CN111879830B - Electrochemical impedance sensor for detecting sucrase and glucose oxidase and logic gate application thereof - Google Patents
Electrochemical impedance sensor for detecting sucrase and glucose oxidase and logic gate application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of an electrochemical sensor based on a silver-sulfhydryl compound and a double-enzyme detection application thereof, which comprises the following steps: firstly, plating a clean bare glassy carbon electrode with a graphene oxide dispersion liquid electrochemical method; mixing GHS and Ag (I) for reaction to obtain GSH-Ag (I) complex, and mixing the complex solution with 0.02 wt% Nafion solution before use; and finally, dripping the compound on the surface of a graphene modified electrode to obtain the electrochemical sensor (GSH-Ag (I)/GO/GCE) required by the invention. The sucrose generates H under the catalysis of sucrase (invertase) and glucose oxidase (GOx) 2 O 2 The sensor catalyzes DAB and H 2 O 2 The reaction generates a precipitate, so that the impedance of the electrode increases. Thus passing through H 2 O 2 The generation of (2) realizes the activity detection of sucrase and glucose oxidase. The method has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.
Description
Technical Field
The invention relates to an electrochemical impedance sensor and logic gate application thereof, in particular to an electrochemical sensor based on a coordination complex for analytical detection of sucrase and glucose oxidase and related logic gate application thereof, belonging to the technical field of functional biological materials and biosensing.
Background
Sucrase (Invertase) also called Invertase can specifically catalyze beta-D-fructofuranoside bond hydrolysis in non-reducing sugar, sucrose is hydrolyzed into two reducing sugars of D-glucose and D-fructose under the catalysis of sucrase, plays an important role in osmotic regulation and signal transduction, and is an enzyme related to glucose. Another class of glucose related enzymes is glucose oxidase (GOx), a typical oxidoreductase with a molecular weight of about 160000 daltons, consisting of two (or four) polypeptide chains, which, during a reduction reaction, first catalyzes the production of D-gluconolactone from β -D-glucose, and then the non-enzymatic hydrolysis of D-gluconolactone to gluconic acid, wherein Flavin Adenine Dinucleotide (FAD) in GOx is reduced to reduced Flavin Adenine Dinucleotide (FADH) 2 ) Reduced GOx-FADH during the oxidation reaction 2 Is re-oxidized to FAD by oxygen and is simultaneously generatedTo hydrogen peroxide (H) 2 O 2 ). In the course of life, these two enzymes attract the attention of many scientists because glucose is closely linked.
In the world today, the development of information science is the time of flight. The information science is rich in connotation, including modern information technology. Modern information technology features communication technology, microelectronics technology and computer technology. The computer technology is wide in content, and the computing base of the computer technology is binary. The binary structure has many characteristics, such as few used elements and only composed of sums; the operation rule is simple and the operation is convenient; suitable for logical operations. However, as science and technology has developed, traditional circuits based on semiconductor technology have faced a serious challenge because the development of such technology is approaching the limit that the technology can tolerate, i.e. the computer serial computing restricts the development of computer parallel computing, so that it is difficult to generate real cognitive ability. It is a challenge to find an efficient and economical logic device. The development and birth of biological computers undoubtedly provides a good choice for the development and research of artificial intelligence. The biological computer is a molecular computer made by using biological chip to replace semiconductor silicon, and mainly includes protein computer and computer. Wherein the operation mechanism of the protein computer provides an opportunity for the analysis and monitoring of the sucrase and the glucose oxidase.
The invention adopts Glutathione (GSH) as a biomolecule ligand, silver ions (Ag (I)) as metal nodes, synthesizes a protein-like GSH-Ag (I) compound by utilizing the interaction of sulfydryl-metal and metal-metal, modifies the compound on the surface of a graphene electrode, and then carries out surface modification on H 2 O 2 Under the conditions, 3-Diaminobenzidine (DAB) can be oxidized to form nonconductive Insoluble Precipitates (IP) and adhere to the electrode surface, resulting in a large increase in the electrochemical impedance value. On the other hand, sucrase can specifically catalyze the conversion of sucrose into D-glucose and D-fructose, while glucose oxidase can specifically convert glucose into gluconic acid and H 2 O 2 . At the moment, the GSH-Ag (I) compound has better electrochemical catalytic activity andcan combine DAB and H 2 O 2 Converted to IP and oxygen. Based on the method, analytical detection of sucrase and glucose oxidase can be realized, and the series of biological reactions are very suitable for designing logic gates. At present, no analytical detection method combining GSH-Ag (I) compound, sucrase reaction, glucose oxidase reaction and electrochemical impedance technology and logic gate application thereof are found.
Disclosure of Invention
The invention aims to solve the technical problem of providing an electrochemical impedance sensor for detecting the sucrase and the glucose oxidase, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost, and a logic gate application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: an electrochemical impedance sensor for detecting sucrase and glucose oxidase and a logic gate application thereof, specifically comprising the following steps:
(1) Preparation of the sensor
Preparation of GSH-Ag (I) complexes: sequentially taking 1-10 mu L of silver nitrate aqueous solution with the concentration of 0.1-20 mM and 1-10 mu L of glutathione aqueous solution with the concentration of 0.1-20 mM, uniformly mixing, adding phosphoric acid buffer solution (10mM, pH7.0, na) 2 HPO 4 /NaH 2 PO 4 ) Preparing 50-120 mu L solution, slowly shaking the solution at 25-40 ℃ for 8-17 min to obtain GSH-Ag (I) compound, uniformly mixing the compound solution with 50-120 mu L0.02 wt% Nafion solution before use, and standing for 5-20 min.
b. Polishing a glassy carbon electrode (GCE, diameter of 3 mm) on chamois leather by using aluminium oxide powder with particle size of 0.3 mu m and 0.05 mu m for 0.5-5 min in sequence, placing the electrode in an ultrasonic cleaner for ultrasonic cleaning by using ultrapure water for 1-5 min after polishing, and then using N to clean the electrode by using the ultrapure water 2 Drying and marking as GCE;
c. utilizing a cyclic voltammetry to set the potential range to be-1.2-0.5V and the sweep speed to be 5-20 mV/s, and electrodepositing 0.5-2 mg/mL graphene dispersion liquid (GO) on a bare glassy carbon electrode to obtain GO/GCE; then 5-15 mul of the solution in the (a) is dripped on GO/GCE, the mixture is kept stand for 20-60 min at room temperature, and the electrode is slowly rinsed by ultrapure water, which is marked as GSH-Ag (I)/GO/GCE.
(2) Electrocatalytic performance test
Placing GSH-Ag (I)/GO/GCE in a container containing 2mM DAB and 2mM H 2 O 2 In a phosphoric acid buffer solution (PBS, 0.1M, pH 7.0) of (1), the electrode was transferred to 5mM [ Fe (CN) after 10 cycles of cyclic voltammetry 6 ] 3-/4- Detects the electrochemical ac impedance response.
(3) Analytical detection of sucrase and glucose oxidase
100 mu L of reaction solution contains 1-50 mM of sucrose and 0.1-10 mM of sucrase 4 U/L and glucose oxidase 0.01-10 3 U/L, phosphoric acid buffer solution (PBS, 0.1M, pH 7.0), placing the reaction solution in water bath at 25-40 ℃ for reaction for 40-120 min; after completion of the reaction, 100. Mu.L of phosphoric acid buffer solution containing 2mM DAB (PBS, 0.1M, pH 7.0) was added, GSH-Ag (I)/GO/GCE was placed in this mixed solution and scanned by cyclic voltammetry for 10 cycles, and the electrode was transferred to 5mM Fe (CN) 6 ] 3-/4- Detects the electrochemical ac impedance response.
The sucrase concentration (final concentration: 0.1-10) was varied 4 U/L) and other steps are the same as above, so that the detection of the sucrase with different concentrations can be realized.
Changing the concentration of glucose oxidase (final concentration: 0.01-10) 3 U/L) and other steps are the same as above, and detection of glucose oxidase with different concentrations can be realized.
Based on the application of the electrochemical impedance sensor for detecting the sucrase and the glucose oxidase and the logic gate thereof, H generated after the sucrase reaction and the glucose oxidase reaction 2 O 2 Cyclic voltammetry (potential range: -1.0- +0.5V, sweep rate: 10 mV. S) -1 ) DAB was oxidized to form nonconductive insoluble IP and attached to the electrode surface, followed by an ac impedance method (frequency range: 10 -2 ～10 5 Hz, amplitude: 2 mV) to obtain a resistance change curve, obtain the corresponding impedance of a series of sucrase or glucose oxidase with different concentrations, establish the quantitative relation between the electrochemical impedance response and the sucrase or glucose oxidase, and determine the sucrase or glucose oxidase in a sample to be tested according to the quantitative relation between the electrochemical impedance response and the sucrase or glucose oxidaseAnd (4) content.
The invention principle is as follows: the invention relates to an electrochemical impedance sensor for detecting sucrase and glucose oxidase and a logic gate application thereof, which synthesizes a special compound (GSH-Ag (I)) based on the interaction of sulfydryl in GSH and Ag (I), wherein the GSH-Ag (I) is prepared by reacting the sulfydryl in GSH with Ag (I) and then reacting the sulfydryl in the GSH with the Ag (I) 2 O 2 When present, DAB can be oxidized to form a non-conductive insoluble IP and attach to the electrode surface, greatly increasing the electrical resistance. Because sucrase can specifically catalyze the conversion of sucrose into D-glucose and D-fructose, and glucose oxidase can specifically convert glucose into gluconic acid and H 2 O 2 DAB and H based on GSH-Ag (I) complexes 2 O 2 The activity detection of the sucrase and the glucose oxidase can be realized by the electrocatalysis effect of the enzyme. Based on the method, a simple, rapid, high-sensitivity, high-selectivity and label-free analytical method for sucrase and glucose oxidase is constructed. Subsequently, sucrase, glucose oxidase, GSH-Ag (I) complex is set as signal outputs 1, 2, 3, electrochemical impedance is used as the signal output, AND an AND-AND-AND Boolean logic gate is constructed.
Compared with the prior art, the invention has the advantages that: the invention relates to an electrochemical impedance sensor for detecting sucrase and glucose oxidase and a logic gate application thereof. GSH-Ag (I) complexes synthesized by GSH and Ag (I), in H 2 O 2 Under the conditions, DAB can be oxidized to form nonconductive insoluble IP and adhere to the electrode surface, greatly increasing the electrical resistance. Because sucrase can specifically catalyze the conversion of sucrose into D-glucose and D-fructose, and glucose oxidase can specifically convert glucose into gluconic acid and H 2 O 2 And the activity detection of sucrase and glucose oxidase can be realized. It is clear that the greater the concentration of sucrase (glucose oxidase) within a certain range of concentrations, the greater the production of H 2 O 2 The more, the more pronounced the impedance response. The experimental result shows that the impedance and the concentration of the sucrase (glucose oxidase) are in a linear relationship in a certain range, so that the detection of the sucrase (glucose oxidase) is realized. Then an AND-AND-AND Boolean logic is constructed based on the composition of sucrase, glucose oxidase AND GSH-Ag (I)And (6) editing the door. The advantages are that:
(1) Good electrocatalytic activity. GSH-Ag (I) complexes synthesized by GSH and Ag (I), in H 2 O 2 Under conditions, DAB can be oxidized to form non-conductive insoluble IP and attach to the electrode surface, resulting in a larger electrochemical impedance response.
(2) High sensitivity. H generated based on sucrase reaction and glucose oxidase reaction 2 O 2 And obtaining a linear correlation equation of the impedance response of the sensor to the concentration of the sucrase as y =8702C Invertase +10806，R 2 =0.9998, the detection limit is 0.05U/L, which indicates that the sensor realizes high-sensitivity detection on sucrase; the linear correlation equation of the impedance response of the sensor to the concentration of glucose oxidase is y =8880C GOx +14750，R 2 And the detection limit is 0.008U/L, which indicates that the sensor realizes high-sensitivity detection on the glucose oxidase.
(3) High specificity. Other enzymes such as acetylcholinesterase (AChE), terminal transferase (TdT), uricase (uricase), exonuclease I (Exo I), pyrophosphatase (PPase) and Lysozyme (LZM) did not interfere with the system.
(4) The result is accurate. The recovery rate is between 90% and 110%.
(5) The preparation and detection methods have the advantages of less reagent consumption and low cost. The invention can realize high-sensitivity detection of the sucrase or the glucose oxidase only by consuming a small amount of materials and reagents.
(6) Three input AND-AND-AND logic gates are constructed by utilizing the sucrase, the glucose oxidase AND the compound, so that the logic operation detection application is realized.
In conclusion, the electrochemical impedance sensor for detecting the sucrase AND the glucose oxidase AND the application of the logic gate thereof are constructed, the electrochemical impedance sensor has the advantages of high sensitivity, good selectivity, simplicity in operation, quickness in analysis, easiness in operation AND the like, can realize the detection of the sucrase or the glucose oxidase with lower concentration, is applied to the design of an AND-AND-AND logic gate, AND has good application prospect.
Drawings
FIG. 1 shows a sensor pair DAB and H according to the invention 2 O 2 An electrochemical impedance plot;
FIG. 2 is a diagram of the feasibility experiment of the sensor of the present invention for the detection of sucrase and glucose oxidase;
FIG. 3 is a graph of the impedance response to sucrase for a sensor of the present invention and a calibration curve;
FIG. 4 is a graph showing a selectivity test of a sensor of the present invention for sucrase;
FIG. 5 is a graph of the impedance response of the sensor of the present invention to glucose oxidase and a calibration curve;
FIG. 6 is a graph showing an experiment of selectivity of a sensor of the present invention for glucose oxidase;
FIG. 7 is an experimental diagram of an AND-AND-AND logic gate constructed by the sensor of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
EXAMPLE 1 preparation of the sensor
Preparation of GSH-Ag (I) complexes: sequentially collecting 10 μ L of 1mM silver nitrate aqueous solution and 10 μ L of 1mM glutathione aqueous solution, mixing, adding phosphate buffer solution (10mM, pH7.0, na) 2 HPO 4 /NaH 2 PO 4 ) Preparing 80 μ L solution, slowly shaking the solution at 30 deg.C for 12min to obtain GSH-Ag (I) complex, mixing the complex solution with 80 μ L0.02 wt% Nafion solution, and standing for 12min.
b. Polishing glassy carbon electrode (GCE, diameter of 3 mm) on chamois leather with aluminum oxide powder with particle size of 0.3 μm and 0.05 μm for 1.5min, ultrasonic cleaning the electrode with ultrapure water in ultrasonic cleaner for 2min, and then cleaning with N 2 Drying, and marking as GCE;
c. utilizing a cyclic voltammetry to set the potential range to be-1.2-0.5V and the sweep rate to be 12V/s, and electrodepositing 1.2mg/mL graphene dispersion liquid (GO) onto a bare glassy carbon electrode to obtain GO/GCE; then 6 mul of the solution in (a) is dripped on GO/GCE, the mixture is kept stand for 32min at room temperature, and the electrode is slowly rinsed by ultrapure water, which is marked as GSH-Ag (I)/GO/GCE.
The prepared modified electrode is added with 2mM DAB and 2mM H 2 O 2 In a phosphoric acid buffer solution (PBS, 0.1M, pH 7.0) of (1), the electrode was transferred to 5mM [ Fe (CN) after 10 cycles of cyclic voltammetry 6 ] 3-/4- Detects the electrochemical ac impedance response. The results are shown in FIG. 1A, where the prepared sensor has a significant impedance response compared to GCE and GO/GCE, and in DAB and H 2 O 2 And (3) the sensor has stronger impedance response under the existing condition (figure 1B), and the two conditions are not available, so that the successful preparation of the sensor can be used for further experiments.
Example 2 sensor feasibility test
A sensor was prepared as in example 1 above, and 100. Mu.L of the reaction solution contained 10mM sucrose and 10mM sucrase 3 U/L, glucose oxidase 400U/L, phosphate buffer solution (PBS, 0.1M, pH 7.0), placing the reaction solution in water bath at 37 ℃ for reaction for 60min; after completion of the reaction, 100. Mu.L of phosphoric acid buffer solution containing 2mM DAB (PBS, 0.1M, pH 7.0) was added, GSH-Ag (I)/GO/GCE was placed in this mixed solution and scanned by cyclic voltammetry for 10 cycles, and the electrode was transferred to 5mM Fe (CN) 6 ] 3-/4- Detects the electrochemical ac impedance response.
Meanwhile, the performance of the sensor in the absence of either or both of sucrase and glucose oxidase was examined, and the results are shown in FIG. 2, which shows that only sucrase and glucose oxidase can produce H in the presence of both sucrase and glucose oxidase 2 O 2 Meanwhile, the sensor is proved to be used for detecting the polysucrase and the glucose oxidase.
Example 3 detection of sucrase
A sensor was prepared as in example 1 above, by changing the concentration of sucrase (0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000, 10000U/L) based on the reaction conditions of example 2 to obtain a series of reaction solutions, followed by addition of 100. Mu.L of phosphoric acid buffer solution containing 2mM DAB (PBS, 0.1M, pH 7.0), further placing GSH-Ag (I)/GO/GCE in this mixed solution, scanning for 10 cycles by cyclic voltammetry, and further transferring the electrode to 5mM Fe (CN) 6 ] 3-/4- Detect a electrochemical ac impedance response.
The results are shown in figure 3 of the drawings,along with the increase of the concentration of sucrase, the response of alternating current impedance is more obvious, and the concentration of the sucrase and the alternating current impedance is between 0.1 and 10 3 The logarithm in the U/L range is in good linear relation, and the linear relation is that y =8702C Invertase +10806, linear correlation coefficient R 2 =0.9998, and the detection limit is 0.05U/L, which indicates that the sensor can realize high-sensitivity detection on sucrase.
Subsequently, the specificity of the sensor for detecting sucrase is examined, and as a result, as shown in fig. 4, the sensor basically has no response to acetylcholinesterase (AChE), terminal transferase (TdT), uricase (uricase), exonuclease I (Exo I), pyrophosphatase (PPase) and Lysozyme (LZM) at the same concentration, and has a significant response to sucrase, which proves that the sensor has good selectivity for detecting sucrase.
Example 4 detection of glucose oxidase
A sensor was prepared as in example 1 above by changing the concentration of glucose oxidase (0.04, 0.1, 0.4, 1, 4, 10, 40, 100, 400, 1000U/L) based on the reaction conditions of example 2 to obtain a series of reaction solutions, followed by addition of 100. Mu.L of 2mM DAB-containing phosphate buffer solution (PBS, 0.1M, pH 7.0), placing GSH-Ag (I)/GO/GCE in this mixed solution, scanning for 10 cycles by cyclic voltammetry, and transferring the electrode to 5mM Fe (CN) 6 ] 3-/4- Detects the electrochemical ac impedance response.
As shown in FIG. 5, the AC impedance response became more pronounced with increasing glucose oxidase concentration, and was well linear with the logarithm of the glucose oxidase concentration in the range of 0.04-400U/L, with the linear relationship being y =8880C GOx +14750，R 2 And the detection limit is 0.008U/L, which indicates that the sensor can realize high-sensitivity detection on glucose oxidase.
Then, the specificity of the sensor for detecting glucose oxidase is examined, and as shown in fig. 6, the sensor basically has no response to acetylcholinesterase (AChE), terminal transferase (TdT), uricase (uricase), exonuclease I (Exo I), pyrophosphatase (PPase) and Lysozyme (LZM) at the same concentration, while the selectivity of the sensor for detecting sucrase is proved to be good when the sensor is obvious to glucose oxidase.
EXAMPLE 5 construction of logic gates
The sensor designed by the invention can be used for detecting the activities of sucrase and glucose oxidase. Three AND-AND boolean logic gates (as in the table below) of inputs (input 1: sucrase; input 2: glucose oxidase; input 3: gsh-Ag (I)) AND one output were designed following the sensor preparation procedure of example 1 above, AND the results are shown in fig. 7. The logic gates for sucrase, glucose oxidase and the complex were successfully constructed.
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 (5)
1. An electrochemical impedance sensor for detecting sucrase and glucose oxidase specifically comprises the following steps:
(1) Preparation of the sensor
Preparation of GSH-Ag (I) complexes: sequentially taking 1-10 mu L of silver nitrate aqueous solution with the concentration of 0.1-20 mM and 1-10 mu L of glutathione aqueous solution with the concentration of 0.1-20 mM, uniformly mixing, adding phosphoric acid buffer solution to prepare 50-120 mu L of solution, and slowly oscillating the solution for 8-17 min at the temperature of 25-40 ℃ to obtain GSH-Ag (I) compound; mixing the complex solution with 50-120 μ L0.02 wt% Nafion solution, standing for 5-20 min;
b. polishing glassy carbon electrode on chamois leather with aluminium oxide powder with grain size of 0.3 micron and 0.05 micron for 0.5-5 min, ultrasonic cleaning electrode in an ultrasonic cleaner with ultrapure water for 1-5 min, and N 2 Drying and marking as GCE;
c. utilizing a cyclic voltammetry to set the potential range to be-1.2-0.5V and the sweep speed to be 5-20 mV/s, and electrodepositing 0.5-2 mg/mL of graphene dispersion GO onto a bare glass carbon electrode to obtain GO/GCE; then 5-15 mu L of the solution obtained in the step (a) is dripped on GO/GCE, the mixture is kept stand for 20-60 min at room temperature, and the electrode is slowly rinsed by ultrapure water and marked as GSH-Ag (I)/GO/GCE;
(2) Electrocatalytic performance test
Placing GSH-Ag (I)/GO/GCE in a container containing 2mM 3, 3-diaminobenzidine DAB and 2mM H 2 O 2 The phosphoric acid buffer solution of (2) was subjected to cyclic voltammetry for 10 cycles, and then the electrode was transferred to 5mM [ Fe (CN) 6 ] 3-/4- Detecting electrochemical alternating current impedance response in solution;
(3) Analytical detection of sucrase and glucose oxidase
100 mu L of reaction solution contains 1-50 mM of sucrose and 0.1-10 mM of sucrose 4 0.01 to 10 portions of U/L sucrase 3 Putting the reaction solution into a water bath at the temperature of between 25 and 40 ℃ for reaction for 40 to 120min by using U/L glucose oxidase and phosphoric acid buffer solution; after completion of the reaction, 100. Mu.L of phosphoric acid buffer solution containing 2mM DAB was added, GSH-Ag (I)/GO/GCE was placed in this mixed solution and scanned by cyclic voltammetry for 10 cycles, and the electrode was transferred to 5mM [ Fe (CN) ] 6 ] 3-/4- Detecting electrochemical alternating current impedance response in solution;
in the range of 0.1 to 10 4 The sucrase concentration is changed within the U/L concentration range, and other steps are the same as above, so that the sucrase detection with different concentrations can be realized;
in the range of 0.01 to 10 3 The concentration of the glucose oxidase is changed within the U/L concentration range, and the detection of the glucose oxidase with different concentrations can be realized by the same steps as above.
2. The sensor of claim 1, wherein: at H 2 O 2 When the electrode is in use, the 3, 3-diaminobenzidine DAB is oxidized by cyclic voltammetry to form non-conductive insoluble IP and is attached to the surface of the electrode, and then the resistance change rule is obtained by an alternating current impedance method.
3. A sensor according to claim 2, wherein the cyclic voltammetry potential range is: -1.0- +0.5V, sweep rate: 10 mV. S -1 (ii) a Frequency range of ac impedance method: 10 -2 ～10 5 Hz, amplitude: 2mV.
4. A sensor according to any one of claims 1 to 3, wherein: for detecting sucrase and glucose oxidase with different concentrations, the linear correlation equation of sucrase concentration is y =8702C Sucrase +10806，R 2 =0.9998, detection limit is 0.05U/L; linear correlation equation for glucose oxidase concentration of y =8880C Glucose oxidase +14750，R 2 =0.9954, detection limit of 0.008U/L.
5. A sensor according to any one of claims 1 to 3, wherein: adopts sucrase, glucose oxidase and GSH-Ag (I) compound as three signal outputs, namely 3, 3-diaminobenzidine DAB and H 2 O 2 AND the impedance signal under the condition is output as a signal to construct an AND-AND-AND Boolean logic gate.
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