CN112525966B - Based on H2O2Non-enzyme pyruvic acid sensing detection method - Google Patents
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Abstract
The invention provides a method based on H2O2The enzyme-free pyruvate sensing detection method comprises the following steps: using a three-electrode system with H in the electrolyte solution2O2(ii) a Adding to the electrolyte solutionAdding sodium pyruvate with different concentrations, and acquiring a standard curve of current response and sodium pyruvate concentration by using a timing current method; the working electrode of the three-electrode system is H2O2A sensing electrode; according to the inclusion of H2O2And comparing the current response value generated by the solution of the sample to be tested of the pyruvic acid with the standard curve to obtain the concentration of the sodium pyruvate. The method is based on pyruvic acid-H2O2The method utilizes a Prussian blue modified electrode to monitor H on line in real time2O2The consumption rate, and thus the pyruvate concentration was quantitatively obtained. The pyruvic acid concentration detection strategy adopting the method has a wider linear detection range, can effectively avoid the problems of poor repeated effect and the like caused by poor stability of the pyruvate oxidase, can realize quick and efficient real-time detection in a complex biological system, and has the detection limit of 0.5 mM.
Description
Technical Field
The invention belongs to the technical field of electrochemical sensing, and particularly relates to a pyruvic acid-H-based sensor2O2The detection method of the enzyme-free pyruvic acid sensing electrode of the decarbonylation reaction.
Background
Pyruvic acid is a key intermediate product starting from the beginning in the biological metabolism process, plays a significant role in the metabolic growth and fermentation stages of strains, and has an important reference value for reflecting the growth and metabolism rules and the fermentation state of microbial cells in the concentration detection. In addition, the concentration of pyruvic acid in blood can be used as one of diagnostic indicators of vitamin B1 deficiency, and has important reference value for diagnosis and treatment of diabetic ketoacidosis and anion gap elevation poisoning. Therefore, the rapid real-time monitoring of the concentration of the pyruvic acid has great guiding function and economic benefit for the fields of biological fermentation production process, medical research and food safety.
The existing methods for measuring pyruvic acid (pyruvate) mainly comprise a 2, 4-dinitrophenylhydrazine method, a lactic acid dehydrogenase method and a high performance liquid chromatography. The nitrobenzene hydrazine method has poor specificity, can form similar phenylhydrazone with keto acids such as acetone, acetoacetic acid and the like, and is easy to interfere; the lactate dehydrogenase method has strong specificity, but the enzyme reagent is expensive, and the detection cost is high; the high performance liquid chromatography is accurate in measurement, but is complex and time-consuming in operation, and is not suitable for online real-time detection.
Compared with the traditional pyruvic acid detection method, the electrochemical sensing-based online detection technology has the characteristics of simplicity in operation, rapidness, high efficiency, automation and the like. Pyruvate biosensors generally use pyruvate oxidase (EC: 1.2.2.3) as a bio-enzyme element, which reacts specifically with pyruvate as a test substance to produce H2O2Signal, channel H2O2The detection electrode collects and converts the signal into an electric signal, thereby realizing the determination of the concentration of the pyruvic acid. Pyruvate oxidase natural enzyme has poor stability and is easy to inactivate, and is one of the important reasons that the development of the existing pyruvate biosensor is limited and is difficult to be widely applied.
Disclosure of Invention
In order to solve the technical problem, the invention provides a catalyst based on H2O2The enzyme-free pyruvate sensing detection method. The enzyme-free electrochemical sensor can effectively avoid the problems that the enzyme of the bioactive substance is easy to inactivate, the electrochemical adaptability of the enzyme element and the electrode is poor, the enzyme element falls off and the like, and greatly prolongs the service life of the biosensor. The method can realize rapid and efficient real-time detection in a complex biological system, and has a wider linear detection range.
In order to achieve the purpose, the invention adopts the following technical scheme:
based on H2O2The enzyme-free pyruvate sensing detection method comprises the following steps:
(1) using a three-electrode system with H in the electrolyte solution2O2(ii) a Adding sodium pyruvate with different concentrations into the electrolyte solution, and obtaining a standard curve of current response and sodium pyruvate concentration by using a chronoamperometry; the working electrode of the three-electrode system is H2O2A sensitive electrode;
(2) according to the inclusion of H2O2And comparing the current response value generated by the solution of the sample to be tested of the pyruvic acid with the standard curve to obtain the concentration of the sodium pyruvate.
The current response is the percent current drop.
Preferably, the step (1) specifically comprises the following steps:
(1-1) constructing a three-electrode system, and recording a current value I corresponding to a current baseline of the electrolyte solution by using a chronoamperometry0；
(1-2) adding H to the electrolyte solution2O2Recording the current value I1；
(1-3) adding sodium pyruvate with different concentrations into the electrolyte solution, and recording the current value I when the sodium pyruvate is added for 50-100 s2；
(1-4) the standard curve was obtained by plotting the percent current decrease versus the sodium pyruvate concentration.
The calculation formula of the current reduction percentage is (I)1-I2)/(I1-I0)×100％。
Preferably, the working electrode of the three-electrode system is a prussian blue modified electrode. More preferably, the prussian blue modified electrode is prepared by a method disclosed in patent CN101532979A, and specifically comprises the following steps:
polishing, performing ultrasonic surface pretreatment on a substrate electrode, and placing the substrate electrode in an aerosol deposition box; anionic solution 0.01-2M K4Fe(CN)6Or K3Fe(CN)6Atomizing the solution and 0.01-0.5M KCl solution into aerosol by an ultrasonic atomizer, introducing the aerosol into an aerosol deposition box, and reacting for a plurality of times; 0.01-2M FeCl of isoconcentrate cation solution3Or (NH)4)2Fe(SO4)2Ultrasonically atomizing with 0.01-0.5M KCl solution to form aerosol, introducing into an aerosol deposition box, and reacting for a certain time; repeating the above aerosol deposition for several times, and oven drying.
Preferably, in the step (1-1), after the current baseline is stabilized, the current value I corresponding to the current baseline of the electrolyte solution is recorded0。
Preferably, H is added in step (1-2)2O2The final concentration of (A) is in the range of 0.1 to 0.5 mM; preferably 0.2 mM.
Preferably, the current value I is recorded when sodium pyruvate is added for 50s in the step (1-3)2。
Preferably, the electrolyte solution is a PBS buffer solution. More preferably, the concentration of the PBS buffer is 50mM and the pH is 6.5.
Preferably, the scanning potential of the chronoamperometry method is-0.05V, which is the preferred Prussian blue electrode response H2O2The optimum potential of (c).
Preferably, said compound contains H2O2And pyruvic acid as biological system. More preferably, the compound contains H2O2And the solution of the sample to be detected of the pyruvic acid is fermentation liquor of the bacteria.
The method has the beneficial effects that:
(1) compared with the traditional pyruvic acid detection method, the electrochemical sensing-based pyruvic acid detection technology can be used for rapidly and efficiently detecting in real time.
(2) Compared with a pyruvic acid detection biosensor based on pyruvate oxidase, the pyruvic acid concentration detection method can effectively avoid the problem of poor stability of the existing pyruvate oxidase, has long service life and wider linear detection range.
(3) The method can realize rapid and efficient real-time detection in a complex biological system, and the detection limit can reach 0.5 mM.
Drawings
FIG. 1 is a curve of current value and time variation of Prussian blue modified electrode in response to sodium pyruvate with different concentrations;
fig. 2 is a linear standard curve of the current reduction percentage of the prussian blue modified electrode responding to different concentrations of sodium pyruvate and the concentration of sodium pyruvate.
Detailed Description
The invention is described in detail below with reference to the drawings and specific examples to help the invention to be better understood. The description of the embodiments is intended to be illustrative of the invention and should not, nor should it be taken to limit the invention to the details set forth in the claims.
Example 1 plotting of the Standard Curve
(1) Preparing 50mM PBS phosphate buffer (pH 6.5) as an electrode buffer;
(2) constructing a three-electrode system, wherein the working electrode is a Prussian blue modified electrode, the electrolyte solution is 50mL of electrode buffer solution, the applied potential is selected to be-0.05V, timing current scanning is carried out, activation is carried out for 30min until the current baseline is stable, and the corresponding current value I is recorded0；
(3) Adding H into electrode buffer solution2O2So that the final concentration is 0.2mM, and recording the current value I after the current value has stabilized1；
(4) Adding electrode buffer solution and sodium pyruvate with different concentration gradients into the electrode buffer solution respectively to enable the final concentration to be 0, 1, 2,4, 6, 8 and 10mM respectively, recording the current change, and accurately recording the current value I when the sodium pyruvate solution is added for 25s, 50s, 100s and 150s2. As shown in figure 1, after the sodium pyruvate is added, the current value slowly decreases, namely the sodium pyruvate continuously consumes H in the detection cell2O2，H2O2The current signal decays. The higher the concentration of sodium pyruvate added, the higher the H content per unit time2O2The faster the signal decays. The linear relationship between the current value and the time is good 50-100 s after the sodium pyruvate is added.
(5) Calculating percent Current decrease (I)1-I2)/(I1-I0) X 100%, and drawing a standard curve of the current reduction percentage-sodium pyruvate concentration. As shown in FIG. 2, the concentration of sodium pyruvate in the range of 0 to 10mM is in a good linear relationship with the percent current decrease. The current values I were recorded at 25s, 50s, 100s and 150s2The linear relationship obtained by plotting is respectively: linear equation (C) 40.29 xW-0.36 and correlation coefficient R20.9841; linear equation C25.27 xW-0.32, correlation coefficient R20.9985; linear equation (C) 16.14 xW-0.49, correlation coefficient R20.9960; linear equation (C) 13.13 XW-0.75, correlation coefficient R20.9777. Wherein W is the percent reduction in current and C is the concentration (mM) in the sodium pyruvate detection cell. The current values I were recorded at 50s and 100s2The obtained linear fitting result is better, and in view of shortening the detection time as much as possible, the current value I is recorded when the object to be detected is added for 50s2Is an optimal choice.
Example 2 practical application
Respectively and exogenously adding sodium pyruvate with the final concentration of 11g/L into fermentation liquor of clostridium acetobutylicum, klebsiella pneumoniae and pseudomonas putida, detecting the sodium pyruvate of a sample according to the detection method provided by the invention, and detecting the sodium pyruvate in 25mL of fermentation liquor containing 0.2mM H2O2And adding 1mL of sample to be detected into the electrode buffer solution, and recording the current change in real time by using the Prussian blue modified electrode. Concentration C of sodium pyruvate in sample to be detected*＝(25.27×W-0.32)×26。
(1) Clostridium acetobutylicum (Clostridium acetobutylicum) is anaerobically fermented at 37 ℃ for 7 days in a 40mL fermentation system, the concentrations of main products of the fermentation liquid are detected by a high performance gas chromatograph, and are respectively 12.1g/L butanol, 6.3g/L butyric acid, 2.0g/L ethanol and 1.6g/L acetone, and sodium pyruvate with the final concentration of 11.0g/L is exogenously added to obtain a sample 1.
(2) Klebsiella Pneumoniae (Klebsiella Pneumoniae) is subjected to anaerobic fermentation at 30 ℃ for 3 days in a 40mL fermentation system, the concentrations of main products of the fermentation liquid detected by a high performance liquid chromatograph are respectively 20.3g/L propylene glycol, 3.4g/L ethanol, 2.4g/L butanediol, 2.1g/L acetic acid and 1.5g/L lactic acid, and sodium pyruvate with the final concentration of 11.0g/L is exogenously added to obtain a sample 2.
(3) Actinobacillus succinogenes (Actinobacillus succinogenes 130Z) is fermented for 3 days at 37 ℃ in a 5L fermentation tank system, the concentration of main products detected by a high performance liquid chromatograph is respectively 58.0g/L succinic acid, 10.1g/L fumaric acid, 8.5g/L acetic acid and 5.3g/L lactic acid, and sodium pyruvate with the final concentration of 11.0g/L is exogenously added to obtain a sample 3.
As shown in table 1, based on sodium pyruvate-H2O2The detection method of the enzyme-free pyruvic acid biosensor adopting the neutralization strategy has good consistency of detection results of the externally added sodium pyruvate in the three fermentation liquors and the maximum phase of detectionThe error is about 2.36%, and the detection of the concentration of the pyruvic acid in the fermentation liquid can be realized. The detection result of the pyruvic acid detection method established by the invention is more accurate and reliable, the method for online detection of pyruvic acid is widened, and the method has wide practical application value.
TABLE 1 detection results of three fermentation broths with Prussian blue modified electrode pre-added with a certain amount of sodium pyruvate
| Sample (I) | Theoretical sodium pyruvate concentration g/L | Actually measured concentration g/L of sodium pyruvate | Relative error% |
| Sample 1 | 11.00 | 11.20 | 1.82 |
| Sample 2 | 11.00 | 11.07 | 0.64 |
| Sample 3 | 11.00 | 11.26 | 2.36 |
Claims (12)
1. Based on H2O2The enzyme-free pyruvate sensing detection method is characterized by comprising the following steps:
(1): (1-1) constructing a three-electrode system by taking the Prussian blue modified electrode as a working electrode, and recording a current value I corresponding to a current baseline of an electrolyte solution by using a chronoamperometry0；
(1-2) adding H to the electrolyte solution2O2Recording the current value I1；
(1-3) adding sodium pyruvate with different concentrations into the electrolyte solution, and recording the current value I when the sodium pyruvate is added for 50-100 s2；
(1-4) drawing the current reduction percentage to the sodium pyruvate concentration to obtain a standard curve;
(2) according to the inclusion of H2O2And comparing the current response value generated by the solution of the sample to be tested of the pyruvic acid with the standard curve to obtain the concentration of the sodium pyruvate.
2. The method of claim 1, wherein the current response is a percent decrease in current.
3. The method for detecting enzyme-free pyruvate sensor according to claim 1, wherein the percentage of decrease in current is calculated by
。
4. The enzyme-free pyruvate sensing detection method according to claim 1, wherein the Prussian blue modified electrode is prepared by the following method:
carrying out surface pretreatment on a substrate electrode and then placing the substrate electrode in an aerosol deposition box; anionic solution 0.01-2M K4Fe(CN)6Or K3Fe(CN)6Atomizing the solution and 0.01-0.5M KCl solution into aerosol by an ultrasonic atomizer, introducing into an aerosol deposition box,reacting for a plurality of times; 0.01-2M FeCl of isoconcentrate cation solution3Or (NH)4)2Fe(SO4)2Ultrasonically atomizing with 0.01-0.5M KCl solution to form aerosol, introducing into an aerosol deposition box, and reacting for a certain time; repeating the aerosol deposition for several times, and drying.
5. The method for detecting enzyme-free pyruvate according to claim 1, wherein H is added in the step (1-2)2O2The final concentration of (b) is in the range of 0.1 to 0.5 mM.
6. The method for detecting enzymatically-free pyruvate according to claim 5, wherein H is added in the step (1-2)2O2Was 0.2 mM.
7. The method for detecting enzyme-free pyruvic acid sensing and detection of claim 1, wherein the current value I is recorded when sodium pyruvate is added for 50s in step (1-3)2。
8. The method for detecting enzyme-free pyruvate sensor according to claim 1, wherein the electrolyte solution is PBS buffer solution.
9. The enzyme-free pyruvate sensing method according to claim 8, wherein the concentration of the PBS buffer is 50mM and the pH is 6.5.
10. The method for detecting enzyme-free pyruvate according to claim 1, wherein the scanning potential of the chronoamperometry is-0.05V.
11. The method for the enzyme-free pyruvate sensing according to claim 1, wherein the H is contained2O2And pyruvic acid as biological system.
12. Root of herbaceous plantThe method for detecting enzymatically-free pyruvate according to claim 11, wherein the H is contained2O2And the solution of the sample to be detected of the pyruvic acid is fermentation liquor of the bacteria.
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