Preparation method and application of cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide released by cancer cells

Abstract

The application relates to a preparation method and application of a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of cancer cell-released nitric oxide, and relates to the field of electrochemical biosensing. Method: I. Synthesis of Co-COF material; II. Preparation of the sensor. The cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide realizes real-time detection of cancer cell-released nitric oxide. The cobalt-based covalent organic framework material synthesized by the application has a highly ordered pore structure, a large specific surface area, good chemical stability, an extended pi conjugated system and rich electrocatalytic active sites; the cobalt-doped covalent organic framework electrochemical sensor for real-time detection of cancer cell-released nitric oxide realizes efficient and specific conversion of the chemical signal of NO into a measurable electrical signal through the synergistic mechanism of the "pore enrichment-metal catalysis-electronic conduction" three-in-one of the Co-COF material, so that high-performance sensing of NO in a complex biological environment is realized.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensing, specifically to a method for preparing and applying a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells. Background Technology

[0002] Nitric oxide (NO), as a key endogenous gaseous signaling molecule, plays a dual role in the complex physiological and pathological networks of mammals. Developing technologies for highly sensitive, real-time, and in-situ detection of NO in biological systems is crucial for elucidating disease mechanisms, evaluating drug efficacy, and advancing precision medicine. While electrochemical sensing technology has become mainstream in the current field of NO detection due to its rapid response, ease of operation, and strong in-situ detection capabilities, it still faces a series of key challenges: metal-based materials suffer from insufficient long-term stability and biocompatibility; enzyme sensors are susceptible to enzyme inactivation and complex preparation processes; optical detection methods are often affected by autofluorescence interference; and the selectivity of existing sensors is generally limited in complex biological microenvironments containing interfering substances such as hydrogen peroxide. These problems make it difficult for traditional NO detection methods to simultaneously achieve high sensitivity, high selectivity, excellent stability, and good biocompatibility in in vivo systems or for long-term monitoring. Summary of the Invention

[0003] To address the shortcomings of the existing technology, this invention provides a method for preparing and applying a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

[0004] This invention constructs a novel electrochemical sensor by designing and synthesizing a cobalt-based covalent organic framework (Co-COF) material. This material utilizes its highly ordered porous structure to promote NO enrichment, accelerates electron transfer through an extended π-conjugated system, and combines the electrocatalytic activity of the cobalt metal center. The sensor also exhibits excellent reproducibility, repeatability, and long-term stability. Most importantly, its practical application value was successfully verified by real-time in-situ monitoring of NO molecules released from stimulated A549 cancer cells. Subsequently, the obtained cobalt-doped covalent organic framework electrochemical sensor was successfully applied to the detection of NO in real serum samples, showing excellent spiked recoveries (102%-106%), confirming its practical application value in biological fluid analysis.

[0005] A method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells, specifically comprising the following steps:

[0006] I. Synthesis of Co-COF materials:

[0007] 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde and cobalt salt were dissolved in glacial acetic acid to obtain a mixed solution; the mixed solution was transferred to a hydrothermal reactor for solvothermal reaction; after the reaction was completed, the solution was cooled to room temperature, washed and dried to obtain Co-COF material;

[0008] II. Sensor fabrication:

[0009] ① Polishing, rinsing, ultrasonic cleaning, and natural drying at room temperature were performed on the glassy carbon electrode using alumina powder of different particle sizes to obtain the pretreated electrode.

[0010] ② Immerse the pretreated electrode in sulfuric acid solution and perform cyclic voltammetry scans within a potential range of -1.0V to 1.0V until a stable cyclic voltammetric curve is obtained. Then rinse the electrode with ultrapure water and dry it at room temperature to obtain the activated electrode.

[0011] ③ Add the Co-COF material to ultrapure water and sonicate to obtain a Co-COF material suspension;

[0012] ④ The Co-COF material suspension was drop-coated onto the activated electrode surface and dried at room temperature to obtain a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

[0013] A cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

[0014] The principle of this invention:

[0015] I. The cobalt-based covalent organic framework material (Co-COF) synthesized in this invention has a highly ordered pore structure, a large specific surface area, good chemical stability, an extended π-conjugated system, and abundant electrocatalytic active sites.

[0016] II. The cobalt-doped covalent organic framework electrochemical sensor prepared in this invention for real-time detection of nitric oxide release from cancer cells utilizes the synergistic mechanism of "pore enrichment-metal catalysis-electron conduction" in Co-COF materials to efficiently and specifically convert the chemical signal of NO into a measurable electrical signal, thereby achieving high-performance sensing of NO in complex biological environments.

[0017] The beneficial effects of this invention are:

[0018] The cobalt-doped covalent organic framework electrochemical sensor prepared in this invention exhibits excellent comprehensive performance: a detection limit as low as 49.8 nM, a wide linear response range of 0.75–83.7 μM, and high sensitivity; it maintains excellent selectivity even when multiple biological interfering substances such as hydrogen peroxide, uric acid, dopamine, and ascorbic acid coexist; the sensor performs stably over a testing period of up to 8 days, making it suitable for long-term monitoring; and it demonstrates good reproducibility and repeatability.

[0019] It exhibits a small relative standard deviation, ensuring reliable data; excellent biocompatibility, making it suitable for in situ detection of live cells without cytotoxicity; and in practical applications, it achieves a recovery rate of 102%–106% in real serum samples, demonstrating excellent clinical testing potential. Furthermore, its simple operation, requiring no complex instruments or expensive reagents, offers a low-cost advantage, making it suitable for widespread application in clinical diagnostics, drug screening, and environmental monitoring. Attached Figure Description

[0020] Figure 1 This is the reaction formula for synthesizing Co-COF materials in step one of Example 1 of the present invention;

[0021] Figure 2 Images (a)-(c) are scanning electron microscope images of the Co-COF material synthesized in step one of Example 1; images (d)-(f) are transmission electron microscope images of the Co-COF material; images (g)-(h) are scanning transmission electron microscope images of the Co-COF material; and corresponding elemental distribution maps of carbon (C), nitrogen (N), oxygen (O) and cobalt (Co).

[0022] Figure 3 Fourier transform infrared spectra of BTA, HBC, COF, and Co-COF;

[0023] Figure 4 The powder X-ray diffraction pattern of the Co-COF material synthesized in step one of Example 1;

[0024] Figure 5 The X-ray photoelectron spectra of the Co-COF material synthesized in step one of Example 1 are shown in the figure. (a) is the XPS scan spectrum and high resolution, (b) is C 1s, (c) is N 1s, (d) is O 1s, and (e) is Co 2p.

[0025] Figure 6 In the middle, (a) is the CV curve of the Co-COF / GCE electrode in 0.1M PBS solution (pH 7.4) saturated with N2 and without 100μM NO, and (b) is the DPV curve of Co-COF / GCE and COF / GCE in 0.1M PBS solution (pH 7.4) saturated with N2 and with 300μM NO.

[0026] Figure 7 (a) shows the cyclic voltammetry curves of the Co-COF / GCE electrode at different NO concentrations in a 0.1M PBS solution (pH 7.4) saturated with N2; (b) shows the calibration curves of the current response with different NO concentrations; (c) shows the cyclic voltammetry curves at different scan rates in a 0.1M PBS solution (pH 7.4) containing 100 μM NO; and (d) shows the linear change of the peak current related to NO oxidation with the scan rate.

[0027] Figure 8 (a) shows the current response of Co-COF / GCE to N2-saturated 0.1M PBS solution (pH 7.4) at 1.0V by gradually adding NO (concentration range 0.75µM to 83.7µM), and (b) shows the calibration curves of the current response versus different NO concentrations.

[0028] Figure 9 (a) shows the current response of Co-COF / GCE in N2-saturated 0.1 M PBS (pH 7.4) under 1.0 V vs. HgO conditions after the sequential addition of NO, KCl, NaCl, NaNO2, CaCl2, Glucose, CuSO4, H2O2, UA, DA, AA, and the second addition of NO; ​​(b) shows the reproducibility of five Co-COF / GCE electrodes in detecting 50 μM NO; (c) shows the repeatability of one Co-COF / GCE electrode in detecting 50 μM NO five times; (d) shows the stability of the Co-COF / GCE electrode in 50 μM NO in N2-saturated 0.1 M PBS (pH 7.4) for 8 days. The error bars represent the standard deviation of three independent measurements.

[0029] Figure 10 The effects of Co-COF / GCE on (a) BEAS-2B cells and (b) A549 cells; (c) real-time in situ detection of nitric oxide release from A549 cancer cells stimulated by different concentrations of acetylcholine (Ach); (d) current response of A549 cancer cells stimulated by different concentrations of Ach (0.5 mM, 1.0 mM, and 1.5 mM); and (e) real-time in situ detection of NO release from A549 cancer cells at different cell concentrations.

[0030] Figure 11 To assess the consistency of triplet detection results at different spiking concentrations using the Bland-Altman plot: (a) 2 μM spiking concentration; (b) 4 μM spiking concentration; (c) 6 μM spiking concentration; (d) 8 μM spiking concentration; (e) 10 μM spiking concentration. Detailed Implementation

[0031] Specific Implementation Method 1: This implementation method is a preparation method for a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells, specifically completed according to the following steps:

[0032] I. Synthesis of Co-COF materials:

[0033] 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde and cobalt salt were dissolved in glacial acetic acid to obtain a mixed solution; the mixed solution was transferred to a hydrothermal reactor for solvothermal reaction; after the reaction was completed, the solution was cooled to room temperature, washed and dried to obtain Co-COF material;

[0034] II. Sensor fabrication:

[0035] ① Polishing, rinsing, ultrasonic cleaning, and natural drying at room temperature were performed on the glassy carbon electrode using alumina powder of different particle sizes to obtain the pretreated electrode.

[0036] ② Immerse the pretreated electrode in sulfuric acid solution and perform cyclic voltammetry scans within a potential range of -1.0V to 1.0V until a stable cyclic voltammetric curve is obtained. Then rinse the electrode with ultrapure water and dry it at room temperature to obtain the activated electrode.

[0037] ③ Add the Co-COF material to ultrapure water and sonicate to obtain a Co-COF material suspension;

[0038] ④ The Co-COF material suspension was drop-coated onto the activated electrode surface and dried at room temperature to obtain a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

[0039] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the cobalt salt mentioned in step one is cobalt acetate tetrahydrate. The other steps are the same as in Specific Implementation Method One.

[0040] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that: the molar ratio of 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde, and cobalt salt in step one is 1:2:4; the mass ratio of 1,2,4,5-phenyltetramine tetrahydrochloride to glacial acetic acid in step one is 0.1 g:1 mL. Other steps are the same as in Specific Implementation Method One or Two.

[0041] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the temperature of the solvothermal reaction in step one is 120°C, and the reaction time is 3 days. The other steps are the same as in Specific Implementation Methods One to Three.

[0042] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that: In step two①, the glassy carbon electrode is polished sequentially using 0.3μm and 0.05μm alumina powders, then rinsed with ultrapure water, and then ultrasonically cleaned in ultrapure water and anhydrous ethanol for 10-15 minutes respectively. After removal, it is allowed to air dry at room temperature to obtain the pretreated electrode. Other steps are the same as in Specific Implementation Methods One to Four.

[0043] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: the scanning rate of the cyclic scan in step two ② is 100 mV / s; and the concentration of the sulfuric acid solution in step two ② is 0.5 mol / L. The other steps are the same as in Specific Implementation Methods One to Five.

[0044] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that: the mass ratio of the Co-COF material to the volume of ultrapure water in step two ③ is 2 mg: 1 mL; the ultrasonic treatment time in step two ③ is 10 min to 20 min. Other steps are the same as in Specific Implementation Methods One to Six.

[0045] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One through Seven in that the volume ratio of the Co-COF material suspension in step two (④) to the surface area of ​​the activated electrode is 10 μL: 0.07 cm². 2 The other steps are the same as those in Specific Implementation Methods One through Seven.

[0046] Specific Implementation Method Nine: This implementation method is prepared according to the preparation method described in any one of Specific Implementation Methods One to Eight.

[0047] Specific Implementation Method 10: This implementation method is a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

[0048] The beneficial effects of the present invention are verified using the following embodiments:

[0049] Example 1: A method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells, specifically comprising the following steps:

[0050] I. Synthesis of Co-COF materials:

[0051] 1,2,4,5-phenyltetramine tetrahydrochloride (BTA), 2,5-dihydroxyterephthalaldehyde (HBC) and cobalt acetate tetrahydrate were dissolved in glacial acetic acid to obtain a mixed solution. The mixed solution was transferred into a hydrothermal reactor and subjected to a solvothermal reaction at 120°C for 3 days. After the reaction was completed, the solution was cooled to room temperature, washed, and dried to obtain the Co-COF material.

[0052] The molar ratio of 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde and cobalt acetate tetrahydrate mentioned in step one is 1:2:4.

[0053] The mass ratio of 1,2,4,5-phenyltetramine tetrahydrochloride to glacial acetic acid in step one is 0.1 g: 1 mL;

[0054] II. Sensor fabrication:

[0055] ① Polish the glassy carbon electrode with 0.3μm and 0.05μm alumina powder in sequence, then rinse it with ultrapure water, and then place the electrode in ultrapure water and anhydrous ethanol in sequence for ultrasonic cleaning for 10min~15min respectively. After taking it out, let it air dry at room temperature to obtain the pretreated electrode.

[0056] ② Immerse the pretreated electrode in a 0.5 mol / L sulfuric acid solution and perform cyclic voltammetry at a scan rate of 100 mV / s within a potential range of -1.0 V to 1.0 V until a stable cyclic voltammetric curve is obtained. Then rinse the electrode with ultrapure water and dry it at room temperature to obtain the activated electrode.

[0057] ③ Add 2 mg of Co-COF material to 1 mL of ultrapure water and sonicate for 10 min to obtain a Co-COF material suspension;

[0058] ④ The Co-COF material suspension was drop-coated onto the activated electrode surface and dried at room temperature to obtain a cobalt-doped covalent organic framework electrochemical sensor (denoted as Co-COF / GCE) for real-time detection of nitric oxide release from cancer cells.

[0059] The volume ratio of the Co-COF material suspension to the surface area of ​​the activated electrode, as described in step 2④, is 10 μL: 0.07 cm². 2 .

[0060] Comparing with Example 1: The preparation method of a covalent organic framework electrochemical sensor is specifically carried out according to the following steps:

[0061] I. Synthesis of COF materials:

[0062] 1,2,4,5-phenyltetramine tetrahydrochloride (BTA) and 2,5-dihydroxyterephthalaldehyde (HBC) were dissolved in glacial acetic acid to obtain a mixed solution. The mixed solution was transferred to a hydrothermal reactor and subjected to a solvothermal reaction at 120°C for 3 days. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain the COF material.

[0063] The molar ratio of 1,2,4,5-phenyltetramine tetrahydrochloride and 2,5-dihydroxyterephthalaldehyde mentioned in step one is 1:2;

[0064] The mass ratio of 1,2,4,5-phenyltetramine tetrahydrochloride to glacial acetic acid in step one is 0.1 g: 1 mL;

[0065] II. Sensor fabrication:

[0066] ① Polish the glassy carbon electrode with 0.3μm and 0.05μm alumina powder in sequence, then rinse it with ultrapure water, and then place the electrode in ultrapure water and anhydrous ethanol in sequence for ultrasonic cleaning for 10min~15min respectively. After taking it out, let it air dry at room temperature to obtain the pretreated electrode.

[0067] ② Immerse the pretreated electrode in a 0.5 mol / L sulfuric acid solution and perform cyclic voltammetry at a scan rate of 100 mV / s within a potential range of -1.0 V to 1.0 V until a stable cyclic voltammetric curve is obtained. Then rinse the electrode with ultrapure water and dry it at room temperature to obtain the activated electrode.

[0068] ③ Add 2 mg of COF material to 1 mL of ultrapure water and sonicate for 10 min to obtain a COF material suspension;

[0069] ④ The COF material suspension is drop-coated onto the activated electrode surface and dried at room temperature to obtain a covalent organic framework electrochemical sensor (denoted as COF / GCE).

[0070] In step 2④, the volume ratio of the COF material suspension to the surface area of ​​the activated electrode is 10 μL: 0.07 cm². 2 .

[0071] Example 1 successfully prepared Co-COF material using a solvothermal reaction. The microstructure of Co-COF was characterized by scanning electron microscopy (SEM). Co-COF exhibited an aggregated structure composed of stacked nanosheets. Figure 2 a–c). Transmission electron microscopy (TEM) analysis ( Figure 2(d–f) The structural features of the material were confirmed at the atomic scale, revealing the well-defined stacked structure and interconnected pores of the ultrathin nanosheets. The observed interlayer spacing indicates π-π stacking interactions between adjacent layers, which promote efficient interlayer electron transport while maintaining structural integrity. Comprehensive elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS). Figure 2 g–h further confirmed the uniform dispersion of Co within the Co-COF framework. Simultaneously, Fourier transform infrared spectroscopy (FTIR) was used. Figure 3 Powder X-ray diffraction ( Figure 4 ) and X-ray photoelectron spectroscopy ( Figure 5 The crystal and chemical structure of Co-COF were characterized.

[0072] from Figure 3 It can be seen that it is located at 1665cm -1 The characteristic stretching vibration peak of C=O at 3301 cm⁻¹ is located at 3301 cm⁻¹. -1 The disappearance of the NH stretching vibration peak at 1647 cm⁻¹, and the disappearance of the peak at 1647 cm⁻¹. -1 The appearance of the C=N stretching vibration peak confirms the formation of Co-COF.

[0073] from Figure 4 It can be seen that the crystal structure of the synthesized Co-COF was characterized by powder X-ray diffraction. Two characteristic diffraction peaks of Co-COF were clearly observed at 2θ=8.7° and 25.1°, which belong to the (100) and (001) crystal planes, respectively; the appearance of the (001) crystal plane confirmed the existence of a π-π layered stacked structure in the Co-COF framework.

[0074] from Figure 5 XPS analysis shows that C, N, O and Co elements are present in the Co-COF framework. High-resolution spectra show the characteristic chemical bonds of each element (such as C=C, C=N, Co-O, etc.), among which the Co-O coordination bond confirms that Co is successfully fixed in the COF framework.

[0075] Electrochemical detection conditions:

[0076] (1) Electrolyte: Use 0.1M PBS (pH 7.4), and pre-purge with high-purity nitrogen for 15 minutes to remove dissolved oxygen;

[0077] (2) Three-electrode system: Co-COF / GCE and COF / GCE are used as working electrodes, HgO electrode is used as reference electrode, and platinum wire electrode is used as counter electrode;

[0078] (3) Detection methods: The electrocatalytic behavior of the modified electrode was characterized by cyclic voltammetry, chronoamperometry, and differential pulse voltammetry, and the quantitative detection of NO was achieved. See Figure 6As shown. Real-time detection of NO was performed using the chronoamperometry (it) method under an applied potential of +1.0 V (vs. HgO). The current response was recorded by gradually adding a known concentration of NO standard solution to the electrolytic cell. (See figure). Figure 8 As shown;

[0079] Figure 6 In the middle, (a) is the CV curve of the Co-COF / GCE electrode in 0.1M PBS solution (pH 7.4) saturated with N2 and without 100μM NO, and (b) is the DPV curve of Co-COF / GCE and COF / GCE in 0.1M PBS solution (pH 7.4) saturated with N2 and with 300μM NO.

[0080] from Figure 6 It can be seen that Co-COF / GCE exhibits significant electrochemical responses under different NO concentrations. In contrast, COF / GCE shows a shift in its anodic peak (located at 0.9 V) under the same conditions, and its peak current is significantly lower, at only 337 μA. Co-COF / GCE demonstrates significant improvements in both current response (6.2-fold increase) and positive potential shift.

[0081] Standard curve plotting and sample testing:

[0082] (1) Plotting the standard curve: NO standard solution was added sequentially to a 0.1M PBS solution (pH 7.4) saturated with nitrogen, and the corresponding steady-state current values ​​were recorded. A standard curve was plotted with NO concentration on the x-axis and current response on the y-axis to obtain the linear regression equation, see [link to curve]. Figure 7 As shown in (b) and (d);

[0083] (2) Serum sample pretreatment: Take human serum sample, centrifuge to remove large molecular interferences such as proteins, take the supernatant and dilute it appropriately with 0.1M PBS solution (pH 7.4) (the volume ratio of supernatant to 0.1M PBS solution (pH 7.4) is 1:99).

[0084] (3) Spiked recovery experiment: Different concentrations of NO standard solution were added to serum samples, and the Co-COF / GCE electrode prepared in Example 1 was used for detection. The recovery rate was calculated based on the standard curve. Figure 8 (See Table 1); Experimental results show that the recovery rate is between 102% and 106%, indicating that the method has high accuracy and practicality.

[0085] Figure 7(a) shows the cyclic voltammetry curves of the Co-COF / GCE electrode at different NO concentrations in a 0.1M PBS solution (pH 7.4) saturated with N2; (b) shows the calibration curves of the current response with different NO concentrations; (c) shows the cyclic voltammetry curves at different scan rates in a 0.1M PBS solution (pH 7.4) containing 100 μM NO; and (d) shows the linear change of the peak current related to NO oxidation with the scan rate.

[0086] from Figure 7 It is evident that Co-COF materials can serve as high-performance electrocatalysts for the quantitative determination of NO. The NO oxidation process on Co-COF / GCE follows a surface-dominated kinetic pathway. With increasing scan rate, the anodic peak potential shifts positively from 1.07 V (vs. HgO) to 1.13 V. No corresponding cathode peak was detected in the CV curve, confirming the irreversible nature of the NO electrocatalytic oxidation.

[0087] Table 1 shows the spiked recovery rate in human serum samples (n=3).

[0088] Table 1

[0089]

[0090] Figure 8 (a) shows the current response of Co-COF / GCE to N2-saturated 0.1M PBS solution (pH 7.4) at 1.0V by gradually adding NO (concentration range 0.75µM to 83.7µM), and (b) shows the calibration curves of the current response versus different NO concentrations.

[0091] from Figure 8 As can be seen, NO was gradually added to the electrolytic cell, and the corresponding current response was monitored in real time. The current rose rapidly after each addition of NO and reached a steady state within 7 seconds. Given the short half-life of NO, this response time demonstrates the sensor's ability to monitor the real-time dynamics of NO release from biological systems.

[0092] Performance verification:

[0093] (1) Selectivity test: In a 0.1M PBS solution (pH 7.4) saturated with N2 containing 10μM NO, 10 times the concentration of the interfering substances (including KCl, NaNO2, NaCl, Glucose, CaCl2, H2O2, CuSO4, UA, DA and AA) were added respectively, and the changes in current response were observed.

[0094] (2) Repeatability and reproducibility: The relative standard deviation (RSD) was 2.27% when the same Co-COF / GCE was used to detect 10 μM NO in 0.1 M PBS solution (pH 7.4) 5 times consecutively; the RSD was 7.08% when the same concentration of NO was detected by 5 independently prepared Co-COF / GCE.

[0095] (3) Stability test: The Co-COF / GCE electrode was stored in a refrigerator at 4℃ and tested every 2 days to verify its stability. The current density measured on the 8th day still maintained about 75% of the initial response value. See Figure 9 As shown in (d).

[0096] Figure 9 (a) shows the current response of Co-COF / GCE in N2-saturated 0.1 M PBS (pH 7.4) under 1.0 V vs. HgO conditions after the sequential addition of NO, KCl, NaCl, NaNO2, CaCl2, Glucose, CuSO4, H2O2, UA, DA, AA, and the second addition of NO; ​​(b) shows the reproducibility of five Co-COF / GCE electrodes in detecting 50 μM NO; (c) shows the repeatability of one Co-COF / GCE electrode in detecting 50 μM NO five times; (d) shows the stability of the Co-COF / GCE electrode in 50 μM NO in N2-saturated 0.1 M PBS (pH 7.4) for 8 days. The error bars represent the standard deviation of three independent measurements.

[0097] from Figure 9 It can be seen that Co-COF / GCE exhibits excellent selectivity, anti-interference ability, reproducibility, repeatability and stability in NO detection.

[0098] The Co-COF / GCE prepared in Example 1 was used for real-time monitoring of nitric oxide release from living cells.

[0099] 1. Cell culture and treatment:

[0100] (1) Cells: Normal human BEAS-2B cells and A549 cancer cells;

[0101] (2) Culture conditions: Cells were cultured in DMEM medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator until the logarithmic growth phase.

[0102] (3) Cell suspension preparation: Wash cells twice with PBS solution (pH 7.4), digest with trypsin, centrifuge to collect cells, and resuspend in PBS solution (pH 7.4) to a concentration of 1×10⁻⁶. 5 cells / mL.

[0103] 2. Real-time monitoring system setup:

[0104] (1) Electrochemical detection cell: an electrolytic cell with a built-in three-electrode system;

[0105] (2) Sensor preparation: Co-COF / GCE prepared in Example 1;

[0106] (3) Cell introduction: Inject the cell suspension into the electrolytic cell and insert the three-electrode system.

[0107] 3. NO release stimulation and detection:

[0108] (1) Background current stabilized: Record the background current until it stabilizes under no-stimulation conditions.

[0109] (2) Drug stimulation: acetylcholine is added to the electrolytic cell to stimulate the release of NO from the cells.

[0110] (3) Real-time recording: The current change is monitored in real time at a potential of +1.0 V using the time-current method to record the dynamic process of NO release.

[0111] 4. Results Analysis:

[0112] (1) Comparison between normal cells and cancer cells: BEAS-2B cells showed a weak current response after stimulation, indicating that the amount of NO released was small; A549 cells showed a significant increase in current under the same stimulation, indicating that cancer cells had an enhanced ability to release NO.

[0113] (2) Quantitative analysis: Based on the standard curve, the real-time concentration change of NO released by cells can be calculated, providing data support for the study of cell signal transduction and disease mechanisms.

[0114] Figure 10 The effects of Co-COF / GCE on (a) BEAS-2B cells and (b) A549 cells; (c) real-time in situ detection of nitric oxide release from A549 cancer cells stimulated by different concentrations of acetylcholine (Ach); (d) current response of A549 cancer cells stimulated by different concentrations of Ach (0.5 mM, 1.0 mM, and 1.5 mM); and (e) real-time in situ detection of NO release from A549 cancer cells at different cell concentrations.

[0115] from Figure 10 It is known that Co-COF has low cytotoxicity and good biocompatibility. Co-COF-based electrochemical biosensors can selectively detect nitric oxide released by A549 cancer cells in real time, which highlights its great potential in practical biomedical applications.

[0116] Figure 11To assess the consistency of triplet assay results at different spiking concentrations using the Bland-Altman plot: (a) 2 μM spiking concentration; (b) 4 μM spiking concentration; (c) 6 μM spiking concentration; (d) 8 μM spiking concentration; (e) 10 μM spiking concentration;

[0117] NO in serum samples was detected using a standard spiking method. Serum samples were diluted 100-fold with 0.1M PBS solution (pH 7.4), and different volumes of saturated NO solution were added to blank serum samples. Subsequently, Co-COF / GCE was used for detection.

[0118] from Figure 11 It can be seen that the relative standard deviations (RSDs) of the detection results of the Co-COF / GCE sensor at spiking levels of 2, 4, 6, 8 and 10 μM were 6.8%, 3.3%, 2.2%, 7.8% and 3.8%, respectively, indicating that the sensor has excellent practical performance in detecting NO in serum samples.

Claims

1. A method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells, characterized in that... The preparation method is specifically carried out according to the following steps: I. Synthesis of Co-COF materials: 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde and cobalt salt were dissolved in glacial acetic acid to obtain a mixed solution; the mixed solution was transferred to a hydrothermal reactor for solvothermal reaction; after the reaction was completed, the solution was cooled to room temperature, washed and dried to obtain Co-COF material; II. Sensor fabrication: ① Polishing, rinsing, ultrasonic cleaning, and natural drying at room temperature were performed on the glassy carbon electrode using alumina powder of different particle sizes to obtain the pretreated electrode. ② Immerse the pretreated electrode in sulfuric acid solution and perform cyclic voltammetry scans within a potential range of -1.0V to 1.0V until a stable cyclic voltammetric curve is obtained. Then rinse the electrode with ultrapure water and dry it at room temperature to obtain the activated electrode. ③ Add the Co-COF material to ultrapure water and sonicate to obtain a Co-COF material suspension; ④ The Co-COF material suspension was drop-coated onto the activated electrode surface and dried at room temperature to obtain a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells.

2. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The cobalt salt mentioned in step one is cobalt acetate tetrahydrate.

3. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The molar ratio of 1,2,4,5-phenyltetramine tetrahydrochloride, 2,5-dihydroxyterephthalaldehyde and cobalt salt in step one is 1:2:4; the mass ratio of 1,2,4,5-phenyltetramine tetrahydrochloride to glacial acetic acid in step one is 0.1 g:1 mL.

4. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The temperature of the solvothermal reaction described in step one is 120°C, and the reaction time is 3 days.

5. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... In step 2①, the glassy carbon electrode is polished with 0.3μm and 0.05μm alumina powders in sequence, then rinsed with ultrapure water, and then ultrasonically cleaned in ultrapure water and anhydrous ethanol for 10min~15min respectively. After removal, it is naturally dried at room temperature to obtain the pretreated electrode.

6. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The scanning rate of the cyclic scan mentioned in step 2② is 100mV / s; the concentration of the sulfuric acid solution mentioned in step 2② is 0.5mol / L.

7. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The mass ratio of the Co-COF material to the volume of ultrapure water in step 2③ is 2 mg: 1 mL; the ultrasonic treatment time in step 2③ is 10 min to 20 min.

8. The method for preparing a cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 1, characterized in that... The volume ratio of the Co-COF material suspension to the surface area of ​​the activated electrode, as described in step 2④, is 10 μL: 0.07 cm². 2 .

9. A cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells, characterized in that... It is prepared according to the preparation method described in any one of claims 1 to 8.

10. The application of the cobalt-doped covalent organic framework electrochemical sensor for real-time detection of nitric oxide release from cancer cells according to claim 9, characterized in that... The electrochemical sensor detects the release of nitric oxide from cancer cells in real time.

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