Electrochemical sensor chip based on nickel porphyrin / CuO nanosheet composite sensitive material, preparation method of electrochemical sensor chip and application of electrochemical sensor chip in construction of living cell NO release monitoring platform

Abstract

The invention discloses an electrochemical sensor chip based on a nickel porphyrin / CuO nanosheet composite sensitive material, a preparation method of the electrochemical sensor chip and application of the electrochemical sensor chip in construction of a living cell NO release monitoring platform, and relates to the technical field of biosensing, electrochemical analysis and biomedical detection. The invention designs an electrochemical sensor chip based on a nickel porphyrin / CuO nanosheet composite sensitive material on the basis of a specific electro-catalytic reaction of NO. The sensor chip can achieve high-performance detection of NO, the detection limit can reach 1 nM, and good selectivity and stability are shown. A living cell NO monitoring platform capable of simulating the physiological environment is constructed by further combining a cell three-dimensional culture model based on GelMA hydrogel, dynamic evaluation of the NO release level of endothelial cells under the action of sugar substitutes, diabetes treatment drugs and the like is achieved, a reliable tool is provided for biological effect analysis of the diabetes drugs and the sugar substitutes, and the method is suitable for popularization and application. The method has an important clinical application prospect in the aspects of diabetes mellitus diet management and drug curative effect evaluation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensing, electrochemical analysis and biomedical detection, and particularly relates to an electrochemical sensor chip based on a porphyrin nickel / CuO nanosheet composite sensitive material, a preparation method thereof and application thereof in constructing a living cell NO release monitoring platform. BACKGROUND

[0002] Diabetes, especially type 2 diabetes mellitus (T2DM), has become a global epidemic chronic metabolic disease and one of the major factors leading to death and disability. The core pathological features of the disease include insulin resistance and islet beta cell dysfunction, and its complications such as cardiovascular disease, kidney disease and neuropathy seriously endanger human health. In the comprehensive management of diabetes, in addition to conventional drug treatment, dietary intervention means represented by sugar substitutes are also increasingly important. However, whether it is hypoglycemic drugs or food additives, the long-term safety and its regulation mechanism on the deep metabolic signaling pathways of the body still need to be further evaluated. Therefore, the development of a technology capable of real-time and accurate monitoring of key physiological indicators has great significance for the management of diabetes, drug efficacy evaluation and food safety research.

[0003] Nitric oxide (NO) is a vital gaseous signaling molecule in the body, playing a core role in maintaining vascular endothelial function, regulating insulin sensitivity and immune response. In diabetic patients, the synthesis and metabolism of NO are key factors leading to vascular endothelial dysfunction and cardiovascular complications. Therefore, the dynamic changes of NO are considered as a key biomarker for evaluating metabolic health and drug effects. However, NO has the characteristics of extremely short half-life (about 5 s), extremely low concentration and high reactivity in the body, which makes it a great technical challenge to detect it in real time, in situ and with high sensitivity.

[0004] At present, common NO detection methods include chemiluminescence, spectrophotometry, fluorescent probe and chromatography, etc. Although these methods have their own advantages, they generally have the following limitations: complex operation process, expensive equipment, slow detection speed, difficult to realize dynamic continuous monitoring, and insufficient sensitivity in dealing with complex biological samples. These shortcomings seriously limit their application in studying the rapid dynamic changes of NO, especially in real-time evaluation of drugs or food additives at the level of living cells.

[0005] Now there are relevant reports show that based on electrochemical sensor method can be achieved by using a new type of nanocomposite high sensitivity real-time monitoring of NO. However, these methods still have some shortcomings, such as detection limit is difficult to meet the physiological level of ultra-low concentration (pM ~ nM) detection requirements, and lack of integration with the three-dimensional cell culture model, difficult to evaluate the biological effects of drugs in real physiological environment, therefore, the need to build a new sensor system in the sensitivity, selectivity and stability performance is better, and can be directly used for live cell pharmacological research for the detection of NO. SUMMARY

[0006] The purpose of the present application is to provide a kind of based on porphyrin nickel / CuO nanosheet composite sensitive material Electrochemical sensor chip and its preparation method and application in the construction of live cell NO release monitoring platform, to solve the problems existing in the prior art described above. The present application is by constructing a kind of integrated high sensitivity, high selectivity and excellent stability based on porphyrin nickel / CuO nanosheet composite sensitive material Electrochemical sensor chip for the detection of NO, and based on the electrochemical sensor chip and three-dimensional cell culture technology Real-time monitoring platform, to evaluate the influence of diabetes treatment drugs (such as GLP-1, insulin) and sugar substitutes (such as erythritol, aspartame, sucralose) on vascular endothelial cell function.

[0007] To achieve the above object, the present application provides the following scheme: One of the technical solutions of the present application: a kind of based on porphyrin nickel / CuO nanosheet composite sensitive material Electrochemical sensor chip, including polymer substrate and the detection unit of setting on the surface of polymer substrate, the components of the detection unit include working electrode (WE), counter electrode (CE), reference electrode (RE) and wire; The surface of the working electrode is modified with porphyrin nickel / CuO nanosheet composite sensitive material; The porphyrin nickel / CuO nanosheet composite sensitive material takes CuO nanosheet as the bottom layer (i.e. takes CuO nanosheet layer as the substrate layer of porphyrin nickel crystal), and the surface is loaded with prismatic porphyrin nickel crystal.

[0008] In the above-mentioned electrochemical sensor chip, the working electrode conducts current in the electrochemical measurement to drive a specific chemical reaction, and the change of its potential directly reflects the characteristics and rate of the electrochemical reaction; the porphyrin nickel in the working electrode surface modified porphyrin nickel / CuO nanosheet composite sensitive material effectively adsorbs NO molecules through its coordination center, and cooperates with the oxidation environment and electron transfer auxiliary effect provided by the CuO nanosheet to realize specific capture and efficient electrocatalytic oxidation of NO molecules. Specifically, the CuO nanosheet has a high specific surface area and excellent electrocatalytic performance, and can realize rapid sensing. However, in a complex biological system, a single CuO nanosheet material faces challenges such as insufficient selectivity and limited anti-interference ability. Porphyrin nickel is a regular prismatic crystal structure with a smooth crystal face and a particle size of tens of microns, reflecting its high crystallinity and good morphology consistency. This regular crystal morphology is conducive to subsequent directional arrangement and stable loading on the electrode interface, and helps to improve the construction efficiency and signal stability of the electrochemical sensor. Moreover, as an organic metal catalyst, porphyrin nickel can interact with NO molecules through its coordination center. The present application realizes high selectivity and high sensitivity detection of NO through the specific coordination capture ability of porphyrin nickel for NO molecules and the electrocatalytic synergistic advantage of CuO nanosheet. It can effectively exclude the influence of common interfering substances such as glucose, chloride ions and nitrite ions. The sensor shows important application value in the field of biomedicine. It can monitor the release dynamics of endogenous NO in real time, and provides an innovative detection method for the safety evaluation of sugar substitutes and the functional evaluation of diabetes treatment drugs.

[0009] The working electrode surface modified porphyrin nickel / CuO nanosheet composite sensitive material has a CuO nanosheet as a bottom layer, and a regular, prismatic porphyrin nickel crystal is loaded on the surface of the CuO nanosheet, thereby constructing a stable, high specific surface area composite sensitive material.

[0010] Optionally, the counter electrode is a carbon electrode arranged on one side of the working electrode, which is used to form a current loop with the working electrode in the electrochemical measurement, so as to ensure that the electrochemical reaction on the working electrode can continue to proceed.

[0011] Optionally, the reference electrode is an Ag / AgCl electrode arranged on the other side of the working electrode, which is used to provide a known and stable reference potential for the electrochemical measurement.

[0012] Further, the wires are respectively connected to the bottom ends of the RE, WE and CE, and are used to form a complete current loop of the electrodes of the sensor in the electrochemical measurement.

[0013] The second technical solution of the present application is a preparation method of the above-mentioned electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material, comprising the following steps: preparing a carbon substrate of a working electrode, a counter electrode, a reference electrode and a wire on a surface of a polymer substrate by silk screen printing; mixing CuO nanosheets with anhydrous ethanol and a Nafion solution to obtain CuO nanosheet ink; and dropping the CuO nanosheet ink on a surface of the carbon substrate of the working electrode to obtain a CuO nanosheet layer after drying. mixing anhydrous ethanol and porphyrin nickel to obtain porphyrin nickel ink; and dropping the porphyrin nickel ink on a surface of the CuO nanosheet layer to obtain a porphyrin nickel / CuO nanosheet composite sensitive material layer after drying, thereby completing preparation of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material.

[0014] Optionally, the drying is performed at room temperature.

[0015] Further, the use amount ratio of the CuO nanosheets, the anhydrous ethanol and the Nafion solution is 8 mg:200 μL:50 μL.

[0016] Further, the use amount ratio of the porphyrin nickel and the anhydrous ethanol is 1 mg:25 ~ 1000 μL, preferably 1 mg:200 μL.

[0017] The thickness of the porphyrin nickel layer has an influence on the performance of the electrochemical sensor chip. In the present application, different ratios of porphyrin nickel / anhydrous ethanol ink are prepared, and it is found that when the ratio of porphyrin nickel and anhydrous ethanol is 1 mg:200 μL, the oxidation current response of NO reaches a peak value. Under this ratio, the porphyrin nickel has the best dispersibility, and the catalytic sites are fully exposed, which is conducive to charge transfer and oxidation reaction. When the ratio is lower than 1 mg:200 μL, the ink has a high viscosity, which leads to agglomeration of the porphyrin nickel and reduces the catalytic activity; and a too high ratio (such as 1 mg:500 μL and 1 mg:1000 μL) makes the coating too thin, reduces the active centers and weakens the current response.

[0018] Further, the dropping amount of the CuO nanosheet ink is 17.6 ~ 44.1 μL / cm 2 (preferably 2 ~ 5 μL of ink is dropped on a surface of a carbon substrate with an area of 11.34 mm 2 ).

[0019] Further, the dropping amount of the porphyrin nickel ink is 17.6 ~ 44.1 μL / cm 2 .

[0020] Further, the preparation step of the CuO nanosheet comprises: mixing a copper source and a tetrabutylammonium hydroxide (TBAOH) solution, stirring to obtain a suspension; centrifugally separating the suspension, drying and calcining annealing the obtained precipitate to obtain the CuO nanosheet.

[0021] Further, the copper source comprises CuCl.

[0022] Further, the concentration (i.e. the mass fraction of solute) of the tetrabutylammonium hydroxide solution is 10 wt%.

[0023] Further, the usage ratio of the copper source and the tetrabutylammonium hydroxide solution is 8 ~ 12 mg: 10 ~ 20 mL.

[0024] Further, the temperature of the calcining annealing treatment is 200 ℃, and the time is 2 ~ 4 h.

[0025] Optionally, the stirring is continuous stirring at room temperature for 5 ~ 10 h.

[0026] Optionally, the rotation speed of the centrifugal separation is 5000 ~ 10000 r / min, and the centrifugal time is 5 ~ 10 min.

[0027] Optionally, the drying of the obtained precipitate is drying at 60 ~ 80 ℃ for 12 ~ 24 h. Further, the morphology of the CuO nanosheet is a two-dimensional sheet structure, and the sheet layer thickness is 20 nm.

[0028] Optionally, the polymer substrate is PET (polyethylene terephthalate).

[0029] Optionally, the detection unit arranged on the surface of the polymer substrate comprises one or more, and each detection unit has the same structure.

[0030] Optionally, the carbon substrate, the counter electrode, the reference electrode and the lead wire of the working electrode prepared on the surface of the polymer substrate by screen printing comprise: 5 ~ 10 g of Ag / AgCl ink is weighed and 5 ~ 10 mL of diluent is added for dilution, and then the diluted Ag / AgCl ink is uniformly printed on the surface of the PET substrate by using a screen printing machine, and after drying at room temperature, a printed RE is obtained; 4 ~ 8 g of conductive carbon paste is weighed and 5 ~ 10 mL of diluent is added for dilution, and then the diluted conductive carbon paste is uniformly printed on the surface of the PET substrate by using a screen printing machine, and after drying at room temperature, a printed carbon substrate and CE of the working electrode are obtained; 5 ~ 15 g of conductive silver paste is weighed, 5 ~ 20 mL of diluent is added for dilution, and then the diluted conductive silver paste is uniformly printed on the surface of the PET substrate by using a screen printing machine, and after drying at room temperature, a printed conductive wire is obtained.

[0031] The third technical scheme of the present application is an application of the above-mentioned electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material in NO detection.

[0032] The fourth technical scheme of the present application is a living cell NO release monitoring platform, which comprises the above-mentioned electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material, and a cell culture chamber constructed on the working electrode region of the electrochemical sensor chip and a GelMA (methacrylated gelatin) hydrogel filled in the cell culture chamber (the hydrogel is used for embedding and three-dimensional culture of living cells, and the GelMA hydrogel is selected because of its excellent photocuring ability, similar biological characteristics to the natural extracellular matrix, and internal reserved RGD peptide sequence and MMP degradation site, which can provide a necessary biomimetic microenvironment for cell adhesion, growth and migration).

[0033] The fifth technical scheme of the present application is a preparation method of the above-mentioned living cell NO release monitoring platform, which comprises the following steps: preparing a cell culture chamber template by 3D printing; mixing PDMS prepolymer with a curing agent, pouring into the cell culture chamber template, and peeling off the PDMS polymer layer after curing to obtain a primary cell culture chamber; bonding the primary cell culture chamber to the working electrode region of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material; injecting a GelMA prepolymer solution containing living cells into the primary cell culture chamber, and curing to form the living cell NO release monitoring platform.

[0034] The present application combines the high-sensitivity NO detection electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material with the three-dimensional cell culture system based on the GelMA hydrogel. By using 3D printing technology and PDMS to construct a sealed cell culture chamber on the sensor chip, and filling the GelMA hydrogel as a three-dimensional scaffold in it, the physiological environment of the extracellular matrix is successfully simulated.

[0035] Optionally, the curing temperature is 50 ~ 70 ℃, and the curing time is 2 ~ 4 h.

[0036] Further, the preparation step of the GelMA prepolymer solution containing living cells comprises: The living cells are cultured in a culture medium to obtain a cell suspension; the cell suspension is centrifuged to obtain a precipitate, the precipitate is resuspended in a GelMA prepolymer solution, and the cell density is adjusted to 5×10 4 ~ 5×10 5 cells / mL to obtain the living cell-containing GelMA prepolymer solution (referred to as GelMA-living cell mixture).

[0037] Further, the concentration of the GelMA prepolymer solution (i.e., the concentration of GelMA in the GelMA prepolymer solution) is 0.5 ~ 12.5 wt%, preferably 5 wt%.

[0038] The hydrogel at a concentration of 5 wt% has a suitable pore size structure, which can support the three-dimensional growth of living cells and maintain their high activity, and effectively promote the diffusion and transmission of nutrients, drug molecules, and NO gas.

[0039] Further, the preparation step of the GelMA prepolymer solution comprises: mixing a photoinitiator with a PBS solution to obtain a photoinitiator solution; mixing GelMA with the photoinitiator solution to obtain the GelMA prepolymer solution.

[0040] Optionally, the photoinitiator comprises Irgacure 2959.

[0041] Optionally, the amount ratio of the photoinitiator to the PBS solution is 45 ~ 55 mg:8 ~ 12 mL.

[0042] Further, the living cells comprise vascular endothelial cells.

[0043] Optionally, the vascular endothelial cells comprise human umbilical vein endothelial cells (HUVEC). HUVEC is an important source of NO synthesis (mainly through endothelial NO synthase eNOS) in vivo, and is closely related to diabetic vascular complications.

[0044] The sixth technical solution of the present application is the application of the above-mentioned living cell NO release monitoring platform in evaluating the influence of diabetes treatment drugs or sugar substitutes on the NO release of living cells.

[0045] Further, the steps of the application comprise: introducing the diabetes treatment drug or sugar substitute to be tested into the cell culture chamber of the living cell NO release monitoring platform, using the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material to monitor the change of the electric signal generated by the NO released by the cells in real time, and based on the change of the electric signal, evaluating the promotion or inhibition of the diabetes treatment drug or sugar substitute on the NO release of the cells.

[0046] The living cell NO release monitoring platform evaluates the biological effects of diabetes treatment drugs or sugar substitutes by evaluating the effects of the diabetes treatment drugs or sugar substitutes on the NO release of living cells.

[0047] Optionally, the diabetes treatment drug comprises glucagon-like peptide-1 (GLP-1) or insulin.

[0048] Optionally, the sugar substitute comprises erythritol, aspartame or sucralose.

[0049] Optionally, before introducing the diabetes treatment drug or sugar substitute to be tested into the cell culture chamber of the living cell NO release monitoring platform, the method further comprises the steps of adding a culture medium into the cell culture chamber for culture, and removing the culture medium after the culture.

[0050] In constructing the monitoring platform, HUVEC cells are embedded in GelMA hydrogel and cultured in the monitoring platform. In detection, after the cell state is stable, the test substance (such as different concentrations of GLP-1, insulin or sugar substitute) is added into the cell culture chamber, and the current signal change caused by NO release is recorded in real time by chronoamperometry using an electrochemical workstation. By analyzing the occurrence frequency and intensity of the current sharp peak, the promotion or inhibition of the test substance on NO synthesis is qualitatively or quantitatively evaluated.

[0051] The present application discloses the following technical effects: The present application realizes the significant improvement of the performance of the electrochemical sensor by constructing the porphyrin nickel / CuO nanosheet composite sensitive material. The composite sensitive material fully utilizes the high specific surface area characteristics of the CuO nanosheet and the specific recognition ability of the porphyrin nickel molecule, and the two produce a synergistic effect, so that the sensor has a detection sensitivity of nanomolar level, excellent selectivity and long-term stability, and can effectively exclude the interference of common substances in complex biological samples.

[0052] The present application creates a highly biomimetic living cell NO release monitoring platform by integrating the optimized sensor and the three-dimensional cell culture system. The 5 wt% concentration of GelMA hydrogel is used as a cell scaffold, which not only maintains the normal physiological function of human umbilical vein endothelial cells, but also ensures the efficient transmission of signal molecules, realizes the in-situ and real-time monitoring of the NO release kinetics of living cells under external stimulation, and provides an experimental model close to the real state in vivo for the research of cell function. The cell NO release monitoring platform of the present application realizes the dynamic evaluation of the NO release level of endothelial cells under the action of sugar substitutes and diabetes treatment drugs, and provides a reliable tool for the biological effect analysis of diabetes drugs and sugar substitutes, and has important clinical application prospects in the management of diabetes diet and the evaluation of drug efficacy.

[0053] In conclusion, the monitoring platform is integrated by constructing the electrochemical sensor based on the porphyrin nickel / CuO nanosheet composite sensitive material and the three-dimensional cell culture platform, and real-time and accurate monitoring of NO released by living cells is realized. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort.

[0055] Figure 1 The schematic diagram of the principle for constructing the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material, constructing the living cell NO detection based on the GelMA hydrogel and the electrochemical sensor chip, and monitoring the influence of different drugs and sugar substitutes on the NO release of living cells in the present application.

[0056] Figure 2 The SEM images of the CuO nanosheet and porphyrin nickel prepared in Example 1, wherein a is the SEM image of the CuO nanosheet, b and c are the SEM images of the porphyrin nickel material at different magnifications.

[0057] Figure 3 The morphology and microstructure characterization results of the CuO nanosheet prepared in Example 1, wherein a is the low-magnification TEM image of the CuO nanosheet, b is the high-magnification TEM image of the CuO nanosheet, c is the SAED image of the CuO nanosheet, d is the HR-TEM image of the CuO nanosheet, e is the HRTEM image of the yellow region shown in d, f is the HRTEM image of the green region shown in d, g is the high-angle annular dark field transmission electron microscopy (HAADF-TEM) image of the TEM morphology of the CuO nanosheet, h and i are the EDS mapping images of the HAADF-TEM image shown in g.

[0058] Figure 4 The structure schematic diagram of the electrochemical sensor chip in Example 1 and the living cell NO release monitoring platform based on the GelMA hydrogel and the electrochemical sensor chip in Example 2, wherein A is a reference electrode (RE), B is a working electrode (WE), C is a counter electrode (CE), D is a wire, and E is a PDMS microchamber.

[0059] Figure 5 The XRD pattern of the CuO nanosheet prepared in Example 1.

[0060] Figure 6The CV test results of the electrochemical sensor chips prepared under different ratios of porphyrin nickel and anhydrous ethanol, wherein a-e are the CV curves of the electrochemical sensor chips prepared under different ratios of porphyrin nickel and anhydrous ethanol, and f is the specific current value obtained according to a-e.

[0061] Figure 7 The electrochemical impedance (EIS) test results of the CuO nanosheet, porphyrin nickel, porphyrin copper / CuO nanosheet composite sensitive material and porphyrin nickel / CuO nanosheet composite sensitive material in Test Example 1.

[0062] Figure 8 The cyclic voltammetry (CV) test results of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material (prepared in Example 1) in Test Example 1.

[0063] Figure 9 The CV test results of the electrochemical sensor chip based on the CuO nanosheet (prepared in Comparative Example 1) in Test Example 1.

[0064] Figure 10 The CV test results of the electrochemical sensor chip based on the porphyrin nickel (prepared in Comparative Example 2) in Test Example 1.

[0065] Figure 11 The CV test results of the electrochemical sensor chip based on the porphyrin copper / CuO nanosheet composite sensitive material (prepared in Comparative Example 3) in Test Example 1.

[0066] Figure 12 The chronoamperometric response results of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material to different concentrations of NO standard solution in Test Example 1, wherein a is the chronoamperometric curve of the porphyrin nickel / CuO nanosheet to NO in the concentration range of 100-1000 μM, b is the fitting curve of the NO concentration (100-1000 μM) and the steady-state current value of the chronoamperometric curve, c is the chronoamperometric curve of the porphyrin nickel / CuO nanosheet to NO in the concentration range of 1-100 nM, and d is the fitting curve of the NO concentration (10-100 nM) and the steady-state current value of the chronoamperometric curve.

[0067] Figure 13 The selectivity and stability test results of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material in Test Example 1, wherein a is the selectivity test results of the electrochemical sensor chip to Glu, GSH, UA, LA, BSA, H2O2, Ca 2+ and NO2 – ​

[0068] Figure 14 Design drawing for cell culture chamber template structure.

[0069] Figure 15 SEM images and infrared spectra of GelMA hydrogels obtained by solidification based on different concentrations of GelMA prepolymer solution (5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%, 15 wt%) in Example 3, wherein a is the SEM image of 15 wt% GelMA, b is the SEM image of 12.5 wt% GelMA, c is the SEM image of 10 wt% GelMA, d is the SEM image of 7.5 wt% GelMA, e is the SEM image of 5 wt% GelMA, and f is the infrared spectra of different concentrations of GelMA.

[0070] Figure 16 NO release current signal diagram of HUVEC cells under L-Arg stimulation and under eNOS inhibitor L-NAME treatment recorded in real time by the monitoring platform in Example 4, wherein a is the chronoamperometric curve of the effect of different concentrations of L-Arg on the NO release level of HUVEC, and b is the chronoamperometric curve of the effect of high glucose on the NO release level of HUVEC.

[0071] Figure 17 Effect of different generations of sugars (erythritol, aspartame, sucralose) on the NO release of HUVEC cells evaluated by the monitoring platform in Example 4, wherein a is the chronoamperometric curve of the effect of normal concentration of the generation of sugar on the NO release level of HUVEC, and b is the chronoamperometric curve of the effect of high concentration of the generation of sugar on the NO release level of HUVEC.

[0072] Figure 18 Chronoamperometric curves of the effect of different concentrations of GLP-1 and insulin on the NO release of HUVEC cells evaluated by the monitoring platform in Example 4, wherein a is the chronoamperometric curve of the effect of different concentrations of GLP-1 on the NO release level of HUVEC, and b is the chronoamperometric curve of the effect of different concentrations of insulin on the NO release level of HUVEC. DETAILED DESCRIPTION

[0073] Various exemplary embodiments of the present application will now be described in detail, which should be considered in a descriptive sense only and not considered to be limiting to the present application described herein, but rather, as presenting certain aspects, features and embodiments of the present application.

[0074] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, an intermediate value of is specifically contemplated. Each smaller range between any stated value or inferred values within the stated range, and each value between any stated values or inferred values within the stated range is also contemplated. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0076] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0077] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0078] It should be noted that the present application does not describe in detail the conventional operation means in the art, and is not the focus of the present application.

[0079] The principle diagram of constructing an electrochemical sensor chip based on a porphyrin nickel / CuO nanosheet composite sensitive material, constructing a living cell NO detection based on a GelMA hydrogel and an electrochemical sensor chip, and monitoring the influence of different drugs and sugar substitutes on the NO release of living cells is shown in Figure 1 .

[0080] The present application integrates a porphyrin nickel / CuO nanosheet composite sensitive material with a three-dimensional cell culture platform (as shown in Figure 1Based on the above-mentioned problems and the actual needs of the real-time monitoring of NO, a high-efficiency NO real-time monitoring platform (or system) was constructed. In the preparation of the electrochemical sensor chip, a high-sensitivity sensor chip was formed by modifying the surface of the screen-printed electrode with a nickel porphyrin / CuO nanosheet composite sensitive material; then, a cell culture chamber (i.e., a PDMS microchamber) was constructed on the chip by using a 3D printing technology, and 5 wt% GelMA hydrogel was filled as a three-dimensional cell scaffold for culturing HUVEC cells. The integrated system was used to successfully realize the real-time dynamic monitoring of the NO release level of living cells under the stimulation of L-Arg (L-arginine), sugar substitutes, and diabetes treatment drugs.

[0081] In the following examples, comparative examples and test examples of the present application, if referring to room temperature, it specifically refers to 20-30 ℃.

[0082] In the following examples, comparative examples and test examples of the present application, each raw material used is a common commercially available product, wherein the nickel porphyrin is specifically tetra-p-methoxyphenyl nickel porphyrin, and the copper porphyrin is specifically tetra-p-methoxyphenyl copper porphyrin.

[0083] Example 1: Preparation of an electrochemical sensor chip based on a nickel porphyrin / CuO nanosheet composite sensitive material This example prepared an electrochemical sensor chip for NO detection based on a nickel porphyrin / CuO nanosheet composite sensitive material, which had a structure as shown in Figure 4 The electrochemical sensor chip included a PET substrate and a detection unit arranged on the surface of the PET substrate, and each detection unit included A. a reference electrode RE, B. a working electrode WE, C. a counter electrode CE, and D. a lead wire; wherein the WE was a carbon electrode coated with a nickel porphyrin / CuO nanosheet composite sensitive material, the CE was a carbon electrode arranged on one side of the WE, the RE was an Ag / AgCl electrode arranged on the other side of the WE, and the lead wire was connected to the bottom end of the RE, WE, and CE, respectively. In this example, the preparation method of the electrochemical sensor chip was as follows: S11. 5 g of Ag / AgCl ink was weighed and diluted with 8 mL of 8260 diluent, and then the diluted Ag / AgCl ink was uniformly printed on the surface of the PET substrate by using a screen printing machine, and a printed RE was obtained after drying at room temperature; S12. 5 g of conductive carbon paste was taken and diluted with 8 mL of 8240 diluent, and then the diluted conductive carbon paste was uniformly printed on the surface of the PET substrate by using a screen printing machine, and a carbon substrate for the WE and the CE were obtained after drying at room temperature; S13. 5 g of conductive silver paste was taken and diluted with 10 mL of 8260 diluent, and then the diluted conductive silver paste was uniformly printed on the surface of the PET substrate by using a screen printing machine, and a printed lead wire structure was obtained after drying at room temperature; S14. 10 mg of CuCl was weighed and mixed with 10 mL of tetrabutylammonium hydroxide (TBAOH) solution with a mass fraction of 10%, and stirred continuously at room temperature for 6 h to obtain a suspension. A black precipitate was obtained by centrifugal separation of the suspension (8000 r / min for 5 min). The black precipitate was transferred to a blast drying oven and dried at 80°C for 12 h. Then, the black powder was transferred to a muffle furnace for calcination annealing treatment: the temperature was raised from room temperature to 200°C at a rate of 5°C / min, and calcined at this temperature for 2 h, and finally cooled to room temperature naturally to obtain CuO nanosheet materials with two-dimensional sheet structure; S15. 8 mg of CuO nanosheet material was mixed with 200 μL of absolute ethanol and 50 μL of Nafion solution (concentration of 5wt%) to prepare a uniformly dispersed CuO nanosheet ink; 2 μL of CuO nanosheet ink was uniformly drop-coated on the surface of the carbon substrate of WE (drop-coating area of about 11.34 mm 2 ), and a CuO nanosheet layer was obtained after drying at room temperature; S16. 1 mg of porphyrin nickel was mixed with 200 μL of absolute ethanol to prepare a uniformly dispersed porphyrin nickel ink; 2 μL of porphyrin nickel ink was uniformly drop-coated on the surface of the CuO nanosheet layer (drop-coating area of about 11.34 mm 2 ), and an electrochemical sensor chip was obtained after drying at room temperature.

[0084] Characterization test: Figure 2 (a) of FIG. 1 shows the SEM image of CuO nanosheet prepared in step S14 of the present embodiment, and the obvious two-dimensional layered stacked nanosheet structure can be observed, with uniform thickness, a planar size of about 0.5 μm × 0.3 μm, and a thickness of about 20 nm.

[0085] Figure 2 (b) and (c) of FIG. 1 show the SEM images of raw material porphyrin nickel. As can be seen, the nickel tetramethoxyphenyl porphyrin presents a relatively regular prismatic crystal structure, with a flat crystal face and a large particle size, and the size of a single particle can reach tens of microns, reflecting its high crystallinity and good morphology consistency. This regular crystal morphology is conducive to the directional arrangement and stable loading at the electrode interface, and helps to improve the construction efficiency and signal stability of the electrochemical sensor.

[0086] The microstructure characteristics of the CuO nanosheet prepared in step S14 of the present embodiment were further explored by using a spherical aberration-corrected transmission electron microscope (AC-TEM) equipped with EDS and SAED light beams, and the transmission electron microscope (TEM) image is as shown in Figure 3a and b in FIG. 6, it can be seen that the prepared sample presents an ultrathin nanosheet morphology, and there are some pores with a size of several nanometers at the edge of the sample. The SAED image as shown in c in FIG. 6 can be observed, and the concentric diffraction rings can be clearly indexed as the (110), (11-1), (020) and (022) crystal planes of monoclinic CuO, which is consistent with the results of the XRD spectrum as shown in d in FIG. 6. Figure 3 a and b in FIG. 6, it can be seen that the prepared sample presents an ultrathin nanosheet morphology, and there are some pores with a size of several nanometers at the edge of the sample. The SAED image as shown in c in FIG. 6 can be observed, and the concentric diffraction rings can be clearly indexed as the (110), (11-1), (020) and (022) crystal planes of monoclinic CuO, which is consistent with the results of the XRD spectrum as shown in d in FIG. 6. Figure 5 a and b in FIG. 6, it can be seen that the prepared sample presents an ultrathin nanosheet morphology, and there are some pores with a size of several nanometers at the edge of the sample. The SAED image as shown in c in FIG. 6 can be observed, and the concentric diffraction rings can be clearly indexed as the (110), (11-1), (020) and (022) crystal planes of monoclinic CuO, which is consistent with the results of the XRD spectrum as shown in d in FIG. 6. Figure 3 d, e and f in FIG. 6 show the HRTEM images of the CuO nanosheets, and two clear and continuous lattice fringes can be observed, with a spacing of 0.275 nm and 0.252 nm, respectively, corresponding to the (110) and (11-1) crystal planes of CuO. Figure 3 g in FIG. 6 shows the HAADF-STEM image of the CuO nanosheets, which clearly presents the structural characteristics of the material. Figure 3 h and i in FIG. 6 show the EDS element mapping images of the CuO nanosheets, which clearly indicate that the material is mainly composed of uniformly distributed Cu and O elements.

[0087] Example 2 Optimization of the ratio of porphyrin nickel to anhydrous ethanol: Using the preparation method of Example 1, by changing the ratio of porphyrin nickel to anhydrous ethanol in step S16 to 1:25, 1:100, 1:500 and 1:1000 (mg:μL), respectively, a plurality of groups of porphyrin nickel inks with different ratios were obtained (the drop-casting amount was kept consistent), and then a plurality of groups of electrochemical sensor chips with different thicknesses and coverages of the porphyrin nickel layer were obtained.

[0088] The electrochemical sensor chips prepared in Example 1 and Example 2 under different ratios of porphyrin nickel to anhydrous ethanol were subjected to CV testing, and the specific testing method was as follows: 50 μL of 0.1 M PBS solution (pH=7.0) or the same kind of PBS solution containing different concentrations of NO (the concentrations of NO were 10, 50, 100, 500 and 1000 μM, respectively, which were obtained by passing NO gas into the PBS solution until saturation, and then diluting the PBS solution to obtain different concentrations of NO) were uniformly drop-cast on the electrochemical sensor chip, and the solution was ensured to cover WE, RE and CE during drop-casting, and then CV testing was performed. The CV testing condition was to perform scanning at a scanning rate of 60 mV / s in a potential range of-0.6 to 0.6 V vs. Ag / AgCl.

[0089] Figure 6The CV test results of the electrochemical sensor chips prepared under different ratios of porphyrin nickel and anhydrous ethanol are shown in FIG. 1, wherein a-e are the CV curves of the electrochemical sensor chips prepared under different ratios of porphyrin nickel and anhydrous ethanol, and f is the specific current value obtained according to a-e. When the ratio is 1:200 (unit: mg / μL), the NO oxidation current is the largest, indicating that the dispersibility of porphyrin nickel is the best at this time, and the catalytic sites are fully exposed. Further, the current values under different ratios are summarized as a column chart (f in FIG. 1), it can be seen that when the ratio is less than 1:200, the loading is too high, which is easy to cause particle aggregation and activity reduction; and when the ratio is too high (such as 1:500, 1:1000), the coating film is too thin, the active center is reduced, and the current is reduced. Therefore, 1:200 is the optimal ratio, which can effectively improve the detection sensitivity and catalytic performance of the porphyrin nickel / CuO electrode to NO. Figure 6

[0090] Comparative Example 1 The same as Example 1, except that step S16 is omitted.

[0091] Comparative Example 2 The same as Example 1, except that steps S14 and S15 are omitted, and the porphyrin nickel ink is directly drop-coated on the carbon substrate surface of WE.

[0092] Comparative Example 3 The same as Example 1, except that the porphyrin nickel in step S16 is replaced by an equal amount of porphyrin copper.

[0093] Test Example 1: Sensor Performance Test In this test example, the electrochemical sensor chips prepared in Example 1 and Comparative Examples 1-3 are taken as test objects, and the electronic transmission ability of the porphyrin nickel / CuO nanosheet composite sensitive material and its electrochemical response ability to NO are verified. The specific electrochemical performance test method and results are as follows: (1) 50 μL of 0.1 M [Fe(CN)6] 3- / 4- solution is uniformly drop-coated on the electrochemical sensor chip, and the solution is ensured to cover WE, RE and CE during drop-coating, and then EIS test is performed. The EIS test condition is that the frequency is 10 5 to 0.1 Hz, and the amplitude is 5 mV.

[0094] The EIS test results are shown in FIG. 2. Figure 7 ​As shown in the results, the semicircle in the high frequency region and the inclined straight line in the low frequency region of the electrochemical sensor modified with different materials are clearly shown, indicating that the above electrode reactions all include charge transfer and diffusion mechanism. Compared with CuO nanosheet, the electrochemical sensor chip modified with porphyrin nickel / CuO nanosheet composite sensitive material has a smaller semicircle radius in the high frequency region, while the porphyrin nickel / CuO nanosheet composite sensitive material has a larger slope in the low frequency region, which indicates that the porphyrin nickel / CuO nanosheet composite sensitive material has a higher current response and a faster electron transfer.

[0095] In addition, the semicircle radius of porphyrin nickel in the high frequency region is significantly larger than that of the porphyrin nickel / CuO nanosheet composite sensitive material in the high frequency region, and the slope of porphyrin nickel in the low frequency region is smaller, indicating that porphyrin nickel has a larger charge transfer resistance and a weaker ion diffusion capacity compared with porphyrin nickel / CuO nanosheet.

[0096] Moreover, compared with the porphyrin nickel / CuO nanosheet composite sensitive material, the porphyrin copper / CuO nanosheet composite sensitive material has poor conductivity and electron transfer efficiency.

[0097] (2) 50 μL of 0.1 M PBS solution (pH=7.0) or the same PBS solution containing NO (NO concentration is 0.9 mM) was uniformly dropped on the electrochemical sensor chip, and the solution was ensured to cover WE, RE and CE during dropping, and then CV test was performed. The CV test condition is to perform at a scan rate of 60 mV / s in the potential range of -0.6 to 0.6 V vs. Ag / AgCl.

[0098] The CV test results of the electrochemical sensor chip prepared in Example 1 are shown in Figure 8 As shown, it can be clearly shown that the porphyrin nickel / CuO nanosheet composite sensitive material has obvious response to NO, and the electrochemical oxidation potential is about 0.89 V vs. Ag / AgCl, and the corresponding current value is about 2.56 mA.

[0099] The CV test results of the electrochemical sensor chip prepared in Comparative Example 1 are shown in Figure 9 As can be seen, compared with the porphyrin nickel / CuO nanosheet composite sensitive material, the CuO nanosheet has a lower oxidation current value (current value is 1.83 mA) in the reaction, and its inherent catalytic active site has a poor catalytic efficiency for NO molecules and a weak catalytic ability.

[0100] The CV test results of the electrochemical sensor chip prepared in Comparative Example 2 are shown in Figure 10As shown, compared with the porphyrin nickel / CuO nanosheet composite sensitive material, the porphyrin nickel has a weaker electrochemical response at the same NO concentration, and generates a lower oxidation current value (1.16 mA) in the reaction, indicating a weaker catalytic ability.

[0101] The CV test results of the electrochemical sensor chip prepared in Comparative Example 3 are as follows: Figure 11 As shown, compared with the porphyrin nickel / CuO nanosheet composite sensitive material, the porphyrin copper / CuO nanosheet has a weaker electrochemical response and a relatively lower oxidation current (1.62 mA) at the same NO concentration, reflecting that the porphyrin copper / CuO nanosheet has relatively poor catalytic activity and insufficient electron transfer efficiency.

[0102] (3) The sensitivity test results of the electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material prepared in Example 1 for NO are as follows: Figure 12 As shown, the electrochemical testing method is chronoamperometry. The sample preparation and application method are the same as in step (2) above, demonstrating the sensitivity of the electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material to NO at the μM and nM levels. Figure 12 In Figure a (chronoamperometry curves within the concentration range of 100–1000 μM), at a potential of 0.8 V vs. Ag / AgCl, the response current of the constructed sensor to NO in the concentration range of 100–1000 μM increases with increasing NO concentration. Figure 12 As shown in b (linear relationship within the concentration range of 100–1000 μM), the NO concentration within the 100–1000 μM range exhibits a good linear correlation with the oxidation current signal (R0). 2 =0.9828). Figure 12 In the figure, 'c' represents the chronoamperometry curve of the constructed sensor for NO concentrations in the range of 10–100 nM. The current value shows a stable upward trend with increasing NO concentration, and the detection limit of the porphyrin nickel / CuO nanosheet composite sensing material for NO is 1 nM. A fitting curve is plotted between NO concentration and the corresponding terminal current value, as shown below. Figure 12 As shown in d, the relationship between NO concentration at the nM level and current value conforms to a quadratic polynomial model (Rd). 2 =0.999).

[0103] (4) The selectivity and stability of the sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material prepared in Example 1 were comprehensively evaluated. The selectivity test method was as follows: chronoamperometry was used to test in a complex biological medium (cell culture solution containing proteins, metabolites and other interference factors) at a potential of 0.8 V vs. Ag / AgCl. When the current was stable, 10 μL of PBS solution of the same concentration (1 mM) of nitric oxide (NO), glucose (Glu), glutathione (GSH), uric acid (UA), lactic acid (LA), bovine serum albumin (BSA), hydrogen peroxide (H2O2), calcium ion (Ca 2+ ) and nitrite ion (NO2 – ) was added every 50 s, respectively. As shown in a of Figure 13 , only when NO was added, the chronoamperometric signal showed a significant response, while no obvious change in current signal was observed when other interference substances were added. Therefore, the sensor based on the porphyrin nickel / CuO nanosheet composite sensitive material showed good selectivity to NO molecules. In order to evaluate the long-term stability of the sensor, the sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material was prepared in batches by the preparation method of Example 1, and was stored in a dry box at 25 ℃. After one month of continuous monitoring (the sample was prepared and applied in the same way as the above step (2), and the concentration of NO was 1.8 mM), the test results are shown in b of Figure 13 . During the one-month monitoring period, the current response value of porphyrin nickel / CuO nanosheet to NO remained above 98% of the initial value, indicating that it had good long-term stability.

[0104] Example 3: Construction of a living cell NO release monitoring platform based on GelMA hydrogel and the above electrochemical sensor chip The preparation steps of the monitoring platform for HUVEC to release NO based on GelMA hydrogel and the above electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material in this example are as follows: S21. Preparation and pretreatment of cell culture chamber template: AutoCAD software was used to draw the chamber template structure design drawing (as shown in Figure 14As shown, the template surface has solid semi-cylindrical protrusions, with one semi-cylinder representing a cell culture chamber template. A photosensitive resin template was fabricated using 3D printing technology (the photosensitive resin used was commercially available RG Yellow 20 Resin, purchased from Boston Micro Fabrication (BMF)). To ensure the accuracy and reliability of the template, it underwent the following pretreatment: the template surface was rinsed with anhydrous ethanol to remove impurities; it was then immersed in anhydrous ethanol and sonicated to remove residual resin from the channels, ensuring accurate channel height and flatness. To prevent bending or deformation of the template in subsequent experiments, double-sided tape was used to fix it to the culture dish for stability. The template was silanized to reduce its surface energy, giving it "PDMS-repellent" properties. This facilitated clean demolding of the PDMS and maintained the integrity of its microstructure. (The silanization process involved adding 10 μL of fluorosilane coupling agent to the template surface, placing the sample in a sealed container, drying it with nitrogen, and then heating it in a 70 °C oven for 10 h. This allowed the fluorosilane coupling agent to volatilize and deposit on the template surface during heating, achieving silanization. After the reaction, the sample was cooled to room temperature.) The treated sample was then stored at room temperature under dry conditions for later use.

[0105] S22. Preparation of the electrode-cell culture chamber (structure as follows) Figure 1 and Figure 4 (As shown): PDMS prepolymer and curing agent are mixed evenly at a ratio of 10:1 (w / w) and poured into a petri dish with a photosensitive resin template fixed in place. The amount of the PDMS prepolymer and curing agent mixture poured in does not exceed the height of the semi-cylindrical protrusion in the template. After the petri dish containing the PDMS prepolymer and curing agent mixture is placed under vacuum to remove bubbles, it is cured at 60°C for 3 hours. After curing, the PDMS polymer layer with microstructure is peeled off (this process is equivalent to the molding process), obtaining the preliminary structure of the chamber (PDMS microchamber). A PDMS microchamber is cut (a PDMS microchamber is a cube with openings at both the top and bottom, with a semi-cylindrical cavity in the middle, a cavity height of 5 mm, and a volume of 200 mm²). 3 ,like Figure 4PDMS microchamber (as shown in FIG. 1) was adhered to the working electrode area of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material prepared in Example 1. To ensure reliable sealing of the chamber and the electrochemical sensor chip, the PDMS microchamber bottom layer and the electrode surface were subjected to oxygen plasma treatment (conditions: 100 W, 60 s, O2flow rate 50 sccm) to enhance surface affinity and adhesion. Considering the thickness of the electrode surface slurry, to achieve complete sealing, an additional circle of PDMS (PDMS prepolymer + curing agent) was cast around the chamber, and continued to be cured at 60°C for 3 h to form a complete cell culture chamber. Finally, the sealing performance was verified by injecting pure water into the chamber and observing under a microscope whether there was leakage. If there was no leakage, it indicated that the chamber had good sealing performance and could be used for subsequent experiments.

[0106] S23. Preparation of GelMA prepolymer solution: 50 mg of photoinitiator Irgacure 2959 was weighed, added to 10 mL of PBS solution, and dissolved at 45°C in a constant temperature environment to obtain a transparent solution of 0.5 wt%. Then, 0.75 g of GelMA solid was weighed, added to 5 mL of 0.5 wt% Irgacure 2959 solution, and dissolved at 45°C for 3 h to obtain a GelMA prepolymer solution with a concentration of 15 wt%. It was diluted with 0.5 wt% Irgacure 2959 solution in stages to obtain GelMA prepolymer solutions with concentrations of 12.5 wt%, 10 wt%, 7.5 wt%, and 5 wt%, respectively. All solutions were stored in a refrigerator at -20°C before use to maintain their stability.

[0107] S24. Three-dimensional culture of HUVEC cells and in-situ monitoring of NO release: The HUVEC complete culture medium was configured as follows: 84 % (v / v) high-sugar DMEM, 15 % (v / v) fetal bovine serum (FBS), and 1 % (v / v) ampicillin. 3 mL of complete culture medium was added to the HUVEC culture bottle (cell density 8×10 4 cells / mL), and cultured in a constant temperature incubator at 37°C and 5 % CO2. When the cell density reached 90 %, the cells were passaged: after the old culture medium was discarded, washed twice with PBS, 1 mL of trypsin was added, and after the cells were detached, 2 mL of fresh complete culture medium was added to terminate the reaction. After gentle blowing and mixing, the cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 3 min. After discarding the supernatant, different concentrations of GelMA prepolymer solution were added to resuspend the cells, and the cell density was adjusted to 5×10 5Cells / mL were used to obtain a GelMA–HUVEC mixture (i.e., a GelMA prepolymer solution containing HUVEC). 150 μL of the GelMA–HUVEC mixture was injected into each chamber and irradiated under a 365 nm UV lamp for 30 s to solidify and form a three-dimensional cell scaffold. Subsequently, 150 μL of complete culture medium was added to each chamber, and the cells were cultured for another 24 h. After culture, the culture medium was removed, and the electrode containing the cells was placed on an electrochemical workstation. Chorometric amperometry was used to detect the NO release at a constant potential of 0.8 V (vs. Ag / AgCl). Once the current signal stabilized, different exogenous stimuli were added to the system, and the dynamic changes in NO release were monitored in real time.

[0108] Characterization tests: The GelMA hydrogel in this embodiment was obtained by curing GelMA prepolymer solutions of different concentrations under UV light for 30 seconds. The SEM images of the GelMA hydrogels cured with different concentrations of GelMA prepolymer are shown below. Figure 15 As shown in Figure ae, with decreasing GelMA concentration, its crosslinking points gradually become sparse, and the inter-chain interactions weaken, resulting in a looser network structure. Under low concentration conditions, the crosslinking reaction is weak, the pore spacing increases, and water molecules and solvents can more easily penetrate into the gel, forming an incomplete network and a larger, irregular pore structure. Furthermore, infrared spectroscopy analysis of GelMA, such as... Figure 15 As shown in f, 1640 cm -1 The characteristic peak at 3300 cm⁻¹ corresponds to the C=O stretching vibration of the amide group. -1 The absorption peak at 3300 cm⁻¹ is related to the O–H and N–H stretching vibrations. Notably, as the GelMA concentration decreases, the absorption peak at 3300 cm⁻¹... -1 The absorption peak intensity is enhanced. This indicates that the cross-linking degree is lower in low-concentration GelMA hydrogels, allowing water molecules to more easily enter the network and interact with hydrophilic groups (–OH, –NH). This result is consistent with SEM observations, suggesting that low-concentration GelMA hydrogels have larger pore sizes and stronger hydration capabilities.

[0109] Example 4: Evaluation of the effect of live cell NO release monitoring platform on NO release monitoring and glucosamine metabolism in HUVEC cells This embodiment uses the live-cell NO release monitoring platform prepared in Example 2 to monitor the effects of different exogenous stimuli on NO release, and monitors the dynamic changes in NO release in real time. The specific test methods and results are as follows: The sample prepared in Example 2 with a GelMA prepolymer solution concentration of 5 wt% was placed in an electrochemical workstation. After the current stabilized at a constant potential of 0.8 V, an exogenous stimulant was added and the release dynamics of NO were monitored in real time using chronoamperometry.

[0110] (1) The effect of high glucose on L-Arg-induced NO release from HUVECs as follows Figure 16 As shown, L-Arg was first selected as a precursor molecule for NO synthesis to stimulate HUVEC cells. Under stimulation with 150 μL of complete culture medium containing 10 mM L-Arg (the complete culture medium was prepared as in Example 3), a characteristic current spike appeared in the chronoamperometry curve. The rapid rise and slow fall of this spike corresponded to the instantaneous release and subsequent diffusion and degradation of NO. To verify that the signal originated from NO, the eNOS inhibitor L-NAME (at a concentration of 10 mM) was used as a control. After treatment, the current spike essentially disappeared, confirming that the signal indeed originated from NO catalyzed by eNOS. Further analysis of the stimulatory effects of different concentrations of L-Arg (1 mM, 5 mM, 10 mM) revealed that as the concentration decreased, the frequency of the current peak decreased, while the intensity of a single peak did not change significantly. This indicates that L-Arg mainly affects the release frequency of NO, rather than the amount released in a single instance. Figure 16 (a) To simulate a hyperglycemic environment, stimulation was performed using 150 μL of complete culture medium solution containing 12 mM glucose. Figure 16 Figure b shows that the peak frequency of the current peak is significantly reduced under high glucose conditions compared with the normal state, indicating that high glucose has a significant inhibitory effect on NO synthesis. This result is consistent with the endothelial dysfunction mechanism observed in diabetic patients in clinical practice.

[0111] (2) Referring to step (1), different sugar substitutes were used for stimulation. Figure 17 This study compared the effects of different sugar substitutes on NO release from L-Arg-induced HUVEC cells, where a and b represent different concentrations of sugar substitutes. Compared to the control group, sucralose had no significant effect on NO release; its stable structure and low metabolic rate may prevent it from directly interfering with eNOS activity. Aspartame slightly increased the frequency of NO release, and its metabolite phenylalanine may indirectly enhance eNOS activity by regulating amino acid metabolism. Erythritol showed the most significant promoting effect, continuously promoting NO release; its mechanism is related to its ability to scavenge free radicals and reduce oxidative stress to protect eNOS activity. In contrast, at a sugar substitute concentration of 15 mM, NO release decreased significantly, with some cases showing no obvious signal, suggesting that high concentrations of sugar substitutes may inhibit endothelial function. This indicates that sugar substitutes affect NO release through different mechanisms, with erythritol being a preferred sweetener in diabetic dietary management due to its significant NO-promoting effect. However, it is worth noting that excessive intake may also have negative effects and even interfere with normal NO release.

[0112] (3) Figure 18The regulatory effects of GLP-1(a) and insulin(b) on NO release from HUVEC cells were investigated. It was found that GLP-1 significantly promoted NO release in a concentration-dependent manner, and the characteristic current spikes increased significantly with the increase of concentration. This process was completely blocked by eNOS inhibitor L-NAME, confirming that GLP-1 induced NO production through the activation of GLP-1R-eNOS pathway. In contrast, insulin had a weaker effect on NO release. Although it also showed a concentration-dependent effect, the number of current spikes induced by insulin was significantly less than that induced by GLP-1. This may be related to the indirect regulation of NO synthesis by insulin through a negative feedback mechanism. It is worth noting that in the insulin resistance state, the decreased sensitivity of endothelial cells to insulin may further inhibit NO release, increasing the risk of cardiovascular disease.

[0113] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. An electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensing material, characterized in that, The device includes a polymer substrate and a detection unit disposed on the surface of the polymer substrate. The components of the detection unit include a working electrode, a counter electrode, a reference electrode, and a wire. The surface of the working electrode is modified with a porphyrin nickel / CuO nanosheet composite sensing material; The porphyrin nickel / CuO nanosheet composite sensitive material has CuO nanosheets as the bottom layer and prismatic porphyrin nickel crystals loaded on the surface.

2. A method for fabricating an electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material as described in claim 1, characterized in that, Includes the following steps: The carbon substrate, counter electrode, reference electrode, and wires of the working electrode are prepared by screen printing on the surface of a polymer substrate. CuO nanosheets were mixed with anhydrous ethanol and Nafion solution to obtain CuO nanosheet ink; The CuO nanosheet ink was drop-coated onto the carbon substrate surface of the working electrode, and after drying, a CuO nanosheet layer was obtained. Porphyrin nickel is mixed with anhydrous ethanol to obtain porphyrin nickel ink; the porphyrin nickel ink is drop-coated onto the surface of the CuO nanosheet layer, and after drying, a porphyrin nickel / CuO nanosheet composite sensitive material layer is obtained, thus completing the fabrication of the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material.

3. The method for fabricating an electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material as described in claim 2, characterized in that, The ratio of CuO nanosheets to anhydrous ethanol and Nafion solution was 8 mg: 200 μL: 50 μL. And / or, the ratio of nickel porphyrin to anhydrous ethanol is 1 mg: 25 ~ 1000 μL; And / or, the droplet application rate of the CuO nanosheet ink is 17.6 ~ 44.1 μL / cm. 2 ; And / or, the drop volume of the porphyrin nickel ink is 17.6 ~ 44.1 μL / cm. 2 .

4. The method for fabricating an electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material as described in claim 2, characterized in that, The preparation steps of the CuO nanosheets include: mixing a copper source with a tetrabutylammonium hydroxide solution and stirring to obtain a suspension; centrifuging the suspension; drying the obtained precipitate; and calcining and annealing to obtain the CuO nanosheets.

5. The application of an electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material as described in claim 1 in NO detection.

6. A live cell NO release monitoring platform, characterized in that, The invention includes the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material as described in claim 1, a cell culture chamber constructed in the working electrode region of the electrochemical sensor chip, and a GelMA hydrogel filling the cell culture chamber.

7. A method for preparing a live cell NO release monitoring platform as described in claim 6, characterized in that, Includes the following steps: A cell culture chamber template was prepared by 3D printing; PDMS prepolymer was mixed with a curing agent and poured into the cell culture chamber template; after curing, the PDMS polymer layer was peeled off to obtain a primary cell culture chamber; the primary cell culture chamber was bonded to the working electrode area of ​​the electrochemical sensor chip based on porphyrin nickel / CuO nanosheet composite sensitive material; a GelMA prepolymer solution containing live cells was injected into the primary cell culture chamber and cured to obtain the live cell NO release monitoring platform.

8. A method for preparing a live cell NO release monitoring platform as described in claim 7, characterized in that, The preparation steps of the GelMA prepolymer solution containing living cells include: Live cells were cultured in a culture medium to obtain a cell suspension; the cell suspension was centrifuged, and the precipitate was resuspended in GelMA prepolymer solution, with the cell density adjusted to 5 × 10⁻⁶ cells / mL. 4 ~ 5×10 5 The GelMA prepolymer solution containing live cells was obtained by dispersing the concentration of cells / mL.

9. The application of the live cell NO release monitoring platform as described in claim 6 in evaluating the effect of diabetes treatment drugs or sugar substitutes on live cell NO release.

10. The application as described in claim 9, characterized in that, The application steps include: introducing the diabetes treatment drug or sugar substitute to be tested into the cell culture chamber of the live cell NO release monitoring platform; using the electrochemical sensor chip based on the porphyrin nickel / CuO nanosheet composite sensitive material to monitor the changes in electrical signals generated by the release of NO from the cells in real time; and evaluating the promoting or inhibiting effect of the diabetes treatment drug or sugar substitute on the release of NO from the cells based on the changes in electrical signals.

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