Non-enzymatic electrochemical sensor, preparation method thereof and application of non-enzymatic electrochemical sensor in creatinine detection

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a non-enzymatic electrochemical sensor as well as a preparation method and application thereof in creatinine detection. The stable copper azole compound is synthesized at room temperature through a one-step method, the preparation is simple, the cost is low, the material has good response to creatinine, a series of enzymes such as creatininase do not need to be used, and the sensor belongs to an enzyme-free creatinine sensor. The electrochemical sensor can be used for detecting creatinine in serum, and is used for renal function evaluation and early diagnosis of kidney diseases. Since the electrochemical sensor does not depend on enzyme, the electrochemical sensor of the present invention is more stable in performance in a wide range of temperature changes, has resistance to environmental factors, and enhances its robustness under various clinical and realistic conditions.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a non-enzymatic electrochemical sensor, its preparation method, and its application in creatinine detection. Background Technology

[0002] The most widely used method for the quantitative detection of creatinine is the Jaffe colorimetric method, which was developed more than a century ago. However, it is susceptible to optical interference and its accuracy is often compromised by variations in temperature and sample pH.

[0003] In recent years, various electrochemical methods for creatinine quantification have been reported, including multi-enzyme-assisted creatinine degradation, electrochemical ratio probes, selective filtration through functionalized membranes, affinity detection based on antibodies or molecularly imprinted polymers, and enzyme-free detection assisted by nanomaterials. While enzyme biosensors possess high specificity, their poor enzyme stability, susceptibility to inactivation, and susceptibility to external factors such as pH and temperature lead to a significant decrease in response sensitivity. Furthermore, the high cost of enzymes hinders their economic viability and widespread adoption, and enzyme immobilization remains a challenge. Therefore, the development of enzyme-free biosensing technologies holds great promise. Affinity-based biosensors exhibit strong stability but require cumbersome sample preparation and lengthy analysis times. Selective membrane methods also possess specificity, but changes in electrode surface morphology can impair response sensitivity. Furthermore, nanomaterial-assisted enzyme-free detection methods also have some problems: the sensor's detection current is low, located in the microampere range, and this low current is easily affected by electronic interference, reducing the accuracy of the detection; the measurable creatinine concentration range is limited, and the actual creatinine level often exceeds the range, so appropriate dilution is required; a broad detection range is needed, which should cover the measurement range of serum creatinine levels. Low detection sensitivity also hinders the detection of creatinine by biosensors.

[0004] Existing electrochemical enzyme-free creatinine detection technologies also suffer from problems such as complex detection systems, complex material synthesis, and narrow detection ranges. For example, Hongming Hou et al. reported a method for detecting creatinine in urine by depositing a dense Cu nanoparticle film on a nickel foam electrode (see "Rapid electrodeposition of Cunanoparticle film on Ni foam as an integrated 3D free-standing electrode for non-invasive and non-enzymatic creatinine sensing", The Analyst, 2024). However, this method requires the deposition of a Cu nanoparticle film on nickel foam, which limits its application scenarios. Furthermore, the detection range is narrow, with a linear range of 0.25-24 mM and a detection limit of 0.17 mM. This necessitates diluting the urine by 7 times and filtering it when using it to detect actual samples, causing inconvenience in practical applications.

[0005] In view of this, there is an urgent need to develop a non-enzymatic electrochemical sensor for creatinine detection that is simple to prepare, low in cost, stable, has a wide detection range, and high detection sensitivity. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel non-enzymatic electrochemical sensor, its preparation method, and its application in creatinine detection. The electrochemical sensor of this invention can be used to detect serum creatinine for renal function assessment and early diagnosis of kidney disease. Because it is enzyme-independent, the electrochemical sensor of this invention exhibits greater stability under a wide range of temperature variations, resistance to environmental factors (such as temperature changes), and enhanced robustness under various clinical and real-world conditions.

[0007] Specifically, the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a non-enzymatic electrochemical sensor, the method comprising the following steps:

[0009] A copper sulfate pentahydrate solution and a 1H-1,2,4-triazole solution were prepared at a molar ratio of 3:1 to 1:3. The copper sulfate pentahydrate solution was slowly poured into the 1H-1,2,4-triazole solution and stirred at room temperature to form a transparent solution. The transparent solution was centrifuged, washed with water, and dried to obtain a blue powdery copper azole framework catalyst, which is the non-enzymatic electrochemical sensor.

[0010] Alternatively, in the above preparation method, the copper sulfate pentahydrate solution and the 1H-1,2,4-triazole solution are prepared at a molar ratio of 1:1.

[0011] Alternatively, in the above preparation method, the copper sulfate pentahydrate solution is prepared by weighing a certain amount of copper sulfate pentahydrate and adding it to water to prepare a copper sulfate aqueous solution of a certain concentration. The molar concentration of the prepared copper sulfate pentahydrate solution only needs to be consistent with the molar concentration of the 1H-1,2,4-triazole aqueous solution.

[0012] For example, weigh 0.0749 g of copper sulfate pentahydrate (0.3 mmol) and add it to 5 mL of water. Weigh 0.0207 g of 1H,1,2,4-triazole (0.3 mmol) and add it to 5 mL of water as well. Then slowly pour the copper sulfate pentahydrate solution into the 1H-1,2,4-triazole solution.

[0013] Alternatively, in the above preparation method, the centrifugation conditions are 8000rpm-16000rpm for 5-20min, and the drying temperature is 50-80℃.

[0014] Alternatively, in the above preparation method, the centrifugation conditions are 10,000 rpm for 10 min and the drying temperature is 60°C.

[0015] In a second aspect, the present invention provides a non-enzymatic electrochemical sensor prepared by the preparation method described in the first aspect above, wherein the non-enzymatic electrochemical sensor is a blue powdered copper azole framework catalyst.

[0016] Alternatively, the non-enzymatic electrochemical sensor has an amorphous stacked morphology, an internal porous structure, and pore sizes distributed in the range of 10–30 nm.

[0017] In a third aspect, the present invention provides the use of the non-enzymatic electrochemical sensor described in the second aspect above in the preparation of creatinine detection reagents.

[0018] Alternatively, in the above-described applications, the reagent is a glassy carbon electrode coated with the electrochemical sensor.

[0019] Alternatively, in the above-described uses, the reagent does not require creatinine enzyme in the detection of creatinine, and the reagent is used to detect creatinine in serum for renal function assessment and early diagnosis of kidney disease.

[0020] Alternatively, in the above-described applications, the reagent exhibits a good logarithmic response to creatinine in the range of 20-2000 μM.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention provides a one-step synthesis of a stable copper azole compound (Cutrz) at room temperature, which is simple and inexpensive. This material exhibits a good response to creatinine without requiring creatinine oxidase or other enzymes, making it an enzyme-free creatinine sensor. The electrochemical sensor of this invention can be used to detect serum creatinine for renal function assessment and early diagnosis of kidney disease. Because it is enzyme-independent, the electrochemical sensor of this invention exhibits greater stability across a wide range of temperature variations, resistance to environmental factors, and enhanced robustness under various clinical and real-world conditions. Attached Figure Description

[0023] Figure 1 X-ray diffraction pattern of Cutrz. Red line: experimental data; blue line: data after background subtraction; black line: standard pattern.

[0024] Figure 2 Scanning electron microscope image of Cutrz.

[0025] Figure 3 : Scanning electron microscopy elemental distribution of Cutrz.

[0026] Figure 4 Transmission electron microscope image of Cutrz.

[0027] Figure 5 Cyclic voltammetric scans of creatinine at different concentrations in 0.1 M phosphate buffer solution on a Cutrz sensor.

[0028] Figure 6 Peak voltammetric current values ​​of creatinine at different concentrations in 0.1M phosphate buffer solution on Cutrz. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Example:

[0033] A method for preparing a non-enzymatic electrochemical sensor includes the following steps: preparing a copper sulfate pentahydrate (CuSO4·5H2O) solution and a 1H-1,2,4-triazole solution at a molar ratio of 1:1; slowly pouring the copper sulfate pentahydrate solution into the 1H-1,2,4-triazole solution; stirring at room temperature to form a transparent solution; centrifuging the transparent solution at 10,000 rpm for 10 min; washing with water; and drying at 60 °C to obtain a blue powdered copper azole framework catalyst (Cutrz), which is the non-enzymatic electrochemical sensor of the present invention.

[0034] The method for preparing copper sulfate pentahydrate solution is to weigh a certain amount of copper sulfate pentahydrate and add it to water to prepare a copper sulfate aqueous solution of a certain concentration. The molar concentration of the prepared copper sulfate pentahydrate solution only needs to be consistent with the molar concentration of the 1H-1,2,4-triazole aqueous solution. For example, weigh 0.0749 g of copper sulfate pentahydrate (0.3 mmol) and add it to 5 mL of water. Weigh 0.0207 g of 1H,1,2,4-triazole (0.3 mmol) and add it to 5 mL of water as well. Then slowly pour the copper sulfate pentahydrate solution into the 1H-1,2,4-triazole solution.

[0035] Experimental results show that the obtained X-ray diffraction pattern of the novel Cutrz composite material ( Figure 1 This demonstrates that the Cutrz material possesses good crystallinity, and the Cutrz material prepared in this invention is consistent with the standard Cutrz structure spectrum. Scanning electron microscopy images (...) Figure 2 As can be seen, Cutrz exhibits an amorphous packing morphology. The elemental distribution map obtained from scanning electron microscopy confirms that Cutrz contains Cu, C, N, and O elements. Figure 3 Furthermore, transmission electron microscopy images reveal that Cutrz possesses a porous structure with pore sizes ranging from 10 to 30 nm. Figure 4 ).

[0036] The Cutrz material was ultrasonically dispersed and drop-coated onto a glassy carbon electrode in water. After drying, the Cutrz-coated glassy carbon electrode was used as the working electrode for detecting creatinine. In creatinine phosphate buffer solutions with concentrations ranging from 20 μM to 2000 μM, an electrochemical workstation was used with a three-electrode system. The Cutrz-coated glassy carbon electrode was used as the working electrode, silver / silver chloride as the reference electrode, and platinum as the counter electrode. Cyclic voltammetry was performed in the range of -0.6 V to 0.6 V at a scan rate of 5 mV / s for three cycles. After stabilization, the oxidation peak current from the last cycle was selected. The oxidation peak current showed a logarithmic response to the creatinine concentration.

[0037] Cyclic voltammetry scans of creatinine at different concentrations in 0.1 M phosphate buffer solution showed that the Cutrz material exhibited good response to creatinine at different concentrations. Figure 5It does not require the use of enzymes such as creatinine enzyme, and is an enzyme-free creatinine sensor.

[0038] This enzyme-free creatinine sensor exhibits high sensitivity, rapid response, and good stability. As an enzyme-free creatinine sensor, it demonstrates a good logarithmic response to creatinine levels in the range of 20-2000 μM. Figure 6 Normal serum creatinine levels range from approximately 40 μM to 150 μM. This method can achieve full coverage of serum creatinine level detection, making it highly suitable for detecting serum creatinine. However, concentrations greater than 500 μM indicate severely impaired kidney function. The electrochemical sensor of this invention covers both normal and abnormal serum creatinine concentrations, thus eliminating the need for dilution in practical measurements and allowing for direct detection. Furthermore, the Cutrz material exhibits excellent selectivity for creatinine.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a non-enzymatic electrochemical sensor, characterized in that: The preparation method includes the following steps: A copper sulfate pentahydrate solution and a 1H-1,2,4-triazole solution were prepared at a molar ratio of 3:1 to 1:

3. The copper sulfate pentahydrate solution was slowly poured into the 1H-1,2,4-triazole solution and stirred at room temperature to form a transparent solution. The transparent solution was centrifuged, washed with water, and dried to obtain a blue powdery copper azole framework catalyst, which is the non-enzymatic electrochemical sensor.

2. The preparation method according to claim 1, characterized in that: The copper sulfate pentahydrate solution and the 1H-1,2,4-triazole solution were prepared at a molar ratio of 1:

1.

3. The preparation method according to claim 1, characterized in that: The centrifugation conditions are a rotation speed of 8000rpm-16000rpm, a time of 5-20min, and a drying temperature of 50-80℃.

4. The preparation method according to claim 1, characterized in that: The centrifugation conditions were 10,000 rpm for 10 minutes and the drying temperature was 60°C.

5. A non-enzymatic electrochemical sensor prepared by the method according to any one of claims 1 to 4, characterized in that: The non-enzymatic electrochemical sensor is a blue powdered copper azole framework catalyst.

6. Use of the non-enzymatic electrochemical sensor according to claim 5 in the preparation of creatinine detection reagents.

7. The use according to claim 6, characterized in that: The reagent is a glassy carbon electrode coated with the electrochemical sensor.

8. The use according to claim 6, characterized in that: The reagent does not require creatinine enzyme in the detection of creatinine. The reagent is used to detect creatinine in serum for the assessment of renal function and the early diagnosis of kidney disease.

9. The use according to claim 6, characterized in that: The reagent has a good logarithmic response to creatinine in the range of 20-2000 μM.

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