CN105784801A - Method for detecting low density lipoprotein cholesterin through double-enzyme concerted catalysis silver deposition - Google Patents

Abstract

The invention discloses a method for detecting low-density lipoprotein cholesterol through double-enzyme synergistic catalysis of silver deposition. Firstly, a sulfhydryl-containing poly-o-aminothiophenol film is formed on the surface of an electrode by electropolymerization, and then gold ions are deposited by a constant potential deposition method. Electroreductive reduction on the surface of the electrode forms gold nanoparticles, and anchors the gold nanoparticles on the surface of the electrode by electropolymerizing the sulfhydryl groups on the membrane, then immobilizes the apoB-100 antibody on the gold nanoparticles, and uses the apoB-100 antibody for low-density The specific recognition of lipoprotein captures low-density lipoprotein to the electrode surface. Under the synergistic action of cholesterol esterase and cholesterol oxidase, the cholesterol in low-density lipoprotein is decomposed and produces a weak reducing agent H ₂ O ₂ , which can make silver ions generate on the surface of gold nanoparticles. reduced and deposited onto the surface of gold nanoparticles. Finally, according to the stripping voltammetric current value of the detected silver element, a standard curve was drawn to realize the detection of low-density lipoprotein cholesterol.

Description

A method for detecting low-density lipoprotein cholesterol by synergistically catalyzing silver deposition with two enzymes

technical field

The invention belongs to the technical field of biological detection, and relates to a method for detecting low-density lipoprotein cholesterol by synergistically catalyzing silver deposition with two enzymes.

Background technique

Low-density lipoprotein cholesterol (LDL-C) is the main component of atherosclerotic plaque and the chief culprit of atherosclerotic vascular disease. Clinical studies have confirmed that there is a positive correlation between elevated LDL-C and the incidence of atherosclerosis and coronary heart disease. Therefore, accurate determination of the content of LDL-C in serum is of great significance for the diagnosis, prevention and treatment of atherosclerosis, hypertension, coronary heart disease and other diseases. The detection methods mainly include ultracentrifugation, electrophoresis, chemical or immunoprecipitation, etc., but the precipitation method currently used in clinical laboratories, the content of triglycerides in serum has a greater interference with its detection. Recently, John J et al. reported a homogeneous method for the detection of LDL-C, and its detection sensitivity and specificity have been greatly improved (John J, Albers, Hal Kennedy, Santica M, Marcovina. Evaluation of a new homogenous method for detection of small dense LDL cholesterol: Comparison with the LDL cholesterol profile obtained by density gradient ultrasound trifugation[J]. Clinica ChimicaActa.412.556-561(2011)); Watanabe et al. used any of the following three reagents or reagent groups One: (1) α-cyclodextrin sulfate, dextran sulfate, magnesium ion, polyoxyethylene-polyoxypropylene block copolyether reagent group; (2) amphoteric surfactants and carboxyl or sulfonic acid group Aliphatic amine reagent group; (3) polycation reagent. Constructed a cholesterol sensor and quantitative method of cholesterol together with oxidoreductase (Kiichi Watanabe, Yukawa Yuko, Nankai Shiro. Cholesterol sensor and quantitative method of cholesterol. China, invention patent, authorized time: 2005.02.02, Authorized patent number: 00802824.9). The instruments used in these methods are expensive, complicated, time-consuming and technically demanding. It is necessary to establish a fast, sensitive and easy-to-operate LDL-C detection method.

Contents of the invention

The purpose of the present invention is to provide a method for detecting low-density lipoprotein cholesterol by synergistically catalyzing silver deposition with two enzymes, which solves the problems of high cost, complicated operation, time-consuming and high technical requirements of the traditional LDL-C detection method.

The technical scheme adopted in the present invention is to carry out according to the following steps:

Step 1: Glassy carbon electrode pretreatment;

(1) Grinding and polishing the surface of the glassy carbon electrode to a mirror surface with Al ₂ O ₃ suspension, and washing the polished electrode with magnetic stirring in pure water, absolute ethanol, and pure water in sequence;

(2) Soak the electrode in the piraha solution, rinse it with pure water, and then wash it with magnetic stirring in pure water;

(3) Place the electrode in H ₂ SO ₄ for cyclic voltammetry scanning to activate the surface of the electrode to obtain an activated glassy carbon electrode, rinse it with pure water;

Step 2: Modification of the electrode;

(1) Immerse the activated glassy carbon electrode in a solution containing o-aminothiophenol and perchloric acid for cyclic voltammetry scanning, and perform magnetic stirring and washing with pure water after scanning;

( ₂ ) Put the electrode into the deposition solution composed of HCl, KCl, and HAuCl4 for constant potential deposition, and then wash it with magnetic stirring with pure water to obtain a glassy carbon electrode modified with gold nanoparticles.

Step 3: Construction of the Biosensing Interface

(1) Add the apoB-100 antibody dropwise to the surface of the glassy carbon electrode modified with gold nanoparticles, and incubate for 0.5-2 hours. After the incubation is completed, wash off the unfixed antibody with pure water, and use glycine-NaOH buffer solution Magnetic stirring washing;

(2) Add BSA solution dropwise on the surface of the electrode to block the reaction for 0.5-2 hours;

(3) carry out magnetic stirring washing with the glycine-NaOH buffer solution containing BSA;

Step 4: Standard curve drawing of LDL-C

(1) Add LDL solution dropwise to the electrode surface obtained in step 3 and fix the ApoB-100 antibody for incubation, and use glycine-NaOH buffer to wash with magnetic agitation to wash away uncaptured LDL;

(2) Drop cholesterol esterase (CHER), cholesterol oxidase (CHOD) and AgNO ₃ solutions on the electrode surface, and incubate in the dark to obtain a working electrode deposited with elemental silver, which is magnetically stirred with glycine-NaOH buffer washing;

(3) put the electrode into the _KNO3 solution, perform a linear scan, and record the stripping voltammetry peak of Ag;

(4) Draw the LDL-C standard curve according to the stripping voltammetric current value of elemental silver, and calculate the sensitivity and detection limit.

Step 5: Detection of samples to be tested

(1) Add 15 μL of the sample to be tested to the surface of the electrode on which the ApoB-100 antibody is immobilized obtained in step 3 to incubate, and wash with magnetic stirring with glycine-NaOH buffer;

( ₂ ) Dropping a solution containing CHER enzyme, CHOD enzyme and AgNO on the surface of the electrode, and incubating in the dark to obtain a working electrode deposited with elemental silver, which is washed by magnetic stirring with a glycine-NaOH buffer;

(3) Put the working electrode into the _KNO3 solution, perform linear scanning, the scanning range is 0.0～+0.8V, the scanning rate is 50-200mV/s, and record the stripping voltammetric current value of Ag;

(4) According to the standard curve described in step 4, the concentration of LDL-C in the sample solution to be tested is obtained.

Further, the particle size of the Al ₂ O ₃ suspension in step 1 is 0.3 μm and 0.05 μm.

Further, the piraha solution in step 1 is a mixture of 30% H ₂ O ₂ and 98% H ₂ SO ₄ with a volume ratio of 3:7.

Further, in step ₁ , place the electrode in _H2SO4 for cyclic voltammetry scanning, rinse it with pure water, and then place the electrode in potassium ferricyanide/potassium ferrocyanide solution for circulation Voltammetric scanning and AC impedance scanning, and finally rinse with pure water and dry for later use.

Further, in the step 2, the concentration of o-aminothiophenol is 5mmol/L, and the concentration of perchloric acid is 1mol/L.

Further, the deposition bottom solution in step 2 is composed of 0.01 mol/L HCl, 0.1 mol/L KCl, and 10 μmol/L HAuCl ₄ respectively in the same volume.

Further, the incubation temperature is 37°C.

Further, the glycine-NaOH is glycine 0.1 mol/L-NaOH buffer solution with pH 8.6.

Further, the linear scan range in step 4 and step 5 is 0.0-+0.8V, and the scan rate is 50-200mV/s.

Among them, step 1 provides a fresh electrode surface for step 2, and provides conditions for forming a complete, stable, firm, and good-conducting polymer film. The formation of the polymeric membrane in step 2 provides a site for the immobilization of the biorecognition molecule apoB-100 antibody in step 3, thereby constituting a biosensing interface that specifically recognizes LDL and facilitates the transfer of electrons. The construction of the biosensing interface in step 3 is an essential key step in the electrochemical detection of LDL-C in step 4. It can be seen that the steps 1-4 support each other and work together to realize the electrochemical detection of LDL-C by using the dual enzymes to catalyze the silver deposition reaction.

The method for detecting LDL-C established by the invention has the advantages of simple operation, short detection time and easy miniaturization.

Description of drawings

Figure 1 Schematic diagram of the electrochemical method for detecting LDL-C based on the principle of dual-enzyme synergistic catalysis of silver deposition reaction;

Figure 2 Cyclic voltammetry characterization diagrams of different modification processes on the electrode surface;

Figure 3 AC impedance characterization diagram of different modification processes on the electrode surface;

Fig. 4(a) The response current of the sensor under different concentrations of LDL-C;

Figure 4(b) Working curve of the sensor.

detailed description

The present invention will be described in detail below in combination with specific embodiments. Figure 1 is a schematic diagram of an electrochemical method for detecting LDL-C based on the principle of dual-enzyme synergistically catalyzed silver deposition reaction. Figure 2 is the cyclic voltammetry characterization diagram of different modification processes on the electrode surface; Figure 3 is the AC impedance characterization diagram of different modification processes on the electrode surface. The implementation steps are as follows:

1. Electrode pretreatment: use Al ₂ O ₃ suspensions with a particle size of 0.3 μm and 0.05 μm to polish the surface of the glassy carbon electrode to a mirror surface, and then magnetically stir the polished electrode in pure water and absolute ethanol respectively Wash for 5 minutes, rinse with pure water and then wash with magnetic stirring in pure water for 5 minutes; soak the electrode in the piraha solution prepared with a volume ratio of 3:7 of 30% H ₂ O ₂ and 98% H ₂ SO ₄ for 1 min, and use pure After rinsing with water, wash with magnetic stirring for 5 minutes; place the electrode in 0.5MH ₂ SO ₄ and perform cyclic voltammetry scanning at a potential of -0.3 to +1.5V for 10 minutes to activate the surface of the electrode. The scanning range is -0.3 to +1.5V, and the scanning rate is 50-100mV/s, rinse with pure water; place the electrode in potassium ferricyanide/potassium ferrocyanide solution for cyclic voltammetry scanning (-0.5～+1.0V, 50mV/s) and AC impedance Scan (0.24V, 1×10 ⁵ Hz, 0.1Hz), rinse with pure water and dry for later use.

2. Electrode modification: The activated glassy carbon electrode was vertically immersed in a solution containing o-aminothiophenol (oATP) (concentration: 5 mmol/L) and perchloric acid (concentration: 1 mol/L) for cyclic voltammetry scanning ( The scanning range is 0.0～+0.8V, the scanning rate is 10-100mV/s, and the number of scanning cycles is 40). After the scanning is completed, use pure water for magnetic stirring and washing for 5 minutes; 0.01mol/L HCl, 0.1mol/L KCl, 10μmol/L HAuCl ₄ ) for constant potential deposition (deposition potential is -0.5V, deposition time is 100-150 seconds), after the deposition is completed, use pure water for magnetic Stirring and washing for 5 minutes, the glassy carbon electrode modified with gold nanoparticles was obtained.

3. Immobilize ApoB-100 antibody: drop 10 μl of 20 μg/ml ApoB-100 antibody on the electrode surface modified with gold nanoparticles, incubate at 37°C for 1 hour, wash off the unfixed antibody with pure water after incubation, and wash with pH8 .6 Glycine-NaOH buffer solution was used for magnetic stirring and washing for 5 minutes; 10 μl of 1% BSA solution was added dropwise on the electrode surface to seal the non-specific adsorption sites on the electrode surface, and the blocking reaction was carried out at 37 ° C for 1 hour. After the reaction was completed, use BSA containing (0.1%) glycine (0.1mol/L)-NaOH buffer solution of pH 8.6 was washed away.

4. Standard curve for detection of LDL-C: drop 10 μl of LDL solution on the surface of the electrode immobilized with ApoB-100 antibody, incubate at 37°C for 30 minutes, after the completion of the immune reaction, use pH8.6 glycine (0.1mol/L)- Wash away the uncaptured LDL with NaOH buffer; add 5 μl of 100 μg/ml CHER enzyme solution, 5 μl of 100 μg/ml CHOD enzyme solution, and 10 μl of 5mMAgNO ₃ solution on the surface of the electrode dropwise, and incubate at 37°C for 30 minutes to obtain a working electrode deposited with elemental silver After the enzyme-catalyzed reaction was completed, the pH8.6 glycine-NaOH buffer solution was used for magnetic stirring and washing for 5 minutes; in the KNO ₃ (1mol/L) solution, the washed electrode was used as the working electrode, the platinum electrode was used as the counter The mercury electrode was used as a reference electrode, and a linear LSV scan was performed on an electrochemical workstation with a scan range of -0.5 to 0.6 V and a scan rate of 50 mV/s to obtain the stripping voltammetric current value of elemental silver. The stripping voltammetric current value (Y) of elemental silver is in a linear relationship with the concentration (X) of LDL-C in the range of 10ng/mL-1000ng/mL, and its linear equation is: Y=0.9459+0.4413X, linear correlation coefficient R ² =0.9982. Figure 4 is the standard curve of the electrochemical method for detecting LDL-C based on the principle of dual-enzyme synergistic catalyzed silver deposition reaction, in which Figure 4(a) is the response current of the sensor at different concentrations of LDL-C; Figure 4(b) is the working curve of the sensor .

5. Detection of LDL-C in actual samples: Add 10 μL LDL-C solution to be tested on the surface of the electrode immobilized with 10 μl 20 μg/ml ApoB-100 antibody, incubate at 37 ° C for 30 min, after the immune reaction is completed, use pH8.6 Glycine-NaOH buffer solution to wash away uncaptured LDL; drop 5 μl 100 μg/ml CHER enzyme solution, 5 μl 100 μg/ml CHOD enzyme solution, 10 μl 5mMAgNO ₃ solution on the surface of the electrode, and incubate at 37°C for 30 minutes to obtain elemental silver deposited After the enzyme-catalyzed reaction is completed, wash with pH 8.6 glycine-NaOH buffer for 5 minutes with magnetic stirring; in 1mol/L KNO ₃ solution, use the washed electrode as the working electrode, the platinum electrode as the counter electrode, and saturate The calomel electrode is used as a reference electrode, and linear scanning is performed on the electrochemical workstation. The scanning range is -0.5～0.6V, and the scanning rate is 50mV/s. ,395.7μA). According to the standard curve Y=0.9459+0.4413X in step 4, the corresponding concentrations of LDL-C in the actual sample solution can be obtained as (51.3ng/mL, 490.3ng/mL, 894.5ng/mL).

Compared with the prior art, the present invention has the following advantages:

1. The principle of dual-enzyme synergistic catalysis of silver deposition is used to realize the electrochemical detection of LDL-C, with short detection time, low background interference and high detection sensitivity.

2. The high molecular polymer membrane can provide a site for the immobilization of the biorecognition molecule apoB-100 antibody, and is conducive to the transfer of electrons.

3. The immune reaction on the surface of gold nanomaterials is an interface reaction system, which can improve the reaction efficiency of ApoB-100 antibody and LDL

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any simple modifications made to the above embodiments according to the technical essence of the present invention, equivalent changes and modifications, all belong to this invention. within the scope of the technical solution of the invention.

Claims (9)

1. a method for the detection of low-density lipoprotein cholesterol by a double-enzyme synergistic catalysis of silver deposition, characterized in that it is carried out according to the following steps: Step 1: Glassy carbon electrode pretreatment; (1) Grinding and polishing the surface of the glassy carbon electrode to a mirror surface with Al ₂ O ₃ suspension, and washing the polished electrode with magnetic stirring in pure water, absolute ethanol, and pure water in sequence; (2) Soak the electrode in the piraha solution, rinse it with pure water, and then wash it with magnetic stirring in pure water; (3) Place the electrode in H ₂ SO ₄ for cyclic voltammetry scanning to activate the surface of the electrode to obtain an activated glassy carbon electrode, rinse it with pure water; Step 2: Modification of the electrode; (1) Immerse the activated glassy carbon electrode in a solution containing o-aminothiophenol and perchloric acid for cyclic voltammetry scanning, and perform magnetic stirring and washing with pure water after scanning; ( ₂ ) Put the electrode into the deposition solution composed of HCl, KCl, and HAuCl4 for constant potential deposition, and then wash it with magnetic stirring with pure water to obtain a glassy carbon electrode modified with gold nanoparticles. Step 3: Construction of the Biosensing Interface (1) Add the apoB-100 antibody dropwise to the surface of the glassy carbon electrode modified with gold nanoparticles, and incubate for 0.5-2 hours. After the incubation is completed, wash off the unfixed antibody with pure water, and use glycine-NaOH buffer solution Magnetic stirring washing; (2) Add BSA solution dropwise on the surface of the electrode to block the reaction for 0.5-2 hours; (3) carry out magnetic stirring washing with the glycine-NaOH buffer solution containing BSA; Step 4: Standard curve drawing of LDL-C (1) Add LDL solution dropwise to the electrode surface obtained in step 3 and fix the ApoB-100 antibody for incubation, and use glycine-NaOH buffer to wash with magnetic agitation to wash away uncaptured LDL; (2) drop cholesterol esterase, cholesterol oxidase and AgNO solution _on the electrode surface, and incubate in the dark to obtain a working electrode deposited with elemental silver, which is washed by magnetic stirring with glycine-NaOH buffer solution; (3) put the electrode into the _KNO3 solution, perform a linear scan, and record the stripping voltammetry peak of Ag; (4) Draw the LDL-C standard curve according to the stripping voltammetric current value of elemental silver, and calculate the sensitivity and detection limit. Step 5: Detection of samples to be tested (1) Add 15 μL of the sample to be tested to the surface of the electrode on which the ApoB-100 antibody is immobilized obtained in step 3 to incubate, and wash with magnetic stirring with glycine-NaOH buffer; ( ₂ ) Dropping a solution containing CHER enzyme, CHOD enzyme and AgNO on the surface of the electrode, and incubating in the dark to obtain a working electrode deposited with elemental silver, which is washed by magnetic stirring with a glycine-NaOH buffer; (3) Put the working electrode into the _KNO3 solution, perform linear scanning, the scanning range is 0.0～+0.8V, the scanning rate is 50-200mV/s, and record the stripping voltammetric current value of Ag; (4) According to the standard curve described in step 4, the concentration of LDL-C in the sample solution to be tested is obtained. 2. A method for detecting low-density lipoprotein cholesterol by co-catalyzing silver deposition with dual enzymes according to claim 1, characterized in that: in the step 1, the particle size of the Al ₂ O ₃ suspension is 0.3 μm and 0.05 μm. 3. according to the method for a kind of double enzyme synergistic catalysis silver deposition detection low-density lipoprotein cholesterol according to claim 1, it is characterized in that: in described step 1, piraha solution is 30% H of volume ratio 3:7 ₂ O ₂ and 98 _{% H2SO4} mixed configuration. 4. according to claim 1, a kind of double-enzyme synergistic catalytic silver deposition detects the method for low-density lipoprotein cholesterol, it is characterized in that: in described step ₁ , electrode is placed in H ₂ SO After carrying out cyclic voltammetry scanning, After rinsing with pure water, place the electrode in potassium ferricyanide/potassium ferrocyanide solution for cyclic voltammetry scanning and AC impedance scanning respectively, and finally rinse with pure water and dry it for later use. 5. according to the method for a kind of double enzyme synergistic catalysis silver deposition detection low-density lipoprotein cholesterol described in claim 1, it is characterized in that: in described step 2, o-aminothiophenol concentration is 5mmol/L, perchloric acid concentration 1mol/L. 6. according to the method for a kind of dual-enzyme synergistic catalyzed silver deposition detection low-density lipoprotein cholesterol according to claim 1, it is characterized in that: in the described step 2, the deposition bottom liquid is respectively made of the same volume of 0.01mol/L HCl, 0.1mol /L KCl, 10μmol/L HAuCl ₄ composition. 7. A method for detecting low-density lipoprotein cholesterol by co-catalyzing silver deposition with dual enzymes according to claim 1, characterized in that: the incubation temperature is 37°C. 8. A method for detecting low-density lipoprotein cholesterol according to claim 1, wherein said glycine-NaOH is glycine 0.1mol/L-NaOH buffer solution with pH 8.6. 9. According to claim 1, the method for detecting low-density lipoprotein cholesterol by a kind of double-enzyme synergistically catalyzed silver deposition is characterized in that: the linear scanning range in the step 4 and step 5 is 0.0～+0.8V, and the scanning rate is 50-200mV/s.