Method for rapidly detecting lactic acid based on self-assembled electroactive single-layer film

Abstract

The invention discloses a method for rapidly detecting lactic acid based on a self-assembled electroactive single-layer film, and belongs to the technical field of microbial electrochemical sensor detection. The problems that an existing lactic acid detection method is tedious, complex, high in detection cost and the like are mainly solved. According to the method, the surface of the electrode is subjected to positive charge modification by utilizing the characteristic that electroactive microorganisms carry negative charges under normal physiological conditions, the self-assembled electroactive single-layer film is prepared through electrostatic adsorption force, and the operation is simple, convenient and rapid; the self-assembled electroactive single-layer film can quickly convert biochemical signals of oxidized lactic acid into electric signals, and the output electric signals can reflect the content of lactic acid. The constructed self-assembled electroactive single-layer film is high in response speed when being used for detecting lactic acid, has relatively high detection sensitivity, and is expected to be clinically applied to risk classification of sepsis patients in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial electrochemical sensor detection, and specifically relates to a method for rapidly detecting lactic acid based on a self-assembled electroactive monolayer film. Background Art

[0002] Lactic acid is a common metabolic intermediate in the human body. Under healthy conditions, the lactic acid produced by the reduction of pyruvate by lactate dehydrogenase is mainly excreted through the liver and kidneys. When the body is undersupplied with oxygen, pyruvate in the body accumulates rapidly and converts into lactic acid, resulting in an increase in lactic acid levels. A large number of studies have shown that changes in lactate levels can reflect the level of anaerobic metabolic activity in patients with sepsis. Therefore, lactate is considered an important marker for risk stratification of patients with sepsis.

[0003] At present, the commonly used methods for detecting lactic acid mainly include colorimetry and high-performance liquid chromatography. The colorimetry method reflects the lactic acid content by measuring the absorbance value of the colored complex formed by lactic acid, but there are problems such as the complex detection process and the accuracy is easily affected by the color interference of the sample. Compared with the colorimetry method, the high-performance liquid chromatography method has higher accuracy and sensitivity, but it relies on large and expensive detection equipment and professional operators, which is not easy to promote widely.

[0004] Microbial electrochemical sensors are expected to solve the above problems. Microbial electrochemical sensors use electroactive microorganisms as sensing elements and utilize the extracellular electron transfer process of microorganisms to transduce biochemical signals into electrical signals to achieve quantitative detection of targets. They have the advantages of simple operation, rapidity and low cost. Shewanella oneidensis When the MR-1 (a model strain of electrochemically active microorganisms) biofilm is used as a sensor element to construct a sensor, the electrons generated when oxidizing lactic acid can be transferred to the electrode through extracellular proteins to generate an electrical signal. Shewanella oneidensis This characteristic of MR-1 reflects the specific content of lactic acid through the intensity of the electrical signal. Shewanella oneidensis As a sensing element, the formation of MR-1 biofilm requires a long incubation time, which makes it difficult to meet the needs of rapid clinical detection. Summary of the invention

[0005] In view of the problems that the existing lactic acid detection methods are complicated and expensive, the present invention provides a method for rapid detection of lactic acid based on self-assembled electroactive monolayer membrane. Shewanella oneidensis MR-1 is a sensor element that uses electrostatic force to self-assemble an electroactive monolayer film to achieve rapid detection of lactate, which is expected to meet clinical detection needs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A method for rapidly detecting lactic acid based on a self-assembled electroactive monolayer film. In a microbial electrochemical system, the electroactive microorganisms are negatively charged, and the electrode is in situ modified with L-arginine using cyclic voltammetry, so that the modified electrode is positively charged. Based on the principle of opposite charges attract, the electroactive microorganisms are rapidly adsorbed to the electrode surface to form a self-assembled electroactive monolayer film. Based on the positive extracellular electron transfer ability of the electroactive microorganisms, the lactic acid content in the sample to be tested can be converted into a bioelectric signal output, and lactic acid is rapidly detected using the self-assembled electroactive monolayer film.

[0007] Furthermore, the microbial electrochemical system includes an electrolytic cell, a working electrode, a counter electrode, a reference electrode and an electrolyte with a pH of 7.4.

[0008] Furthermore, the working electrode in the microbial electrochemical system is 1 cm 2 Carbon cloth, counter electrode is 1 cm 2 Platinum electrode, reference electrode is Ag / AgCl electrode.

[0009] Furthermore, the electrolyte in the microbial electrochemical system comprises L-arginine, HEPES buffer, NaCl, NaHCO3 and NH4Cl.

[0010] Furthermore, each liter of electrolyte in the microbial electrochemical system contains 1.74 g L-arginine, 7.2 g HEPES buffer, 10 g NaCl, 2.5 g NaHCO3 and 1.0 g NH4Cl.

[0011] Furthermore, the electroactive microorganism is Shewanella oneidensis MR-1.

[0012] Furthermore, the parameters of the cyclic voltammetry are specifically as follows: a scanning potential range of -2V to 2V, a scanning rate of 5 mV / s, and a number of cycles of 15.

[0013] Furthermore, based on the principle of opposite charges attracting each other, the electroactive microorganisms are rapidly adsorbed to the electrode surface to form a self-assembled electroactive monolayer film. Specifically, the bacterial suspension of the electroactive microorganisms is centrifuged, then resuspended with a buffer solution, the resuspended bacterial suspension is added to the microbial electrochemical system, allowed to stand, and the self-assembled electroactive monolayer film is quickly prepared using electrostatic force.

[0014] Furthermore, the electroactive microorganisms are first expanded and cultured to a logarithmic growth phase or a stationary phase.

[0015] Furthermore, based on the positive extracellular electron transfer ability of the electroactive microorganisms, the lactic acid content in the sample to be tested can be converted into a bioelectric signal output, and the rapid detection of lactic acid using the self-assembled electroactive monolayer membrane is specifically as follows: a constant potential of +0.4 V is applied to the microbial electrochemical system and the lactic acid to be tested is added, and the lactic acid content is reflected by measuring the current peak signal.

[0016] Compared with the prior art, the present invention has the following advantages: The present invention modifies the electrode surface with positive charge, and the microorganisms use electrostatic adsorption force to quickly construct a self-assembled electroactive monolayer membrane. There is no need for biofilm startup and lengthy incubation time, and the operation is simple. The constructed self-assembled electroactive monolayer membrane has a fast response speed when detecting lactic acid and has a high detection sensitivity. It is expected to be clinically used for risk stratification of sepsis patients in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of a three-electrode electrochemical system in Example 1 of the present invention; Figure 2 1 is the EIS graph of the carbon cloth before and after modification in Example 1 of the present invention; Figure 3 This is the current change of detecting different concentrations of lactic acid in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] In order to gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description of the present invention. However, the present invention has multiple implementations and is not limited to the specific examples listed herein. The presentation of these examples is intended to deepen the comprehensive understanding of the disclosure of the present invention.

[0019] A method for rapid detection of lactic acid based on a self-assembled electroactive monolayer film comprises the following steps: (1) A microbial electrochemical system containing a working electrode, a counter electrode, and a reference electrode was constructed in an ultra-clean workbench. The working electrode was 1 cm 2 Carbon cloth, counter electrode is 1 cm 2 Platinum electrode, the reference electrode is Ag / AgCl electrode, 30 mL of electrolyte is added to the microbial electrochemical system, each liter of electrolyte contains 1.74 g L-arginine, 7.2 g HEPES, 10 g NaCl, 2.5 g NaHCO3 and 1.0 g NH4Cl; (2) The microbial electrochemical system was connected to a potentiostat, and cyclic voltammetry was used to in situ electropolymerize L-arginine on the surface of the working electrode. The scanning range of the cyclic voltammetry was -2 V to 2 V, the scanning rate was 5 mV / s, the number of cycles was 15, and the initial scanning direction was forward scanning; (3) Yes Shewanella oneidensis MR-1 was cultured to the logarithmic growth phase or the stationary phase, 20 mL of bacterial suspension was centrifuged at 8000 rpm for 10 min, and resuspended in 10 mM HEPES buffer to OD600 = 1.5; (4) Add the resuspended bacterial suspension to the microbial electrochemical system and let it stand for 5 min to quickly prepare a self-assembled electroactive monolayer film using electrostatic force; (5) A constant potential of +0.4 V is applied to the microbial electrochemical system and the lactic acid to be tested is added. The detection content is reflected by measuring the current peak signal, thereby achieving rapid detection of lactic acid.

[0020] Example 1

[0021] Three identical three-electrode electrochemical systems were constructed in the clean bench. Figure 1 ), the three electrodes are working electrode, counter electrode and reference electrode. The working electrode is 1 cm 2 Square carbon cloth (WOS1009, Taiwan Carbon Energy Company, China) was soaked in acetone overnight before use, dried and then subjected to high-temperature ammoniation treatment. The counter electrode and reference electrode were 1 cm 2 Square platinum sheet (Pt210, Tianjin Aida Hengsheng Technology Co., Ltd., China) and Ag / AgCl electrode (R0303, Tianjin Aida Hengsheng Technology Co., Ltd., China; 222.4mV vs. standard hydrogen electrode). The electrolyte was prepared with the following formula: 1.74 g L-arginine, 7.2 g HEPES, 10 g NaCl, 2.5 g NaHCO3 and 1.0 g NH4Cl per liter of electrolyte. The electrochemical system was connected to a multichannel potentiostat (CHI1030C, Shanghai Chenhua Instrument Co., Ltd., China) and cyclic voltammetry was used to in situ electropolymerize L-arginine on the working electrode surface. The scanning range of the cyclic voltammetry was -2 V to 2 V, the scanning rate was 5 mV / s, the number of cycles was 15, and the initial scanning direction was forward scanning.

[0022] Store in a refrigerator at 80°C Shewanella oneidensis Take out MR-1, put it in a 37℃ water bath to melt quickly, spread the strain on LB solid medium, culture it at 30℃ overnight, and activate it repeatedly 3 times. Transfer the activated pure bacterial single colony to 100 mL LB liquid medium for expansion culture. When the bacteria are in the stable period, divide the bacterial suspension into sterile centrifuge tubes for centrifugation at a speed of 10000rpm / min for 5 min. Discard the supernatant and resuspend the bottom bacterial mud with 10 mM sterile HEPES to OD 600 =1.5. The resuspended bacterial suspension was added to the electrochemical system and placed in a constant temperature incubator (HPS-500, Harbin Donglian Electronic Technology Development Co., Ltd., China) at 22°C for 5 min. The working electrode potential was set to +0.4 V (vs. Ag / AgCl electrode). After the current baseline was stable, different concentrations of lactic acid were added and the changes in the electrical signal were recorded and observed.

[0023] from Figure 2 It can be seen that compared with the unmodified carbon cloth, the Nyquist curve radius of the modified carbon cloth is smaller, which indicates that the charge transfer internal resistance of the carbon cloth modified with L-arginine is lower, which is more conducive to electron transfer between the electrode and the microorganism, and produces obvious electrical signals.

[0024] from Figure 3 It can be found that when there is no lactic acid in the electrochemical system, the baseline current is very low. When lactic acid (5 μM, 10 μM, 15 μM, 20 μM and 25 μM) is added to the electrochemical system by injection, the current will appear quickly, and the current increases with the increase of lactic acid concentration. When the lactic acid concentration is 5 μM, the peak current is 4.7 μA. When the lactic acid concentration increases to 25 μM, the current can reach 12.73 μA. Using prism to linearly fit the lactic acid concentration and peak current, y=0.3963x+3.002 is obtained, where y is the peak current and x is the lactic acid concentration. The coefficient of determination of the equation reaches 0.991, which shows that the lactic acid concentration and the peak current have a good linear relationship, and the lactic acid concentration can be accurately detected using the self-assembled electroactive monolayer. Compared with the detection technology based on chromatography and high-performance liquid chromatography in previous studies, the self-assembled electroactive monolayer membrane in this study does not require complicated sample pretreatment processes and expensive detection instruments, meeting the needs of instant detection. At the same time, it does not require expensive photoelectric signal transduction elements, and has the advantages of simple operation and low cost.

[0025] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technical personnel in the field, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

Claims

1. A method for rapid detection of lactic acid based on self-assembled electroactive monolayer film, characterized in that: In the microbial electrochemical system, the electroactive microorganisms are negatively charged. The electrode is modified with L-arginine in situ using cyclic voltammetry, and the modified electrode is positively charged. Based on the principle of opposite charges attracting each other, the electroactive microorganisms are rapidly adsorbed to the electrode surface to form a self-assembled electroactive monolayer film. Based on the positive extracellular electron transfer ability of electroactive microorganisms, the lactic acid content in the sample to be tested can be converted into bioelectric signal output, and lactic acid can be quickly detected using self-assembled electroactive monolayer films.

2. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: The microbial electrochemical system comprises an electrolytic cell, a working electrode, a counter electrode, a reference electrode and an electrolyte with a pH value of 7.

4.

3. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 2, characterized in that: The working electrode in the microbial electrochemical system is 1 cm 2 Carbon cloth, counter electrode is 1 cm 2 Platinum electrode, reference electrode is Ag / AgCl electrode.

4. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: The electrolyte in the microbial electrochemical system comprises L-arginine, HEPES buffer, NaCl, NaHCO3 and NH4Cl.

5. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: Each liter of electrolyte in the microbial electrochemical system contains 1.74 g L-arginine, 7.2 g HEPES buffer, 10 g NaCl, 2.5 g NaHCO3 and 1.0 g NH4Cl.

6. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: The electroactive microorganism is Shewanella oneidensis MR-1.

7. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: The parameters of the cyclic voltammetry method are specifically as follows: a scanning potential range of -2V to 2V, a scanning rate of 5 mV / s, and 15 cycles.

8. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: Based on the principle of opposite charges attracting each other, the electroactive microorganisms are rapidly adsorbed to the electrode surface to form a self-assembled electroactive monolayer film. Specifically, the bacterial suspension of the electroactive microorganisms is centrifuged, then resuspended with a buffer solution, the resuspended bacterial suspension is added to the microbial electrochemical system, allowed to stand, and the self-assembled electroactive monolayer film is rapidly prepared using electrostatic force.

9. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: The electroactive microorganisms are first cultured on an expanded scale to a logarithmic growth phase or a stationary phase.

10. The method for rapid detection of lactic acid based on self-assembled electroactive monolayer according to claim 1, characterized in that: Based on the positive extracellular electron transfer ability of electroactive microorganisms, the lactic acid content in the sample to be tested can be converted into a bioelectric signal output, and the self-assembled electroactive monolayer film is used to quickly detect lactic acid. Specifically, a constant potential of +0.4 V is applied to the microbial electrochemical system and the lactic acid to be tested is added, and the lactic acid content is reflected by measuring the current peak signal.

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