CN114563459A - Design and application research of 'binary' biological logic gate based on zinc oxide nanoparticles - Google Patents
Design and application research of 'binary' biological logic gate based on zinc oxide nanoparticles Download PDFInfo
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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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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 catalyzes and decomposes sucrose to generate glucose, the glucose is catalyzed by glucose oxidase to generate gluconic acid, and ZnO nanoparticles (ZnO NPs) introduced based on the glucose are degraded into Zn when meeting acid2+The gene can be used as a factor of DNAzyme cutting amplification reaction, a DNAzyme signal amplification strategy mediated by ZnO NPs is constructed, the gene is modified on an Au electrode, MB is used as signal molecule output, a sensor is successfully prepared, and electrochemical logic analysis and detection of sucrose, glucose oxidase and sucrase are realized. Based on the method, a series of 'binary' biological logic gates (YES, NO, AND AND INHIBIT) of ZnO NPs are constructed so as to realize sucrose AND GOXAnd INV analysis and monitoring, have great significance in the biomedical field. To date, no "binary" bio-logic gate design based on ZnO NPs has been seen and applied to GOXAnd INV-related bio-signaling pathway studies.
Description
Technical Field
The invention relates to an electrochemical biological sensing method and application thereof, in particular to preparation of an electrochemical sensor based on zinc oxide nanoparticles and application research thereof in design of a binary logic circuit related to sucrose, sucrase and glucose oxidase, belonging to the technical field of functional biological materials and biological sensing.
Background
The sugar is food andthe source of sweetness for beverages has considerable commercial value in the food and beverage industry. Over-consumption of sugar-containing beverages can lead to excessive weight gain and diabetes, as well as an increased risk of chronic health consequences. Diabetes research and clinical practice, the international union of diabetes emphasizes that 4.5 million adults worldwide suffer from diabetes, with higher incidence rates in both children and young adults. Therefore, continuous, accurate and rapid detection of the concentration of glucose in blood and detection of the content of sugar in food are important for the control and prevention of diabetes. For the above reasons, many researchers have been working on developing and researching high-performance glucose sensors, including sucrase (INV) and Glucose Oxidase (GO)X) And their bio-signal pathways have been studied very limitedly. Developing a novel approach to implement GO XAnd INV mediated biological signal pathway research has great significance for clinical diagnosis and drug development.
Logic gates are an essential component of electronic and digital circuits, whose logical operations are performed by generating one or more logical output signals, represented by two binary states (0, 1), using one or more logical input signals, and are indispensable components in the digital world. The development of the field has been promoted by the idea that supramolecules, organic molecules, nucleic acids, proteins, polymers, etc. can be used as input signals for analog logic operations, that small switches of molecular logic gates can be designed using input/output states, and that biosensors are used for medical diagnostics as molecular logic gates. Molecular logic System Pair INV and GO that is simple to develop and easy to program and operateXThe research on mediated biological signal paths has important significance, and is beneficial to the detection of biological molecules, the construction of molecular devices and the development of biological computing technology.
DNAzymes are nucleic acids isolated from combinatorial oligonucleotide libraries by in vitro screening, but have properties similar to proteinases, have higher catalytic hydrolytic cleavage activity on specific substrates, are more stable than enzymes, and can be denatured and renatured many times without losing catalytic activity on substrates. The present invention addresses INV and GO involved in bio-signal pathways XMany 'binary' logic gates are designedThe method adopts the property that zinc oxide nano particles (ZnO NPs) are degraded when meeting acid, and GO passes through a biological signal pathXThe solution after the catalytic reaction with INV enzyme presents certain acidity, and ZnO NPs are degraded when meeting acid to release Zn2+，Zn2+Can be used as a factor for DNAzyme cutting 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 nanoparticles is constructed so as to realize sucrose AND GOXAnd INV analysis and monitoring, have important meaning in the biomedical field. To date, no "binary" bio-logic gate design based on zinc oxide nanoparticles has been seen and applied to GOXAnd INV-related bio-signaling 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 nanoparticles, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.
The technical scheme adopted by the invention for solving the technical problems is as follows: the 'binary' biological logic gate design and application research 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 of Zn (NO)3)·6H2Adding O and 50-100 mg of polyethylene glycol (PEG) into 10-15 mL of ultrapure water, and ultrasonically mixing uniformly; solution B: adding 70-88 mg of KOH into 7-8 mL of ultrapure water to form the catalyst. And controlling the temperature to be 40-50 ℃, dropwise adding the solution B into the solution A, carrying out ultrasonic heat preservation for 10-20 min after dropwise adding is finished, centrifuging, washing, and re-dispersing in 1-5 mL of ultrapure water to obtain the ZnO NPs.
(2) Preparation of sucrase and glucose oxidase reaction solution
Preparing an enzyme reaction solution (the total volume is 50-100 mu L): contains 50-100 μ M sucrose, 100-600 mU/mL sucrase, 100-400 mU/mL glucose oxidase, 0.1-1 mM NaCl, and appropriate amount of O2(O in solution)2With O in air2Reaching equilibrium), and reacting the mixed solution at 30-37 DEG C1～2h。
(3) Preparation of electrochemical biosensor
a. Polishing gold electrode (Au, diameter of 1-3 mm) on chamois leather with aluminium oxide powder with particle size of 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 cleaning with N2Drying, and marking as an electrode 1;
preparing a Zn-DNAzyme solution (the total volume is 10-100 mu L): the method comprises the steps of preparing a mixed solution containing 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) phosphine solution (TCEP), 10-50 mM of KCl and 1-10 mM of 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES), placing the mixed solution at 30-37 ℃ for reaction for 0.1-1 h, then dropping 1-5 mu L of the reaction solution on Au for overnight at 4 ℃, and then treating an MCH electrode to replace a non-specific adsorption probe, wherein the electrode is marked as electrode 2. 1-5 μ L of Zn in (1) 2+The solution is dripped on the surface of the electrode 2 to react for 0.1 to 1 hour at the temperature of 30 to 37 ℃, and the electrode 3 is marked. Then, DNA3 is dripped on the surface of the electrode 3, and the reaction is carried out for 0.1-1 h at the temperature of 30-37 ℃, and the electrode is marked as an electrode 4. Subsequently, a signal solution (total volume 10-100. mu.L) was prepared: the electrode 5 is prepared by dropping 1 to 5. mu.L of a mixture containing 0.1 to 1mM methyl blue solution (MB), 0.1 to 1. mu.M DNA4, 0.1 to 1. mu.M DNA5, 10 to 50mM KCl and 1 to 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES) onto the surface of the electrode 4, and reacting at 30 to 37 ℃ for 1 to 2 hours. In the preparation process from the electrode 1 to the electrode 5, each time a modification step is completed, the electrode is slowly rinsed with ultrapure water to remove unreacted or incompletely grafted reagents.
c. In order to construct a sucrase or glucose oxidase electrochemical biosensor, 1-2 muL of ZnO NPs solution prepared in (1) and 2-18 muL of enzyme reaction solution in (2) are mixed and reacted at 30-37 ℃ for 1-2 h. In the preparation process of the electrode 3 in the step b, 1.5 to 5 mu L of the reaction solution is dripped onto the electrode 2 to react for 0.5 to 1 hour at the temperature of between 30 and 37 ℃, and other experimental steps are the same as those in the step b. The quantitative monitoring of the sucrose, the glucose oxidase and the 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 the enzyme reaction system in the same way in other steps.
This patent relates to five pieces of DNA (1, 2, 3, 4 and 5) whose sequences are:
by utilizing the design and application research of the 'binary' biological logic gate based on the zinc oxide nanoparticles, the MB in the signal unit is detected by adopting a square wave voltammetry method, the potential range is set to be-0.4 to-0.1V, the amplitude is 15Hz, and Zn is used2+The formation of (2) leads the completion of the whole system, and the enzyme reaction solution can promote Zn2+Thus, a series of different concentrations of sucrose, INV and GO can be obtainedXCorresponding current magnitude, establishing current response and cross, INV and GOXThe quantitative relation between the two, namely determining the cross, the INV and the GO in the sample to be detected according to the quantitative relation between the twoXThe content of (b).
The invention principle is as follows: the invention relates to a 'binary' biological logic gate design and application research based on zinc oxide nano particles, which firstly adopts the property that ZnO NPs are degraded when meeting acid and reacts with enzyme solution to release Zn2+The gene can be used as a factor for DNAzyme cleavage amplification reaction, a ZnO NPs-mediated DNAzyme signal amplification strategy is constructed, the gene is modified on Au, MB is used as a signal molecule to be output, and the sensor is successfully prepared. Since the solution is acidic after the reaction of sucrose, glucose oxidase and sucrase, Zn can be released by mixing the solution with ZnO NPs based on the acidity 2+Therefore, the method realizes the analysis and detection of the sucrose, the glucose oxidase and the sucrase, and constructs a simple, rapid, high-sensitivity, high-selectivity and label-free electrochemical logic analysis method.
Compared with the prior art, the invention has the advantages that: the invention constructs the design of 'binary' biological logic gate based on zinc oxide nano particles and the GO thereofXAnd the study of INV-related biological signal pathways. First, Zn is released by mixing ZnO NPs with an enzyme reaction solution2+Capable of cleaving its DNAzyme; secondly, the HCR reaction is used for nucleic acid amplification, MB is embedded as a signal molecule, and square wave voltage is adoptedAnn method for detecting different concentrations of sucrose, INV and GO by using sensorXThe electrochemical response of (2). It is clear that ZnO NPs, sucralose, INV, and GO are present in the overall analysis strategyXOn the basis of the fact that the method is not applicable, YES, AND, INHIBIT AND AND-AND-INHIBIT logic gates are 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 pair GO thereofXAnd the research of the biological signal path related to the INV respectively obtains three linear equations: the linear correlation equation of current response to sucrose concentration is that y is 0.934lgC sucrose+1.721，r20.9995 with detection limit of 0.019 μ M; the linear correlation equation of the current response to the INV concentration is that y is 0.861lgCINV+1.35，r20.9950, the detection limit is 0.047 mU/mL; current response pair GOXConcentration linear correlation equation is that y is 0.680lgCGOx+1.098，r2The detection limit was 0.012mU/mL, 0.9976. Illustrating the sensor pairs cross, INV and GOXHigh sensitivity detection is realized.
(2) Setting ZnO NPs, Cross, INV or GOXAs signal input, the MB current signal is output as a signal, and 4 YES logic gates are constructed.
(3) Setting INV and GOXAs signal inputs 1 AND 2, the MB current signal is outputted as a signal, AND logic gates are constructed.
(4) ZnO NPs and EDTA are set as signal inputs 1 and 2, and MB current signals are set as signal outputs, so that an INHIBIT logic gate is constructed.
(5) The preparation and detection method has the advantages of less reagent dosage and low cost. The invention can realize cross, INV and GO only by consuming a small amount of materials and reagentsXHigh sensitivity detection.
(6) Based on cross, INV and GOXInterrelated and interdependent roles in bio-signal pathways that contribute to both cross, INV, and GOXAnd (3) application research of related biological signal paths.
In summary, the present invention is based on the "binary" bio-logic gate design of zinc oxide nanoparticles and its pair GO XAnd INV phaseThe research on the biological signal path has the advantages of high sensitivity, good selectivity, simple operation, rapid analysis, easy operation and the like, and can realize low concentration of sucrose, INV and GOXThe detection has good application prospect.
Drawings
FIG. 1 is a diagram of an electrocatalytic experiment of a sensor of the present invention on MB;
FIG. 2 shows the sensor pairs cross, INV and GO of the present inventionXAnalyzing the detected calibration curve graph;
FIG. 3 is the construction of a ZnO NPs "YES" logic gate of the present invention;
FIG. 4 is a construction of a cross "YES" logic gate of the present invention;
FIG. 5 shows GO in the present inventionXConstructing a YES logic gate;
FIG. 6 is a diagram illustrating the construction of INV "YES" logic gates in the present invention;
FIG. 7 is INV/GO of the present inventionXConstructing an 'AND' logic gate;
FIG. 8 is the construction of the ZnO NPs/EDTA "INHIBIT" logic gate of the present invention;
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
EXAMPLE 1 preparation of ZnO NPs composites and Sensors
(1) Preparation of Zinc oxide nanoparticles (ZnO NPs)
Solution A: 50mg of Zn (NO)3)·6H2Adding O and 100mg polyethylene glycol (PEG) into 15mL of ultrapure water, and ultrasonically mixing uniformly; solution B: formed by adding 88mg of KOH into 8mL of ultrapure water. And controlling the temperature to be 50 ℃, dropwise adding the solution B into the solution A, carrying out ultrasonic heat preservation for 20min after dropwise adding is finished, centrifuging, washing, and re-dispersing 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. mu.L): contains 100 mU M sucrose, 600mU/mL sucrase, 400mU/mL glucose oxidase, 1mM NaCl, and appropriate amount of O2(O in solution)2With O in the air2Equilibrium is reached), the mixed solution is placed at 37 ℃ for reaction for 2 h.
(3) Preparation of electrochemical biosensor
a. Polishing gold electrode (Au, diameter of 3mm) on chamois leather with aluminum oxide powder with particle size of 0.05 μm for 5min, ultrasonic cleaning the electrode with ultrapure water in ultrasonic cleaner for 5min, and then cleaning with N2Drying, 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 tris (2-carboxyethyl) phosphine solution (TCEP), 50mM KCl and 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES), the mixed solution was left to react at 37 ℃ for 1h, followed by dropping 5. mu.L of the reaction solution onto Au overnight at 4 ℃ after which the nonspecific adsorption probe was replaced with an MCH-treated electrode, which was designated as electrode 2. Adding 5 mu L of Zn in the step (1)2+The solution was dropped onto the surface of the electrode 2 and reacted at 37 ℃ for 1 hour, which was designated as electrode 3. Then, DNA3 was dropped onto the surface of the electrode 3, and the reaction was carried out at 37 ℃ for 1 hour to obtain an electrode 4. Subsequently, a signal solution (total volume 100 μ L) was prepared: mu.L of the mixture containing 1mM methyl blue solution (MB), 1. mu.M DNA4, 1. mu.M DNA5, 50mM KCl and 10mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES) was dropped onto the surface of electrode 4, and the mixture was reacted at 37 ℃ for 2 hours, which was designated as electrode 5. In the preparation process from the electrode 1 to the electrode 5, each time a modification step is completed, the electrode is slowly rinsed with ultrapure water to remove unreacted or incompletely grafted reagents.
As can be seen from FIG. 1, MB is able to output an electrochemical signal well as the hybridization chain reaction proceeds, indicating that the sensor was successfully prepared.
EXAMPLE 2 feasibility test
To construct a sucrase or glucose oxidase electrochemical biosensor, 2. mu.L of the ZnO NPs solution prepared in example 1(1) and 18. mu.L of the enzyme reaction mixture in example 2(2) were mixed and reacted at 37 ℃ for 2 hours according to the above-described sensor preparation procedure of example 1. In the preparation process of the electrode 3 in the b, 5 μ L of the reaction solution is dripped on the electrode 2 to react for 1h at 37 ℃, and other experimental steps are the same as the b. The content of sucrose in the enzyme reaction system is changed (0, 0.1, 0.5, 1, 5, 10, 30, 50, 70, 90, 110,130. 150, 180, 200, 400, 600, 800, 1000 μ M), sucrase content (0, 0.5, 1, 5, 10, 30, 50, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000mU/mL) and 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, 500mU/mL), and other steps are the same as above, quantitative monitoring of sucrose, glucose oxidase, sucrose can be achieved. The results are shown in FIGS. 2A, 2B and 2C, and it can be seen that the sensor pairs suclose, INV and GO XHas a good linear relation with the concentration, and the linear correlation equation of the current response of the sensor to the concentration of the cross is that y is 0.934lgCsucrose+1.712，r20.9995 with detection limit of 0.019 μ M; the linear correlation equation of the current response to the INV concentration is that y is 0.861 lgCINV+1.35，r20.9950, detection limit of 0.047mU/mL, current response to GOXConcentration linear correlation equation is that y is 0.680lgCGOx+1.098，r2The detection limit was 0.012mU/mL, 0.9976. Illustrating sensor pairs cross, INV, and GOXHigh sensitivity detection is realized.
Example 3 construction of ZnO NPs "YES" logic gates
The electrochemical response of sensors constructed from the polymers prepared in the presence and absence of ZnO NPs to MB was compared as prepared from the ZnO NPs of example 1 to construct "YES" logic gates for the ZnO NPs, as shown in the following table:
the results are shown in FIG. 3, demonstrating good electrochemical response to MB in the presence of ZnO NPs, consistent with the "YES" logic gate characteristics.
Example 4 construction of Cross "YES" logic gates
The electrochemical response of the polymer-constructed sensors to MB was compared with the electrochemical response of the prepared polymer-constructed sensors in the presence and absence of sucralose prepared as in the ZnO NPs preparation of example 1 to construct a sucralose "YES" logic gate, as shown in the following table:
the results are shown in fig. 4, demonstrating a good electrochemical response to MB in the presence of sucrose, consistent with the "YES" logic gate feature.
Example 5 construction of INV "YES" logic gates
The electrochemical response of sensors constructed from polymers made in the presence and absence of INV to MB was compared as prepared from ZnO NPs of example 1 to construct "YES" logic gates for INV, as follows:
the results are shown in FIG. 5, demonstrating a good electrochemical response to MB in the presence of INV, consistent with the "YES" logic gate feature.
Example 6 GOXConstruction of "YES" logic gates
Preparation of ZnO NPs as in example 1, comparison with GOXAnd the absence of GOXThe electrochemical response of the sensor constructed by the prepared polymer to the MB is used for constructing GOXIs "YES" logic gate, as follows:
the results are shown in FIG. 6, demonstrating GOXWhen present, there is a good electrochemical response to MB, consistent with the "YES" logic gate characteristics.
Example 7 INV/GOXConstruction of an "AND" logic gate
Preparation of ZnO NPs as in example 1, comparison of INV (input 1) and GOX(input 2) two inputs electrochemical of Polymer-constructed sensor couple MBIn response, construct INV/GOXAND logic gates, as follows:
the results are shown in FIG. 7, demonstrating INV and GOXAND when the current exists at the same time, the current has good electrochemical response to MB, AND accords with the characteristics of an AND logic gate.
EXAMPLE 8 construction of ZnO NPs/EDTA "INHIBIT" logic gates
The electrochemical response of sensors constructed from the polymer prepared as in example 1 for the preparation of ZnO NPs, comparing the two inputs of ZnO NPs (input 1) and EDTA (input 2) to MB, was constructed as the "INHIBIT" logic gate for ZnO NPs/EDTA, as shown in the following table:
the results are shown in fig. 8, demonstrating that with ZnO NPs, without EDTA, there is a good electrochemical response to MB, consistent with the "INHIBIT" logic gate feature.
Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Variations, modifications, additions, or substitutions by one of ordinary skill in the art which fall within the spirit of the invention are also within the scope of the invention.
Claims (3)
1. The invention relates to a 'binary' biological logic gate design and application research based on zinc oxide nano particles, which is characterized in that the mechanism is as follows: firstly, the nature of ZnO nano particles (ZnO NPs) which are degraded when meeting acid is adopted, and Zn released by the reaction of the ZnO nano particles and an enzyme solution2+The gene can be used as a factor of DNAzyme cleavage amplification reaction, a ZnO NPs-mediated DNAzyme signal amplification strategy is constructed, the gene is modified on an Au electrode, MB is used as a signal molecule to be output, and the sensor is successfully prepared. The saccharose is utilized to catalyze and decompose the saccharose to generate the glucose, the glucose is catalyzed by the glucose oxidase to generate the gluconic acid, and the ZnO NPs are reacted in the acidic environment Releasing Zn2+The concentrations of sucrose, glucose oxidase and sucrase are respectively fixed, and the response of the compound to MB is used as signal output to realize electrochemical logic analysis and detection of sucrose, glucose oxidase and sucrase. Based on the method, a series of 'binary' biological logic gates (YES, NO, AND AND INHIBIT) of ZnO NPs are constructed, so that the analysis AND monitoring of sucrose, INV AND GOx are realized, AND the method has important significance in the field of biomedicine. To date, no report has been found on the design and application of "binary" bio-logic gates based on ZnO NPs to INV and GOx related bio-signal pathway studies.
2. The design and application research of the "binary" biological logic gate based on zinc oxide nanoparticles as claimed in claim 1, wherein: ZnO NPs, DNAzyme and hybrid chain reaction are firstly linked with biological signal channels supported by glucose oxidase and sucrase, so that sensitive analysis and detection of sucrose, glucose oxidase and sucrase are realized.
3. The research on the design and application of the zinc oxide nano-particle-based 'binary' biological logic gate of claims 1-2, wherein the research results show that: the biological signal path mediated by cross, INV and GOx is designed by using a method of voltammetry (the potential range is-0.4 to-0.1V, the amplitude is 15Hz) to carry out 'binary' logic gate design, all logic gates take MB electrochemical signals as signal output, and different logic gates have different input signals: firstly, ZnO NPs are used as signal input to construct a 'YES' logic gate; secondly, constructing a YES logic gate by taking the cross as signal input; constructing a YES logic gate by taking INV as signal input; fourthly, taking the GOx as the signal input to construct a YES logic gate; constructing an AND logic gate by taking INV AND GOx as signal inputs; sixthly, constructing an INHIBIT logic gate by taking ZnO NPs and EDTA as signal inputs.
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