Summary of the invention
The objective of the invention is to solve the weak point that above-mentioned prior art exists, provide a kind of and have real-time detection, the response time is short, highly sensitive, linear response range is big, antijamming capability is strong, can be recycled and the biological nano material sensors of low production cost and preparation method thereof, method of application.
The present invention realizes through following technical scheme:
A kind of method for making of biological nano material sensors, its special character is may further comprise the steps:
1, CNT is fixed
CNT is dissolved in the Nafion solution that concentration is 0.5-10%; The CNT that contains the 0.5-10 milligram in every milliliter of Nafion solution; This mixed liquor of ultrasonic dispersion 1-3 hour; Obtain the black even dispersion liquid, get dispersion liquid and evenly be covered in the electrode tip surface that platinum electrode or platinum alloy electrode or platinum black plating obtain, normal temperature leaves standstill to drying;
Electrode is divided into top (part to be finished) and tail end, and electrode is connected on the various device through tail end, and the top is a probe segment;
Said Nafion solution is teflon and perfluor-3, the multipolymer of 6-diepoxy-4-methyl-7-decene-sulfuric acid;
Said platinum alloy electrode is platinum/iridium alloy electrode or other platinum alloy electrodes;
It is following that said platinum black is electroplated the method for making its electrode that obtains:
The electrode tail end is connected to the negative pole of potentiostat; One end of platinum line is connected to the positive pole of potentiostat, the top and the platinum line of electrode placed electroplate liquid, apply 5-15V voltage between electrode and platinum line with potentiostat; Switched on 0.1-5 minute; Platinum black can be formed at the electrode tip surface, and electrode is taken out, and leaves standstill to drying under 0-30 ℃;
Said electrode is platinum electrode, platinum/iridium alloy electrode or other platinum alloy electrodes;
The principal ingredient of said electroplate liquid is that concentration is the platinum acid chloride solution of 0.1-1.0%;
Also can add lead acetate in the said electroplate liquid, lead acetate accounts for the 0.0001-0.002% of electroplate liquid gross mass;
2, enzyme is fixed
Method 1: oxidase is dissolved in makes mixed solution in the damping fluid; Wherein, oxidase accounts for the 0.1-10% of mixed solution gross mass, with above-mentioned steps 1) electrode tip that obtains immerses in this mixed solution; Normal temperature leaves standstill; Oxidase is adsorbed in the nano material on electrode tip surface, and electrode is taken out, and is the biological nano material sensors electrode that makes;
Said mixed solution adopts distilled water or phosphate solution as damping fluid, and phosphate buffer can be by every 6-10 g NaCl, 1.0-1.5g Na
2HPO
4, 0.15-0.25g KCl and 0.15-0.25g KH
2PO
4Be dissolved in and join in the 1L distilled water;
Said oxidase is any one in glucose oxidase, LO, alcohol oxidase enzyme, dglutamic oxidase, the atriphos mixed enzyme; Correspondence makes glucose sensor electrode, lactic acid sensor electrode respectively; The alcohol sensor electrode, glutaminic acid sensor electrode and atriphos sensor electrode;
The atriphos mixed enzyme be by glycerokinase and glycerol-3-phosphate oxidase by mass ratio 3: (1-9) mix;
Perhaps method 2:
The electrode tip that above-mentioned steps 1 is obtained is immersed in the mixed solution of being made up of oxidase, glutaraldehyde, damping fluid, leave standstill 10 minutes-2 hours after, taking-up is dried, and is the biological nano material sensors electrode that makes;
0.1-10%, the glutaraldehyde that oxidase accounts for the mixed solution gross mass accounts for the 0.5-10% of mixed solution gross mass,
In order to protect the oxidase in the mixed solution, also adding in the said mixed solution has bovine serum albumin, bovine serum albumin to account for the 0.1-5% of mixed solution gross mass;
Said oxidase is any one in glucose oxidase, LO, alcohol oxidase enzyme, dglutamic oxidase, the atriphos mixed enzyme; Correspondence makes glucose sensor electrode, lactic acid sensor electrode respectively; The alcohol sensor electrode, glutaminic acid sensor electrode and atriphos sensor electrode;
The atriphos mixed enzyme be by glycerokinase and glycerol-3-phosphate oxidase by mass ratio 3: (1-9) mix;
The biological nano material sensors electrode that adopts said method to obtain.
A kind of method of application of biological nano material sensors electrode, its special character is may further comprise the steps:
Biological nano material sensors electrode, measurement of glucose will be carried out on the Electrochemical Detection platform when waiting for test sample article concentration; The Electrochemical Detection platform is a contrast electrode with the Ag/AgCl electrode, and biological nano material sensors electrode is a working electrode, under the normal temperature, carry out in the damping fluid (referring to distilled water or phosphate solution); During measurement; Constant electric current current potential+0.5V is put between biological nano material sensors electrode and the Ag/AgCl contrast electrode ampere response of record biological nano material sensors electrode pair concentration known sample, the rate that meets with a response (calibration process); Be directly proportional with sample concentration according to current value then and carry out the quantitative measurement of testing sample; Need therebetween constantly to stir damping fluid, when biological nano material sensors electrode does not use, store down at subzero 20 ℃ to 20 ℃ above freezing;
Damping fluid is distilled water or phosphate buffer, and phosphate buffer can be by every 6-10 g NaCl, 1.0-1.5g Na
2HPO
4, 0.15-0.25g KCl and 0.15-0.25g KH
2PO
4Be dissolved in and join in the 1L distilled water.
A kind of biological nano material sensors electrode of the present invention and preparation method thereof, the CNT of employing and platinum black nano material have the function of catalytic electrochemical reaction, and CNT and platinum black nano material are fixed in sensor surface; Can significantly improve sensor's response time, sensitivity, linear response range and antijamming capability; And then greatly strengthen the scope of application of sensor, and the existing blood-glucose meter of comparing, this sensor electrical has real-time detection; Response time is as short as 0.8 second; Highly sensitive, the minimum 10 μ M glucose that detect, linear response range big (10 μ M –, 22 mM glucose); Antijamming capability is strong, can be recycled and advantage such as low production cost.
Embodiment
Below provide embodiment of the present invention, be used for the present invention is done further explanation.
Embodiment 1
The method for making of a kind of biological nano material sensors electrode-glucose sensor electrode of present embodiment may further comprise the steps:
If no specified otherwise, all solution are prepared by deionized water (resistivity 18.2 M Ω * cm).
1), CNT is fixed
It is in 0.5% the Nafion solution that CNT is dissolved in concentration; Nafion solution is teflon and perfluor-3, and the multipolymer of 6-diepoxy-4-methyl-7-decene-sulfuric acid contains 0.5 milligram CNT in every milliliter of Nafion solution; This mixed liquor of ultrasonic dispersion 1 hour; Obtain the black even dispersion liquid, get dispersion liquid and evenly be covered in platinum/iridium alloy electrode tip surface, normal temperature leaves standstill to drying; Electrode is divided into top (part to be finished) and tail end, and electrode is connected on the various device through tail end, and the top is a probe segment;
2), enzyme is fixed
Glucose oxidase is dissolved in the distilled water; Wherein, glucose oxidase accounts for 0.1% of mixed solution gross mass, with above-mentioned steps 1) electrode tip that obtains immerses in this mixed solution; Normal temperature leaves standstill; Glucose oxidase is adsorbed in the nano material on electrode tip surface, and electrode is taken out, and promptly gets biological nano material sensors electrode-glucose sensor electrode;
A kind of method of application of biological nano material sensors electrode-glucose sensor electrode may further comprise the steps:
Biological nano material sensors electrode-glucose sensor electrode, measurement of glucose will be carried out on the Electrochemical Detection platform when waiting for test sample article concentration; The Electrochemical Detection platform is a contrast electrode with the Ag/Agcl electrode, and biological nano material sensors electrode is a working electrode, measures under 25 ℃, carries out in the distilled water damping fluid; During measurement; Constant electric current current potential+0.5V is put between biological nano material sensors electrode and the Ag/Agcl contrast electrode, the ampere response of record biological nano material sensors electrode pair concentration known sample, rate meets with a response; Be directly proportional with sample concentration according to current value then and carry out the quantitative measurement of testing sample; Need therebetween constantly to stir distilled water, when biological nano material sensors electrode does not use, under subzero 20 ℃, be stored in the oxidase phosphate buffer;
Phosphate buffer can be by every 6g NaCl, 1.0g Na
2HPO
4, 0.15g KCl and 0.15g KH
2PO
4Be dissolved in and join in the 1L distilled water.
Embodiment 2
The method for making of a kind of biological nano material sensors-lactic acid sensor electrode may further comprise the steps:
1, CNT is fixed
It is in 5% the Nafion solution that CNT is dissolved in concentration; Nafion solution is teflon and perfluor-3, and the multipolymer of 6-diepoxy-4-methyl-7-decene-sulfuric acid contains 5 milligrams CNT in every milliliter of Nafion solution; This mixed liquor of ultrasonic dispersion 2 hours; Obtain the black even dispersion liquid, get dispersion liquid and evenly be covered in the electrode tip surface that the platinum black plating obtains, normal temperature leaves standstill to drying; Electrode is divided into top (part to be finished) and tail end, and electrode is connected on the various device through tail end, and the top is a probe segment;
It is following that platinum black is electroplated the method for making its electrode that obtains:
The platinum electrode tail end is connected to the negative pole of potentiostat, an end of platinum line is connected to the positive pole of potentiostat, the top and the platinum line of electrode placed electroplate liquid; The principal ingredient of electroplate liquid is that concentration is 0.1% platinum acid chloride solution; Also can add lead acetate in the electroplate liquid, lead acetate accounts for 0.0001% of electroplate liquid gross mass, applies 5-15V voltage between electrode and platinum line with potentiostat; Switched on 0.1-5 minute; Platinum black can be formed at the electrode tip surface, and electrode is taken out, and leaves standstill to drying under 0-30 ℃;
2, enzyme is fixed
The electrode tip that above-mentioned steps 1 is obtained is immersed in the mixed solution of being made up of LO, glutaraldehyde, damping fluid, leave standstill 10 minutes-2 hours after, taking-up is dried, and is the biological nano material sensors electrode that makes; LO account for the mixed solution gross mass 0.1%, glutaraldehyde accounts for 0.5% of mixed solution gross mass, in order to protect the LO in the mixed solution, also adding in the mixed solution has bovine serum albumin, bovine serum albumin to account for 0.1% of mixed solution gross mass;
The biological nano material sensors electrode-lactic acid sensor electrode that adopts said method to obtain.
The method of application of a kind of biological nano material sensors electrode-lactic acid sensor electrode may further comprise the steps:
Biological nano material sensors electrode-lactic acid sensor electrode, measurement of glucose will be carried out on the Electrochemical Detection platform when waiting for test sample article concentration; The Electrochemical Detection platform is a contrast electrode with the Ag/AgCl electrode, and biological nano material sensors electrode is a working electrode, under the normal temperature, carry out in the damping fluid; During measurement; Constant electric current current potential+0.5V is put between biological nano material sensors electrode and the Ag/AgCl contrast electrode ampere response of record biological nano material sensors electrode pair concentration known sample, the rate that meets with a response (calibration process); Be directly proportional with sample concentration according to current value then and carry out the quantitative measurement of testing sample; Need therebetween constantly to stir damping fluid, when biological nano material sensors electrode does not use, store down at subzero 20 ℃;
Damping fluid is distilled water or phosphate buffer, and phosphate buffer can be by every 6-10 g NaCl, 1.0-1.5g Na
2HPO
4, 0.15-0.25g KCl and 0.15-0.25g KH
2PO
4Be dissolved in and join in the 1L distilled water.
Embodiment 3
The method for making of a kind of biological nano material sensors electrode-alcohol sensor electrode of present embodiment may further comprise the steps:
1), CNT is fixed
It is in 10% the Nafion solution that CNT is dissolved in concentration; Nafion solution is teflon and perfluor-3, and the multipolymer of 6-diepoxy-4-methyl-7-decene-sulfuric acid contains 10 milligrams CNT in every milliliter of Nafion solution; This mixed liquor of ultrasonic dispersion 3 hours; Obtain the black even dispersion liquid, get dispersion liquid and evenly be covered in platinum/iridium alloy electrode tip surface, normal temperature leaves standstill to drying; Electrode is divided into top (part to be finished) and tail end, and electrode is connected on the various device through tail end, and the top is a probe segment;
2), enzyme is fixed
Alcohol oxidase enzyme is dissolved in makes mixed solution in the phosphate buffer; Wherein, alcohol oxidase enzyme accounts for 10% of mixed solution gross mass, with above-mentioned steps 1) electrode tip that obtains immerses in this mixed solution; Normal temperature leaves standstill; Oxidase is adsorbed in the nano material on electrode tip surface, and electrode is taken out, and is the biological nano material sensors electrode-alcohol sensor electrode that makes;
Phosphate buffer can be by per 10 g NaCl, 1.5g Na
2HPO
4, 0.25g KCl and 0.25g KH
2PO
4Be dissolved in and join in the 1L distilled water;
The method of application of a kind of biological nano material sensors electrode-alcohol sensor electrode may further comprise the steps:
Biological nano material sensors electrode, measurement of glucose will be carried out on the Electrochemical Detection platform when waiting for test sample article concentration; The Electrochemical Detection platform is a contrast electrode with the Ag/Agcl electrode, and biological nano material sensors electrode is a working electrode, measures under (25 ± 0. 5) ℃, carries out in the phosphate buffer; During measurement; Constant electric current current potential+0.5V is put between biological nano material sensors electrode and Ag or the Agcl contrast electrode, the ampere response of record biological nano material sensors electrode pair concentration known sample, rate meets with a response; Be directly proportional with sample concentration according to current value then and carry out the quantitative measurement of testing sample; Need therebetween constantly to stir phosphate buffer, when biological nano material sensors electrode does not use, under 20 ℃ above freezing, be stored in the oxidase phosphate buffer; Wherein, phosphate buffer is by per 8 g NaCl, 1.15 g Na
2HPO
4, 0.2 g KCl and 0.2 g KH
2PO
4Be dissolved in and join in the 1L distilled water.
Embodiment 4
The method for making of a kind of biological nano material sensors-glutaminic acid sensor electrode of present embodiment may further comprise the steps:
1), CNT is fixed
It is that Nafion solution is teflon and perfluor-3, the multipolymer of 6-diepoxy-4-methyl-7-decene-sulfuric acid in 8% the Nafion solution that CNT is dissolved in concentration; Contain 9 milligrams CNT in every milliliter of Nafion solution, this mixed liquor of ultrasonic dispersion 3 hours obtains the black even dispersion liquid, gets dispersion liquid and evenly is covered in the electrode tip surface that the platinum black plating obtains, and normal temperature leaves standstill to drying; Electrode is divided into top (part to be finished) and tail end, and electrode is connected on the various device through tail end, and the top is a probe segment;
It is following that platinum black is electroplated the method for making its electrode that obtains:
The electrode tail end is connected to the negative pole of potentiostat; One end of platinum line is connected to the positive pole of potentiostat; The top and the platinum line of electrode are placed electroplate liquid; The principal ingredient of electroplate liquid is that concentration is 1.0% platinum acid chloride solution, also can add lead acetate in the electroplate liquid, and lead acetate accounts for 0.002% of electroplate liquid gross mass; Apply 15V voltage between electrode and platinum line with potentiostat, switched on 5 minutes, platinum black can be formed at the electrode tip surface, and electrode is taken out, and leaves standstill to drying under 18 ℃;
2), enzyme is fixed
The electrode tip that above-mentioned steps 1 is obtained is immersed in the mixed solution of being made up of dglutamic oxidase, glutaraldehyde, damping fluid, leave standstill 2 hours after, taking-up is dried, and is the biological nano material sensors electrode that makes; Dglutamic oxidase account for the mixed solution gross mass 10%, glutaraldehyde accounts for 10% of mixed solution gross mass, in order to protect the dglutamic oxidase in the mixed solution, also adding in the mixed solution has bovine serum albumin, bovine serum albumin to account for 5% of mixed solution gross mass;
The biological nano material sensors electrode-glutaminic acid sensor electrode that adopts said method to obtain.
The method of application of a kind of biological nano material sensors electrode-glutaminic acid sensor electrode may further comprise the steps:
Biological nano material sensors electrode, measurement of glucose will be carried out on the Electrochemical Detection platform when waiting for test sample article concentration; The Electrochemical Detection platform is a contrast electrode with the Ag/AgCl electrode, and biological nano material sensors electrode is a working electrode, measures under (25 ± 0. 5) ℃, carries out in the distilled water damping fluid; During measurement; Constant electric current current potential+0.5V is put between biological nano material sensors electrode and the Ag/AgCl contrast electrode, the ampere response of record biological nano material sensors electrode pair concentration known sample, rate meets with a response; Be directly proportional with sample concentration according to current value then and carry out the quantitative measurement of testing sample; Need therebetween constantly to stir the distilled water damping fluid, when biological nano material sensors electrode does not use, under 10 ℃, be stored in the oxidase phosphate buffer;
Biological nano material sensors electrode of the present invention can be applied to following occasion:
1, glucose sensor electrode is used for diabetes diagnosis equipment, through measuring the blood sugar concentration diagnosing diabetes;
2, glucose sensor electrode is used for diabetes patient's watch-dog, monitors the concentration of glucose in the blood in real time;
3, glucose sensor electrode is used for research equipment, and when the Self-referencing workstation collocation of this sensor electrode and Applicable electronics, inc. was used, test sample (like cell) was to the absorption rate of glucose in real time; Be flow (mole glucose/(second * square centimeter)); This technology can be widely used in cancer physiology, cancer therapy drug efficient, diabetes pancreas islet physiology; Microorganism and wastewater treatment, and the research of aspect such as meat fiber nutrition.