Electrochemiluminescence sensor for accurately detecting uric acid based on AIE-MOF and preparation method and detection method thereof

Abstract

The invention discloses a high-sensitivity electrochemical luminescence sensor for detecting uric acid as well as a preparation method and a detection method thereof, and belongs to the technical field of detection. A zirconium-based metal organic framework (Zr-TPE MOF) and urate oxidase (UOx) are sequentially dispensed on the surface of a working electrode to serve as modification materials, the Zr-TPE MOF realizes luminous intensity multiplication in an aggregation state through an intramolecular motion limitation (RIM) mechanism, and UOx can react with a to-be-detected sample uric acid in a buffer solution to generate H2O2; the latter can co-react to promote redox electron transfer on the surface of the competitive electrode so as to influence luminescence. The luminescent material based on the novel tetraphenyl ethylene (TPE) derivative has the advantages of high sensitivity, low background signal, strong specificity, simplicity and convenience in operation and the like, and has obvious advantages compared with a previously developed sensor for detecting uric acid. According to the invention, quantitative detection of uric acid can be realized, and an innovative solution is provided for clinical high-sensitivity uric acid detection and early warning of metabolic syndrome.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nano-functional materials, biomedical analysis and detection, and electrochemiluminescence sensing technology, and provides an electrochemiluminescence sensor for highly sensitive detection of uric acid, a preparation method thereof, and a detection method thereof. Background Art

[0002] Uric acid (UA), as an important indicator for measuring serum uric acid levels, is of great significance for preventing metabolic diseases such as gout. Hyperuricemia and gout have seriously affected public health. Therefore, the research and development of medical instruments for uric acid detection have a huge market prospect. High uric acid can have various effects on human health, including gout, kidney stones, cardiovascular diseases, metabolic syndrome, mental health, etc. Therefore, uric acid (UA), as an important indicator for human disease detection, is of great significance for preventing metabolic diseases such as gout.

[0003] Although traditional uric acid detection methods such as chromatography and spectroscopy are effective, and a variety of instruments for uric acid detection have been developed, they are mainly invasive detections, and at the same time, there are limitations such as long detection time, complex operation, expensive equipment, low patient acceptance, and inability to perform immediate detection. Therefore, it is highly necessary to develop a new type of non-invasive and highly sensitive immediate detection sensor. In recent years, electrochemiluminescence sensors have shown great potential in non-invasive uric acid detection due to their fast analysis speed, simplicity, low cost, and ability to perform on-site measurement and monitoring. Therefore, this study aims to construct an electrochemiluminescence (ECL) sensor based on a new type of aggregation-induced emission (AIE) material to achieve highly sensitive detection of uric acid, and evaluate the application effect of the sensor in actual samples to verify its reliability and practicability.

[0004] The present invention prepared and characterized a new type of AIE tetraphenylethylene (TPE) derivative Zr-TPE MOF luminescent material. First, the Zr-TPE MOF derivative was drop-coated on the working electrode. After infrared drying, uricase was added dropwise and naturally dried. Appropriate electrochemiluminescence detection parameters were set to specifically identify uric acid in PB buffer, thereby preparing an electrochemiluminescence sensor with high sensitivity, low background signal, strong specificity, and simple operation. The present invention adopts electrochemiluminescence technology, and the detection cost is low. It is a fast, inexpensive, and sensitive detection method. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method and a detection method for an electrochemiluminescence sensor for efficiently detecting uric acid, so as to overcome the problems and deficiencies of the existing uric acid detection, such as long detection time, complex operation, expensive equipment, low acceptance degree of patients, and inability to perform instant detection. First, based on existing literature, the detection conditions of the TPE derivative Zr-TPE MOF are optimized in the present invention: Zr-TPE MOF synthesized by the hydrothermal method is drop-coated on the surface of the working electrode, and the scanning voltage is optimized to be -0.4V to -2.0V, the number of scanning cycles is 12 cycles, and the high voltage of the photomultiplier tube is 650V. Then, uricase at 0.1U / μL is modified on the surface of the working electrode to specifically detect uric acid. The sensor prepared by the present invention can be used for the highly selective and highly sensitive simultaneous detection of uric acid according to the intensity of the ECL peak value.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a preparation method for an electrochemiluminescence sensor for efficiently detecting uric acid. A TPE derivative Zr-TPE MOF with excellent AIE performance is selected as the core luminescent material, and a Zr-TPE MOF sensitive film is modified on the surface of the working electrode GCE. A uricase molecular film is modified on the outer layer of the Zr-TPE MOF. The uricase molecular film provides an action target for the template molecule uric acid. The above electrode is placed in a 0.1mol.L -1 K 2 S 2 O 8 -containing 0.1mol.L -1 phosphate buffer solution, and a MPI-II type electrochemiluminescence detector is used to detect uric acid until a stable electrochemiluminescence peak is obtained.

[0008] The specific steps for modifying the TPE derivative Zr-TPE MOF on the surface of the working electrode are as follows: 30mg of zirconium tetrachloride, 15mg of tetraphenylethylene, 0.2mL of CH 3 COOH and 1.8mL of DMF are used to synthesize Zr-TPE MOF by the hydrothermal method; a part is respectively diluted to 1.0mg / mL with absolute ethanol, 10μL of Zr-TPE MOF is drop-coated on the surface of the working electrode, dried at infrared for 15min, and then 10μL of uricase is added dropwise thereto and dried naturally.

[0009] A potassium persulfate PB phosphate buffer solution with 0.1M and PH = 7.4 is prepared, incubated in a uric acid solution containing 100nM, 1μM, 10μM, 100μM, 1mM for 15min, and then put into the PB phosphate buffer solution for detecting the uric acid content. Immerse a three - electrode system such as a working electrode in a buffer solution. The reaction conditions for electropolymerization are a scanning voltage of - 0.4 to 2.0 V, 12 scanning cycles, and a photomultiplier tube voltage of 650 V; uricase oxidizes uric acid to produce H 2 O 2 which can compete with the intermediate for electron transfer, resulting in a decrease in the ECL signal. The ECL signals are 3500, 4400, 5600, 6500, and 7300 for 100 nM - 1 mM respectively.

[0010] The detection linear range of the AIE electrochemiluminescence sensor for uric acid in the embodiments of the present invention is 100 nM to 1 mM; the linear regression equation is y = - 490.99x + 10984.32, and the correlation coefficient R 2 is 0.9983.

[0011] The electrochemiluminescence sensor in the embodiments of the present invention has good selectivity and anti - interference ability. The selectivity and anti - interference ability of the sensor were tested. Sample solutions were selected as urea, glucose, ascorbic acid (AA), NaCl, and KCL with the same concentration as uric acid. The above - mentioned Zr - TPE MOF coated with uricase was placed into the above solutions and incubated for 15 - 25 min, and then electrochemiluminescence detection was carried out. The sensor only had an obvious electrochemiluminescence response to uric acid, indicating good selectivity; at the same time, the anti - interference ability of the electrochemiluminescence sensor was verified. The electrochemistry sensor was placed into solutions of UA + urea, UA + glucose, UA + AA, UA + NaCl, and UA + KCL. The concentrations of urea, glucose, and AA were 20 times that of UA, and the concentrations of NaCl and KCL were 100 times that of UA. After incubation for 2 - 6 minutes, the ECL luminescence response was detected with an electrochemiluminescence detector. The results showed that the response of the sensor to uric acid did not change, indicating excellent anti - interference ability of the sensor.

[0012] The Zr - TPE MOF material was characterized by XRD. It was observed that compared with the simulated diagram, the main peak positions and peak shapes of Zr - TPE MOF were consistent, which proved the good synthesis of Zr - TPE MOF and its successful modification on the electrode surface.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) The present invention can be used for highly sensitive, rapid, and accurate detection of uric acid.

[0015] (2) The electrochemiluminescence material prepared by the present invention can specifically recognize uric acid, thus improving the specificity of the sensor of the present invention.

[0016] (3) The sensor in the present invention is simple to prepare, low - cost, easy to miniaturize and integrate, consumes less samples, is easy to miniaturize and integrate, and has a wide range of applications.

[0017] (4) Compared with other methods for detecting uric acid, the present invention has the advantages of low background signal, high sensitivity, low detection limit, etc., thus providing a potential new method for monitoring hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 、 2 It is a parameter optimization data graph of the drop-coated Zr-TPE MOF concentration in Example 2 of the present invention

[0020] Figure 3 、 4 It is a linear equation graph of detecting uric acid by an electrochemiluminescence sensor in Example 7 of the present invention

[0021] Figure 5 It is a selective detection graph of uric acid in Example 5 of the present invention.

[0022] Figure 6 It is an anti-interference detection graph of uric acid in Example 6 of the present invention.

[0023] Figure 7 It is an XRD characterization graph of Zr-TPE MOF in Example 1 of the present invention

[0024] Figure 8 It is an XPS spectrum of Zr-TPE MOF in Example 1 of the present invention

[0025] Figure 9 It is an SEM graph of Zr-TPE MOF in Example 1 of the present invention DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following specific embodiments further illustrate the present invention. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. All kinds of reagents, reaction conditions, detection methods, etc. used in the following embodiments are regarded as reagents, reaction conditions and detection methods commonly used in the art unless otherwise specified.

[0027] Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0028] In the following examples, the experimental materials used can be obtained from conventional biochemical reagent companies without special instructions. The present invention will be further described below in conjunction with examples.

[0029] Example 1

[0030] Preparation of Zr-TPE MOF

[0031] The working electrode surface was modified with TPE derivative Zr-TPE MOF. The specific steps were as follows: 30 mg of zirconium tetrachloride, 15 mg of tetraphenylethylene, 0.2 mL of CH 3 COOH and 1.8 mL of DMF were used to synthesize Zr-TPE MOF by hydrothermal method.

[0032] Example 2

[0033] Preparation of electrochemiluminescence sensor

[0034] Part of Zr-TPE MOF was diluted to 1.0 mg / mL with absolute ethanol, and 10 μL of Zr-TPE MOF was dropped onto the surface of the working electrode and dried under infrared for 15 min.

[0035] Example 3

[0036] Preparation of electrochemiluminescence sensing system

[0037] On the basis of Examples 1 and 2, a potassium persulfate PB phosphate buffer solution with 0.1 M and pH = 7.4 was prepared, and the dried working electrode, reference electrode, and counter electrode were immersed in the prepared buffer solution to form a sensor detection system.

[0038] Example 4

[0039] Detection and comparison of the luminescence intensity of Zr-TPE MOF

[0040] 10 μL of 1.0 mg / mL Zr-TPE MOF solution was dropped onto the electrode surface, and the electrode was detected using an electrochemiluminescence instrument under the conditions of a voltage of -0.4 V to -2.0 V, 12 scanning cycles, and a photomultiplier tube voltage of 650 V.

[0041] Example 5

[0042] The selective detection of uric acid was carried out as follows:

[0043] The sample solutions were respectively 100 nM uric acid, glucose, urea, ascorbic acid (AA), NaCl, and KCl added to the phosphate buffer solution prepared in Example 3. 10 μL of uricase was drop-coated on the electrochemiluminescence sensor in Example 3. After drying, it was placed in the above solution and incubated for 15 - 25 minutes, and electrochemiluminescence detection was carried out. The voltage range was -0.4 V to -2.0 V, the number of scanning cycles was 12, and the photomultiplier tube was 700 V. The sensor had an obvious electrochemiluminescence response only to uric acid and had good selectivity.

[0044] Example 6

[0045] Detection of the anti-interference property of uric acid, the specific steps are as follows:

[0046] The sample solution was a uric acid solution containing urea, glucose, AA, NaCl, and KCl. The working electrode of the electrochemiluminescent biosensor was inserted into the above solutions respectively and incubated for 2 - 6 minutes. The concentrations of the interfering substances urea, glucose, and AA were 20 times that of UA, and the concentrations of NaCl and KCl were 100 times that of UA. The ECL luminescence response was detected with a chemiluminescence detector. The response of the electrochemiluminescence sensor to uric acid did not change compared with that without interfering substances, and the sensor had excellent anti-interference property.

[0047] Example 7

[0048] The electrochemiluminescence sensor of the present invention was used for the detection of uric acid at different concentrations. The specific steps are as follows:

[0049] Uric acid solutions with concentrations of 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM were respectively prepared. The sensor coated with Zr-TPE MOF solution and uricase was dried and immersed in the buffer solution and incubated for 15 minutes. According to the relationship between the obtained luminescence response and the concentration of the uric acid standard solution, a working curve was plotted. The linear detection range of the electrochemistry sensor for uric acid was 100 nmol·L -1 ~1 mmol·L -1 ; The linear regression equation was y = -490.99x + 10984.32, and the correlation coefficient R 2 was 0.9983.

[0050] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For a person skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention. Any reference signs in the claims should not be construed as limiting the claimed invention.

[0051] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. A person skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by a person skilled in the art. Analysis of Uric Acid in Real Serum Samples in Table 1

Claims

1. A method for preparing an electrochemiluminescent sensor for rapid and simple detection of uric acid, characterized in that include: A three-electrode detection system is used, including a working electrode, a reference electrode, and a counter electrode; A tetraphenylethylene (TPE) derivative Zr-TPE MOF with excellent AIE performance was selected as the core luminescent material. The surface of the glassy carbon electrode was modified with Zr-TPE MOF particles. Urate oxidase was added to the modified material, which can specifically undergo redox reaction with uric acid in the test solution to enhance the electrochemiluminescence signal.

2. The method for preparing the TPE derivative according to claim 1, wherein 30 mg (0.03691 mM) to 60 mg (0.07381 mM) zirconium tetrachloride (ZrCl4), 30 mg (0.1287 mM) TPE and 0.6 mL glacial acetic acid (AA)) are dissolved in 6 mL N,N-dimethylformamide (DMF); the mixture is then transferred into a reaction vessel and reacted in an oil bath at 120°C for 24 hours. After the reaction is completed, the product is washed three times with anhydrous ethanol and stored in anhydrous ethanol as a backup material for subsequent characterization and electrochemiluminescence detection. Preferably, the optimal molar ratio of ZrCl4 to TPE is 1:1.

74.

3. The method for preparing an electrochemiluminescent sensor for highly sensitive detection of uric acid according to claim 1, characterized in that: The surface of the working electrode is modified with a TPE derivative Zr-TPE MOF prepared by a hydrothermal method, and the specific steps are: taking 6-12 μL of a Zr-TPE MOF solution with a concentration of 0.2-2.0 mg / mL and dropping it on the surface of the working electrode for deposition, obtaining an electrode modified with Zr-TPE MOF particles after drying, using an MPI-Ⅱ type electrochemiluminescence detector to scan 12 circles under the conditions of a voltage range of -0.2--0.5--1.6--2.0 V, and a photomultiplier tube high voltage of 600-800 V, the electrochemiluminescence peak of the GCE electrode modified with the material in a phosphate buffer solution of 100 mM (K2S2O8) pH=7-8 is about 10 times that of a bare electrode, and the ECL is about 5000 a.u. Preferably, the optimal drop coating volume is 10 μL. Preferably, the optimal drop coating concentration is 1.0 mg / mL. Preferably, the optimal voltage range is -0.4 to -2.0V. Preferably, the optimal photomultiplier tube high voltage is 650V.

4. Use of the electrochemiluminescence sensor prepared by the method according to any one of claims 1 to 4 in detecting uric acid.

5. A method for detecting uric acid using an electrochemiluminescent sensor, characterized in that include: 10U / mg urate oxidase was added to the electrode surface at a drop size of 5-15μL and dried naturally. The electrochemiluminescent sensor obtained by the above method was placed in PB buffer solutions containing uric acid concentrations of 100nM, 1μM, 10μM, 100μM, and 1mM, respectively, for adsorption, and then electrochemical detection was performed.

6. As described in claim 5, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM uric acid are added to 100 nM phosphate buffer, respectively, and adsorption is carried out for 15 to 25 minutes. Preferably, the optimal adsorption time of uric acid is 15 minutes.

7. The detection method according to claim 6, characterized in that: The linear range of the electrochemical sensor for uric acid detection is 100 nmol·L -1 ~1mmol·L -1 ; The linear regression equation is I ECL =-986.46lgC UA +588.70, correlation coefficient R 2 is 0.9984.

8. The detection method according to claim 5, characterized in that: The selectivity and anti-interference of the sensor were tested (n=3); 15mL of sample solutions of 100μM urea (0.9mg), 100μM glucose (2.7mg), 100μM ascorbic acid (AA) (2.6mg), and 100μM KCL (5.6mg) were prepared, and the electrochemical sensor was placed in the above solution for incubation for 15-25 minutes, and electrochemiluminescence detection was performed. The ECL of glucose was 7686a.u., the ECL of urea was 7680a.u., the ECL of AA was 8055a.u., and the ECL of KCL was 7968a.u.; while the ECL of uric acid was 4467a.u., which were 0.58, 0.58, 0.55, and 0.56 times that of glucose, urea, AA, and KCL, respectively, indicating that the sensor only had an obvious electrochemiluminescence quenching response to uric acid, and had good selectivity. At the same time, the electrochemical sensor was placed in a 15 mL solution containing UA + urea, UA + glucose, UA + AA, and UA + KCL. The concentration of UA was set to 100 μM, the concentrations of the interfering substances urea (18 mg), glucose (54 mg), and AA (52 mg) were 20 times that of UA, i.e. 2 mM, and the concentration of the interfering substance KCL was 100 times that of UA, i.e. 10 mM (112 mg). After incubation for 15 to 25 minutes, the ECL luminescence response was detected using an MPI-Ⅱ electrochemiluminescence detector. The ECL of UA + urea was 4456 a.u., the ECL of UA + glucose was 4253 a.u., the ECL of UA + AA was 4392 a.u., and the ECL of UA + KCL was 4326 a.u. The results showed that the interfering substances did not respond significantly to uric acid, and the sensor had excellent anti-interference ability.

9. The detection method according to claim 5, characterized in that: Five groups of electrochemiluminescence sensors were prepared under the same conditions and their ECL signals were detected. The results showed that the ECL signals of the five groups of sensors were between 4400 and 5000 a.u., with an RSD of 4.6%, which showed good repeatability.

10. According to the detection method of claim 5, the uric acid content in actual human serum samples was detected. In order to verify the practicality of the proposed ECL biosensor in actual samples, 5 serum samples with different uric acid concentrations (100nM, 1μM, 10μM, 100μM, 1mM) were prepared and tested. The recovery rate of the ECL sensing platform was 66.8% to 109.6%, and the RSD was 2.6% to 4.9%. The results show that the proposed sensor can be used to accurately analyze the uric acid content in complex samples.

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