5812g again; 20mmol), reaction is one hour.Afterwards, cooling is also filtered, and gets the filtrate evaporate to dryness, does moving phase with normal hexane, and the gained solid is crossed 100～200 purpose silicagel columns, gets second section product, and it is FcSe that evaporate to dryness gets orange red solid
3(2.2788g, 5.4mmol), productive rate 27%.
FcSeSeFc can prepare by the following method: same FcSe
3Course of reaction, different be silicagel column after, get the 3rd section product, evaporate to dryness gets the rufous solid, productive rate 48%.
FcSeCH
2SeFc can prepare by the following method: gets the there-necked flask of 100ml, vacuumizes, and logical nitrogen, 95% ethanol of adding 60ml adds Fc again
2Se
20.528g (1mmol), ice bath adds NaBH down
40.1513g (4mmol), room temperature reaction 2h, the dibromopropane 0.43ml of adding 1mmol, solution are by the orange red dark yellow that becomes, and reaction is spent the night under the room temperature.Be spin-dried for solvent, CH is used in washing
2Cl
2Extract three times, after the extract drying, cross 300-400 purpose silicagel column, with 3: 1 (normal hexanes: methylene chloride) do the eluent wash-out, get second section red oil, be placed as solid, promptly.
Claims (10)
1. glucose electrode based on the assorted ferrocene of electron mediator selenium, this glucose electrode comprises mediator layer and enzyme reaction layer, it is characterized in that described mediator layer is the assorted ferrocene mediator layer of selenium, wherein, the assorted ferrocene of mediator selenium is selected FcSeSeFc, FcSeCH for use
2SeFc and FcSe
3In a kind of, the assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator.
2. the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 1 is characterized in that composite modified dose of mediator is to prepare with the assorted ferrocene acetonitrile solution of selenium, water phase surfactant mixture and Nafion aqueous solution.
3. the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 2, it is characterized in that described surfactant is a kind of among polyvinyl pyrrolidone, department class 60, polysorbas20 or the OP, the quality percentage composition is 2.7%～12.5% of composite modified dose an of mediator.
4. the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 2 is characterized in that in the described Nafion aqueous solution that Nafion quality percentage composition is 1.35%～8.33% of composite modified dose an of mediator.
5. the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 1 is characterized in that described enzyme reaction layer is prepared by glucose oxidase, bovine serum albumin(BSA) and glutaraldehyde cross-linking.
6. the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 5 is characterized in that described enzyme reaction layer is 1～3: 2～3 by volume ratio: 1～2 glucose oxidase solution, mass percentage concentration are that 1% bovine serum albumin solution and mass percentage concentration are that 1% glutaraldehyde solution is mixed with and obtains; Wherein, glucose oxidase solution is a glucose oxidase solution of making 2000～2500 unit of activity/mL with the phosphate buffered solution of pH 6.0～7.0; Mass percent concentration is that 1% bovine serum albumin solution and mass percent concentration are that 1% glutaraldehyde solution is respectively with the phosphate buffered solution preparation of pH 6.0～7.0.
7. the preparation method of the described glucose electrode based on the assorted ferrocene of electron mediator selenium of a claim 1 comprises the pre-service of electrode, it is characterized in that this method may further comprise the steps:
A. drip composite modified dose of the assorted ferrocene electron mediator of selenium on pretreated glass-carbon electrode surface, make it cover glass-carbon electrode fully, dry, obtain the glass-carbon electrode of the assorted ferrocene layer of surface coverage electron mediator selenium; The assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator.
B. will be modified at behind glucose oxidase, bovine serum albumin(BSA) and the glutaraldehyde cross-linking on the assorted ferrocene layer of selenium, at 0～4 ℃ of down dry 18～24h.
8. the preparation method of the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 7 comprises the pre-service of electrode, it is characterized in that this method specifically may further comprise the steps:
A. drip composite modified dose of the assorted ferrocene electron mediator of selenium on pretreated glass-carbon electrode surface, make it cover glass-carbon electrode fully, dry, obtain the glass-carbon electrode of surface coverage electron mediator layer;
The assorted ferrocene electron mediator of this selenium prepares for composite modified dose by the following method: select FcSeSeFc, FcSeCH for use
2SeFc, FcSe
3In a kind ofly be made into the assorted ferrocene acetonitrile solution of selenium with acetonitrile; With selenium assorted ferrocene acetonitrile solution, water phase surfactant mixture and Nafion aqueous solution; Wherein, the assorted ferrocene quality percentage composition of selenium is 8%～9.6% of composite modified dose an of mediator;
B. glucose oxidase is dissolved in the glucose oxidase solution that is made into 2000～2500 unit of activity/mL in the phosphate buffer of pH 6.0～7.0; Being mixed with mass percent concentration respectively with the phosphate buffered solution of pH 6.0～7.0 is that 1% bovine serum albumin solution and mass percent concentration are 1% glutaraldehyde solution;
Volume ratio is 1～3: 2～3: after 1～2 glucose oxidase solution, 1% bovine serum albumin solution and 1% glutaraldehyde solution are crosslinked, dropping is on the glass-carbon electrode of overlay electronic mediator layer and it is covered fully, with electrode at 0～4 ℃ of down dry 18～24h.
9. the preparation method of the glucose electrode based on the assorted ferrocene of electron mediator selenium according to claim 8, it is characterized in that described surfactant is a kind of among polyvinyl pyrrolidone, department class 60, polysorbas20, the OP, the quality percentage composition is 2.7%～12.5% of composite modified dose an of mediator; Described Nafion quality percentage composition is 1.35%～8.33% of composite modified dose an of mediator.
10. according to the preparation method of claim 7 or 8 described glucose electrodes based on the assorted ferrocene of electron mediator selenium, the pre-service that it is characterized in that described glass-carbon electrode is to pass through following steps: with the glass-carbon electrode polishing, be the Al of 1.0 μ m, 0.3 μ m and 0.05 μ m more successively with particle diameter at first
2O
3Water slurry is polished to minute surface, is at last 40～60% HNO successively in volumetric concentration
3Ultrasonic cleaning in aqueous solution, absolute ethyl alcohol and the distilled water.
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| CN102914571A (en) * | 2012-09-13 | 2013-02-06 | 华南师范大学 | Glucose detection device and glucose detection method |
| CN103630593A (en) * | 2012-08-21 | 2014-03-12 | 苏州宇钿医疗器械有限公司 | Two-electrode glucolase electrode sensor |
| CN105301086A (en) * | 2015-12-05 | 2016-02-03 | 桂林理工大学 | Preparation method and application of glucose oxidase biosensor |
| CN110200637A (en) * | 2019-06-12 | 2019-09-06 | 北京怡成生物电子技术股份有限公司 | A kind of enzyme electrode and its preparation method and application based on the fixed modification of multistep |
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| CN1875114A (en) * | 2003-10-29 | 2006-12-06 | 新加坡科技研究局 | Biosensor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103630593A (en) * | 2012-08-21 | 2014-03-12 | 苏州宇钿医疗器械有限公司 | Two-electrode glucolase electrode sensor |
| CN102914571A (en) * | 2012-09-13 | 2013-02-06 | 华南师范大学 | Glucose detection device and glucose detection method |
| CN105301086A (en) * | 2015-12-05 | 2016-02-03 | 桂林理工大学 | Preparation method and application of glucose oxidase biosensor |
| CN110200637A (en) * | 2019-06-12 | 2019-09-06 | 北京怡成生物电子技术股份有限公司 | A kind of enzyme electrode and its preparation method and application based on the fixed modification of multistep |
| CN110200637B (en) * | 2019-06-12 | 2021-12-17 | 北京怡成生物电子技术股份有限公司 | Enzyme electrode based on multi-step fixed modification and preparation method and application thereof |
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| CN102297886B (en) | 2013-06-26 |
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