CN100594379C - Carbon nano tube modified blood sugar biosensor - Google Patents
Carbon nano tube modified blood sugar biosensor Download PDFInfo
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- CN100594379C CN100594379C CN200710069779A CN200710069779A CN100594379C CN 100594379 C CN100594379 C CN 100594379C CN 200710069779 A CN200710069779 A CN 200710069779A CN 200710069779 A CN200710069779 A CN 200710069779A CN 100594379 C CN100594379 C CN 100594379C
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Abstract
The invention provides a blood sugar biosensor of carbon nanometer tube, it includes floor, three electrode system, insulation, forming the reaction lumen body around three electrode system, setting the conversion zone in the reaction lumen body, laying over the cover plate on the reaction lumen body, the conversion zone includes, enzyme and electron transport agent, the enzyme is the glc dh pyrrole and quinone for coenzyme. The transducer's corresponding sensitivity improves evidently, it has not infection for measuring the dextrose deepness in the blood with the oxygen pressure, thickness inthe measuring site and oxygen content of blood sample. The invention has the excellence of easy manufacture artwork, low cost, suited to volume-produce and the low sample quantity etc.
Description
Technical field
The invention belongs to the development field of biology sensor, specifically be meant a kind of carbon nano tube modified, be glucose dehydrogenase blood sugar biosensor of coenzyme and preparation method thereof with the pyrro-quinoline quinone (PQQ).
Background technology
Along with China enters informationized society, population structure is tending towards aging, people strengthen day by day to the concern of health, living standards of the people improve year by year, the morbidity rate of diabetes also improves thereupon, 13 regional total prevalence rates are 0.67% in China, and the big city is about about 1%, and morbidity rate significantly raises after 40 years old.Use glucose oxidase to be subjected to the influence of oxygen content in aerial oxygen pressure, concentration and the blood sample of measure field as the glucose sensor accuracy of catalyzer.
Professor Iijima of Japanese NEC in 1991 develops the nanoscale molecular carbon structure of coaxial tubulose---carbon nano-tube (CNTs), having caused concern widely in the whole world. carbon nano-tube is the carbon pipe that is curled and formed by the graphitic carbon atomic layer, the branch that Single Walled Carbon Nanotube (SWNTs) and multi-walled carbon nano-tubes (MWNTs) are arranged, the former is formed by mono-layer graphite sheet convolution, the latter is formed by multilayer graphite flake convolution, is bound by the pentagon of carbon atom in two ends.Electricity that carbon nano-tube is good and mechanical property attract more and more researchers to be applied to sensor field.The carbon nano tube modified glass carbon of currently reported usefulness, graphite, adamas, tantalum electrode etc. are realized the detection to materials such as glucose, antibody, oxygen.Use carbon nano tube modified electrode can increase electrode surface area on the one hand, on the other hand can the accelerated electron transfer rate, thus the response and the sensitivity that improve sensor.
Serigraphy belongs to porous printing, is silk fabrics, synthetic fabrics or wire gauze are stretched tight on screen frame, adopts manual method of carving paint film or photochemistry plate-making to make screen printing forme.Modern screen printing technique then is to utilize photosensitive material to make screen printing forme (making the silk screen hole of picture and text part on the screen printing forme is through hole, and the silk screen hole of non-graphic part is plugged) by photomechanical method.By the extruding of scraper plate, printing ink is transferred on the stock by the mesh of picture and text part during printing, formed the picture and text the same with original copy.Screen printing apparatus is simple, easy to operate, and it is simple and easy and with low cost to print, make a plate, and adaptability is strong.Film evaporation technology film forming is utilized the principle of high temperature evaporation or plasma sputtering, makes the coating by vaporization material be deposited in substrate surface and forms film, compares with serigraphy, and film forming is even, but price is higher.
Application for a patent for invention 200510049284.4 discloses a kind of full blood glucose biological sensor of modified by oriented carbon nano tube, the bottom that it is characterized in that responding layer is the aligned carbon nanotube as second electron medium, and its upper strata is glucose oxidase and the potpourri formed as the reagent of first electron medium, stabilizing agent, damping fluid.This technical scheme adopts glucose oxidase as catalyzer, and its accuracy is subjected to the influence of the oxygen content in aerial oxygen pressure, concentration and the blood sample of measure field, and measurement result instability, applicability are not high.
Summary of the invention
The invention provides a kind of be not subjected to that oxygen content influences in atmosphere and the sample, cost is low, highly sensitive to be used for the carbon nano tube modified of measure glucose concentration be the blood sugar biosensor of the glucose dehydrogenase of coenzyme with the pyrro-quinoline quinone (PQQ).
A kind of carbon nano tube modified blood sugar biosensor, comprise substrate, three-electrode system, insulation course, be formed with reaction cavity around the three-electrode system, be provided with responding layer in the reaction cavity, be coated with cover plate on the reaction cavity, described responding layer comprises carbon nano-tube, enzyme and electron transfer mediator, and it is the glucose dehydrogenase of coenzyme that described enzyme adopts with the pyrro-quinoline quinone (PQQ).
Described three-electrode system is by contrast electrode, working electrode, form electrode, working electrode with electrode is contacted with lead-in wire; Described contrast electrode adopts silver/silver chloride, carbon, gold, platinum or titanium; Described to electrode and working electrode employing carbon, gold, platinum or titanium.
Also comprise the enzymatic activity adjuvant in the described responding layer; the enzymatic activity adjuvant comprises activity increase agent and live keeping agent; described activity increase agent adopts calcium ion or damping fluids such as citric acid, phthalic acid, and described live keeping agent adopts a kind of of trehalose, shitosan, bovine serum albumin(BSA).
Described responding layer also comprises hydrophilic macromolecule, comprises one or more of carboxymethyl cellulose, neopelex, Triton X-100, polyvinyl alcohol (PVA), polyvinylpyrrolidone, gelatin and derivant thereof, cetyl trimethyl ammonium bromide.
Described electron transfer mediator is selected a kind of in the potassium ferricyanide, ferrocene and derivant thereof, methylene blue, Prussia orchid, the N-Methyl pyrrolidone for use.
Adopt damping fluid, sodium chloride solutions such as citric acid or phthalic acid to guarantee stable contrast electrode current potential.
Described reaction cavity one side has the pore that is used for sample introduction.
The present invention also provides a kind of preparation method of above-mentioned biology sensor.
A kind of method for preparing above-mentioned blood sugar biosensor may further comprise the steps:
The first step adopts method for printing screen or film evaporation technology to form three-electrode system in polypropylene or other macromolecular material substrates, makes the sensor electrode substrate;
Second step was dissolved in sonic oscillation in the concentrated sulphuric acid and the red fuming nitric acid (RFNA) mixed solution with carbon nano-tube, after filtration washing is neutrality, obtained the stable suspension of carbon nano-tube;
The 3rd step dripped the carbon nano tube suspension that makes in the reaction chamber of electrode basement, carried out the lucifuge drying;
The 4th step was dissolved in enzyme, electron transfer mediator, enzyme live keeping agent and sodium chloride in the damping fluid by finite concentration, made enzyme solutions;
The 5th step dripped the enzyme solutions that makes in the reaction chamber of electrode basement, and the lucifuge drying covers cover plate on reaction cavity, promptly make biology sensor.
Wherein, in second step, adding in the described carbon nano-tube has neopelex, lauryl sodium sulfate or cetyl trimethyl ammonium bromide solution, carries out sonic oscillation.
The certain density glucose sample solution that in the reaction cavity of the biology sensor for preparing, drips, responding layer can be dissolved, and in liquid-phase reaction system, by glucose dehydrogenase catalysis glucose, produces an electronics; Electron transfer mediator obtains electronics and is reduced; Also the electron transfer mediator of ortho states is diffused into electrode surface, under the certain current potential of working electrode, go back the ortho states electron transfer mediator can with working electrode generation electron transport, get back to original state; Electrode obtains electronics simultaneously, produces corresponding electric current; Concentration of glucose linear dependence in this current value and the fluid to be measured obtains the sensor response current thus, extrapolates the glucose solution concentration in the sample solution.
Biology sensor of the present invention adopts carbon nano tube modified electrode, and it is long-pending not only can to increase the electrode surface in contact on physical arrangement, and can accelerate electron transport speed, improves electron transport efficient, thereby improves the response sensitivity of biology sensor.
Employing is that the glucose dehydrogenase of coenzyme is as catalyzer with the pyrro-quinoline quinone (PQQ), accuracy of measurement is not subjected to the influence of oxygen content in the atmosphere, and be different from NAD+ and do need add NAD+ solution when coenzyme is measured, it can be modified at pyrro-quinoline quinone (PQQ) in the glucose dehydrogenase.Compare with the sensor that uses glucose oxidase to measure glucose, use the sensor of pyrro-quinoline quinone (PQQ) as the glucose dehydrogenase of coenzyme, its response and oxygen to substrate glucose is irrelevant, thereby be not subjected to the influence that oxygen content changes in dissolved oxygen DO in the blood or the measure field atmosphere, can measure the concentration of glucose more accurately, obtain high performance sensor.
Description of drawings
Fig. 1 is the vertical view of biology sensor of the present invention;
Fig. 2 is the longitudinal section of biology sensor of the present invention;
Fig. 3 is the cyclic voltammetry curve figure of electrode;
Fig. 4 is the current-responsive graph of a relation of biology sensor to glucose solution.
Embodiment
As shown in Figure 1, 2, a kind of carbon nano tube modified blood sugar biosensor, comprise electrical insulating property substrate 1 that polypropylene and other macromolecular materials form, by screen printing technique or film evaporation technology in substrate 1, form contrast electrode 2a, lead-in wire 2d and with the contact portion 2e of detecting instrument, and form working electrode 2b and to electrode 2c, this working electrode 2b with electrode 2c is contacted with the 2d that goes between.And then, in substrate 1, print insulating material, form insulation course 5.Insulation course 5 covers working electrode 2b and to the outer peripheral portion of electrode 2c, makes working electrode 2b, the exposed portions serve area of electrode 2c and contrast electrode 2a is kept certain.
In the substrate 1 that has formed electrode system, formed the responding layer 4 that comprises carbon nano-tube, enzyme catalyst and electron transfer mediator etc.Colloid 3 and cover plate 6 cooperate formation reaction cavity 3a.
During detection, the certain density glucose sample solution that in the reaction cavity 3a of biology sensor, drips, responding layer 4 can be dissolved, and in liquid-phase reaction system, by glucose dehydrogenase catalysis glucose, produces an electronics; Electron transfer mediator obtains electronics and is reduced; Also the electron transfer mediator of ortho states is diffused into electrode surface, under the certain current potential of working electrode, go back the ortho states electron transfer mediator can with working electrode generation electron transport, get back to original state; Electrode obtains electronics simultaneously, produces corresponding electric current; Concentration of glucose linear dependence in this current value and the fluid to be measured obtains the sensor response current thus, extrapolates the glucose solution concentration in the sample solution.
As shown in Figure 3, A is the cyclic voltammetry curve of biology sensor of the present invention among the figure, and B is the cyclic voltammetry curve of the electrode modified of carbon-free nanoscale pipe, show add carbon nano-tube after, the reversibility of the reaction of the potassium ferricyanide improves.
As shown in Figure 4, A is biology sensor current-responsive of the present invention relation among the figure, and B is the electrode current response relation that the carbon-free nanoscale pipe is modified, show add carbon nano-tube after, the response sensitivity of sensor is improved.
Embodiment:
On polypropylene substrate, coat silver-colored pastel by screen printing technique and form the pseudo-contrast electrode of silver/silver chloride; Then, printing conductive carbon pastel in substrate forms working electrode and to electrode; The printing insulation course makes working electrode, certain to the exposed portions serve area maintenance of electrode and pseudo-contrast electrode.
Using the neopelex of 4.8mg/ml and volume ratio is the multi-walled carbon nano-tubes suspending liquid of 0.5% Triton X-100 mixed liquor preparation 2.5mg/ml, and concussion is 2 hours in ultrasonic, the formation steady suspension.In every plate electrode reaction chamber, drip this carbon nano tube suspension of 3 microlitres with pipettor, placed the drying box lucifuge dry 24 hours, be prepared into carbon nano tube modified electrode system.
Then, on carbon nanotube layer, drip the citrate buffer solution (pH6.0 contains 0.2M sodium chloride) of the trehalose of the lime chloride of the potassium ferricyanide, 1mM of glucose dehydrogenase that pyrro-quinoline quinone (PQQ) that 2 microlitres comprise 4780U/ml is a coenzyme, 0.1M and 24mg/ml.Form 4 sensitive layers after the lucifuge drying, make glucose sensor.
Measuring process is that preparing as concentration of glucose is the sample solution of 0.5～35mM.This sample solution drips on responding layer 4, simultaneously, on working electrode, add 0.5V constant potential (with respect to the pseudo-contrast electrode of silver/silver chloride), after responding layer is supplied with this sample solution that comprises glucose, glucose in the sample solution passes through dehydrogenase oxidoreductase, meanwhile, the potassium ferricyanide in the responding layer is reduced into potassium ferrocyanide.Apply constant potential in the time of 20 seconds, measure working electrode and the current value between the electrode.
Measure the current value of the sample of various different concentration of glucose, with the concentration of glucose is that transverse axis, response current value are the longitudinal axis, draw the response characteristics figure of sensor, its result shown in the A of Fig. 4, the electrode (as the B of Fig. 4) that its sensor response gradient is obviously modified greater than the carbon-free nanoscale pipe.
After keeping in Dark Place 1 month under the biology sensor normal temperature of the present invention, response attenuation is not obvious.
Claims (7)
1. method for preparing carbon nano tube modified blood sugar biosensor is characterized in that may further comprise the steps:
The first step adopts method for printing screen or film evaporation technology to form three-electrode system in polypropylene or other macromolecular material substrates, makes the sensor electrode substrate;
Second step was dissolved in sonic oscillation in the concentrated sulphuric acid and the red fuming nitric acid (RFNA) mixed solution with carbon nano-tube, after filtration washing is neutrality, obtained the stable suspension of carbon nano-tube;
The 3rd step dripped the carbon nano tube suspension that makes in the reaction chamber of electrode basement, carried out the lucifuge drying;
The 4th step will be that glucose dehydrogenase, electron transfer mediator, enzyme live keeping agent and the sodium chloride of coenzyme is dissolved in the damping fluid by finite concentration with the pyrro-quinoline quinone (PQQ), make enzyme solutions;
The 5th step dripped the enzyme solutions that makes in the reaction chamber of electrode basement, and the lucifuge drying covers cover plate on reaction cavity, promptly make biology sensor;
Described biology sensor comprises substrate (1), three-electrode system (2), insulation course (5), three-electrode system (2) is formed with reaction cavity (3) on every side, be provided with responding layer (4) in the reaction cavity (3), be coated with cover plate (6) on the reaction cavity (3), it is characterized in that: described responding layer (4) comprises carbon nano-tube, enzyme and electron transfer mediator, and it is the glucose dehydrogenase of coenzyme that described enzyme adopts with the pyrro-quinoline quinone (PQQ).
2. the method for claim 1 is characterized in that: described three-electrode system (2) is by contrast electrode (2a), working electrode (2b), form electrode (2c), working electrode (2b) with electrode (2c) is contacted with lead-in wire (2d); Described contrast electrode adopts silver/silver chloride, carbon, gold, platinum or titanium; Described to electrode and working electrode employing carbon, gold, platinum or titanium.
3. method as claimed in claim 2 is characterized in that: adopt damping fluid, sodium chloride solutions such as citric acid or phthalic acid to guarantee stable contrast electrode current potential.
4, the method for claim 1 is characterized in that: in second step, adding in the described carbon nano-tube has neopelex, lauryl sodium sulfate or cetyl trimethyl ammonium bromide solution, carries out sonic oscillation.
5. the method for claim 1 is characterized in that: described enzyme live keeping agent is a kind of of trehalose, shitosan, bovine serum albumin(BSA).
6. the method for claim 1 is characterized in that: described electron transfer mediator is selected a kind of in the potassium ferricyanide, ferrocene and derivant thereof, methylene blue, Prussia orchid, the N-Methyl pyrrolidone for use.
7. the method for claim 1, it is characterized in that: described reaction cavity one side has the pore that is used for sample introduction.
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