CN111208184A - Non-enzymatic glucose sensing and detecting system - Google Patents
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Abstract
The invention discloses a non-enzymatic glucose sensing detection system and a preparation process thereof, wherein a simple and low-temperature hydrothermal process is utilized to manufacture a zinc oxide nanowire with good quality and few defects, a conductive ink layer additionally added and attached to a bottom layer enhances the sensing capability of the zinc oxide nanowire, the zinc oxide nanowire has good quality in physical property, has good sensitivity and relatively high coefficient of determination, the measurement range is superior to that of detection paper, and the more accurate glucose concentration within 3mMol/L can be measured.
Description
Technical Field
The invention relates to the field of new materials, in particular to a non-enzymatic glucose sensing detection system and a preparation process thereof.
Background
Glucose sensors can be classified into enzymatic and non-enzymatic types. The enzyme type is to oxidize glucose by using glucose oxidase, and then to display data by electric signals through a sensor device so as to obtain the content; the non-enzymatic type is to simulate the action of enzyme by using electrochemical reaction, and then the effect of oxidizing glucose is achieved by electron movement, and then the effect is displayed by a sensor system.
It was found by enzymatic glucose biosensors using a glucosidase enzyme (e.g. glucose oxidase) that their chemical instability, complexity and poor reproducibility are major obstacles to their widespread use and cannot be used below pH2 and above pH8 because both environments can cause the enzyme to affect its activity or even lose its effect, and also directly lose its properties at temperatures above 40 ℃, and more importantly, because washing affects the properties of the enzyme, so that the enzyme-based measurements are all disposable. The non-enzymatic glucose sensor does not need to consider the factors of enzyme, and also has the advantages of low limit value, convenient storage and lower manufacturing cost, thereby greatly expanding the application range of the non-enzymatic glucose sensor, and being accurate and stable relative to the enzymatic glucose sensor no matter the environment or the limit, and therefore, the development of a novel non-enzymatic glucose sensing detection system and a preparation process thereof is very necessary.
Disclosure of Invention
Accordingly, in view of the disadvantages in the related art, examples of the present invention are provided to substantially solve one or more problems due to limitations and disadvantages of the related art, to substantially improve safety and reliability, and to effectively protect equipment.
According to the technical scheme provided by the invention, the invention discloses a preparation process of a non-enzymatic glucose sensing detection system, wherein the preparation process of the non-enzymatic glucose sensing detection system comprises the following preparation steps: A. cleaning the processing substrate assembly; B. preparing a seed solution; C. preparing a growth solution; D. adding conductive ink; E. and depositing the zinc oxide nanowire.
Further, the substrate assembly comprises a paper-based chip and a silicon substrate, and when the substrate assembly is prepared, the paper-based chip and the silicon substrate are cleaned respectively, and then the paper-based chip is attached to the silicon substrate and placed into the transparent box. The paper-based chip cleaning treatment comprises the following steps: taking the paper-based chip out of the packaging paper, washing with deionized water, and drying with a nitrogen spray gun; the silicon substrate cleaning process includes the steps of: 1) soaking the silicon substrate in acetone, cleaning for 10 minutes by using an ultrasonic oscillator, removing residual oil stains on the substrate, and taking out after the cleaning is finished; 2) soaking the silicon substrate in methanol, cleaning the silicon substrate for 10 minutes by using an ultrasonic oscillator, removing residual organic matters on the substrate, and taking out the silicon substrate after the cleaning is finished; 3) then soaking in deionized water, oscillating for 10 minutes, removing residual cleaning liquid, and drying by blowing with a nitrogen spray gun.
Further, the preparation process of the seed solution comprises the following steps: 1) adding 0.12 g of sodium hydroxide into 100ml of methanol to prepare a sodium hydroxide solution; 2) adding 0.328 g of zinc acetate into 150ml of methanol to prepare a zinc acetate solution; 3) stirring the zinc acetate solution in an electromagnetic heating stirrer, heating to 60 ℃, and then dripping the sodium hydroxide solution into the zinc acetate solution at the speed of 50ml/h by using a constant-pressure delivery pump; 4) after dropping 100ml, the solution was heated at 60 ℃ for three hours, and stirred for 10 hours to prepare a seed solution of zinc oxide nanowires.
Further, the preparation process of the growth solution comprises the following steps: 0.7435 g of zinc nitrate hexahydrate and 0.35 g of hexamethylenetetramine are added into 100ml of deionized water and stirred uniformly.
Further, the adding of the conductive ink comprises the following steps: 1) taking the cleaned substrate assembly out of the transparent box, and covering a part of the paper-based chip by using a heat-resistant adhesive tape; 2) operating the substrate assembly by using a puncher, and punching a hole on the paper-based chip to expose a working electrode of the paper-based chip; 3) 0.8ul of conductive ink is absorbed by a mechanical micro-dropping pipe and dropped on the working electrode; 4) and then putting the electrode into an oven, setting the temperature of the oven at 85 ℃, baking the electrode for 30 minutes, and then taking the electrode out, so as to form a conducting layer on the working electrode.
Further, the step of depositing the zinc oxide nanowire comprises the following steps: 1) uniformly stirring the seed solution by using an electromagnetic heating stirrer, and uniformly melting the sediment at the bottom into the solution; 2) sucking 2ul of seed solution by using a mechanical micro-dropper, dripping the seed solution on a working electrode with a conductive ink layer, baking the working electrode in an oven for 3 minutes, setting the temperature of the oven to be 85 ℃, taking out the working electrode, dripping 2ul of seed solution on the working electrode, baking the working electrode in the oven with the same temperature for 3 minutes, and repeating the steps for four times to finish the preparation of the seed layer; 3) pouring the growth solution into a culture dish, facing the electrode of the paper-based chip with the seed layer to the water surface, and connecting the back surface of the substrate assembly with a support; 4) placing the substrate assembly in a growth solution to make the liquid level higher than the substrate assembly; 5) coating a culture dish by using aluminum foil paper, and then putting the culture dish into an oven, wherein the temperature of the oven is set to be 85 ℃ or 95 ℃, and the deposition time is 4 hours; 6) after 4 hours, taking out the substrate assembly, removing the heat-resistant adhesive tape, enabling the substrate assembly to face upwards, and immediately washing residual salts on the surface by using deionized water; 7) then putting the mixture into a culture dish and then putting the culture dish into an oven to be dried at the temperature of 37 ℃, thus obtaining the zinc oxide nanowire.
Further, the area of the hole is the same as the area of the working electrode.
Furthermore, the invention also discloses a non-enzymatic glucose sensing detection system which is prepared by the preparation process.
The invention provides a non-enzymatic glucose sensing detection system and a preparation process thereof, a simple and low-temperature hydrothermal process is utilized to manufacture a zinc oxide nanowire with good quality and few defects, a conductive ink layer additionally added and attached to a bottom layer enhances the sensing capability of the zinc oxide nanowire, the zinc oxide nanowire has good quality in physical property, has good sensitivity and relatively high coefficient of determination, the measurement range is better than that of detection paper, and the accurate glucose concentration within 3mMol/L can be measured.
Drawings
FIG. 1 is a schematic diagram of electron transfer between zinc oxide and glucose according to the present invention.
Fig. 2 is a schematic process diagram of a process for depositing nanowires according to the present invention.
FIG. 3 is a schematic diagram of the seed solution of the present invention.
FIG. 4 is a schematic view of the growth solution of the present invention.
Figure 5 is a schematic diagram of the present invention of growing zinc oxide nanowires on a paper-based chip.
FIG. 6 is a schematic view of a sensing assembly according to the present invention.
FIG. 7 is a graph showing the test results of the present invention.
Detailed Description
The present invention will be further described with reference to the following specific examples.
The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention. The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments.
A non-fermented glucose sensor is mainly characterized in that an electrode material with catalytic capability is used for replacing a glucosyl ferment. Among many metal oxide materials, zinc oxide is a semiconductor material with high energy gap and also has high exciton confinement energy of 60meV, so that it has the advantages of thermal stability and chemical stability, etc. and the one-dimensional nanostructure has large surface area and unique quantum tunneling effect, so that it is used as the catalyst of glucose.
Fig. 1 is a schematic diagram of electron transfer between zinc oxide and glucose, when zinc oxide is in contact with NaOH, the conduction band and the valence band of zinc oxide bend downward to form energy barriers because the chemical potential of the electrolyte is equal to the fermi level of zinc oxide. The electrons released by the oxidation of glucose can be conducted in three ways through reaching the zinc oxide conduction band by an energy barrier: when the ambient temperature of the solution rises, the carriers have enough energy and can directly cross the energy barrier to conduct to the conduction band, and thermionic emission is realized; when the concentration of glucose in the solution is increased, the change of the environmental electric field bends the energy band, and the carrier has the opportunity to be conducted to the conduction band in the state of energy barrier reduction; or electrons bounce to the conductive layer using intrinsic defects, causing tunneling.
The conductive ink is added, the conductive ink can be firstly dripped on a substrate which is pre-deposited, the seed solution can be dripped on the area with the ink layer after drying, and the nanowires deposited on the later layer have good conductivity.
FIG. 2 is a schematic view of the nanowire deposition of the present invention, showing that the conductive ink layer, the seed layer, and the nanowire are sequentially disposed on the substrate.
The first embodiment is as follows: substrate assembly cleaning process
The substrate assembly used in the invention comprises a paper-based chip and a silicon substrate, and cleaning treatment is carried out in order to prevent the substrate assembly from being influenced by impurities on the surface of the substrate assembly when the seed layer is spread. The paper-based chip and the silicon substrate are processed separately in different modes. And (3) opening the paper-based chip from the packaging paper, taking out a new paper-based chip, washing the new paper-based chip with deionized water, drying the new paper-based chip by using a nitrogen spray gun, and placing the new paper-based chip in a clean transparent box so as to avoid surface pollution. And the silicon substrate is soaked in acetone, cleaned for 10 minutes by using an ultrasonic oscillator, cleaned of residual oil stains on the silicon substrate, taken out after completion, soaked in methanol, cleaned for 10 minutes by using the ultrasonic oscillator similarly, cleaned of residual organic matters on the silicon substrate, taken out, soaked in deionized water, vibrated for 10 minutes, removed of residual cleaning liquid, dried by using a nitrogen spray gun, and placed in a clean transparent box so as to avoid surface pollution.
And then attaching the cleaned paper-based chip to a silicon substrate to form a substrate assembly, and putting the substrate assembly into a transparent box. The subsequent casting, painting, and heating of the material droplets on the substrate assembly are all performed in a clean room.
Example two: preparation of seed solutions and growth solutions
FIG. 3 is a schematic diagram of a seed solution prepared by using methanol as a solvent and sodium hydroxide and zinc acetate as solutes. Specifically, 0.12 g of sodium hydroxide is added into 100ml of methanol to prepare a sodium hydroxide solution; and adding 0.328 g of zinc acetate into 150ml of methanol to prepare a zinc acetate solution, stirring the zinc acetate solution in an electromagnetic heating stirrer, heating to 60 ℃, dripping the solution into the zinc acetate solution at the speed of 50ml per hour by using a constant pressure delivery pump, continuously heating at 60 ℃ for three hours after dripping 100ml, continuously stirring for 10 hours, and converting the solution from clear to turbid, wherein the solution is the seed solution of the zinc oxide nanowire.
The growth solution is a solution needed in a hydrothermal method, and is a solution in which a salt containing zinc ions is dissolved into a alkalescence solution. A molar solution of zinc ions provided by zinc nitrate hexahydrate and hydroxide ions provided by hexamethylenetetramine, the reaction being as given by the formula:
C6H12N4+6H2O ↔ 4NH3+6HCHO；
NH3+ H2O ↔ NH4 ++ OH−；
Zn(NO3)2↔ Zn2++2(NO3)2-；
Zn2++4NH3↔ [Zn (NH3)4]2+；
Zn2++2OH−↔ Zn(OH)2↔ ZnO+H2O。
wherein, the alkalescent hexamethylenetetramine can generate ammonia and formaldehyde, and the ammonia can decompose water to obtain hydroxyl ions (OH)−) And zinc nitrate dissociates zinc ions (Zn) in aqueous solution2+) Both form Zn (OH)2Namely growth elementary crystal of the zinc oxide, and can be used as an acid-base buffer in the solution. The hydrolysis rate of hexamethylenetetramine is pH and temperature dependent, while ammonia (NH)3) Stable release of OH during the reaction−The preparation is carried out slowly to promote Zn (OH)2The crystallization quality reduction caused by the over-fast ZnO precipitation is avoided.
Fig. 4 is a schematic diagram for preparing a growth solution, specifically, 0.7435 g of zinc nitrate hexahydrate and 0.35 g of hexamethylenetetramine are added into 100ml of deionized water and uniformly dissolved in the water, and the concentration of the growth solution required by the present invention can be 0.025mol/L or 0.075 mol/L.
Example three: adding conductive ink
After the substrate assembly is cleaned, a zinc oxide nanowire material is required to grow on a working electrode of a paper-based chip, three electrodes (the working electrode, a counter electrode and a reference electrode) are arranged on the paper-based chip, so that a part of the paper-based chip is covered by heat-resistant glue, the substrate assembly is operated by a puncher, a hole is punched with the paper-based chip, the hole is attached to the working electrode on the paper-based chip, the area of the hole is the same as that of the working electrode, only the working electrode is exposed, the other two electrodes are covered, and then conductive ink, seed solution and growth solution can be used after being added, dropped and soaked.
The conductive ink is used for increasing the conductivity between the nanowire and the working electrode and improving the current value for sensing the concentration of glucose.
The present invention uses transparent conductive ink added to the sensor to increase the sensed value. The conductive ink is water-based or oil-based, and is attached to the surface to be detected on the sensor by screen printing or coating, and then the required finished product is deposited.
0.8ul of conductive ink is absorbed by a mechanical micropipette and is dripped on the working electrode, then the working electrode is put into an oven, the temperature of the oven is set to be 85 ℃, the working electrode is taken out after 30 minutes, and a conductive layer can be formed on the working electrode.
Example four: depositing zinc oxide nanowires
The seed solution is uniformly stirred by an electromagnetic heating stirrer, after precipitates at the bottom are uniformly blended into the solution, 2ul of seed solution is absorbed by a mechanical micro-burette and is dripped on a working electrode with a conductive ink layer, the working electrode is placed in an oven for 3 minutes, the oven is set to be 85 ℃, the seed solution is dripped on the working electrode after being taken out, the seed solution is placed in the oven with the same temperature for 3 minutes, and the steps are repeated for four times, so that the complete seed layer can be completed.
The growth solution is poured into a culture dish, then the three electrodes of the paper-based chip with the seed layer face the water surface, the back surface of the substrate component is connected with the support without covering the working electrode, and the nanowire grows in a space. Then the substrate assembly is put into the growth solution, the liquid level is higher than that of the substrate assembly, the culture dish is coated by the aluminum foil paper, water vapor is not leaked out, and the substrate assembly is put into an oven, wherein the oven has two temperatures, namely high temperature and low temperature, the high temperature is 95 ℃, the low temperature is 85 ℃, and the deposition time is 4 hours. And 4 h, taking out the substrate assembly, removing the heat-resistant adhesive tape, immediately washing residual salts on the surface by using deionized water when the electrode faces upwards, putting the electrode in a clean culture dish, and then putting the culture dish in a 37 ℃ oven to dry the culture dish, thus obtaining the zinc oxide nanowire.
In order to verify the sensing effect, the invention also makes an optimized selection for the material for electrochemically catalyzing glucose, and the standard of the selection is that in the electrochemical cyclic voltammetry, the obviously outstanding current value is required by the invention.
The invention utilizes two screen printing electrodes for comparison, namely a common zinc oxide nanowire deposition chip (ZnO/SPCE) and a zinc oxide nanowire chip (ZnO/INK/SPCE) modified by adding conductive INK, and the zinc oxide nanowire is prepared to grow for four hours in the environment of 0.025M of growth solution concentration and high temperature of 95 ℃.
Both chip samples were then immersed in 1mM glucose solution for comparison, and swept from 0.0 to 1.0 using cyclic voltammetry in electrochemistry at a rate of 50mV/s, as shown in FIG. 6. From fig. 6, it can be known that the zinc oxide nanowire chip (ZnO/INK/SPCE) modified by adding the conductive INK has a current degree higher by more than about four times than that of the common deposited zinc oxide nanowire chip (ZnO/SPCE).
Because the film with the conductive ink is used as the base layer, the electrocatalysis of the zinc oxide nanowire and the glucose is more sensitive, and the current value is more obvious.
The zinc oxide is a material with high energy gap and high exciton binding energy, is a material with room temperature stability, has the characteristics of higher body surface area and rapid electron transfer as a nano material, and has great potential in electrochemistry and biosensors. According to the invention, the zinc oxide nanowire with good quality and few defects can be manufactured by utilizing a simple and low-temperature hydrothermal process.
In addition, compared with invasive blood measurement, non-invasive detection is becoming the mainstream alternative, and the glucose concentration can be directly sensed by using body fluids such as saliva, urine, sweat and the like, so that the sensing detection system of the invention is mainly aimed at the urine correlation value.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. The preparation process of the non-enzymatic glucose sensing detection system is characterized by comprising the following preparation steps of:
A. cleaning the processing substrate assembly;
B. preparing a seed solution;
C. preparing a growth solution;
D. adding conductive ink;
E. and depositing the zinc oxide nanowire.
2. The process according to claim 1, wherein the substrate assembly comprises a paper-based chip and a silicon substrate, and the paper-based chip and the silicon substrate are cleaned respectively before being attached to the silicon substrate and then placed in a transparent box.
3. The process of claim 1, wherein the paper-based chip cleaning process comprises the steps of: and taking the paper-based chip out of the packaging paper, washing with deionized water, and drying by using a nitrogen spray gun.
4. The process of claim 1, wherein the cleaning of the silicon substrate comprises the steps of:
1) soaking the silicon substrate in acetone, cleaning for 10 minutes by using an ultrasonic oscillator, removing residual oil stains on the silicon substrate, and taking out after the cleaning is finished;
2) soaking the silicon substrate in methanol, cleaning the silicon substrate for 10 minutes by using an ultrasonic oscillator, removing residual organic matters on the silicon substrate, and taking out the silicon substrate after the cleaning is finished;
3) then soaking in deionized water, oscillating for 10 minutes, removing residual cleaning liquid, and drying by blowing with a nitrogen spray gun.
5. The process of claim 1, wherein the seed solution is prepared by the steps of:
1) adding 0.12 g of sodium hydroxide into 100ml of methanol to prepare a sodium hydroxide solution;
2) adding 0.328 g of zinc acetate into 150ml of methanol to prepare a zinc acetate solution;
3) stirring the zinc acetate solution in an electromagnetic heating stirrer, heating to 60 ℃, and then dripping the sodium hydroxide solution into the zinc acetate solution at the speed of 50ml/h by using a constant-pressure delivery pump;
4) after dropping 100ml, the solution was heated at 60 ℃ for three hours, and stirred for 10 hours to prepare a seed solution of zinc oxide nanowires.
6. The process of claim 1, wherein the growth solution is prepared by the steps of:
0.7435 g of zinc nitrate hexahydrate and 0.35 g of hexamethylenetetramine are added into 100ml of deionized water and stirred uniformly.
7. The process of claim 1, wherein the addition of the conductive ink comprises the steps of:
1) taking the cleaned substrate assembly out of the transparent box, and covering a part of the paper-based chip by using a heat-resistant adhesive tape;
2) operating the substrate assembly by using a puncher, and punching a hole on the paper-based chip to expose a working electrode of the paper-based chip;
3) 0.8ul of conductive ink is absorbed by a mechanical micro-dropping pipe and dropped on the working electrode;
4) and then putting the electrode into an oven, setting the temperature of the oven at 85 ℃, baking the electrode for 30 minutes, and then taking the electrode out, so as to form a conducting layer on the working electrode.
8. The process of claim 1, wherein depositing the zinc oxide nanowires comprises the steps of:
1) uniformly stirring the seed solution by using an electromagnetic heating stirrer, and uniformly melting the sediment at the bottom into the solution;
2) 2ul of seed solution is absorbed by a mechanical micro-dropper to be dripped on the working electrode with the conductive ink layer,
baking in an oven for 3 minutes, setting the temperature of the oven to be 85 ℃, taking out, then dripping 2ul of seed solution on the working electrode, baking in the oven with the same temperature for 3 minutes, repeating for four times, and finishing the preparation of the seed layer;
3) pouring the growth solution into a culture dish, facing the electrode of the paper-based chip with the seed layer to the water surface, and connecting the back surface of the substrate assembly with a support;
4) placing the substrate assembly in a growth solution to make the liquid level higher than the substrate assembly;
5) coating a culture dish by using aluminum foil paper, and then putting the culture dish into an oven, wherein the temperature of the oven is set to be 85 ℃ or 95 ℃, and the deposition time is 4 hours;
6) after 4 hours, taking out the substrate assembly, removing the heat-resistant adhesive tape, enabling the substrate assembly to face upwards, and immediately washing residual salts on the surface by using deionized water;
7) then putting the mixture into a culture dish and then putting the culture dish into an oven to be dried at the temperature of 37 ℃, thus obtaining the zinc oxide nanowire.
9. The process according to claim 1, wherein the area of the hole is the same as the area of the working electrode.
10. A non-enzymatic glucose sensing system prepared by the process of any one of claims 1-9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113358725A (en) * | 2021-05-17 | 2021-09-07 | 西安交通大学 | Flexible electrochemical glucose sensor electrode and preparation method thereof |
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