CN114354709A - Reagent kit for detecting total bile acid in urine by electrochemical method and application thereof - Google Patents

Reagent kit for detecting total bile acid in urine by electrochemical method and application thereof Download PDF

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CN114354709A
CN114354709A CN202210006213.XA CN202210006213A CN114354709A CN 114354709 A CN114354709 A CN 114354709A CN 202210006213 A CN202210006213 A CN 202210006213A CN 114354709 A CN114354709 A CN 114354709A
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urine
bile acid
total bile
kit
electrode
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张晓清
李彦茹
丁敏
吕文静
朱阳
宋紫苹
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International Institute Of In Vitro Diagnostics Chongqing Medical University
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Abstract

The invention discloses a kit for detecting total bile acid in urine by an electrochemical method and application thereof. The reagent kit for detecting the total bile acid in the urine by the electrochemical method comprises the following reagents: phosphate buffer solution, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), oxidized coenzyme I (NAD)+) Ruthenium terpyridyl (Ru (bpy)3 2+) And screen printing electrodes. The reagent kit for detecting the total bile acid in the urine by the electrochemical method adopts a screen printing electrode combined with an I-t curve, and realizes the rapid quantitative detection of the total bile acid in the urine according to the linear relation between the electrochemical response signal and the logarithm of the concentration of the substance to be detected. The electrochemical detection kit is simple, convenient and quick, has better repeatability, stability and accuracy, can realize quick quantitative detection of the content of total bile acid in urine, and provides a new choice for noninvasive real-time monitoring of liver and gall diseases.

Description

Reagent kit for detecting total bile acid in urine by electrochemical method and application thereof
Technical Field
The invention belongs to the field of electrochemical detection, and mainly relates to a kit for detecting total bile acid in urine by an electrochemical method and application thereof.
Background
Bile acid is a kind of 24-carbon cholanic acid, which is synthesized in the liver by using cholesterol as a raw material. Bile acid has important physiological functions, can promote the digestion and absorption of fat, can be used as a signal molecule to activate various receptors and mediate various signal regulation pathways, and plays an important role in glucose, fat, self-metabolism and energy metabolism of a human body. When liver diseases occur, the damage of liver cells can cause the synthesis and excretion disorder of bile acid, and the synthesis of bile acid is obviously reduced. Dysfunction of bile acid uptake by hepatocytes slows the rate of bile acid clearance from the blood, resulting in increased levels of bile acids in the blood and urine. Serum bile acid is a clinical biochemical routine detection item, and the change of the concentration of the serum bile acid can sensitively reflect liver function. The research shows that urine bile acid has strong correlation with serum bile acid level. Because the urine specimen can be obtained without invasive operation, the urine bile acid can replace serum bile acid to be used as an index for noninvasive and real-time monitoring of liver and gall diseases.
At present, the total bile acid is clinically determined mainly by an enzyme cycle method, and the principle is that the bile acid is subjected to 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and Thio-NAD+Under the action of the said catalyst, 3 alpha-ketosteroid and Thio-NADH are produced. The newly generated 3 alpha-ketosteroid generates bile acid and NAD under the action of 3 alpha-HSD and NADH+. Thus, the detection signal of a trace amount of bile acid is amplified in the course of multiple enzyme cycles, and the amount of bile acid can be determined by amplifying the produced Thio-NADH and measuring the change in absorbance of the Thio-NADH. The method is widely applied in clinic, but the detection sensitivity is in the mu mol/L level, and needs to be improved, and a special instrument for detection is expensive and has larger volume.
The electrochemical analysis method is to realize qualitative and quantitative analysis of the object to be detected according to the electrochemical property of the object to be detected in the solution and through the relationship between the parameters of current, potential, resistance and the like and the concentration of the object to be detected. The electrochemical method for measuring the total bile acid in the urine has the following advantages: simple operation, strong mobility, high detection speed, small dosage of kit samples, high sensitivity, good selectivity and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the urine total bile acid detection kit with high sensitivity and convenient use and the application thereof.
The principle of the reagent kit for detecting the total bile acid in urine by the electrochemical method is as follows: adding 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and oxidized coenzyme I (NAD) on the surface of working electrode of Screen Printing Carbon Electrode (SPCE)+) And ruthenium terpyridyl (Ru (bpy)3 2+) Phosphate buffer of (4). Applying a certain constant oxidation potential, Ru (bpy) in the solution3 2+Oxidized to Ru (bpy) at the electrode surface3 3+And adding a urine sample to be detected after the baseline is stable, and specifically catalyzing bile acid in the urine by the 3 alpha-HSD to generate 3 alpha-ketosteroid and NADH. NADH loses electrons on the surface of the electrode and is oxidized into NAD+Simultaneous Ru (bpy)3 3+Is reduced to Ru (bpy)3 2+. Under a certain potential condition, Ru (bpy)3 2+Oxidized again to Ru (bpy) at the electrode surface3 3+So as to generate electrochemical signals, and the indirect electrochemical quantitative analysis of the total bile acid content in the urine can be carried out on an I-t curve.
The SPCE comprises a polyethylene terephthalate (PET) substrate with a printed electrode, an external insulating film on the PET substrate and a lead interface on the substrate. The device is characterized in that three electrodes, namely a carbon working electrode, an Ag/AgCl reference electrode and a carbon counter electrode, are printed on the substrate. The three electrodes form a circular working area, and each electrode is connected with the interface through a lead printed under the insulating film. And washing the SPCE by using ultrapure water, and airing at room temperature for later use.
Wherein, more preferably, the activity of the 3 alpha-HSD in the detection mixed solution is 1.2kU/L, and the Ru (bpy)3 2+At a concentration of 7mmol/L, the NAD+The concentration of the phosphate buffer solution is 8mmol/L, the concentration of the phosphate buffer solution is 50.0mmol/L, the pH value is 8.5, and the detection potential is 1.20V.
Wherein the Phosphate Buffer Solution (PBS) is prepared by mixing 0.1mol/L disodium hydrogen phosphate solution and 0.1mol/L sodium phosphate solution.
1. The specific use method of the kit for detecting the total bile acid in urine by the electrochemical method comprises the following steps:
1) sample pretreatment: 10 μ L urine and 990 μ L ultrapure water were added to 1.5mL EPUniformly mixing in a vortex mode in the tube to obtain a primary diluted urine sample; then, 10. mu.L and 990. mu.L of ultrapure water of the urine sample diluted once are added into 1.5mL of EPUniformly mixing in a vortex mode in the tube to obtain a secondary diluted urine sample;
2) respectively taking 8 mu L of 100.0mmol/L NAD+，12μL 10.0kU/L 3α-HSD，7μL 100.0mmol/L Ru(bpy)3 2+23 mu L of ultrapure water and 50 mu L of 100.0mmol/L PBS solution with the pH value of 8.5 are mixed to prepare a detection solution;
3) dripping 50 mu L of the detection liquid obtained in the step 2) into an SPCE working area, wherein the SPCE is connected with an electrochemical workstation. Carrying out current-time (I-t) curve analysis under constant potential, adding 5.0 mu L of the urine sample to be detected obtained in the step 1) on the surface of the working electrode when the baseline is constant, and indirectly and quantitatively determining the total bile acid content in the urine sample to be detected by calculating the increased peak current in the I-t curve, wherein the constant potential parameter is set to be 1.20V.
2. The reagent kit for detecting the total bile acid in urine by the electrochemical method has the following beneficial effects:
1) according to the kit for detecting the total bile acid in the urine by the electrochemical method, the total bile acid in the urine is quantitatively determined by using the screen printing carbon electrode and the electrochemical workstation, and a detection instrument is simple to operate and low in cost;
2) the screen printing electrode used in the kit for detecting the total bile acid in urine by the electrochemical method has low manufacturing cost, simple and quick manufacturing process and can realize batch production, and the SPCE is a disposable electrode, so that the time and energy loss of polishing of the traditional column electrode can be avoided, and the problem of experimental cross contamination possibly caused in the repeated use process of the column electrode can be effectively avoided;
3) the reagent kit for detecting the total bile acid in the urine by the electrochemical method can eliminate the matrix interference of the urine sample only by dilution treatment, and has simple, convenient and quick operation;
4) the kit for detecting the total bile acid in the urine by the electrochemical method has the advantages of quick response, high sensitivity, better repeatability and accuracy, and capability of quickly and accurately quantifying the total bile acid in the urine;
5) the reagent kit for detecting the total bile acid in the urine by the electrochemical method can realize the content determination of the total bile acid in the urine, monitor the urine bile acid level of a patient under the non-invasive condition and assist the clinical diagnosis of the hepatobiliary diseases.
Drawings
FIG. 1 is a schematic diagram of the principle of the electrochemical detection of total bile acid in urine according to the present invention.
FIG. 2 is a standard graph of oxidation peak current versus total bile acid concentration in urine according to the present invention.
FIG. 3 is a diagram of the correlation analysis between the total bile acid in urine detected by the electrochemical detection kit of the present invention and the total bile acid in serum detected by the fifth generation enzyme cycling method.
Detailed Description
The invention provides a reagent kit for detecting total bile acid in urine by an electrochemical method and a using method thereof, and the invention is explained in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The electrochemical workstation of the present invention is exemplified by CHI852C electrochemical workstation, which is available from Shanghai Chenghua instruments, Inc.
Example 1: preparation of the Silk-Screen printing carbon electrode
The screen printing carbon electrode is formed by printing carbon paste, Ag/AgCl paste and insulating paste on a PET substrate in sequence. The method specifically comprises the following steps:
1) cleaning a PET substrate, printing carbon slurry on the PET substrate after drying, manufacturing a carbon working electrode and a carbon paste auxiliary electrode, and drying at normal temperature;
2) printing silver paste containing silver chloride on the PET substrate to prepare an Ag/AgCl reference electrode, and drying at normal temperature;
3) printing insulating paste on the PET substrate to cover the wires while avoiding the round working area;
4) the working electrode, the auxiliary electrode and the reference electrode form a circular working area, each electrode is connected with an interface through a lead under an insulating film, and then dried at 30-40 ℃ for storage and standby;
5) and (4) washing the electrode by using ultrapure water, and airing at room temperature for later use.
Example 2: the reagent kit for detecting the total bile acid in urine by the electrochemical method comprises the following specific steps:
1) pretreating a urine sample: 10 μ L urine and 990 μ L ultrapure water were added to 1.5mL EPUniformly mixing in a vortex mode in the tube to obtain a primary diluted urine sample; then, 10. mu.L and 990. mu.L of ultrapure water of the urine sample diluted once are added into 1.5mL of EPUniformly mixing in a vortex mode in the tube to obtain a secondary diluted urine sample;
2) respectively taking 8 mu L of 100mmol/L NAD+，12μL 10kU/L 3α-HSD，7μL 100mmol/L Ru(bpy)3 2+23 μ L of ultrapure water and 50 μ L of a 100.0mmol/L PBS solution at pH 8.5, 1.5mL of E was addedPVortex and mix evenly in the tube to obtain a detection mixed solution;
3) and (3) dropwise adding 50 mu L of the mixed solution obtained in the step (2) into a working area of the SPCE, connecting the SPCE with an electrochemical workstation, carrying out current-time (I-t) curve analysis under a constant potential of 1.20V, adding 5 mu L of the urine sample to be detected obtained in the step (1) to the surface of the working electrode when the baseline is constant, calculating the peak current increased in the I-t curve, wherein the increase of the oxidation peak current and the logarithm of the concentration of the urine bile acid are in a linear relation in a certain range, and thus carrying out indirect quantitative determination on the total bile acid in the urine sample to be detected.
Example 3:
this example examines the correlation between the oxidation peak current and the urine bile acid concentration of the electrochemical detection kit, and the result is shown in fig. 2. Adding a series of bile acid standard solutions with concentration (1.0-100.0 mu mol/L) into urine matrix, and passing through a passage of 104After dilution by fold, the assay is performed under optimal assay conditions. In the range of 1.0 to 100.0. mu. mol/LIn the concentration range, the oxidation peak current and the logarithm of the concentration of the bile acid form a good linear relation, and the regression equation is as follows: 2.3065lgc +8.182, R20.9922. LOD is 0.2 μmol/L (S/N ═ 3).
Example 4:
this example is to examine the precision of the reagent kit of the present invention for measuring total bile acid in urine. Low, medium and high concentration bile acid standard solutions are added into the urine substrate to prepare working solutions with final concentrations of 5.0 mu mol/L, 40.0 mu mol/L and 80.0 mu mol/L respectively. Through 104After dilution, the concentration of total bile acids in the urine is determined under optimal conditions. The working solutions of the respective concentrations were repeatedly measured 5 times a day and continuously measured 5 days a day, and the intra-day and inter-day Relative Standard Deviations (RSD) of the respective concentrations were calculated to examine the intra-day and inter-day precision of the method, and the results are shown in table 1. The standard deviation in the day is lower than 9.2%, and the standard deviation in the day is lower than 9.5%, which shows that the electrochemical detection kit has better repeatability.
TABLE 1 precision test
Figure BDA0003456868470000041
Example 5:
this example is a study of the recovery rate of total bile acid in urine measured by the kit of the present invention. The bile acid standard solution was added to the urine matrix to prepare a working solution containing low (5.0. mu. mol/L), medium (40.0. mu. mol/L) and high (80.0. mu. mol/L) bile acids, and each concentration was measured in parallel for 5 times to conduct a recovery test. The relative recovery rate (concentration of bile acid in urine measured after adding standard-concentration of bile acid in urine measured before adding standard)/concentration of bile acid standard substance added x 100.0%, the results are shown in table 2. The average recovery rate of total bile acid in urine is 97.2-101.9%, and the relative standard deviation is less than or equal to 9.5%, which shows that the electrochemical detection kit has better accuracy.
TABLE 2 recovery rate experiment
Figure BDA0003456868470000042
Example 6:
this example is to examine the correlation between the determination of total bile acid in urine by the electrochemical detection kit of the present invention and the determination of total bile acid in serum by the fifth-generation enzyme method commonly used in clinic. The total bile acid content in 50 serum samples corresponding to the urine samples of 50 subjects and the fifth generation enzyme method are respectively detected by using the kit disclosed by the invention, and the result is shown in figure 3. The result shows that the result of the electrochemical detection kit for detecting the total bile acid in urine has good correlation (r is 0.9800, p is 0.000) with the result of the existing method for detecting the total bile acid in serum, which indicates that the electrochemical detection kit can be used for detecting the content of the total bile acid in urine.

Claims (8)

1. A reagent kit for detecting total bile acid in urine by an electrochemical method is characterized in that: comprises 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), oxidized coenzyme I (NAD)+) Ruthenium terpyridyl (Ru (bpy)3 2+) Phosphate buffered saline and screen printed electrode (SPCE).
2. The kit for electrochemically detecting total bile acid in urine according to claim 1, wherein the screen-printed electrodes comprise a polyethylene terephthalate (PET) substrate with electrodes printed thereon, an external insulating layer printed on the PET substrate, and a wire interface at one end of the substrate, and is characterized in that the substrate is further printed with three electrodes, namely a carbon working electrode, an Ag/AgCl reference electrode and a carbon counter electrode, the three electrodes form a circular working area, and each electrode is connected with the interface through a wire printed under the insulating film.
3. The kit for electrochemically detecting total bile acid in urine according to claim 1, wherein the activity of the 3 α -HSD is 10 kU/L.
4. According to claim1, the kit for detecting the total bile acid in urine by the electrochemical method, wherein the NAD+The concentration of (B) was 100.0 mmol/L.
5. The kit for electrochemically detecting total bile acid in urine according to claim 1, wherein the amount of Ru (bpy)3 2+The concentration of (B) was 100.0 mmol/L.
6. The kit for electrochemically detecting total bile acid in urine according to claim 1, wherein the concentration of the phosphate buffer is 100.0mmol/L, and the pH value is 8.5.
7. The kit for electrochemically detecting total bile acid in urine according to claim 6, wherein the Phosphate Buffer Solution (PBS) is prepared by mixing a 100.0mmol/L disodium hydrogen phosphate solution and a 100.0mmol/L sodium phosphate solution.
8. The application of the kit for detecting the total bile acid in the urine by the electrochemical method according to the claims 1 to 6 is characterized by comprising the following specific steps:
1) pretreating a urine sample: 10 μ L urine and 990 μ L ultrapure water were added to 1.5mL EPUniformly mixing in a vortex mode in the tube to obtain a primary diluted urine sample; then, 10. mu.L and 990. mu.L of ultrapure water of the urine sample diluted once are added into 1.5mL of EPUniformly mixing in a vortex mode in the tube to obtain a secondary diluted urine sample;
2) respectively taking 8 mu L of 100.0mmol/L NAD+，12μL 10.0kU/L 3α-HSD，7μL 100.0mmol/LRu(bpy)3 2+23 μ L of ultrapure water and 50 μ L of a 100.0mmol/L PBS solution at pH 8.5, 1.5mL of E was addedPVortex and mix evenly in the tube to obtain a detection mixed solution;
3) dropwise adding 50 μ L of the mixed solution obtained in the step 2 into a working area of the SPCE, connecting the SPCE with an electrochemical workstation, performing current-time curve analysis under a constant potential of 1.20V, and adding 5 μ L of 10 μ L obtained in the step 1) when the baseline is constant4Multiple diluted urine samples in the workAnd calculating the peak current in the I-t curve on the surface of the electrode, wherein the oxidation peak current and the logarithm of the concentration of the urine bile acid are in a linear relation in a certain range, so that the total bile acid content in the urine sample to be detected is indirectly and quantitatively determined.
CN202210006213.XA 2022-01-05 2022-01-05 Reagent kit for detecting total bile acid in urine by electrochemical method and application thereof Pending CN114354709A (en)

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