CN112162023A - Lactate dehydrogenase electrochemical detection method based on signal amplification of multiwalled carbon nanotube and gold nanoparticle - Google Patents
Lactate dehydrogenase electrochemical detection method based on signal amplification of multiwalled carbon nanotube and gold nanoparticle Download PDFInfo
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Abstract
An electrochemical detection method of lactate dehydrogenase based on signal amplification of a multiwalled carbon nanotube and a gold nanoparticle belongs to the technical field of life science analysis. The detection principle is as follows: the method comprises the steps of sequentially modifying a multi-wall carbon nano tube, gold nanoparticles and a lactate dehydrogenase antibody on the surface of a glassy carbon electrode, combining the antibody when the lactate dehydrogenase appears, then catalyzing lactic acid in a substrate solution to be oxidized by the lactate dehydrogenase fixed on the surface of the electrode to generate pyruvic acid, measuring the current intensity by a differential pulse voltammetry method to obtain a linear equation between the concentration and the current intensity of the lactate dehydrogenase, and calculating to realize the sensitive detection of the lactate dehydrogenase. The invention provides a rapid and sensitive detection method of lactate dehydrogenase, which has good application value and is convenient to popularize.
Description
Technical Field
The invention discloses a lactate dehydrogenase electrochemical detection method based on signal amplification of a multiwalled carbon nanotube and a gold nanoparticle, and belongs to the technical field of life science analysis.
Background
Lactate dehydrogenase, a key enzyme in the glycolysis process, is present in the cytoplasm of almost all tissue cells of the body and catalyzes the production of pyruvate from lactate. The content of lactate dehydrogenase varies depending on various genetic factors, dysfunction of organ tissues, and causes of various diseases. The lactate dehydrogenase activity increases when some tissues are inflamed by trauma or by infection with pathogenic bacteria. Due to the increased permeability of cell membranes in the process of inflammation, the lactate dehydrogenase in cells is released, so that the content of the lactate dehydrogenase in blood or tissue fluid is increased. In addition, the content of the lactate dehydrogenase is also significant for diagnosing myocardial infarction, liver and gallbladder diseases, certain malignant tumors and leukemia, so that the development of a simple and efficient lactate dehydrogenase detection method has important use value.
At present, the detection methods of lactate dehydrogenase in China include a colorimetric method, a continuous detection method, a western blot method and the like, wherein the colorimetric method is most commonly used. The colorimetric method usually utilizes the color change generated by pyruvic acid and phenylhydrazine to measure the absorbance of the pyruvic acid and phenylhydrazine, but phenylhydrazine chemical substances such as common 2, 4-dinitrophenylhydrazine are unstable in chemical property, are flammable dangerous chemicals, have certain potential safety hazards during storage and use, and have large errors in detection results due to unstable properties.
The multi-walled carbon nanotube has good conductivity and larger specific surface area, and is widely applied to the research of modified electrodes, the activity of biomolecules can be kept by modifying gold nanoparticles on the surface of the carbon nanotube, the direct and rapid electron transfer between enzyme and a sensor is effectively promoted, and the detection speed of the electrochemical biosensor is improved. The gold nanoparticles can improve the immobilization rate of molecules on the surface, can keep the activity of a detection object immobilized on the gold nanoparticles, and provide a proper matrix for the immobilization of biological molecules on the surface of an electrode.
An electrochemical biosensor is a kind of instrument which is sensitive to biological substances and can convert the concentration of substances to be detected into an electric signal for detection. It uses biological macromolecule (such as antibody) as recognition element, and converts the signal generated by the combination of recognition element and the substance to be detected into electric signal change, so as to realize the analysis and detection of the substance to be detected. The electrochemical detection method has the advantages of sensitivity, strong specificity and the like, does not need complex instruments and equipment, and can detect in complex system samples.
Disclosure of Invention
In order to overcome the defects of the prior detection technology, the invention aims to fix the lactate dehydrogenase on the surface of the glassy carbon electrode through the recognition and combination action of a lactate dehydrogenase antibody by modifying the glassy carbon electrode and realize the detection of the lactate dehydrogenase by utilizing an electrochemical biosensing technology.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an electrochemical detection method for lactate dehydrogenase based on signal amplification of a multiwalled carbon nanotube and a gold nanoparticle comprises the following steps:
(1) pretreatment of glassy carbon electrodes
Sequentially grinding the surface of the glassy carbon electrode by using 1000-mesh, 2000-mesh and 5000-mesh sand paper, polishing the glassy carbon electrode into a mirror surface by using 1.0 mu m and 0.3 mu m alumina suspension, and thoroughly washing by using double distilled water; then ultrasonic cleaning is carried out in 95% ethanol and ultra-clean water for 5min respectively, the electrode is placed in 0.5M sulfuric acid solution for 30 cycles of cyclic voltammetry scanning to remove impurities on the surface of the electrode, the scanning potential is set to be-0.3V to +1.50V and is set to be 0.5 V.s-1Until a stable cyclic voltammogram is obtained, the glassy carbon electrode surface is rinsed with distilled water and dried.
(2) Modification of multiwall carbon nanotubes
Weighing 10mg of carboxylated multi-walled carbon nanotubes, weighing 5mL of deionized water, adding the multi-walled carbon nanotubes into the deionized water, performing ultrasonic dispersion for 30min to obtain a 2mg/mL multi-walled carbon nanotube suspension, and storing at 4 ℃ for later use. Adding the multiwall carbon nanotube suspension with the volume of 5 mu L to the surface of the glassy carbon electrode, drying in an incubator at 37 ℃ for 1h, washing off redundant multiwall carbon nanotubes with water, and drying.
(3) Modification of gold nanoparticles
2mg/mL chloroauric acid was dissolved in 0.5M sulfuric acid and stored in a refrigerator at 4 ℃ until use. And (3) placing the modified glassy carbon electrode in the step (2) in 5mL of 2mg/mL chloroauric acid solution for electrochemical deposition, wherein the electrochemical deposition potential is-200 mV, and the electrochemical deposition time is 120 s.
(4) Modification of lactate dehydrogenase antibodies
And (3) placing the modified electrode surface in 100 mu L of lactate dehydrogenase antibody solution with the concentration of 0.41 mu g/mL for modifying the antibody, wherein the modification time is 12h, the modification temperature is 4 ℃, and washing and drying.
(5) Removal of non-specific adsorption
And (3) dropwise adding 5 mu L of 0.1% bovine serum albumin solution on the surface of the electrode in the step (4) to block the non-specific adsorption sites on the surface of the electrode, incubating in an incubator at 37 ℃ for 1h, washing and drying for later use.
(6) Incubation of the solutions to be tested
And (3) dropwise adding 5 mu L of solution to be detected on the surface of the glassy carbon electrode modified in the step (5), incubating for 1h in a 37 ℃ incubator, washing and drying the surface of the electrode, and using for electrochemical determination.
(7) Electrochemical assay
The surface of the glassy carbon electrode in (6) above was placed in a substrate solution containing 0.8mM lithium lactate and 0.3mM oxidized coenzyme I phosphate buffered saline solution and having a pH of 6.5 and a volume of 5mL, and detected by differential pulse voltammetry. The phosphate buffered saline solution was 1L solution containing 8.5g of sodium chloride, 2.2g of disodium hydrogen phosphate and 0.2g of sodium dihydrogen phosphate, and the voltage ranged from-0.5V to + 0.3V.
According to the invention, the specific surface area of the electrode is increased through modification of the multi-wall carbon nano tube, and the conductivity of the electrode is improved through modification of the gold nano particles, so that an electric signal is effectively amplified. The detection system utilizes the characteristics that lactate dehydrogenase can catalyze lactic acid to generate pyruvic acid and generate electron transfer, and can reduce oxidized coenzyme I to increase the current on the surface of an electrode, determines the content of the lactate dehydrogenase by analyzing the current intensity, establishes a linear relation between the concentration of the lactate dehydrogenase and the current, and realizes efficient and sensitive detection of the lactate dehydrogenase.
Drawings
FIG. 1 is a schematic diagram of the construction principle of the detection system.
FIG. 2 is a diagram showing the results of the feasibility test of the test system: (A) a characterization diagram of a glassy carbon electrode modification process (B) a differential pulse voltammogram for feasibility verification of the detection system.
FIG. 3 is a graph showing the results of detection of lactate dehydrogenase at various concentrations: (A) differential pulse voltammogram (B) for detecting lactate dehydrogenase at different concentrations is a linear relationship graph of lactate dehydrogenase and corresponding current intensity.
The specific implementation mode is as follows:
example 1 characterization of electrode modification and validation of feasibility of detection System
Firstly, a detection system is constructed on the surface of the electrode, as shown in FIG. 1. The electrode modifications were then characterized in the same manner as described in the summary (1) to (7). As shown in fig. 2(a), the bare electrode shows good current response after pretreatment (curve a), after multi-wall carbon modification (curve b), the electron transfer on the surface of the electrode is blocked, the current response on the surface of the electrode is weakened, and the gold nanoparticles after gold plating modification (curve c) obviously enhance the current response on the surface of the electrode. Since the lactate dehydrogenase antibody (curve d) and lactate dehydrogenase (curve e) have negative charges in the solution, the modification of the lactate dehydrogenase antibody (curve d) and the lactate dehydrogenase (curve e) affects electron transfer on the surface of the electrode, and thus the current response is slightly weakened, indicating that the multi-walled carbon nanotube, the gold nanoparticle, the lactate dehydrogenase antibody and the lactate dehydrogenase are successfully modified on the surface of the electrode. As shown in fig. 2B, the current intensity was weak when the detection was performed without adding lactate dehydrogenase (curve a), but the current was significantly increased when the detection was performed with the addition of lactate dehydrogenase (curve B), and the feasibility of the electrochemical detection method for lactate dehydrogenase was fully demonstrated by comparative experiments.
Example 2 sensitivity test
The experimental procedures are the same as the contents (1) to (7), and lactate dehydrogenase standard solutions with the concentrations of 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 mu g/mL are selected for detection. As shown in FIG. 3(A), the current on the electrode surface increased with the increase in the lactate dehydrogenase concentration in the solution. As shown in FIG. 3(B), the linear detection equation of the biosensor is shown in the graph of the linear results obtained according to the current generated by lactate dehydrogenase solutions with different concentrations: y25.78807 +4.50166X (R)20.99186), detection range 0.001-0.5 μ g/mL. Calculating the lowest detection limit value of the biosensor by using the signal-to-noise ratio equal to 3, and obtaining the lowest detection limit value of the biosensor as follows: 0.00039 μ g/mL (n ═ n)10). The method can effectively and sensitively detect the content of the lactate dehydrogenase in the solution, and has great application potential in the technical field of life science analysis.
Claims (8)
1. An electrochemical detection method of lactate dehydrogenase based on signal amplification of a multiwalled carbon nanotube and a gold nanoparticle comprises the following detection principles: the method comprises the steps of sequentially modifying a multi-wall carbon nano tube, gold nanoparticles and a lactate dehydrogenase antibody on the surface of a glassy carbon electrode, combining the antibody when the lactate dehydrogenase appears, then catalyzing lactic acid in a substrate solution to be oxidized by the lactate dehydrogenase fixed on the surface of the electrode to generate pyruvic acid, measuring the current intensity by a differential pulse voltammetry method to obtain a linear equation between the concentration and the current intensity of the lactate dehydrogenase, and calculating to realize the sensitive detection of the lactate dehydrogenase.
2. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multi-walled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the pretreatment steps of the glassy carbon electrode are as follows: sequentially grinding the surface of the glassy carbon electrode by using 1000-mesh, 2000-mesh and 5000-mesh sand paper, polishing the glassy carbon electrode into a mirror surface by using 1.0 mu m and 0.3 mu m alumina suspension, and thoroughly washing by using double distilled water; then ultrasonic cleaning is carried out in 95% ethanol and ultra-clean water for 5min respectively, the electrode is placed in 0.5M sulfuric acid solution for 30 cycles of cyclic voltammetry scanning to remove impurities on the surface of the electrode, the scanning potential is set to be-0.3V to +1.50V and is set to be 0.5 V.s-1Until a stable cyclic voltammogram is obtained, the glassy carbon electrode surface is rinsed with distilled water and dried.
3. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multiwalled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the multiwalled carbon modification step of the glassy carbon electrode is as follows: weighing 10mg of carboxylated multi-walled carbon nanotubes, weighing 5mL of deionized water, adding the multi-walled carbon nanotubes into the deionized water, performing ultrasonic dispersion for 30min to obtain a 2mg/mL multi-walled carbon nanotube suspension, adding the 5 muL multi-walled carbon nanotube suspension to the surface of an electrode, drying in a 37 ℃ incubator for 1h, washing off redundant multi-walled carbon nanotubes with water, and drying.
4. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multiwalled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the gold nanoparticles of the glassy carbon electrode are modified as follows: dissolving 2mg/mL chloroauric acid in 0.5M sulfuric acid solution, soaking the surface of the glassy carbon electrode in the chloroauric acid solution for electrochemical deposition, applying the potential of the electrochemical deposition to-200 mV for 120s, cleaning and drying.
5. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multi-walled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the step of modification of the lactate dehydrogenase antibody of the glassy carbon electrode is as follows: soaking the surface of the glassy carbon electrode modified by the gold nanoparticles in a lactic dehydrogenase antibody solution with the volume of 100 mu L and the concentration of 0.41 mu g/mL for modifying the antibody, wherein the modification time is 12h, the modification temperature is 4 ℃, and the surface of the modified electrode is cleaned and dried.
6. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multi-walled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the surface sealing step of the glassy carbon electrode is as follows: and dropwise adding 5 mu L of 0.1% bovine serum albumin solution on the surface of the antibody modified electrode to seal the non-specific adsorption sites on the surface of the electrode, placing the electrode in an incubator at 37 ℃ for incubation for 1h, and washing and drying the electrode for later use.
7. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multi-walled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the steps of lactate dehydrogenase modification are as follows: dripping 5 mu L of solution to be detected on the surface of the sealed glassy carbon electrode, incubating for 1h in a 37 ℃ incubator, washing and drying the surface of the electrode, and performing electrochemical detection in a substrate solution.
8. The electrochemical detection method for lactate dehydrogenase based on signal amplification of multi-walled carbon nanotubes and gold nanoparticles as claimed in claim 1, wherein the electrochemical detection steps are as follows: a glassy carbon electrode obtained after carrying out claim 7 was placed in a substrate solution containing 0.8mM lithium lactate, 0.3mM oxidized coenzyme I phosphate buffered saline solution and having a pH of 6.5 and a volume of 5mL and detected by differential pulse voltammetry. The phosphate buffered saline solution was 1L solution containing 8.5g of sodium chloride, 2.2g of disodium hydrogen phosphate and 0.2g of sodium dihydrogen phosphate, and the voltage ranged from-0.5V to + 0.3V.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5147781A (en) * | 1986-03-04 | 1992-09-15 | Judith Rishpon | Enzyme electrode and assay for determining LDH5 |
| CN104502583A (en) * | 2015-01-09 | 2015-04-08 | 江苏大学 | Carbon nano tube/nanogold composite membrane electrochemical immunosensor and application thereof |
| CN110346311A (en) * | 2019-07-15 | 2019-10-18 | 三诺生物传感股份有限公司 | A kind of lactic dehydrogenase detection reagent |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5147781A (en) * | 1986-03-04 | 1992-09-15 | Judith Rishpon | Enzyme electrode and assay for determining LDH5 |
| CN104502583A (en) * | 2015-01-09 | 2015-04-08 | 江苏大学 | Carbon nano tube/nanogold composite membrane electrochemical immunosensor and application thereof |
| CN110346311A (en) * | 2019-07-15 | 2019-10-18 | 三诺生物传感股份有限公司 | A kind of lactic dehydrogenase detection reagent |
Non-Patent Citations (1)
| Title |
|---|
| 王运斗: "一种新型酶生物传感器电极", 《国外医学.生物医学工程分册》 * |
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