CN102636540A - Glucose detection sensor, and preparation and application methods thereof - Google Patents
Glucose detection sensor, and preparation and application methods thereof Download PDFInfo
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Abstract
The invention discloses a glucose detection sensor, and preparation and application methods thereof. The sensor comprises a three-electrode system, wherein a graphene/chitosan/glucose oxidase deposition modified glassy carbon electrode is used as a working electrode, a platinum sheet is used as a counter electrode, and a calomel electrode is used as a reference electrode. In the detection process, by using the graphene/chitosan/glucose oxidase deposition modified glassy carbon electrode as the working electrode, the platinum sheet as the counter electrode and the calomel electrode as the reference electrode, an electrochemical work station is utilized to determine the glucose content. Compared with the traditional method, the invention has the advantages of higher sensitivity and higher specificity, and the low detection limit is 4.0*10<-7> mol/L; and meanwhile, the glucose sensor has the advantages of simple preparation process, stable performance and short sample detection time, can be recycled, and is convenient to operate.
Description
Technical field:
The present invention relates to a kind of easy, quick, specific glucose detection sensor and preparation and method of application.
Background technology:
Diabetes are a kind of worldwide pandemics, and it is high especially that a middle-aged person more than 40 years old suffers from the rate of dying, and in Japan, the diabetic accounts for nearly 10% in the population more than 40 years old.The reason that forms diabetes is can not produce enough insulin or cell in the health not respond insulin and make human body produce very high blood sugar, and its complication is more, comprises angiocardiopathy, renal failure, blind etc.Therefore, realization is significant for treatment and control of diabetes to easy, quick, the specific detection of concentration of glucose in the blood of human body.Wherein, has the research part that highly sensitive glucose detection material and method are blood sugar test.Be applied to detect the main zymetology colourimetry of method and the electrochemical method of glucose at present; The electrochemical analysis method that direct electron shifts between glucose oxidase and electrode surface recently more and more causes people's attention; But there are two in this method problem to be solved is arranged: at first, because the existence of protein insulation crust makes the electron transfer of enzyme become difficult; Secondly, directly be adsorbed on the easy sex change of enzyme and the inactivation of electrode surface, more than all be unfavorable for realizing to easy, quick, the specific detection of glucose.The present invention is based on the glucose detection sensor of step electrodeposition process preparation, use electrochemical methods to realize easy, quick, the specific detection of glucose.
Summary of the invention:
The purpose of this invention is to provide a kind of glucose detection sensor of easy, quick, environmental protection and the method and the method for application of one of which step prepared by electrodeposition.
A kind of glucose detection sensor comprises: Graphene, shitosan and glucose oxidase deposition modified glassy carbon are working electrode, and platinized platinum is to electrode, and mercurous chloride electrode is the three-electrode system of contrast electrode.
The preparation method of described Graphene, shitosan and glucose oxidase deposition modified glassy carbon is following:
5mg～20mg graphite oxide joins in the chitosan solution of 5mL～20mL 0.2wt%, and sonic oscillation 3h～4h obtains graphene oxide-chitosan solution;
Under magnetic agitation speed 200rpm～500rpm condition, 5mL～10mL 4mg/mL～10mg/mL glucose oxidase solution and graphene oxide-chitosan solution mix, and form graphene oxide-shitosan-glucose oxidase solution;
Graphene oxide-gather chitose-glucose oxidase solution is being working electrode with the glass-carbon electrode; Platinized platinum is to electrode; Mercurous chloride electrode is under the three-electrode system of contrast electrode, carries out the potentiostatic method electro-deposition, obtains Graphene, shitosan and glucose oxidase deposition modified glassy carbon.
Described potentiostatic method electrodeposition condition is: sedimentation potential-0.5V～-1.5V, sedimentation time 300s～500s.
Described glass-carbon electrode is the process pre-service before carrying out the potentiostatic method electro-deposition:
Glass-carbon electrode before the electro-deposition in advance on chamois leather the pasty liquid with 0.3 μ m and 0.05 μ m alumina powder be polished to " minute surface ", respectively clean 5min～10min with ethanol, ultrapure water ultrasound wave respectively after rinsing well with deionized water.
With pretreated glass-carbon electrode through carrying out the potentiostatic method electro-deposition after the following process again: the pH value is 6～9, concentration is in the phosphate buffer of 0.02mol/L～0.10mol/L; In-0.6V～+ the 0.6V scanning voltage between; With 0.05V/s～0.2V/s sweep velocity scanning 2～8 circles, the potential difference (PD) at a pair of redox peak is at 60mV～90mV on the cyclic voltammetry curve that obtains;
The potentiostatic method electro-deposition is carried out under magnetic agitation speed 200rpm～500rpm condition.
The preparation method of above-mentioned glucose detection sensor:
Graphene, shitosan and grape carbohydrate oxidase are deposited modified glassy carbon as working electrode; The platinized platinum conduct is to electrode; Mercurous chloride electrode constitutes three-electrode system as contrast electrode, and the preparation method of described Graphene, shitosan and grape carbohydrate oxidase deposition modified glassy carbon is following:
(1) 5mg～20mg graphite oxide joins in the chitosan solution of 5mL～20mL 0.2wt%, and sonic oscillation 3h～4h obtains graphene oxide-chitosan solution;
Under magnetic agitation speed 200rpm～500rpm condition, 5mL～10mL 4mg/mL～10mg/mL glucose oxidase solution and graphene oxide-chitosan solution mix, and form graphene oxide-shitosan-glucose oxidase solution;
(2) glass-carbon electrode before the electro-deposition in advance on chamois leather the pasty liquid with 0.3 μ m and 0.05 μ m alumina powder be polished to " minute surface ", respectively clean 5min～10min with ethanol, ultrapure water ultrasound wave respectively after rinsing well with deionized water;
Then with glass-carbon electrode through carrying out the potentiostatic method electro-deposition after the following process again: the pH value is 6～9, concentration is in the phosphate buffer of 0.02mol/L～0.10mol/L; In-0.6V～+ the 0.6V scanning voltage between; With 0.05V/s～0.2V/s sweep velocity scanning 2～8 circles, the potential difference (PD) at a pair of redox peak is at 60mV～90mV on the cyclic voltammetry curve that obtains;
(3) under magnetic agitation speed 200rpm～500rpm condition, graphene oxide-shitosan-glucose oxidase solution that step (1) obtains; Handling the glass-carbon electrode that obtains with step (2) is working electrode; Platinized platinum is to electrode, and mercurous chloride electrode is in the three-electrode system of contrast electrode, uses electrochemical workstation to carry out the potentiostatic method electro-deposition; Mode of deposition is: sedimentation potential-0.5V～-1.5V, sedimentation time 300s～500s; Graphene, shitosan and glucose oxidase on the glass-carbon electrode surface, are obtained Graphene, shitosan and glucose oxidase deposition modified glassy carbon by a step electro-deposition.
The method of application of above-mentioned glucose detection sensor:
Prepare the glucose sample solution with phosphate buffer; After this, be working electrode with Graphene, shitosan and glucose oxidase deposition modified glassy carbon again, platinized platinum is to electrode, mercurous chloride electrode is under the three-electrode system of contrast electrode, uses electrochemical workstation; Adopt electric current-time response working method, the glucose of various criterion concentration is detected, the production standard working curve, thus realize determination of glucose.
Specifically be that the phosphate buffer that 4mL～20mL, pH value are 5～8, concentration is 0.03mol/L～0.20mol/L is mixed with the glucose of 4mmol～20mmol in the above-mentioned method of application, preparation concentration is the glucose sample solution of 1mol/L; Get the glucose sample solution of 0.8 μ L～above-mentioned 1mol/L for preparing of 1.2 μ L then at every turn, measure in the phosphate buffer that join 4mL～6mL, pH value continuously and be 5～8, concentration is 0.03mol/L～0.20mol/L; During detection, under magnetic agitation speed 150rpm～400rpm condition, carry out.Testing conditions is: the utilization voltage range is 0.2V～0.7V.
The principle of the invention is following:
Why redox reaction can take place on the glass-carbon electrode surface in glucose oxidase, is because it contains activated centre-flavin adenine dinucleotide (FAD) (FAD).Under certain voltage, it is the glucose lactone with glucose oxidase that FAD can obtain two electronics, and self is reduced to FADH
2The FADH of ortho states also
2Can lose two electronics again, again by O
2Molecular oxidation is FAD.At last, through H to producing in the reaction
2O
2Carry out Electrochemical Detection, thereby realize the specific quantitative test of glucose (its mechanism is as shown in Figure 2).
The phosphate buffer that the pH value is 6～9, concentration is 0.02mol/L～0.10mol/L is as supporting electrolyte.Electric current-time curve under magnetic agitation speed 150rpm～400rpm, voltage in+0.2V～+ 1.0V detects the response current of potential measurement modified electrode to glucose solution down.Fig. 3: A adds the current-responsive figure of 0.2mM glucose on Graphene-shitosan-glucose oxidase modified glassy carbon continuously; B, the glucose that adds variable concentrations can cause that current value changes on the collection of illustrative plates.Interfering ion detects glucose influence little (as shown in table 1) to this method.
The possible chaff interference of table 1 is to the influence of glucose detection
| Chaff interference | Current ratio |
| Ascorbic acid | 1.01 |
| Uric acid | 0.84 |
| Citric acid | 0.97 |
| Paracetamol | 1.07 |
Graphene-shitosan-glucose oxidase the Stability Analysis of Structures of synthesizing among the present invention, the detection signal favorable reproducibility, chemical property is outstanding; The immobilization that is configured to glucose oxidase of Graphene-shitosan provides the bio-compatible microenvironment of an excellence, makes glucose oxidase keep original activity and selectivity preferably.
Through the present invention, we have realized concentration of glucose specificity, fast detecting in the serum appearance.Compare with classic method, have more high sensitivity and specificity, detectability is low to moderate 4.0 * 10
-7Mol/L; Simultaneously, simple, the stable performance of glucose sensor preparation and can reuse, the sample detection time is short, easy to operate.To sum up, explain that the inventive method is a kind of new method of easy, quick, specific detection glucose.
Description of drawings
Fig. 1: Graphene-shitosan electron microscope picture;
A, Graphene-shitosan transmission electron microscope; B, Graphene-shitosan scanning electron microscope;
Fig. 2: the schematic diagram that the present invention detects;
Fig. 3: A adds the current-responsive figure of 0.2mM glucose on Graphene-shitosan-glucose oxidase modified glassy carbon continuously; B, the glucose that adds variable concentrations can cause that current value changes on the collection of illustrative plates.
Embodiment
Further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
We have realized Hospital of Hunan University is provided easy, quick, the specific detection of concentration of glucose in the blood serum sample to utilize the present invention.
Prepare glucose detection sensor of the present invention:
(1) the 10mg graphite oxide joins in the chitosan solution of 10mL 0.2wt%, and sonic oscillation 3.5h obtains graphene oxide-chitosan solution;
Under the magnetic agitation speed 300rpm condition, 6mL 5mg/mL glucose oxidase solution and graphene oxide-chitosan solution mix, and form graphene oxide-shitosan-glucose oxidase solution;
(2) glass-carbon electrode before the electro-deposition in advance on chamois leather the pasty liquid with 0.3 μ m and 0.05 μ m alumina powder be polished to " minute surface ", respectively clean 10min with ethanol, ultrapure water ultrasound wave respectively after rinsing well with deionized water; Then with glass-carbon electrode through carrying out the potentiostatic method electro-deposition after the following process again: the pH value is 7, concentration is in the phosphate buffer of 0.05mol/L; In-0.6V～+ the 0.6V scanning voltage between; With 0.05V/s sweep velocity scanning 6 circles, the potential difference (PD) at a pair of redox peak is 80mV on the cyclic voltammetry curve that obtains;
(3) under magnetic agitation speed 300rpm condition, graphene oxide-shitosan-glucose oxidase solution that step (1) obtains; Handling the glass-carbon electrode that obtains with step (2) is working electrode; Platinized platinum is to electrode, and mercurous chloride electrode is in the three-electrode system of contrast electrode, uses electrochemical workstation to carry out the potentiostatic method electro-deposition; Mode of deposition is: sedimentation potential-1.0V, sedimentation time 400s; Graphene, shitosan and glucose oxidase on the glass-carbon electrode surface, are obtained Graphene, shitosan and glucose oxidase deposition modified glassy carbon by a step electro-deposition.
When detecting the glucose content in the serum
The phosphate buffer that 10mL, pH value are 7, concentration is 0.1mol/L is mixed with the glucose of 10mmol, and preparation concentration is the glucose sample solution of 1mol/L; Get the glucose sample solution of the 1mol/L that 1 μ L prepares at every turn, join 5mL, pH value continuously and be 7, concentration is in the phosphate buffer of 0.1mol/L; With Graphene, shitosan and glucose oxidase deposition modified glassy carbon is working electrode, and platinized platinum is to electrode, and mercurous chloride electrode is under the three-electrode system of contrast electrode, uses electrochemical workstation; Adopt electric current-time response working method, detect through the glucose sample to various criterion concentration, the production standard working curve uses that the pH value is 7, concentration is diluted 100 times as the phosphate buffer of 0.05mol/L with the serum testing sample; Thereby realize mensuration to concentration of glucose in the blood serum sample; Testing conditions is: magnetic agitation speed is 300rpm, and utilization voltage is 0.5V.Concentration of glucose is evaluated as 4.8 * 10 in the blood serum sample
-3Mol/L.The result 4.6 * 10 that its result and inductivity coupled plasma mass spectrometry are measured
-3Mol/L does not have significant difference.
When said method detects, can also use that the pH value is 7, concentration as the phosphate buffer of 0.05mol/L with 100 times of normal human serum diluted samples; Get the blood serum sample solution of 1 μ L dilution at every turn, prepare the blood serum sample of various criterion concentration in the phosphate buffer that join 5mL, pH value continuously and be 7, concentration is 0.05mol/L; With above-mentioned identical operations production standard working curve and detection serum testing sample; Its concentration of glucose is evaluated as 4.8 * 10
-3Mol/L.The result 4.6 * 10 that its result also measures with inductivity coupled plasma mass spectrometry
-3Mol/L does not have significant difference.
Claims (10)
1. a glucose detection sensor is characterized in that, comprising: Graphene, shitosan and glucose oxidase deposition modified glassy carbon are working electrode, and platinized platinum is to electrode, and mercurous chloride electrode is the three-electrode system of contrast electrode.
2. glucose detection sensor according to claim 1 is characterized in that, the preparation method of described Graphene, shitosan and glucose oxidase deposition modified glassy carbon is following:
5mg～20mg graphite oxide joins in the chitosan solution of 5mL～20mL 0.2wt%, and sonic oscillation 3h～4h obtains graphene oxide-chitosan solution;
Under magnetic agitation speed 200rpm～500rpm condition, 5mL～10mL 4mg/mL～10mg/mL glucose oxidase solution and graphene oxide-chitosan solution mix, and form graphene oxide-shitosan-glucose oxidase solution;
Graphene oxide-gather chitose-glucose oxidase solution is being working electrode with the glass-carbon electrode; Platinized platinum is to electrode; Mercurous chloride electrode is under the three-electrode system of contrast electrode, carries out the potentiostatic method electro-deposition, obtains Graphene, shitosan and glucose oxidase deposition modified glassy carbon.
3. glucose detection sensor according to claim 2 is characterized in that, the potentiostatic method electrodeposition condition is: sedimentation potential-0.5V～-1.5V, sedimentation time 300s～500s.
4. glucose detection sensor according to claim 2 is characterized in that, glass-carbon electrode is the process pre-service before carrying out the potentiostatic method electro-deposition:
Glass-carbon electrode before the electro-deposition in advance on chamois leather the pasty liquid with 0.3 μ m and 0.05 μ m alumina powder be polished to " minute surface ", respectively clean 5min～10min with ethanol, ultrapure water ultrasound wave respectively after rinsing well with deionized water.
5. glucose detection sensor according to claim 4; It is characterized in that; With pretreated glass-carbon electrode through carrying out the potentiostatic method electro-deposition after the following process again: the pH value is 6～9, concentration is in the phosphate buffer of 0.02mol/L～0.10mol/L; In-0.6V～+ the 0.6V scanning voltage between, with 0.05V/s～02V/s sweep velocity scanning 2～8 circles, the potential difference (PD) at a pair of redox peak is at 60mV～90mV on the cyclic voltammetry curve that obtains;
The potentiostatic method electro-deposition is carried out under magnetic agitation speed 200rpm～500rpm condition.
6. the preparation method of each described glucose detection sensor of claim 1-5 is characterized in that,
Graphene, shitosan and grape carbohydrate oxidase are deposited modified glassy carbon as working electrode; The platinized platinum conduct is to electrode; Mercurous chloride electrode constitutes three-electrode system as contrast electrode, and the preparation method of described Graphene, shitosan and grape carbohydrate oxidase deposition modified glassy carbon is following:
(1) 5mg～20mg graphite oxide joins in the chitosan solution of 5mL～20mL 0.2wt%, and sonic oscillation 3h～4h obtains graphene oxide-chitosan solution;
Under magnetic agitation speed 200rpm～500rpm condition, 5mL～10mL 4mg/mL～10mg/mL glucose oxidase solution and graphene oxide-chitosan solution mix, and form graphene oxide-shitosan-glucose oxidase solution;
(2) glass-carbon electrode before the electro-deposition in advance on chamois leather the pasty liquid with 0.3 μ m and 0.05 μ m alumina powder be polished to " minute surface ", respectively clean 5min～10min with ethanol, ultrapure water ultrasound wave respectively after rinsing well with deionized water; Then with glass-carbon electrode through carrying out the potentiostatic method electro-deposition after the following process again: the pH value is 6～9, concentration is in the phosphate buffer of 0.02mol/L～0.10mol/L; In-0.6V～+ the 0.6V scanning voltage between; With 0.05V/s～02V/s sweep velocity scanning 2～8 circles, the potential difference (PD) at a pair of redox peak is at 60mV～90mV on the cyclic voltammetry curve that obtains;
(3) under magnetic agitation speed 200rpm～500rpm condition, graphene oxide-shitosan-glucose oxidase solution that step (1) obtains; Handling the glass-carbon electrode that obtains with step (2) is working electrode; Platinized platinum is to electrode, and mercurous chloride electrode is in the three-electrode system of contrast electrode, uses electrochemical workstation to carry out the potentiostatic method electro-deposition; Mode of deposition is: sedimentation potential-0.5V～-1.5V, sedimentation time 300s～500s; Graphene, shitosan and glucose oxidase on the glass-carbon electrode surface, are obtained Graphene, shitosan and glucose oxidase deposition modified glassy carbon by a step electro-deposition.
7. the method for application of each described glucose detection sensor of claim 1-5 is characterized in that,
Prepare the glucose sample solution with phosphate buffer; After this, be working electrode with Graphene, shitosan and glucose oxidase deposition modified glassy carbon again, platinized platinum is to electrode, mercurous chloride electrode is under the three-electrode system of contrast electrode, uses electrochemical workstation; Adopt electric current-time response working method, the glucose of various criterion concentration is detected, the production standard working curve, thus realize determination of glucose.
8. method of application according to claim 7 is characterized in that,
The phosphate buffer that 4mL～20mL, pH value are 5～8, concentration is 0.03mol/L～0.20mol/L is mixed with the glucose of 4mmol～20mmol, and preparation concentration is the glucose sample solution of 1mol/L;
Get the glucose sample solution of 0.8 μ L～above-mentioned 1mol/L for preparing of 1.2 μ L at every turn, measure in the phosphate buffer that join 4mL～6mL, pH value continuously and be 5～8, concentration is 0.03mol/L～0.20mol/L.
9. method of application according to claim 7 is characterized in that,
During detection, under magnetic agitation speed 150rpm～400rpm condition, carry out.
10. method of application according to claim 7 is characterized in that,
Testing conditions is: the utilization voltage range is 0.2V～0.7V.
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