Microfluidic electrochemical sensor based on monatomic catalyst as well as preparation method and application of microfluidic electrochemical sensor

Abstract

The invention discloses a microfluidic electrochemical sensor based on a monatomic catalyst as well as a preparation method and application of the microfluidic electrochemical sensor. The sensor takes an iron monatomic catalyst (FeSA-GC) as a core sensing element, and the key structural feature of the sensor is that a monatomic FeN4 site is embedded into a graphene-like carbon matrix. A uric acid (UA) sensor, a glucose (Glu) sensor and a pH value (pH) sensor are successfully constructed by modifying the surface of an integrated array electrode with a monatomic catalyst and combining diversified strategies such as direct catalysis, enzyme coupling and polymer hybridization, and sensitive detection of different biomarkers can be realized. Meanwhile, the specially designed micro-fluidic chip integrates multiple functions of sample filtration, enrichment, detection and the like, and efficient integrated operation of the detection process can be realized. The microfluidic electrochemical sensor disclosed by the invention has the advantages of high sensitivity, good selectivity, simplicity and convenience in operation and the like, and has better application potential in the fields of bedside instant detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic electrochemical sensor technology, and in particular relates to a microfluidic electrochemical sensor based on a single-atom catalyst, its preparation method, and its application in the detection of various biomarkers. Background Technology

[0002] Biomarkers play a crucial role in disease diagnosis and health management. For example, changes in uric acid (UA) levels directly reflect the risk of gout attacks and disease progression; glucose (Glu) is an indispensable monitoring indicator in the clinical management of diabetes; and pH is an important basis for assessing acid-base imbalances. Accurate detection of these biomarkers is of great significance in disease monitoring and treatment evaluation.

[0003] Currently, methods for detecting biomarkers mainly include high-performance liquid chromatography (HPLC), capillary electrophoresis, colorimetric analysis, and fluorescence analysis. While these methods offer high sensitivity and accuracy, their applications have significant limitations: firstly, the instruments are large, with high purchase and maintenance costs, and high reagent consumption, leading to high overall testing costs; secondly, the detection requires stringent experimental environments and highly skilled personnel, necessitating professional staff in standard laboratories to complete sample pretreatment, instrument calibration, and data analysis, making them unsuitable for convenient scenarios such as point-of-care testing. Therefore, there is an urgent need to develop a new method that balances high sensitivity, low detection cost, and portability to overcome the limitations of traditional detection technologies and drive biomarker detection towards a more efficient and practical direction.

[0004] Electrochemical sensors, with their advantages of easy miniaturization and integration, fast response speed, and simple operation, have been widely used in biomarker detection. Furthermore, microfluidics, an analytical technique characterized by micrometer-scale fluid manipulation, can integrate key operational units in biochemical analysis, such as sample filtering, enrichment, and detection, onto a micrometer-scale chip platform, achieving integrated analysis. This type of technology also boasts advantages such as low sample consumption, small device size, and high portability. Combining electrochemical sensors with microfluidics to construct novel microfluidic electrochemical sensors can fully leverage their synergistic effects, providing a more efficient and applicable solution for biomarker detection.

[0005] To further enhance the detection performance of microfluidic electrochemical sensors, the design of sensing materials is crucial. In recent years, single-atom catalysts (SACs) have developed rapidly, becoming a highly promising new type of sensing material. In SACs, metal active sites are uniformly dispersed on the support surface in the form of isolated atoms and stably anchored through coordination bonds, forming a catalytic structure that combines high activity and stability. Compared to traditional nanoparticle or nanobulk catalysts, SACs exhibit many advantages, providing new insights for breakthroughs in microfluidic electrochemical sensing performance. For example, the atomic utilization rate of SACs is close to 100%, fundamentally solving the problem of wasted active sites caused by metal atom aggregation in traditional catalysts. The high density and well-defined active sites on the surface of SACs can significantly improve the electronic conduction efficiency and kinetics of electrochemical reactions by optimizing electron transfer paths and lowering reaction energy barriers, thereby greatly enhancing catalytic activity. Furthermore, the special interactions between metal atoms and the substrate can provide an unsaturated coordination environment for the metal atoms, which is beneficial for improving catalytic activity and selectivity. Therefore, SACs, with their unique advantages, show great application prospects in fields such as bioassay and analysis. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a microfluidic electrochemical sensor based on a single-atom catalyst, its preparation method, and its applications in the detection of various biomarkers. The single-atom catalyst (FeSA@GC) used in the sensor of this invention is characterized by a single-atom FeN4 site embedded in a graphene-like carbon matrix. This structure combines the advantages of atomically dispersed active centers and a highly conductive carbon matrix, exhibiting excellent catalytic activity and rapid electron transfer kinetics. By modifying the surface of an integrated array electrode with the single-atom catalyst and combining it with diverse strategies such as direct catalysis, enzyme coupling, and polymer hybridization, sensitive detection of various biomarkers can be achieved. Simultaneously, a specially designed microfluidic chip integrates multiple functions such as sample filtration, enrichment, and detection, enabling efficient and integrated operation of the detection process. The microfluidic electrochemical sensor of this invention has advantages such as high sensitivity, high selectivity, and ease of operation, and has good application potential in fields such as point-of-care testing.

[0007] The technical solution adopted in this invention is: a method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst, comprising the following steps:

[0008] (1) Preparation of single-atom catalysts

[0009] Dicyandiamide, 1-(2-cyanoethyl)-2-phenylimidazole, and ferric nitrate nonahydrate were dissolved in an organic solvent at room temperature. After stirring and dissolving, the organic solvent was evaporated by heating. The resulting solid was thoroughly ground and placed in a crucible, which was then placed in a muffle furnace for a first calcination to obtain carbon-rich iron-doped graphitic carbon nitride Fe-g-C3N4. Subsequently, a second calcination was carried out under a nitrogen atmosphere to finally obtain FeSA@GC.

[0010] (2) Fabrication of array sensing electrodes

[0011] Multiple array electrodes, including at least one working electrode, a shared counter electrode, and a reference electrode, are laser-etched on an indium tin oxide (ITO) glass substrate; wherein the reference electrode is prepared by dropping Ag / AgCl ink onto the ITO and curing it at 70°C for 30 minutes.

[0012] (3) Electrochemical sensors are prepared by modifying the working electrode, including at least one of the following sensors;

[0013] UA sensor preparation: FeSA@GC was dispersed in chitosan solution and ultrasonically treated to form a suspension; 4 μL of the FeSA@GC suspension was drop-coated onto the surface of the ITO working electrode and dried to obtain the FeSA@GC / ITO electrode, which is the UA sensor.

[0014] Glu sensor fabrication: Glu oxidase was further immobilized on a FeSA@GC / ITO electrode by covalent coupling to obtain a Glu sensor.

[0015] pH sensor fabrication: A pH sensor was prepared by electrochemically modifying poly(o-phenylenediamine) on the surface of a FeSA@GC / ITO electrode using cyclic voltammetry (CV).

[0016] (4) Fabrication of microfluidic electrochemical sensors

[0017] A microfluidic chip is prepared, which is provided with a filter, an inlet channel, an outlet channel and a detection chamber; the electrochemical sensor prepared in step (3) is bonded to the microfluidic chip, and each array electrode is placed below the detection chamber.

[0018] Furthermore, the organic solvent is methanol. Ethanol, etc., may also be used.

[0019] Furthermore, the conditions for the first calcination are: heating to 550°C at a heating rate of 2.5°C / min and holding at that temperature for 4 hours; the conditions for the second calcination are: heating to 800°C at a heating rate of 5°C / min for carbonization treatment and holding at that temperature for 2 hours.

[0020] Furthermore, the specific steps for further immobilizing Glu oxidase on the FeSA@GC / ITO electrode via covalent coupling include: first, adding 4 μL of a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the FeSA@GC / ITO electrode; after drying for 3 hours, then dropping 4 μL of the enzyme solution onto the electrode surface to obtain the Glu sensor (GO). x / FeSA@GC / ITO); the enzyme solution is obtained by passing 10 mg GO x It was prepared by dissolving it in 1 mL of 0.2% chitosan solution.

[0021] Further, the specific steps for electrochemically modifying the FeSA@GC / ITO electrode surface with poly(o-phenylenediamine) are as follows: 4 μL of 0.02M o-phenylenediamine phosphate buffer solution is drop-coated onto the FeSA@GC / ITO electrode surface, followed by CV polymerization. The parameters are set as follows: potential window -0.6V to 0.2V, scan rate 50mV / s, continuous scan for 10 cycles, and the resulting pH sensor is denoted as PoPD / FeSA@GC / ITO.

[0022] Furthermore, a syringe filter is provided at the front end of the sample inlet channel of the microfluidic chip, which contains a mixed cellulose ester membrane modified with 3-aminopropyltriethoxysilane.

[0023] Furthermore, several micropillar structures are disposed within the microfluidic detection cavity.

[0024] This invention also provides a microfluidic electrochemical sensor based on a single-atom catalyst, comprising a microfluidic chip as the upper layer and an electrochemical sensor as the lower layer, wherein the microfluidic chip and the electrochemical sensor are bonded together; the microfluidic chip is provided with a filter, an inlet channel, an outlet channel, and a detection chamber; the electrochemical sensor is provided with multiple array electrodes, including at least one working electrode, a shared counter electrode, and a reference electrode; each array electrode is positioned below the detection chamber; wherein the reference electrode is coated with Ag / AgCl ink, and the working electrode is coated with FeSA@GC suspension. The FeSA@GC is prepared by further immobilizing Glu oxidase in a flotation solution and further electrochemically modifying poly(o-phenylenediamine). The FeSA@GC is prepared by dissolving dicyandiamide, 1-(2-cyanoethyl)-2-phenylimidazolium, and ferric nitrate nonahydrate in an organic solvent at room temperature. After stirring and dissolving, the organic solvent is evaporated by heating. The resulting solid is thoroughly ground and placed in a crucible, then subjected to a first calcination in a muffle furnace to obtain carbon-rich iron-doped graphitic carbon nitride Fe-g-C3N4. Subsequently, a second calcination is performed under a nitrogen atmosphere to finally obtain FeSA@GC.

[0025] Finally, this invention provides the application of the microfluidic electrochemical sensor based on a single-atom catalyst in the detection of UA, Glu, and pH.

[0026] The beneficial technical effects of this invention include:

[0027] 1. Significantly improved detection performance, achieving high sensitivity and high selectivity detection.

[0028] Leveraging the superior catalytic activity and rapid electron transfer kinetics of single-atom catalysts, and combining diverse strategies such as direct catalysis, enzyme coupling, and polymer hybridization, high sensitivity and high selectivity can be achieved for the properties of different biomarkers.

[0029] 2. Integrated testing process significantly improves testing efficiency.

[0030] The designed microfluidic chip integrates sample filtration, enrichment and detection functions, eliminating the cumbersome sample pretreatment steps in traditional detection and greatly improving detection efficiency.

[0031] 2. The wide range of application scenarios promotes the practical application and portability of testing technologies.

[0032] The designed microfluidic electrochemical sensor is easy to operate, requiring no complex instruments or specialized skills, thus reducing the technical requirements for operators. The single-atom catalyst boasts high metal atom utilization, lowering material costs. Its efficient detection performance and integrated design make it suitable not only for precise laboratory testing but also for rapid on-site detection, driving the development of biomarker detection technology towards practicality and portability. Attached Figure Description

[0033] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0034] Figure 1 This is a schematic diagram illustrating the principle of the present invention;

[0035] Figure 2 For the morphological characterization of single-atom catalysts, the figures are as follows: a: Transmission electron microscope image; b: Scanning transmission electron microscope image and corresponding energy dispersive spectroscopy (EDS); c: Spherical aberration electron microscope image; d: Gaussian function fitting map of atomic overlap.

[0036] Figure 3For the atomic structure analysis of single-atom catalysts, the figures are as follows: a: Normalized Fe K-edge X-ray absorption near-edge structure spectra (XANES) of Fe foil, FeO, Fe2O3, and FeSA@GC; b: Corresponding Fourier transform extended X-ray absorption fine structure spectra (FT-EXAFS); c: Fourier transform extended X-ray absorption fine structure spectra in R space; d: Wavelet transform plots of the X-ray absorption near-edge structure spectra of Fe foil, FeO, Fe2O3, and FeSA@GC.

[0037] Figure 4 The figures show a schematic diagram and a physical image of a microfluidic electrochemical sensor. In the figures, a: schematic diagram of the microfluidic electrochemical sensor; b: physical image of the microfluidic electrochemical sensor; c: schematic diagram of the microfluidic chip dimensions.

[0038] Figure 5 For the functional verification of the microfluidic chip, the figure shows: a: sample detection image combined with smartphone; b: fluorescence intensity curves before and after filtration of fluorescently labeled ovalbumin; c: linear velocity distribution obtained by simulation calculation in XZ axis space; d: streamline distribution obtained by simulation calculation in three-dimensional space.

[0039] Figure 6 The microfluidic electrochemical sensor is used for UA detection. In the figure, a: schematic diagram of UA detection; b: DPV response curves at different UA concentrations, inset: linear fitting curve of UA concentration and DPV response (n=3); c: DPV response curve in the presence of interfering substances; d: DPV response value in the presence of interfering substances (n=3); e: DPV response values ​​of five independent sensors to UA (n=3); f: DPV response value of the sensor in 10 repeated tests.

[0040] Figure 7 The figure shows a microfluidic electrochemical sensor for Glu detection. a: Schematic diagram of Glu detection; b: IT response curves at different Glu concentrations; inset: linear fitting curve of Glu concentration versus IT response (n=3); c: IT response curves for different interfering substances; d: IT response values ​​for different interfering substances (n=3); e: IT response values ​​of five independent sensors for Glu (n=3); f: IT response values ​​of the sensor in 10 repeated tests.

[0041] Figure 8 For microfluidic electrochemical sensors used for pH detection, the figures are as follows: a: Schematic diagram of pH detection; b: CV response curves at different pH values; inset: linear fitting curve of pH value and CV response (n=3); c: CV response curve in the presence of interfering substances; d: CV response value in the presence of interfering substances (n=3); e: CV response values ​​of five independent sensors to pH (n=3); f: CV response value of the sensor in 10 repeated tests.

[0042] Figure 9 For the application of microfluidic electrochemical sensors in serum sample detection, the figures are as follows: a: CV response curves of serum samples at different pH values; b: DPV response curves obtained by adding standard UA to serum samples; c: It response curves obtained by adding standard Glu to serum samples; d: Correlation analysis between pH values ​​measured by the sensor and pH meter values; e: Spiked recovery rate of UA in serum samples; f: Spiked recovery rate of Glu in serum samples. Detailed Implementation

[0043] Example 1: Fabrication of a microfluidic electrochemical sensor based on a single-atom catalyst

[0044] (1) Preparation of single-atom catalysts

[0045] At room temperature, 16 g of dicyandiamide, 2 g of 1-(2-cyanoethyl)-2-phenylimidazole, and 24 mg of ferric nitrate nonahydrate were dissolved in 400 mL of methanol. After stirring for 10 hours, the system temperature was raised to 80 °C to evaporate the methanol. The resulting solid was thoroughly ground and placed in a crucible, then heated in a muffle furnace to 550 °C at a heating rate of 2.5 °C / min and held for 4 hours to obtain carbon-rich iron-doped graphitic carbon nitride (Fe-g-C3N4). Subsequently, Fe-g-C3N4 was heated to 800 °C under a nitrogen atmosphere at a heating rate of 5 °C / min for carbonization treatment and held for 2 hours to obtain the final iron single-atom catalyst (FeSA@GC).

[0046] (2) Fabrication of array sensing electrodes

[0047] The sensor's electrodes comprise a five-electrode array: three working electrodes (3 mm in diameter) for detecting UA, Glu, and pH, respectively, along with a shared counter electrode (CE) and a reference electrode (RE). The electrodes are fabricated using laser etching on an indium tin oxide (ITO) glass substrate. This sensor electrode system is an integrated three-electrode system, where modified ITO serves as the working electrode (WE), Ag / AgCl / ITO as the reference electrode (RE), and bare ITO as the counter electrode (CE). The reference electrode is prepared by dropping Ag / AgCl ink onto ITO and curing it at 70°C for 30 minutes.

[0048] (3) Fabrication of electrochemical sensors

[0049] UA sensor preparation: 2 mg FeSA@GC was dispersed in 1 mL of chitosan solution (0.2%) and sonicated for 10 minutes to form a homogeneous suspension. Subsequently, 4 μL of the FeSA@GC suspension was drop-coated onto the surface of an ITO electrode and dried to obtain the FeSA@GC / ITO electrode, i.e., the UA sensor.

[0050] Glu sensor fabrication: Glu oxidase (GO) was further immobilized on a FeSA@GC / ITO electrode. x To construct a Glu sensor, 4 μL of a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS, 40 mM) was added dropwise and dried for 3 hours to fully activate the carboxyl groups on the electrode surface. Subsequently, 10 mg of GO was added... x An enzyme solution was prepared by dissolving the enzyme in 1 mL of 0.2% chitosan solution. 4 μL of this enzyme solution was then drop-coated onto the surface of the working electrode to prepare the Glu sensor (GO). x / FeSA@GC / ITO). Store at 4°C before use.

[0051] pH sensor fabrication: A pH sensor was fabricated by electrochemically modifying the FeSA@GC / ITO electrode surface with poly(o-phenylenediamine) (PoPD) using cyclic voltammetry (CV). The specific steps were as follows: 4 μL of 0.02 M o-phenylenediamine in phosphate-buffered saline (PBS) solution was drop-coated onto the electrode surface, followed by CV polymerization. The parameters were set as follows: potential window -0.6 V to 0.2 V, scan rate 50 mV / s, and continuous scanning for 10 cycles. The resulting sensor was designated PoPD / FeSA@GC / ITO.

[0052] (4) Fabrication of microfluidic electrochemical sensors

[0053] To achieve integrated sample processing, enrichment, and detection, thereby enhancing its practical value, the fabricated array sensor was further integrated with a meticulously designed microfluidic chip. This chip, made of polyethylene terephthalate (PET) using 3D printing technology, includes an inlet channel, an outlet channel, and a circular detection chamber. The fluid channel has a cross-sectional dimension of 1 mm (width) × 0.5 mm (height); the detection chamber has a diameter of 12 mm and contains a series of micropillars with a diameter of 1.5 mm and a height of 0.2 mm. Furthermore, to remove macromolecules (such as proteins), the chip incorporates a syringe filter, the core of which is a mixed cellulose ester membrane (0.22 μm pore size). This membrane is chemically functionalized by treatment with ammonia / hydrogen peroxide solution and grafting with 3-aminopropyltriethoxysilane.

[0054] Example 2, Analysis of Single-Atom Catalysts

[0055] Morphological characterization of single-atom catalysts, such as Figure 2 As shown, it exhibits a typical graphene-like morphology, with high-density bright spots uniformly doped into the carbon matrix, directly demonstrating the existence of atomic-level active sites. Further analysis of the atomic structure of the single-atom catalyst, such as... Figure 3 As shown, the XANES energy of FeSA@GC lies between FeO and Fe2O3, indicating that the average valence state of Fe atoms is between FeO and Fe2O3. II and Fe III Between. In the corresponding FT-EXAFS spectrum, FeSA@GC only appears between. A main peak appeared at the [specific location], and no Fe-Fe interaction peaks were detected. This result confirms the atomic-level dispersion of Fe. Furthermore, by fitting the EXAFS spectrum to R-space and K-space, and combining it with wavelet transform techniques, the quantitative structural parameters around the central Fe atom were precisely determined, ultimately verifying the existence of the FeN4 active site.

[0056] Example 3: Structure of a microfluidic electrochemical sensor

[0057] like Figure 4 As shown, the microfluidic electrochemical sensor has a microfluidic chip on top and an electrochemical sensor on the bottom, with the microfluidic chip and the electrochemical sensor bonded together. The microfluidic chip is equipped with a filter, an inlet channel, an outlet channel, and a circular detection chamber, which can also be designed in an elliptical or other shape. The inlet and outlet channels are connected to the detection chamber. The electrochemical sensor has five array electrodes, including three working electrodes, one shared counter electrode, and one reference electrode. The electrode preparation method can refer to the method in Example 1. Each array electrode is placed below the detection chamber so that the liquid being detected can contact the electrode.

[0058] A series of micropillars are arranged inside the detection chamber, which can induce vertical fluid flow and generate local micro-vortices. This hydrodynamic effect can enhance the interaction between the reactants and the sensing interface (equivalent to an enrichment effect), which helps to improve the sensitivity of electrochemical detection.

[0059] Example 4: Functional Verification of Microfluidic Chips

[0060] Integrating the fabricated microfluidic electrochemical sensor into a smartphone enables user-friendly operation and real-time data access. Prior to actual testing, the function of the microfluidic chip was evaluated using fluorescently labeled ovalbumin (OVA-FITC). The fluorescence intensity of the OVA-FITC solution significantly decreased after filtration through a syringe filter, indicating that the filter effectively removed protein molecules. Subsequently, computational fluid dynamics simulations were used to investigate the fluid behavior within the microfluidic chip. Figure 5As shown, the results indicate that the fluid velocity is relatively high in the main channel, but decreases significantly after entering the detection chamber; furthermore, the flow velocity around the cylindrical structure within the detection chamber exhibits a significant difference. The streamline distribution further suggests that the cylindrical structure can induce vertical flow and generate local micro-vortices. This hydrodynamic effect can enhance the interaction between the reactants and the sensing interface (equivalent to an enrichment effect), contributing to improved sensitivity of electrochemical detection.

[0061] Example 5: Detection of UA by a microfluidic electrochemical sensor

[0062] like Figure 6 As shown, known concentrations of UA were added to PBS solutions (0 μM, 5 μM, 10 M, 20 μM, 50 μM, 100 μM, 200 μM, 400 μM) as the target analyte, and differential pulse voltammetry (DPV) curves were scanned between 0.1 and 0.5 V. The relationship between peak current intensity and UA concentration was detected by the electrochemical sensor, and a linear fitting curve was obtained. The detection limit was estimated to be 3.84 μM (detection limit = 3σ / k, where σ is the standard deviation of the background signal in the blank sample, and k is the slope of the fitted curve). The prepared sensor was exposed to PBS solution containing 5 times the amount of interfering substances (Glu, Urea, Ascorbic Acid AA, Dopamine DA) and 200 μM UA. The obtained DPV response values ​​showed no significant difference, indicating good selectivity. Five sensors were independently prepared under the same conditions, and each sensor tested a PBS buffer solution containing 200 μM UA. The relative standard deviation (RSD) of the obtained responses was 7.41%, indicating that the sensors prepared in different batches have good reproducibility. After 10 consecutive tests in a PBS buffer solution containing 200 μM UA, the response deviation did not exceed 9%, indicating that the prepared UA sensor has good testing stability.

[0063] Example 6: Detection of Glu by a Microfluidic Electrochemical Sensor

[0064] like Figure 7As shown, known concentrations of Glu as the target analyte were added to PBS solutions (0 μM, 50 μM, 100 M, 200 μM, 500 μM, 1000 μM, 2000 μM), and chronocurrent (IT) curves were scanned at a working voltage of 0.5 V. The relationship between the chronocurrent intensity and Glu concentration was detected using the electrochemical sensor, yielding a linear fitting curve. The detection limit was estimated to be 14.37 μM. The prepared sensor was exposed to solutions containing 5 times the amount of interfering substances (Glu, UA, DA, AA, KCl, Urea) and their mixtures, and the obtained IT response values ​​showed no significant difference, indicating good selectivity. Five sensors were independently prepared under the same conditions, and each sensor detected PBS buffer solution containing 500 μM Glu. The RSD of the obtained response was 6.65%, indicating good reproducibility among different batches of Glu sensors. In PBS buffer solution containing 500 μM UA, the response deviation was less than 10% after 10 consecutive tests, indicating that the prepared Glu sensor has good testing stability.

[0065] Example 7: pH detection using a microfluidic electrochemical sensor

[0066] like Figure 8 As shown, the pH values ​​of the PBS solution were adjusted (pH=6, pH=6.5, pH=7, pH=7.5, pH=8), and cyclic voltammetry curves were scanned between 0.7 and 0.2 V. The relationship between the oxidation peak potential and pH value was detected using the electrochemical sensor, and a linear fitting curve was obtained. The prepared sensor was exposed to a solution containing 5 times the amount of interfering substances (Glu, UA, DA, AA, Urea, K). + Na + The CV peak potentials obtained from the pH sensors and their mixtures showed no significant difference, indicating good selectivity. Five sensors were independently prepared under the same conditions, each detecting a PBS buffer solution at pH 7.0. The relative standard deviation of the peak potential response was 1.08%, indicating good reproducibility among different batches of pH sensors. After 10 consecutive tests in a PBS buffer solution at pH 7.0, the peak potential deviation did not exceed 2%, indicating good testing stability of the prepared pH sensor.

[0067] Example 8: Microfluidic electrochemical sensor for serum sample detection application

[0068] like Figure 9As shown, the performance of the microfluidic electrochemical sensor was validated through spiked recovery experiments in human serum samples (diluted 20-fold). The sensor can sensitively detect changes in serum pH, and the results show good agreement with those obtained using a conventional pH meter. Furthermore, significant UA and Glu signals were successfully detected in the serum samples. The recovery rates of UA were 90.62%–102.74%, and those of glucose were 97.22%–107.89%. These results collectively demonstrate the application potential of this microfluidic electrochemical sensor in practical sample analysis.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst, characterized in that, Includes the following steps: (1) Preparation of single-atom catalysts Dicyandiamide, 1-(2-cyanoethyl)-2-phenylimidazolium, and ferric nitrate nonahydrate were dissolved in an organic solvent at room temperature. After stirring and dissolving, the organic solvent was evaporated by heating. The resulting solid was thoroughly ground and placed in a crucible, which was then placed in a muffle furnace for a first calcination to obtain carbon-rich iron-doped graphitic carbon nitride Fe-g-C3N4. Subsequently, a second calcination was carried out under a nitrogen atmosphere to finally obtain FeSA@GC. (2) Fabrication of array sensing electrodes Multiple array electrodes, including at least one working electrode, a shared counter electrode, and a reference electrode, are laser-etched on an indium tin oxide (ITO) glass substrate; wherein the reference electrode is prepared by dropping Ag / AgCl ink onto the ITO and curing it at 70°C for 30 minutes. (3) Electrochemical sensors are prepared by modifying the working electrode, including at least one of the following sensors; UA sensor preparation: FeSA@GC was dispersed in chitosan solution and ultrasonically treated to form a suspension; 4 μL of the FeSA@GC suspension was drop-coated onto the surface of the ITO working electrode and dried to obtain the FeSA@GC / ITO electrode, which is the UA sensor. Glu sensor fabrication: Glu oxidase was further immobilized on a FeSA@GC / ITO electrode via covalent coupling to obtain a Glu sensor; pH sensor fabrication: A pH sensor was prepared by electrochemically modifying poly(o-phenylenediamine) on the surface of a FeSA@GC / ITO electrode using cyclic voltammetry (CV). (4) Fabrication of microfluidic electrochemical sensors A microfluidic chip is prepared, which is provided with a filter, an inlet channel, an outlet channel and a detection chamber; the electrochemical sensor prepared in step (3) is bonded to the microfluidic chip, and each array electrode is placed below the detection chamber.

2. The method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst according to claim 1, characterized in that: The conditions for the first calcination are: heating to 550°C at a heating rate of 2.5°C / min and holding at that temperature for 4 hours; the conditions for the second calcination are: heating to 800°C at a heating rate of 5°C / min for carbonization treatment and holding at that temperature for 2 hours.

3. The method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst according to claim 1, characterized in that: The specific steps for further immobilizing Glu oxidase on the FeSA@GC / ITO electrode via covalent coupling include: First, adding 4 μL of a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the FeSA@GC / ITO electrode, drying for 3 hours, and then dripping 4 μL of the enzyme solution onto the electrode surface to obtain the Glu sensor (GO). x / FeSA@GC / ITO); the enzyme solution is obtained by passing 10 mg GO x It was prepared by dissolving it in 1 mL of 0.2% chitosan solution.

4. The method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst according to claim 1, characterized in that: The specific steps for electrochemically modifying the FeSA@GC / ITO electrode surface with poly(o-phenylenediamine) are as follows: 4 μL of 0.02M o-phenylenediamine phosphate buffer solution is drop-coated onto the FeSA@GC / ITO electrode surface, followed by CV polymerization. The parameters are set as follows: potential window -0.6V to 0.2V, scan rate 50mV / s, continuous scan for 10 cycles. The resulting pH sensor is denoted as PoPD / FeSA@GC / ITO.

5. The method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst according to claim 1, characterized in that: A syringe filter is provided at the front end of the sample inlet channel of the microfluidic chip. This filter contains a mixed cellulose ester membrane modified with 3-aminopropyltriethoxysilane.

6. The method for preparing a microfluidic electrochemical sensor based on a single-atom catalyst according to claim 1, characterized in that: Several micropillar structures are arranged inside the detection cavity.

7. A microfluidic electrochemical sensor based on a single-atom catalyst, characterized in that: The upper layer is a microfluidic chip, and the lower layer is an electrochemical sensor, which are bonded together. The microfluidic chip is equipped with a filter, an inlet channel, an outlet channel, and a detection chamber. The electrochemical sensor is equipped with multiple array electrodes, including at least one working electrode, a shared counter electrode, and a reference electrode. Each array electrode is positioned below the detection chamber. The reference electrode is coated with Ag / AgCl ink, and the working electrode is coated with FeSA@GC suspension, or further immobilized Glu oxygen. The FeSA@GC is prepared by further electrochemical modification of poly(o-phenylenediamine) with enzymes, or by further electrochemical modification. The FeSA@GC is prepared by dissolving dicyandiamide, 1-(2-cyanoethyl)-2-phenylimidazolium, and ferric nitrate nonahydrate in an organic solvent at room temperature. After stirring and dissolving, the organic solvent is evaporated by heating. The resulting solid is thoroughly ground and placed in a crucible, then placed in a muffle furnace for a first calcination to obtain carbon-rich iron-doped graphitic carbon nitride Fe-g-C3N4. Subsequently, a second calcination is performed under a nitrogen atmosphere to finally obtain FeSA@GC.

8. The microfluidic electrochemical sensor based on a single-atom catalyst according to claim 7, characterized in that: The filter contains a mixed cellulose ester membrane modified with 3-aminopropyltriethoxysilane.

9. The microfluidic electrochemical sensor based on a single-atom catalyst according to claim 7, characterized in that: The detection chamber is equipped with several micropillar structures.

10. The application of the microfluidic electrochemical sensor based on a single-atom catalyst as described in claim 7 in the detection of UA, Glu and pH.

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