CN114563459B - Binary biological logic gate design and application research based on zinc oxide nano particles - Google Patents
Binary biological logic gate design and application research based on zinc oxide nano particles Download PDFInfo
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 6
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- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims description 4
- MCPLVIGCWWTHFH-UHFFFAOYSA-L methyl blue Chemical compound [Na+].[Na+].C1=CC(S(=O)(=O)[O-])=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[NH+]C=2C=CC(=CC=2)S([O-])(=O)=O)C=2C=CC(NC=3C=CC(=CC=3)S([O-])(=O)=O)=CC=2)C=C1 MCPLVIGCWWTHFH-UHFFFAOYSA-L 0.000 claims description 4
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- 241001481789 Rupicapra Species 0.000 claims description 3
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- 239000010985 leather Substances 0.000 claims description 3
- 238000006395 oxidase reaction Methods 0.000 claims description 3
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 3
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- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims 1
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- 150000003445 sucroses Chemical class 0.000 claims 1
- 108091027757 Deoxyribozyme Proteins 0.000 abstract description 7
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- 229960004793 sucrose Drugs 0.000 abstract 4
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 abstract 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 abstract 1
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- 125000000143 2-carboxyethyl group Chemical group [H]OC(=O)C([H])([H])C([H])([H])* 0.000 description 1
- QEDXSHCYPROEOK-UHFFFAOYSA-N 3-phosphanylpropanoic acid Chemical compound OC(=O)CCP QEDXSHCYPROEOK-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- Hematology (AREA)
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- Biochemistry (AREA)
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Abstract
The invention relates to a binary biological logic gate design and application research based on zinc oxide nano particles, which comprises the following specific steps: firstly, saccharose is catalyzed to decompose sucrose to produce glucose, the glucose is catalyzed by glucose oxidase to produce gluconic acid, and ZnO nano particles (ZnO NPs) introduced based on the glucose are degraded into Zn when meeting acid 2+ The ZnO NPs-mediated DNAzyme signal amplification strategy can be constructed as a factor of DNAzyme cleavage amplification reaction, and is modified on an Au electrode, MB is used as a signal molecule to be output, so that the sensor is successfully prepared, and electrochemical logic analysis and detection of sucrose, glucose oxidase and sucrase are realized. Based on this, a series of "binary" biological logic gates (YES, NO, AND and INHIBIT) for ZnO NPs were constructed in order to realize sucrose, GO X And analysis and monitoring of INV, has important significance in the field of biomedicine. To date, no "binary" bio-logic gate design based on ZnO NPs has been seen and applied to GO X And INV-related biosignal pathway studies.
Description
Technical Field
The invention relates to an electrochemical biosensing method and application thereof, in particular to preparation of an electrochemical sensor based on zinc oxide nano particles and application research of the electrochemical sensor in design of a related binary logic circuit of sucrose, sucrase and glucose oxidase, belonging to the technical field of functional biological materials and biosensing.
Background
Sugar is a sweet source of food and beverages and has considerable commercial value in the food and beverage industry. Overdrinking of sugar-containing beverages can lead to excessive weight gain and diabetes, and also increase the risk of chronic health consequences. Diabetes research and clinical practice, international diabetes is combined with emphasis that 4.5 million adults worldwide have diabetes, and that children and young adults have a high incidence. Therefore, continuous, accurate and rapid detection of the concentration of glucose in blood and detection of the sugar content in food are important for the control and prevention of diabetes. For the above reasons, a plurality of familiesResearchers have been working on developing and studying high performance glucose sensors, and on the sucrose enzymes (INV), glucose Oxidase (GO) X ) And their biological signaling pathways are under very limited study. Development of a novel method for realizing GO X And INV-mediated biological signal pathway research are of great significance to clinical diagnosis and drug development.
Logic gates are the basic components of electronic and digital circuits whose logic operations are performed by using one or more logic input signals to generate one or more logic output signals, represented in two binary states (0, 1), are indispensable components in the digital world. Supermolecules, organic molecules, nucleic acids, proteins, polymers, etc. can be used as input signals for analog logic operations, and the idea of using biosensors as molecular logic gates for medical diagnosis has driven the development of this field by using input/output states to design small switches for molecular logic gates. Molecular logic system pair INV and GO with simple development and easy programming and operation X The mediated biological signal path has important significance, and is beneficial to the detection of biological molecules, the construction of molecular devices and the development of biological computing technology.
DNAzyme is a nucleic acid isolated from a combinatorial oligonucleotide library by in vitro screening, but has properties similar to those of a protease, has higher catalytic hydrolytic cleavage activity on a specific substrate, is more stable than an enzyme, and can be denatured and renatured for multiple times without losing catalytic activity on the substrate. The present invention is directed to INV and GO involved in biosignal pathway X A number of binary logic gates are designed, and the method adopts the property of zinc oxide nano particles (ZnO NPs) that the zinc oxide nano particles degrade when encountering acid, and GO is experienced in a biological signal path X The solution shows certain acidity after the catalytic reaction with the INV enzyme, and at the moment, znO NPs are degraded by acid to release Zn 2+ ，Zn 2+ Can be used as a factor of DNAzyme cleavage amplification reaction, AND based on the factor, a binary biological logic gate (YES, NO, AND, INHIBIT AND AND-INHIBIT) of a series of zinc oxide nano particles is constructed to realize sucrose AND GO X And analysis and monitoring of INV, has important significance in the field of biomedicine. To date, no radicals are foundBinary bio-logic gate design on zinc oxide nanoparticles and application thereof to GO X And INV-related biosignal pathway studies.
Disclosure of Invention
The invention aims to solve the technical problem of providing a binary biological logic gate design and application research based on zinc oxide nano particles, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.
The technical scheme adopted for solving the technical problems is as follows: the design and application research of the binary biological logic gate based on the zinc oxide nano particles comprises the following specific steps:
(1) Preparation of Zinc oxide nanoparticles (ZnO NPs)
Solution a: 10-50 mg Zn (NO) 3 )·6H 2 Adding O and 50-100 mg of polyethylene glycol (PEG) into 10-15 mL of ultrapure water, and uniformly mixing by ultrasonic waves; solution B: 70-88 mg KOH is added into 7-8 mL ultra-pure water to form the product. Controlling the temperature to be 40-50 ℃, dripping the solution B into the solution A, carrying out ultrasonic heat preservation for 10-20 min after dripping, centrifuging, washing, and redispersing in 1-5 mL of ultrapure water to obtain the ZnO NPs.
(2) Preparation of sucrase and glucose oxidase reaction solution
Enzyme reaction liquid preparation (total volume is 50-100 mu L): contains 50-100 mU M sucrose, 100-600 mU/mL sucrase, 100-400 mU/mL glucose oxidase, 0.1-1 mM NaCl and proper amount of O 2 (O in solution) 2 With O in air 2 Reaching equilibrium), and placing the mixed solution at 30-37 ℃ for reaction for 1-2 h.
(3) Preparation of electrochemical biosensor
a. Polishing gold electrode (Au, diameter 1-3 mm) on chamois leather with aluminum oxide powder with particle diameter 0.01-0.05 μm for 0.5-5 min, placing the electrode in ultrasonic cleaner, ultrasonic cleaning with ultrapure water for 1-5 min, and then N 2 Blow-drying, and marking as an electrode 1;
Zn-DNAzyme solution preparation (total volume 10-100. Mu.L): comprises 0.1-1 mu M of DNA1 (Zn-DNAzyme), 0.1-1 mu M of DNA2 (Zn-sub DNA), 0.1-1 mM of tris (2-carboxyethyl)Radical) phosphine solution (TCEP), 10 to 50mM KCl and 1 to 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES), the mixed solution was reacted at 30 to 37 ℃ for 0.1 to 1 hour, then 1 to 5 μl of the reaction solution was dropped onto Au overnight at 4 ℃, after which the electrode was treated with MCH to replace the non-specific adsorption probe, designated as electrode 2. 1 to 5 mu L of Zn in (1) 2+ The solution is dripped on the surface of the electrode 2 and reacts for 0.1 to 1 hour at the temperature of 30 to 37 ℃ and is marked as the electrode 3. Then, the DNA3 was dropped onto the surface of the electrode 3, and reacted at 30 to 37℃for 0.1 to 1 hour, which was designated as electrode 4. Subsequently, a signal solution (total volume 10 to 100. Mu.L) was prepared: comprises 0.1-1 mM methyl blue solution (MB), 0.1-1 mu M DNA4, 0.1-1 mu M DNA5, 10-50 mM KCl and 1-10 mM 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution (HEPES), and 1-5 mu L of the mixed solution is dripped on the surface of the electrode 4 to react for 1-2 hours at 30-37 ℃ to obtain the electrode 5. In the course of preparation of the electrodes 1 to 5, each modification step was completed by slowly rinsing the electrodes with ultrapure water to remove unreacted or ungrafted complete reagent.
c. For constructing the sucrase or glucose oxidase electrochemical biosensor, 1 to 2. Mu.L of the ZnO NPs solution prepared in (1) and 2 to 18. Mu.L of the enzyme reaction solution prepared in (2) are mixed and reacted at 30 to 37 ℃ for 1 to 2 hours. In the preparation process of the electrode 3 in the step b, 1.5-5 mu L of the reaction liquid is dripped on the electrode 2 and reacts for 0.5-1 h at the temperature of 30-37 ℃, and other experimental steps are the same as the step b. The quantitative monitoring of sucrose, glucose oxidase and sucrose can be realized by changing the sucrose content (0-1000 mU M), the sucrase content (0-1000 mU/mL) and the glucose oxidase content (0-500 mU/mL) in an enzyme reaction system and other steps as above.
This patent relates to five DNA (1, 2,3,4 and 5) sequences:
by using the design and application research of the binary biological logic gate based on the zinc oxide nano particles, the MB in the signal unit is detected by adopting square wave voltammetry, the potential range is set to be-0.4 to-0.1V, the amplitude is 15Hz, and the Zn is used for the detection of the MB 2+ Is a guide to the generation of (1)The whole system is completed, and the enzyme reaction solution can promote Zn 2+ Thus a series of different concentrations of sucrose, INV and GO can be obtained X Corresponding current magnitude, establishing current response and cross, INV and GO X Quantitative relation between the two, and determining the cross, the INV and the GO in the sample to be detected according to the quantitative relation between the two X Is contained in the composition.
The principle of the invention: the invention relates to a binary biological logic gate design and application research based on zinc oxide nano particles, which firstly adopts the property of ZnO NPs degrading when encountering acid, and reacts with enzyme solution to release Zn 2+ The ZnO NPs-mediated DNAzyme signal amplification strategy can be constructed as a factor of DNAzyme cleavage amplification reaction, and is modified on Au, MB is used as a signal molecule to be output, so that the sensor is successfully prepared. Since the solution after the reaction of sucrose, glucose oxidase and sucrase is acidic, zn can be released by mixing the solution with ZnO NPs based on the acidity 2+ Therefore, the analysis and detection of sucrose, glucose oxidase and sucrase are realized, and a simple, rapid, high-sensitivity, high-selectivity and label-free electrochemical logic analysis method is constructed.
Compared with the prior art, the invention has the advantages that: the invention constructs a binary biological logic gate design based on zinc oxide nano particles and a logic gate alignment method thereof X And INV-related biosignal pathway. First, zn is released by mixing ZnO NPs with an enzyme reaction solution 2+ Capable of cleaving its DNAzyme; secondly, performing nucleic acid amplification by utilizing HCR reaction, simultaneously embedding MB as a signal molecule, and detecting different concentrations of the cross, the INV and the GO by adopting a square wave voltammetry detection sensor X Is a function of the electrochemical response of the battery. Obviously, in the whole analysis strategy, znO NPs, cross, INV and GO X In the absence of unavailability, based on this YES, AND, INHIBIT AND AND-AND-INHIBIT logic gates were constructed. The advantages are that:
(1) High sensitivity. The invention relates to a binary biological logic gate design based on zinc oxide nano particles and a GO thereof X And the research of the INV related biological signal path respectively obtains three linear equations: current response to cross concentrationThe linear correlation equation is y=0.934 lgC sucrose +1.721，r 2 =0.9995, limit of detection 0.019 μm; the linear correlation equation of the current response to the concentration of INV is y=0.861 lgC INV +1.35，r 2 = 0.9950, limit of detection 0.047mU/mL; current response GO X The linear correlation equation of the concentration is y=0.680 lgC GOx +1.098，r 2 = 0.9976, the limit of detection is 0.012mU/mL. Explaining the sensor pair cross, INV and GO X High sensitivity detection is achieved.
(2) Setting ZnO NPs, cross, INV or GO X As signal inputs, MB current signals are output as signals, constructing 4 YES logic gates.
(3) Setting INV and GO X As signal inputs 1 AND 2, mb current signals are output as signals, constructing AND logic gates.
(4) ZnO NPs and EDTA are set as signal inputs 1 and 2, MB current signals are set as signal outputs, and INHIBIT logic gates are constructed.
(5) The preparation and detection method has the advantages of less reagent consumption and low cost. The invention can realize the sterilization of the sucrose, the INV and the GO by only consuming a small amount of materials and reagents X Is a high sensitivity detection of (1).
(6) Based on the sucrose, INV and GO X The roles of mutual connection and interdependence in a biological signal path are helpful for realizing the roles of cross, INV and GO X Application study of related biological signal paths.
In summary, the present invention relates to a binary bio-logic gate design based on zinc oxide nanoparticles and a logic gate for GO X The research of the biological signal path related to the INV has the advantages of high sensitivity, good selectivity, simple operation, quick analysis, easy operation and the like, and can realize the low concentration of the cross, the INV and the GO X Has good application prospect.
Drawings
FIG. 1 is a diagram of an electrocatalytic experiment of a sensor of the present invention versus MB;
FIG. 2 shows the sensor pairs of the present invention, including cross, INV, and GO X Analyzing the detected calibration curve graph;
FIG. 3 is a diagram of the construction of a ZnO NPs "YES" logic gate in the present invention;
FIG. 4 is a schematic diagram of the construction of a cross "YES" logic gate in accordance with the present invention;
FIG. 5 shows GO in the present invention X Construction of a YES logic gate;
FIG. 6 is a construction of an INV "YES" logic gate in the present invention;
FIG. 7 is a diagram of INV/GO in the present invention X Construction of an AND logic gate;
FIG. 8 is a schematic diagram of the construction of a ZnO NPs/EDTA "INHIBIT" logic gate in the present invention;
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
Example 1 preparation of ZnO NPs complexes and sensors
(1) Preparation of Zinc oxide nanoparticles (ZnO NPs)
Solution a: 50mg Zn (NO) 3 )·6H 2 Adding O and 100mg of polyethylene glycol (PEG) into 15mL of ultrapure water, and uniformly mixing by ultrasonic; solution B: 88mg of KOH was added to 8mL of ultrapure water to form. Controlling the temperature to be 50 ℃, dropwise adding the solution B into the solution A, carrying out ultrasonic heat preservation for 20min after the dropwise adding, centrifuging, washing, and redispersing in 5mL of ultrapure water to obtain the ZnO NPs.
(2) Preparation of sucrase and glucose oxidase reaction solution
Enzyme reaction solution preparation (total volume 100 μl): comprises 100 mU M sucrose, 600mU/mL sucrase, 400mU/mL glucose oxidase, 1mM NaCl and proper amount of O 2 (O in solution) 2 With O in air 2 Equilibrium is reached) and the mixed solution is allowed to react at 37℃for 2h.
(3) Preparation of electrochemical biosensor
a. Polishing gold electrode (Au, diameter 3 mm) on chamois leather with aluminum oxide powder with particle diameter of 0.05 μm for 5min, placing the electrode in ultrasonic cleaner, ultrasonic cleaning with ultrapure water for 5min, and then N 2 Blow-drying, and marking as an electrode 1;
Zn-DNAzyme solution preparation (total volume 100. Mu.L): containing 1. Mu.M DNA1 (Zn-DNAzyme), 1. Mu.M DNA2 (Zn-sub DNA), 1mM tri-DNA(2-carboxyethyl) phosphine solution (TCEP), 50mM KCl and 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), this mixed solution was reacted at 37℃for 1 hour, followed by dropping 5. Mu.L of the reaction solution onto Au at 4℃overnight, after which the nonspecific adsorption probe was replaced with the MCH-treated electrode, which was designated as electrode 2. mu.L of Zn in (1) 2+ The solution was dropped onto the surface of electrode 2 and reacted at 37℃for 1 hour, which was designated as electrode 3. Then, the DNA3 was dropped onto the surface of the electrode 3, and reacted at 37℃for 1 hour, which was designated as electrode 4. Subsequently, a signal solution (total volume of 100 μl) was prepared: 1mM methyl blue solution (MB), 1. Mu.M DNA4, 1. Mu.M DNA5, 50mM KCl and 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES), and 5. Mu.L of the mixture was dropped onto the surface of electrode 4, and reacted at 37℃for 2 hours, which was designated as electrode 5. In the course of preparation of the electrodes 1 to 5, each modification step was completed by slowly rinsing the electrodes with ultrapure water to remove unreacted or ungrafted complete reagent.
As can be seen from fig. 1, MB is able to output electrochemical signals well as the hybridization chain reaction proceeds, indicating successful sensor fabrication.
Example 2 feasibility experiment
In order to construct a sucrase or glucose oxidase electrochemical biosensor according to the above-described sensor preparation procedure of example 1, 2. Mu.L of the ZnO NPs solution prepared in example 1 (1) was mixed with 18. Mu.L of the enzyme reaction solution of example 2 (2) and reacted at 37℃for 2 hours. In the preparation of electrode 3 in b, 5. Mu.L of the reaction solution was dropped onto electrode 2 and reacted at 37℃for 1 hour, and the other experimental steps were the same as in b. The quantitative monitoring of sucrose, glucose oxidase and sucrose can be realized by changing the sucrose content (0, 0.1, 0.5, 1, 5, 10, 30, 50, 70, 90, 110, 130, 150, 180, 200, 400, 600, 800, 1000mU M), the sucrase content (0, 0.5, 1, 5, 10, 30, 50, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mU/mL) and the glucose oxidase content (0, 0.1, 0.5, 1, 5, 10, 15, 20, 30, 50, 70, 90, 110, 150, 200, 250, 300, 350, 400, 450, 500 mU/mL) in an enzyme reaction system. The results are shown in FIGS. 2A, 2B and 2C, and it can be seen that the sensor pairs are cross, INV and GO X Is good in current response and concentrationThe current response of the sensor is given by y=0.934 lgC in relation to the linear correlation equation of the cross concentration sucrose +1.712，r 2 =0.9995, limit of detection 0.019 μm; the linear correlation equation of the current response to the concentration of INV is y=0.861 lgC INV +1.35，r 2 = 0.9950, detection limit of 0.047mU/mL, current response to GO X The linear correlation equation of the concentration is y=0.680 lgC GOx +1.098，r 2 = 0.9976, the limit of detection is 0.012mU/mL. Explaining the sensor pairs cross, INV and GO X High-sensitivity detection is realized.
EXAMPLE 3 construction of ZnO NPs "YES" logic Gate
The "YES" logic gate for ZnO NPs was constructed as per the preparation of ZnO NPs of example 1, comparing the electrochemical response to MB of the resulting polymer-constructed sensor in the presence and absence of ZnO NPs, as shown in the following table:
the results are shown in FIG. 3, which demonstrates good electrochemical response to MB in the presence of ZnO NPs, consistent with the "YES" logic gate characteristics.
Example 4 construction of a cross "YES" logic Gate
The "YES" logic gate for the preparation of ZnO NPs as in example 1 was constructed as compared to the electrochemical response to MB of the resulting polymer-constructed sensor in the presence and absence of crossover, as shown in the following table:
the results are shown in FIG. 4, which demonstrates good electrochemical response to MB in the presence of crossover, consistent with the "YES" logic gate feature.
Example 5 construction of INV "YES" logic Gate
The "YES" logic gate for INV was constructed as per the preparation of ZnO NPs of example 1, comparing the electrochemical response of the prepared polymer-built sensor to MB in the presence and absence of INV, as shown in the following table:
the results are shown in FIG. 5, which demonstrates good electrochemical response to MB in the presence of INV, consistent with the "YES" logic gate feature.
Example 6 GO X Construction of "YES" logic gates
Preparation of ZnO NPs as in example 1, comparative presence of GO X And no GO is present X Electrochemical response of the polymer-built sensor to MB is obtained, and GO is built X The "YES" logic gate of (c):
the results are shown in FIG. 6, demonstrating GO X When present, has good electrochemical response to MB, and accords with the characteristic of a 'YES' logic gate.
Example 7 INV/GO X Construction of AND logic gates
Preparation of ZnO NPs as in example 1, comparison of INV (input 1) and GO X (input 2) two inputs make electrochemical response of Polymer-built sensor to MB, build INV/GO X The "AND" logic gate of (2) is as follows:
the results are shown in FIG. 7, demonstrating INV and GO X AND when the two-phase-change type organic light emitting diode AND the organic light emitting diode exist simultaneously, the organic light emitting diode has good electrochemical response to MB, AND accords with the AND logic gate characteristic.
EXAMPLE 8 construction of ZnO NPs/EDTA "INHIBIT" logic gates
The electrochemical response of the polymer-built sensor to MB was made as per the preparation of ZnO NPs of example 1, comparing the two inputs ZnO NPs (input 1) and EDTA (input 2), and an "INHIBIT" logic gate of ZnO NPs/EDTA was constructed as follows:
the results are shown in FIG. 8, which demonstrates that ZnO NPs, in the absence of EDTA, have good electrochemical response to MB, consistent with the "INHIBIT" logic gate characteristics.
Of course, the above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions that would be within the spirit and scope of the invention would be within the purview of one of ordinary skill in the art.
Claims (6)
1. A method for electrochemical analysis of sucrose, glucose oxidase and sucrase based on zinc oxide nanoparticles, comprising the steps of:
(1) Preparation of ZnO NPs as zinc oxide nanoparticles
Solution a: 10-50 mg Zn (NO) 3 )·6H 2 Adding O and 50-100 mg of polyethylene glycol into 10-15 mL of ultrapure water, and uniformly mixing by ultrasonic; solution B: adding 70-88 mg KOH into 7-8 mL ultrapure water to form the product; controlling the temperature to be 40-50 ℃, dripping the solution B into the solution A, carrying out ultrasonic heat preservation for 10-20 min after dripping, centrifuging, washing, and redispersing in 1-5 mL of ultrapure water to obtain ZnO NPs;
(2) Preparation of sucrase and glucose oxidase reaction solution
Preparing an enzyme reaction solution: the total volume is 50-100 mU L, and the total volume comprises 50-100 mU M sucrose, 100-600 mU/mL sucrase, 100-400 mU/mL glucose oxidase, 0.1-1 mM NaCl and proper amount of O 2 To make O in solution 2 With O in air 2 The mixed solution is placed at 30-37 ℃ to react for 1-2 h when the balance is reached;
(3) Preparation of electrochemical biosensor
a. Polishing gold electrode Au with diameter of 1-3 mm on chamois leather with aluminum oxide powder with particle diameter of 0.01-0.05 μm for 0.5-5 min, placing the electrode in an ultrasonic cleaner, ultrasonically cleaning with ultrapure water for 1-5 min, and then using N 2 Blow-drying, and marking as an electrode 1;
Zn-DNAzyme solution preparation: the total volume is 10-100 mu L, 0.1-1 mu M DNA1, 0.1-1 mu M DNA2, 0.1-1 mM tri (2-carboxyethyl) phosphine solution, 10-50 mM KCl and 1-10 mM 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution are contained, the mixed solution is placed at 30-37 ℃ to react for 0.1-1 h, then 1-5 mu L of reaction liquid is dripped on Au at 4 ℃ overnight, and then a non-specific adsorption probe is replaced by a mercapto hexanol MCH treatment electrode, which is marked as an electrode 2; 1 to 5 mu L of Zn 2+ Dropping the solution onto the surface of the electrode 2, and reacting at 30-37 ℃ for 0.1-1 h, namely the electrode 3; then, dropping the DNA3 into the surface of the electrode 3, and reacting for 0.1-1 h at 30-37 ℃ to obtain an electrode 4; subsequently, a signal solution was prepared: the total volume is 10-100 mu L, 0.1-1 mM methyl blue MB solution, 0.1-1 mu M DNA4, 0.1-1 mu M DNA5, 10-50 mM KCl and 1-10 mM 4-hydroxyethyl piperazine ethane sulfonic acid buffer solution are contained, 1-5 mu L of mixed solution is dripped on the surface of the electrode 4, and the reaction is carried out for 1-2 hours at the temperature of 30-37 ℃ and is marked as electrode 5; in the preparation process of the electrodes 1 to 5, each time a modification step is completed, the electrodes are slowly rinsed by ultrapure water to remove unreacted or ungrafted complete reagent;
c. construction of sucrose, sucrase or glucose oxidase electrochemical biosensor
Mixing 1-2 mu L of ZnONPs solution prepared in the step (1) with 2-18 mu L of enzyme reaction solution prepared in the step (2) and reacting for 1-2 hours at 30-37 ℃ to obtain reaction solution; in the preparation process of the electrode 3 in the step b, 1.5-5 mu L of the reaction liquid is dripped on the electrode 2 and reacts for 0.5-1 h at the temperature of 30-37 ℃, and other operations are the same as the step b;
changing the sucrose content in the enzyme reaction liquid to be 0-1000 mU M, the sucrase content to be 0-1000 mU/mL and the glucose oxidase content to be 0-500 mU/mL, and realizing the quantitative monitoring of sucrose, sucrase or glucose oxidase by other steps as above;
wherein, the DNA1 sequence is: HS-TTT CCA CCA CTT TTT ACC CAC TAT rA GGA AGT CTG AAT TTT GTG GTG G;
the DNA2 sequence is: TAT CAG ACT TCT CCG AGC CGG TCG AAA TAG TGG GT;
the DNA3 sequence is: ACT AAA AGG GTC TGA GGG ATA GTG GGT AAA AAG TGG TGG;
the DNA4 sequence is: CCC TCA GAC CCT TTT AGT TAC TCC CCC AGG TGC;
the DNA5 sequence is: ACT AAA AGG GTC TGA GGG GCA CCT GGG GGA GTA.
2. The method according to claim 1, wherein the detection of methyl blue MB in the signal cell is performed by square wave voltammetry, with a potential ranging from-0.4 to-0.1V and an amplitude of 15Hz, due to Zn 2+ The formation of (C) leads to the completion of the whole system, and the enzyme reaction solution can promote Zn 2+ Therefore, the current corresponding to a series of sucrose, sucrase or glucose oxidase with different concentrations can be obtained, the quantitative relation between the current response and the sucrose, the sucrase and the glucose oxidase is established, and the contents of the sucrose, the sucrase and the glucose oxidase in the sample to be detected are determined according to the quantitative relation between the current response and the sucrose, the sucrase and the glucose oxidase.
3. The electrochemical analysis method according to claim 1 or 2, wherein the linear correlation equation of the current response of the sensor to the concentration of sucrose is y = 0.934lgC Sucrose +1.712，r 2 =0.9995, limit of detection 0.019 μm.
4. The electrochemical analysis method according to claim 1 or 2, characterized in that the linear correlation equation of the current response of the sensor to the sucrase concentration is y = 0.861lgC Sucrase +1.35，r 2 = 0.9950, the limit of detection was 0.047mU/mL.
5. The electrochemical analysis method according to claim 1 or 2, wherein the linear correlation equation of the current response of the sensor to the glucose oxidase concentration is y=0.680 lgC Glucose oxidationEnzymes +1.098，r 2 = 0.9976, the limit of detection is 0.012mU/mL.
6. Use of a "binary" biological logic gate based on zinc oxide nanoparticles, characterized in that it comprises the following steps: constructing a sucrose, sucrase or glucose oxidase electrochemical biosensor according to the method of claim 1 or 2, using MB electrochemical signal as signal output, different logic gates having different input signals: (1) constructing a 'YES' logic gate by taking ZnO NPs as signal input; (2) the sucrose is taken as a signal input to construct a YES logic gate; (3) the sucrase is taken as a signal input to construct a 'YES' logic gate; (4) constructing a 'YES' logic gate by taking glucose oxidase as a signal input; (5) the sucrase AND glucose oxidase are taken as signal inputs to construct an AND logic gate; (6) ZnO NPs and EDTA are used as signal inputs to construct an INHIBIT logic gate.
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