CN111879836B - Electrochemical method for detecting uric acid and urate oxidase and application thereof - Google Patents
Electrochemical method for detecting uric acid and urate oxidase and application thereof Download PDFInfo
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Abstract
The invention discloses a silver ion-based methodThe preparation method and the application of the electrochemical sensor of the seed and the glutathione comprise the following specific steps: firstly, plating a graphene oxide dispersion liquid on a clean bare glassy carbon electrode by an electrochemical method, synthesizing a GSH-Ag (I) compound by utilizing the interaction between GSH and Ag (I), uniformly mixing the compound solution with 100 mu L of 0.02 wt% Nafion solution before use, standing for 80min, and dripping on the surface of a graphene modified electrode to prepare GSH-Ag (I)/GO/GCE, wherein the electrode pair is H 2 O 2 Has stronger electrocatalytic response. Catalytic production of H from UA using UOx 2 O 2 Fixation of the concentration of UOx or UA, respectively, by the complex to H 2 O 2 As a signal output, a highly sensitive detection of UA or UOx is achieved. The method has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.
Description
Technical Field
The invention relates to an electrochemical method and application thereof, in particular to preparation of an electrochemical sensor based on a biological metal compound and application thereof in uric acid and urate oxidase analysis sensing, belonging to the technical field of functional biomaterials and biosensing.
Background
Uric acid (UA, 2, 4, 6-trihydroxy purine) is mainly decomposed by nucleic acid and other purine compounds decomposed by cell metabolism and purine in food through the action of enzyme, is the final product of purine derivatives in human metabolism, and has a normal UA level of 240-520 mu M in serum and a normal UA level of 1.4-4.4 mM in urinary excretion. Research shows that the change of uric acid content in human bodies is closely related to a plurality of cardiovascular diseases and metabolic diseases in clinic, such as gout, obesity, hyperuricemia and Lesch-Nyhan syndrome, so uric acid can be used as a valuable clinical diagnosis index and attracts wide attention of scientists, and detection methods comprise a spectroscopic method, a fluorescence method, a liquid chromatography method, an enzyme method and the like. Although these detection methods have high sensitivity and selectivity, they have the disadvantages of time consumption, expensive materials and complicated pretreatment, and electrochemical methods have been the focus of scientific attention due to their advantages of easy operation, high sensitivity and fast response. Uricase (UOx), a biological enzyme closely related to uric acid, can rapidly oxidize uric acid into allantoic acid, and the allantoic acid is not absorbed by renal tubules and excreted, so the activity of the enzyme in a human body has a great influence on the diagnosis and treatment of uric acid related diseases. The development of a new electrochemical method for realizing sensitive detection of uric acid and urate oxidase has very important significance for clinical diagnosis and drug development.
The bio-metal complex is a coordination polymer material formed by utilizing intramolecular and intermolecular interaction connection of metal ions and biomolecules, wherein coordination polymers of coinage metals (such as Au, Ag and Cu) and sulfhydryl biomolecules attract considerable attention. Generally, such complexes are based on supramolecular polymeric materials formed by the interaction of thiol groups of biomolecules with metal ions (thiol-metal/metal-metal). Glutathione (GSH) is an amino acid derivative with special biological functions, has three dissociable protons and ten atoms participating in coordination, belongs to a small molecular peptide substance containing sulfydryl, and is an ideal biological molecular model for forming a compound. So far, there are many reports about interaction between GSH and metal ions, but the application of a biological metal complex formed by GSH and metal ions to the analysis and detection of uric acid and urate oxidase has not been reported yet, and the biological metal complex has great potential.
The invention designs an electrochemical method for detecting uric acid and urate oxidase, which adopts GSH as a biological organic ligand, silver ions (Ag (I)) as metal nodes, and synthesizes GSH-Ag (I) compound simply and greenly through the interaction of sulfydryl in the GSH and Ag (I). Based on the redox activity of metal ions, the invention discovers that the compound is opposite to hydrogen peroxide (H) 2 O 2 ) Has better electrocatalysis. In addition, UOX rapidly oxidizes UA to allantoic acid and H 2 O 2 Thus modifying the electrode and its pair H by the complex 2 O 2 The invention constructs a new electrochemical method to realize the analysis and monitoring of uric acid and urate oxidase, and provides a new idea for clinical diagnosis and drug development related to uric acid.
Disclosure of Invention
The invention aims to provide an electrochemical method for detecting uric acid and urate oxidase, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost, and an application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: an electrochemical method for detecting uric acid and urate oxidase and application thereof, specifically comprises the following steps:
(1) preparation of GSH-Ag (I) complexes
Sequentially taking 1-10 μ L of silver nitrate aqueous solution with concentration of 0.1-20 mM and 1-10 μ L of glutathione aqueous solution with concentration of 0.1-20 mM, mixing uniformly, adding phosphoric acid buffer solution (10mM, pH 7.0, Na) 2 HPO 4 /NaH 2 PO 4 ) Preparing 50-120 mu L of solution, slowly shaking the solution at 25-40 ℃ for 8-17 min to obtain GSH-Ag (I) compound, uniformly mixing the compound solution with 50-120 mu L of 0.02 wt% Nafion solution before use, and standing for 5-20 min.
(2) Preparation of electrochemical biosensor
a. Polishing a glassy carbon electrode (GCE, the diameter of which is 3mm) on chamois leather by using aluminium oxide powder with the grain diameter of 0.3 mu m and 0.05 mu m for 0.5-5 min in sequence, placing the electrode in an ultrasonic cleaner for ultrasonic cleaning by using ultrapure water for 1-5 min after polishing, and then using N 2 Drying, and marking as GCE;
b. utilizing a cyclic voltammetry to set the potential range to be-1.2-0.5V and the sweep speed to be 5-20 mV/s, and electrodepositing 0.5-2 mg/mL graphene dispersion liquid (GO) on a bare glassy carbon electrode to obtain GO/GCE; then 5-15 mu L of the solution in the formula (1) is dripped on GO/GCE, standing at room temperature for 20-60 min, and the electrode is slowly rinsed by ultrapure water, which is marked as GSH-Ag (I)/GO/GCE.
(3) Analysis and detection of uric acid and urate oxidase
The reaction solution with a total volume of 2mL comprises UA (0-5 mM), UOx (0-2000U/L), and phosphate buffer solution (Na) 2 HPO 4 /NaH 2 PO 4 0.1M, pH 8.0), reacting at 25-45 ℃ for 2-10 min, and then detecting the electrochemical response of GSH-Ag (I)/GO/GCE.
Based on the reaction liquid, the UA concentration (0.001-1 mM) is changed, and other steps are the same as above, so that the UA can be detected;
based on the reaction liquid, the detection of the UOx can be realized by changing the concentration of the UOx (1-2000U/L) in other steps.
Electrochemical method for detecting uric acid and urate oxidase by using sameThe application comprises setting potential at +0.4V by current-time method (Amperometric i-t Curve), and using prepared electrochemical sensor pair H 2 O 2 The electrochemical response of (2) is to obtain a series of current magnitudes corresponding to different concentrations of UA (UOx) in an electrolyte solution containing a UOx reaction solution, establish a quantitative relation between the current response and the UA (UOx), and determine the content of UA (UOx) in the sample to be detected according to the quantitative relation between the current response and the UA (UOx).
The invention principle is as follows: the invention relates to an electrochemical method for detecting uric acid and urate oxidase and application thereof, which comprises the steps of firstly adopting GSH as a biological ligand, Ag (I) as a metal node, and synthesizing a probe capable of detecting H based on the interaction between sulfhydryl-metal and metal-metal 2 O 2 And (3) a biological metal compound (GSH-Ag (I) compound) with an electrocatalytic effect, and dripping the polymer on the GO modified electrode to successfully prepare the sensor. Catalytic production of H from UA using UOx 2 O 2 Fixation of UOx or UA concentration, respectively, by complex pair H 2 O 2 As a signal output, enabling the analytical detection of UA or UOx. Based on the method, a simple, rapid, high-sensitivity, high-selectivity and label-free UA (UOx) electrochemical analysis method is constructed.
Compared with the prior art, the invention has the advantages that: the invention discloses an electrochemical method for detecting uric acid and urate oxidase and application thereof. First, GSH-Ag (I) complex is simply synthesized in green by the interaction of sulfydryl in GSH with Ag (I) and Ag (I), and the complex is used for H 2 O 2 Has obvious catalytic effect. Secondly, a complex modified electrode is utilized, and the electrochemical response of the sensor to different concentrations of UA (UOx) is detected by adopting a current-time method. It is clear that within a certain range of concentration, the greater the concentration of UA, the greater the H produced 2 O 2 The more, the more pronounced the current response; similarly, the greater the UOx concentration, the more pronounced the current response. The experimental result shows that the current magnitude and the concentration of UA (UOx) are in a linear relationship in a certain range, so that the UA (UOx) detection is realized. The advantages are that:
(1) high catalytic activity. GSH-Ag (I) complex pairs prepared from GSH and Ag (I) 2 O 2 Has high catalytic activity and can be used for preparing novel electrochemical sensors.
(2) High sensitivity. The invention discloses an electrochemical sensor prepared on the basis of GSH-Ag (I) compound, and H generated by catalyzing UA (UA with UOx 2 O 2 Two linear equations are obtained: the linear correlation equation of the current response to the UA concentration is that y is 53.47C UA +0.01, r ═ 0.9939, limit of detection 0.3 μ M; the linear correlation equation of the current response to the concentration of the UOx is 18.07lgC UOx +0.05, r ═ 0.9980, limit of detection 0.5U/L; the sensor can realize high-sensitivity detection on UA (UOx).
(3) High specificity. And for UA detection: other control substances such as Urea (Urea), Glucose (Glucose), Caffeine (Caffeine) and Ascorbic Acid (AA) do not interfere with the system; detection of UOx: acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase) and Lysozyme (LZM) have no interference to the system;
(4) the result is accurate. The recovery rate is between 90% and 110%.
(5) The preparation and detection method has the advantages of less reagent dosage and low cost. The invention can realize high-sensitivity detection of UA (UOx) with only a small consumption of materials and reagents.
In conclusion, the electrochemical sensor with the GSH-Ag (I) composite substrate is used for detecting UA (UOx), has the advantages of high sensitivity, good selectivity, simplicity in operation, rapidness in analysis, easiness in operation and the like, can realize detection of UA (UOx) with lower concentration, and has good application prospect.
Drawings
FIG. 1 shows a sensor pair H according to the invention 2 O 2 An electrochemical response map of (a);
FIG. 2 is a diagram of experimental feasibility of the sensor of the present invention for UA (UOx) assay;
FIG. 3 is a graph of the current response versus concentration of the sensor of the present invention for different concentrations UA (UOx);
FIG. 4 is a diagram showing the specificity test of the sensor of the present invention for UA (UOx).
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
Example 1 preparation of GSH-Ag (I) complexes
Sequentially mixing 10 μ L of 10mM silver nitrate aqueous solution and 10 μ L of 10mM glutathione aqueous solution, adding phosphate buffer solution (10mM, pH 7.0, Na) 2 HPO 4 /NaH 2 PO 4 ) Preparing 100 μ L solution, slowly shaking the solution at 30 deg.C for 12min to obtain GSH-Ag (I) compound, mixing the compound solution with 100 μ L0.02 wt% Nafion solution, and standing for 80min before use.
EXAMPLE 2 preparation of electrochemical biosensor
a. Firstly polishing a glassy carbon electrode (GCE, diameter of 3mm) on chamois leather with aluminium oxide powder with particle size of 0.3 μm, 0.1 μm and 0.05 μm for 2min, placing the electrode in an ultrasonic cleaner, ultrasonically cleaning in ultrapure water for 2min, and then cleaning with N 2 Drying to obtain a bare glassy carbon electrode, and marking as GCE;
b. utilizing a cyclic voltammetry to set the potential range to be-1.2-0.5V and the sweep rate to be 10mV/s, and electrodepositing 1.2mg/mL graphene dispersion liquid (GO) onto a bare glassy carbon electrode to obtain GO/GCE; then 4 mul of the solution of example 1 was applied dropwise to GO/GCE, left to stand at room temperature for 30min, and the electrode was rinsed slowly with ultra pure water, which was recorded as GSH-Ag (I)/GO/GCE.
The electrochemical response of the above-described electrode to the PBS (0.1M, pH 7.0) electrolyte solution was measured by adding 0.025mM H per 100s 2 O 2 . As shown in FIG. 1, it can be seen that the prepared sensor is compared with the other two electrodes, pair H 2 O 2 The electrochemical response of (a) is evident. Description of the sensor pairs H 2 O 2 Has good electrocatalytic activity.
EXAMPLE 3 feasibility experiment
The preparation procedure of the sensors of examples 1 and 2 was followed, and a total volume of 2mL of reaction solution including UA (0.001, 0.01, 0.1mM), UOX (500U/L), phosphate buffer solution (Na) 2 HPO 4 /NaH 2 PO 4 0.1M, pH 8.0), at 37 ℃ for 3min, and then for GSH-Ag (I)/GO/GCEThe electrochemical response of (3) is detected. The results are shown in fig. 2A, and it can be seen that GSH-ag (i)/GO/GCE has good electrochemical response to UA with different concentrations, and can be applied to detection of UA.
Subsequently, according to the sensor preparation procedures of examples 1 and 2 above, a total volume of 2mL of reaction solution including UA (1mM), UOX (500U/L), phosphate buffer solution (Na) 2 HPO 4 /NaH 2 PO 4 0.1M, pH 8.0), reacted at 37 ℃ for 3min, then used for electrochemical response detection of GSH-ag (i)/GO/GCE. As a result, as shown in FIG. 2B, the sensor showed substantially no response at a UOx concentration of 0, whereas the sensor showed a significant response at a UOx concentration of 500U/L.
The above results demonstrate that the sensor has good electrochemical response to UA and UOx, and can be applied to detection of UA (UOx).
Example 4 detection of UA and UOx
The sensor preparation steps of examples 1 and 2 and the response of example 3 to UA (UOX) were carried out by changing the concentration of UA (0.001-1 mM) or UOX (1-2000U/L) and detecting the response to the reaction solution with the sensor, and the results are shown in FIG. 3. As shown in fig. 3A, the current response of the sensor to UA (uox) is in good linear relation with concentration, and the linear correlation equation of the current response of the sensor to UA concentration is that y is 53.47C UA +0.01, r ═ 0.9939, linear range 0.001-1 mM, limit of detection 0.3 μ M; as shown in fig. 3B, the linear correlation equation of current response to UOx concentration is 18.07lgC UOx And the linear range is 1-2000U/L, the detection limit is 0.5U/L, and the result shows that the sensor realizes high-sensitivity detection on UA (UOx).
Example 4 specific assay
To verify the specificity of the sensor, according to the preparation steps of the sensors of the above examples 1, 2 and 3, the reaction solution of UA is added with other interferents of the same concentration, such as Urea (Urea), Glucose (Glucose), Caffeine (Caffeine), Ascorbic Acid (AA), acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase) and Lysozyme (LZM) of the same concentration as that of UOx, and the specificity of the sensor to UA (UOx) is detected. The results are shown in fig. 4, which illustrates that the sensor has good specificity for detection of UA (e.g., fig. 4A) (UOx (e.g., fig. 4B)).
Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Variations, modifications, additions and substitutions which may occur to those skilled in the art and which fall within the spirit and scope of the invention are also considered to be within the scope of the invention.
Claims (5)
1. An electrochemical method for detecting uric acid and urate oxidase is characterized by comprising the following specific steps:
(1) preparation of GSH-Ag (I) complexes
Sequentially taking 1-10 mu L of silver nitrate aqueous solution with the concentration of 0.1-20 mM and 1-10 mu L of glutathione aqueous solution with the concentration of 0.1-20 mM, uniformly mixing, adding phosphoric acid buffer solution to prepare 50-120 mu L of solution, slowly shaking the solution at 25-40 ℃ for 8-17 min to obtain GSH-Ag (I) compound, uniformly mixing the compound solution with 50-120 mu L of 0.02 wt% Nafion solution before use, and standing for 5-20 min;
(2) preparation of electrochemical biosensor
a. Polishing the glassy carbon electrode GCE on chamois leather by using aluminium oxide powder with the grain diameter of 0.3 mu m and 0.05 mu m for 0.5-5 min in sequence, placing the electrode in an ultrasonic cleaner for ultrasonic cleaning by using ultrapure water for 1-5 min after polishing, and then using N to clean the electrode by using the ultrapure water 2 Drying, and marking as GCE;
b. setting a potential range of-1.2-0.5V and a sweep speed of 5-20 mV/s by using a cyclic voltammetry, and electrodepositing 0.5-2 mg/mL graphene GO dispersion liquid onto a bare glassy carbon electrode to obtain GO/GCE; dripping 5-15 mu L of the solution obtained in the step (1) on GO/GCE, standing at room temperature for 20-60 min, and slowly washing an electrode with ultrapure water, wherein the solution is marked as GSH-Ag (I)/GO/GCE;
(3) analysis and detection of uric acid and urate oxidase
The reaction solution with the total volume of 2mL comprises 0-5 mM uric acid UA, 0-2000U/L urate oxidase UOx and a phosphate buffer solution, the reaction is carried out for 2-10 min at the temperature of 25-45 ℃, and then the reaction solution is used for electrochemical response detection of GSH-Ag (I)/GO/GCE.
2. The electrochemical method according to claim 1, wherein the UA concentration of uric acid is varied within the range of 0.001 to 1mM, the potential is set to +0.4V by a current-time method, and the electrochemical sensor pair H is prepared by 2 O 2 The electrochemical response of the method is to obtain a series of current magnitudes corresponding to uric acid UA with different concentrations in an electrolyte solution containing a UOx reaction solution, establish a quantitative relation between the current response and the uric acid UA, and determine the content of the uric acid UA in the sample to be detected according to the quantitative relation between the current response and the uric acid UA.
3. The electrochemical process of claim 2, wherein the linear correlation equation for current response to UA uric acid concentration is y-53.47C UA +0.01, r-0.9939, linear range 0.001-1 mM, limit of detection 0.3. mu.M.
4. The electrochemical method according to claim 1, wherein the concentration of urate oxidase UOx is varied within a range of 1 to 2000U/L, a potential is set to +0.4V by a current-time method, and H is measured by using the prepared electrochemical sensor pair 2 O 2 The electrochemical response of the method is to obtain a series of current magnitudes corresponding to urate oxidase UOx with different concentrations in an electrolyte solution containing urate UA reaction liquid, establish a quantitative relation between the current response and urate oxidase UOx, and determine the urate oxidase UOx content in a sample to be detected according to the quantitative relation between the current response and urate oxidase UOx.
5. The electrochemical process of claim 4, wherein the linear dependence of the current response on urate oxidase UOx concentration is y-18.07 lgC UOx +0.05, r equals 0.9980, linear range is 1-2000U/L, detection limit is 0.5U/L.
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