CN104597100B - Method for detecting triglyceride in serum - Google Patents
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Abstract
The invention belongs to the field of bioanalytical chemistry, and relates to an electrochemical method for detecting triglyceride in serum. The invention utilizes the characteristic that dopamine is automatically polymerized to form polydopamine under the alkalescent condition, on one hand, the formed polydopamine is used for fixing enzyme molecules, on the other hand, nanogold generated by in-situ reduction of chloroauric acid is used as a signal amplification element, and Cyclic Voltammetry (CV) is used as an electrochemical detection method. The developed electrochemical sensing strategy for detecting the triglyceride in the serum has the advantages of high sensitivity, simplicity in operation and the like, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the field of bioanalytical chemistry, and relates to a method for detecting triglyceride in serum, in particular to a method for detecting triglyceride in serum with high sensitivity by utilizing electrochemical sensing strategy that polydopamine is used for efficiently fixing biological enzyme molecules on one hand, and chloroauric acid is reduced in situ in the polymerization process of dopamine to play a role in signal amplification on the other hand.
Background
Triglyceride (TG), also known as neutral fat, is esterified from three molecules of fatty acids and one molecule of glycerol and is the main source of energy in the body. Triglyceride levels are not only positively correlated with Total Cholesterol (TC), Body Mass Index (BMI) and blood Glucose (GLU), but also strongly correlated with obesity. The detection of triglycerides in human blood is very important because of its high concentration (normal range male 40-160 mg-dL)-1And 35-135 mg.dL for women-1) Can cause hyperlipidemia, and the increased concentration of triglyceride is associated with increased risk of atherosclerosis, and the timely detection of lipid is helpful for diagnosing and treating diabetes, nephropathy, liver obstruction and other diseasesVarious endocrine disorders are metabolized. However, there are many problems in the measurement of triglyceride in serum and its clinical application, such as biological variation, influence of free glycerol on the measurement, and imperfect standardized system of measurement. The prior art mainly depends on a large-scale biochemical analyzer for measuring triglyceride in serum, the price is high, time is consumed in the measuring process, the efficiency is greatly reduced, and the electrochemical analysis method is easy to operate and low in price and is favored by analysis workers.
Dopamine is a neurotransmitter, and in 2007, American scientists take dopamine as a monomer for the first time, polydopamine is formed through simple self-polymerization reaction under the alkalescent condition, a bionic polydopamine film can be formed on the surfaces of various materials, the polydopamine film has super-strong adhesion characteristics, various organic and inorganic materials can be fixed, and the polydopamine film has wide application prospects in the field of biosensors.
The prior art does not disclose a technical scheme for detecting the activity of lipase by using dopamine as a carrier for immobilizing lipase and in-situ reduction of chloroauric acid to form nanogold as a signal enhancement element.
Disclosure of Invention
The technical purpose of the present invention is to provide a simple, inexpensive and effective electrochemical assay for detecting triglycerides in serum. The method firstly uses polydopamine for immobilizing lipase molecules and reduces chloroauric acid in situ in the polymerization process to generate nanogold for signal amplification.
The purpose of the invention can be realized by the following technical scheme:
a method for detecting triglycerides in serum, comprising the steps of:
(1) pretreating an ITO electrode;
(2) putting the treated ITO electrode obtained in the step (1) into a dopamine solution, and adjusting the pH value to an alkaline condition by using a buffer solution;
(3) adding chloroauric acid into the system in the step (2), uniformly mixing, and reacting for a period of time to obtain a polydopamine-nanogold modified ITO electrode;
(4) dripping a lipase solution on the surface of the polydopamine-nanogold-modified ITO electrode obtained in the step (3), and drying in the shade at low temperature to obtain a polydopamine-nanogold-lipase electrode;
(5) obtaining a triglyceride detection standard curve: placing a polydopamine-nanogold-lipase modified electrode in a buffer solution containing triglyceride with gradient concentration for reacting for a certain time, cleaning the electrode with ultrapure water, and placing the electrode in a solution containing potassium ferricyanide for electrochemical scanning to obtain cyclic voltammetry peak currents corresponding to the triglyceride with different concentrations; the triglyceride standard curve was plotted using the reduction peak current as ordinate and the triglyceride concentration as abscissa.
(6) Determination of the recovery of triglycerides in serum: removing the inherent triglyceride in the serum of a healthy person, adding the triglyceride with different concentrations, performing electrochemical detection under the same operation condition of drawing a standard curve in the step (5), comparing the obtained data on the standard curve in the step (5), and calculating the recovery rate of the triglyceride.
The method of the present invention, the pretreatment of the ITO electrode in step (1) is understood to be any method of the prior art for pretreating an ITO electrode, and the present invention is not limited thereto.
Wherein, the concentration of the dopamine solution in the step (2) is 2mg/mL, the buffer solution is Tris-HCl buffer solution, and the pH value is 8.5.
Wherein the final concentration of the chloroauric acid in the step (3) is 5 mu mol/L; the reaction time was 4 h.
Wherein, the concentration of the lipase dripped on the modified electrode surface in the step (4) is 2 mg/mL.
Wherein, in the step (5), the final concentration of the potassium ferricyanide is 5 mmol/L. The electrochemical scan is a cyclic voltammetry scan.
Wherein, in the step (5), the buffer solution is phosphate buffer solution, and the pH value is 7.5.
Wherein, in the step (6), the method for removing the inherent triglyceride in the serum of the healthy person comprises the following steps: 1mL of human serum was taken, 2mg of lipase was added thereto, incubated at 37 ℃ overnight, and then 2mL of 15% trichloroacetic acid solution was added to denature and precipitate the lipase, and centrifuged at 12000 rpm for 10 min. Sucking supernatant, dropwise adding 0.1mol/L NaOH solution into the supernatant, and adjusting pH to 7.0 to obtain human serum without inherent triglyceride.
More specifically, the method for detecting triglyceride in serum comprises the following steps:
(1) pretreating an ITO electrode;
(2) putting the treated ITO electrode obtained in the step (1) into a dopamine solution, and adjusting the pH value to an alkaline condition by using a buffer solution, wherein the concentration of the dopamine solution is 2 mg/mL; the buffer solution is Tris-HCl buffer solution, and the pH value is 8.5.
(3) And (3) adding chloroauric acid into the system in the step (2), uniformly mixing and reacting for a period of time to obtain the polydopamine-nanogold modified ITO electrode. Wherein the final concentration of the chloroauric acid is 5 mu mol/L; the reaction time is 4 h;
(4) and (4) dripping a lipase solution on the surface of the polydopamine-nanogold-modified ITO electrode obtained in the step (3), and drying in the shade at low temperature to obtain the polydopamine-nanogold-lipase electrode. Wherein the concentration of the lipase is 2 mg/mL;
(5) obtaining a triglyceride detection standard curve: placing a polydopamine-nanogold-lipase modified electrode in a buffer solution containing triglyceride with gradient concentration for reacting for a certain time, cleaning the electrode with ultrapure water, and placing the electrode in a solution containing potassium ferricyanide for electrochemical scanning to obtain cyclic voltammetry peak currents corresponding to the triglyceride with different concentrations; the triglyceride standard curve was plotted using the reduction peak current as ordinate and the triglyceride concentration as abscissa. Wherein the final concentration of the potassium ferricyanide is 5 mmol/L; the electrochemical scan is a cyclic voltammetry scan.
(6) Determination of the recovery of triglycerides in serum: firstly removing the inherent triglyceride in the serum of a healthy person, then adding the triglyceride with different concentrations, and determining the recovery rate. The method for removing the inherent triglyceride in the serum of a healthy person comprises the following steps: 1mL of human serum was taken, 2mg of lipase was added thereto, incubated at 37 ℃ overnight, and then 2mL of 15% trichloroacetic acid solution was added to denature and precipitate the lipase, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was aspirated, 0.1mol/L NaOH solution was added thereto, and pH was adjusted to 7.0, whereby human serum from which intrinsic triglycerides were removed was obtained.
The invention has the beneficial effects that:
the technical scheme of the invention utilizes the property that dopamine is subjected to autopolymerization reaction under the alkalescent condition to form bionic membrane polydopamine, and can fix lipase molecules on one hand and reduce chloroauric acid into nano gold in situ in the polymerization process of dopamine on the other hand due to the super strong adsorption property, so that the movement of electrons on the surface of an electrode is accelerated, and the signal amplification effect is achieved.
Due to the high selectivity of enzyme molecules to substrates, triglyceride can be specifically detected, and Cyclic Voltammetry (CV) is used as an electrochemical detection method. The developed electrochemical sensing strategy for detecting the triglyceride in the serum has the advantages of high sensitivity, simple operation, low detection cost and the like. Therefore, the method is beneficial to popularization and application.
Drawings
FIG. 1 is a schematic diagram of a sensing strategy established by the present invention;
fig. 2 is a Scanning Electron Microscope (SEM) image of a polydopamine-nanogold/ITO electrode.
FIG. 3 is a diagram of: a) a bare ITO electrode; b) modifying the surface of the ITO electrode with nano gold coated by polydopamine; c) dripping 20 mu L of 2mg/mL lipase solution on the surface of the polydopamine-nanogold/ITO electrode, and drying in the shade; containing 5mmol/L [ Fe (CN)6]3-/4-(1: 1) and 0.1mol/L KCl in phosphate buffer solution (10mmol/L, pH 7.4).
FIG. 4 shows a graph containing a)50mg/dL, respectively; b)100 mg/d; c)150 mg/dL; d)200 mg/dL; e)250 mg/dL; f)300 mg/dL; g)400 mg/dL; h)500 mg/dL; i) CV diagram measured at 600 mg/dL. The solution was a phosphate buffer solution (pH7.5, 50mmol/L) containing 5mmol/L potassium ferricyanide.
FIG. 5 is a standard graph of triglyceride with the inset being a linear relationship between the concentration of triglyceride and the CV reduction peak current.
FIG. 6 is a selective experimental method for detecting triglycerides in solution: adding 1 mmol/L into solution containing triglyceride according to the ratio of each component in serum-1Urea (Urea), 0.2 mmol. L-1Uric Acid (UA), 5 mmol. L-1Cholesterol (Chol), 5 mmol. multidot.L-1Glucose (Glu), the results are presented as the magnitude of the reduction peak current.
Detailed Description
Example 1
(1) Preparation of polydopamine-nanogold/ITO electrode
Immersing the pretreated ITO electrode into 1mL of dopamine solution with the concentration of 2mg/mL, adding 0.1mol/L of Tris-HCl buffer solution into the solution, adjusting the pH value of the solution to 8.5, adding 0.1mol/L chloroauric acid solution into the solution by using a liquid-transferring gun to enable the final concentration of the chloroauric acid solution to be 5 mu mol/L, mixing the solution uniformly, reacting for 4 hours at room temperature, taking out the ITO electrode by using tweezers, and cleaning the ITO electrode for three times by using double distilled water to obtain the polydopamine-nanogold modified ITO electrode.
(2) Modification of lipase on the surface of polydopamine-nanogold/IT electrode
And (2) dropwise adding 20 mu L of 2mg/mL lipase solution on the surface of the polydopamine-nanogold/ITO electrode obtained in the step (1), uniformly coating by using a gun head, then naturally drying in the shade for 24 hours at 4 ℃, cleaning for three times by using phosphate buffer solution (pH7.5, 50mmol/L), cleaning for three times by using distilled water, and blow-drying by using high-purity nitrogen to obtain the polydopamine-nanogold/ITO electrode modified by the lipase, namely the lipase/polydopamine-nanogold/ITO electrode, and storing in a refrigerator when not used.
Example 2
Electrochemical detection of triglycerides
The modified bio-enzyme electrode in example 1 is placed in a solution containing triglyceride with a certain concentration, the reaction is carried out for 25min at 37 ℃, the electrode is cleaned by ultrapure water after the reaction is finished, potassium ferricyanide solution is added into the reaction solution to enable the final concentration to be 5mmol/L, then electrochemical cyclic voltammetry scanning is carried out to obtain CV peak currents (shown in figure 4) corresponding to the triglyceride with different concentrations, and then a standard curve (shown in figure 5) of the triglyceride is drawn by taking the reduction peak current as an ordinate and the concentration of the triglyceride as an abscissa.
Example 3
Determination of the triglyceride content in serum
The content of triglyceride in serum is measured by diluting the treated human serum 50 times by standard addition method, adding triglyceride (50, 100, 150, 200mg/mL) with different concentrations, measuring corresponding current signal value, and comparing with the standard curve obtained in example 2 to obtain triglyceride recovery rate.
| Triglyceride concentration (mg.dL)-1) | The amount measured by the method (mg.dL)-1) | Recovery (%) |
| 50 | 49.2 | 98.4 |
| 100 | 102.3 | 102.3 |
| 150 | 145.4 | 96.9 |
| 200 | 205.6 | 102.8 |
Example 4
Alternating current impedance (EIS) characterization
The impedance standard solution is 5mmol/L of [ Fe (CN)6]3-/4-(1: 1) and 0.1mol/LKCl phosphate buffer solution (10mmol/L, pH7.4), in FIG. 3, a, b, c are impedance diagrams of a bare ITO electrode, a polydopamine-nanogold/ITO electrode, and a lipase/polydopamine-nanogold/ITO electrode, respectively. Wherein, the radius of the impedance represents the magnitude of the resistance, i.e., the larger the radius, the larger the impedance value, say [ Fe (CN)6]3-/4-The weaker the electron transfer ability with the electrode, the experimental results show that the impedance value is further increased as the lipase is modified onto the electrode surface because the protein is insulating and not conducting.
The selectivity experiment shows that the triglyceride sensor constructed by the inventor has good selectivity on triglyceride, and common interferents such as urea, uric acid, cholesterol, glucose and the like which are used for detecting triglyceride can only generate negligible electrochemical signals (< 3%), and further, the method can be used for detecting triglyceride in serum with high selectivity, and other interferents can not generate influence.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for detecting triglycerides in serum, comprising the steps of:
(1) pretreating an ITO electrode;
(2) putting the treated ITO electrode obtained in the step (1) into a dopamine solution, and adjusting the pH value to an alkaline condition by using a buffer solution;
(3) adding chloroauric acid into the system in the step (2), uniformly mixing, and reacting for a period of time to obtain a polydopamine-nanogold modified ITO electrode;
(4) dripping a lipase solution on the surface of the polydopamine-nanogold-modified ITO electrode obtained in the step (3), and drying in the shade at low temperature to obtain a polydopamine-nanogold-lipase electrode;
(5) obtaining a triglyceride detection standard curve: placing a polydopamine-nanogold-lipase modified electrode in a buffer solution containing triglyceride with gradient concentration, incubating for a certain time at a proper temperature, washing the electrode with ultrapure water, and placing the electrode in a solution containing potassium ferricyanide to perform electrochemical scanning to obtain cyclic voltammetry peak currents corresponding to the triglyceride with different concentrations; drawing a triglyceride standard curve by using the reduction peak current as an ordinate and the triglyceride concentration as an abscissa;
(6) determination of the recovery of triglycerides in serum: removing the inherent triglyceride in the serum of a healthy person, adding the triglyceride with different concentrations, performing electrochemical detection under the same operation condition of drawing a standard curve in the step (5), comparing the obtained data on the standard curve in the step (5), and calculating the recovery rate of the triglyceride.
2. The method for detecting triglycerides in serum according to claim 1,
the concentration of the dopamine solution in the step (2) is 2 mg/mL; the buffer solution is Tris-HCl buffer solution, and the pH value is 8.5.
3. The method for detecting triglycerides in serum according to claim 1,
the final concentration of the chloroauric acid in the step (3) is 5 mu mol/L; the reaction time was 4 h.
4. The method for detecting triglycerides in serum according to claim 1,
dripping a lipase solution with the concentration of 2mg/mL on the modified electrode surface in the step (4); low temperature drying in shade at 4 deg.c.
5. The method of detecting triglycerides in serum according to claim 1, wherein said step is carried out
(5) The medium reaction temperature is 40 ℃, and the reaction time is 25 min.
6. The method for detecting triglycerides in serum according to claim 1,
in the step (5), the final concentration of the potassium ferricyanide is 5 mmol/L.
7. The method for detecting triglycerides in serum according to claim 1,
the buffer solution used was phosphate buffer solution with a pH of 7.5.
8. The method for detecting triglycerides in serum according to claim 1,
in the step (6), the method for removing the inherent triglyceride in the serum of the healthy person comprises the following steps: taking 1mL of human serum, adding 2mg of lipase into the human serum, incubating at 37 ℃ overnight, then adding 2mL of 15% trichloroacetic acid solution to denature and precipitate the lipase, rotating at 12000 r/min, centrifuging for 10min, sucking supernatant, dropwise adding 0.1mol/L NaOH solution into the supernatant, and adjusting the pH to 7.0 to obtain the human serum without inherent triglyceride.
9. The method for detecting triglycerides in serum according to claim 1,
the method for detecting triglycerides in serum and the electrochemical scan for the determination of the recovery of triglycerides in serum described in step (6) are cyclic voltammetric scans.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005114164A2 (en) * | 2004-05-14 | 2005-12-01 | Bayer Healthcare Llc | Voltammetric systems for assaying biological analytes |
| CN1858592A (en) * | 2005-04-30 | 2006-11-08 | 北京怡成生物电子技术有限公司 | Test sheet |
| CN102212609A (en) * | 2011-04-01 | 2011-10-12 | 哈尔滨商业大学 | Method for detecting lipase heterogeneous catalytic reaction with biosensor |
| US20120135509A1 (en) * | 2009-07-27 | 2012-05-31 | SureSensors Ltd. | Sensor devices |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005114164A2 (en) * | 2004-05-14 | 2005-12-01 | Bayer Healthcare Llc | Voltammetric systems for assaying biological analytes |
| CN1858592A (en) * | 2005-04-30 | 2006-11-08 | 北京怡成生物电子技术有限公司 | Test sheet |
| US20120135509A1 (en) * | 2009-07-27 | 2012-05-31 | SureSensors Ltd. | Sensor devices |
| CN102212609A (en) * | 2011-04-01 | 2011-10-12 | 哈尔滨商业大学 | Method for detecting lipase heterogeneous catalytic reaction with biosensor |
Non-Patent Citations (2)
| Title |
|---|
| Electrochemical growth of flowerlike gold nanoparticles on polydopamine modified ITO glass for SERS application;Weichun Ye 等;《Electrochimica Acta》;20091111;第55卷;第2004-2006页 * |
| 甘油酯基于脂肪酶修饰电极的生物传感;郭春景;《中国优秀硕士学位论文全文数据库（电子期刊） 工程科技I辑》;20130615(第06期);第16-26页 * |
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