A DNase I-coordinated silicon channel dual-mode sensor, its construction method, and its application.

Abstract

This application discloses a DNase I-synergistic silicon channel dual-mode sensor, its construction method, and its application, relating to the field of biosensor technology. The sensor includes a working electrode, a reference electrode, and a counter electrode. The working electrode is a vertically ordered mesoporous silica nanochannel film-modified electrode. The vertically ordered mesoporous silica nanochannel film has a negatively charged array of mesoporous nanochannels; the pore size of the nanochannels is 2 nm to 3 nm. This application utilizes a vertically ordered mesoporous silica nanochannel film-modified electrode, leveraging the size exclusion effect of its ultra-small nanochannels and the electrostatic enrichment effect of its surface negative charge on cation probes. This allows for efficient enrichment and detection of signal molecules without complex sample pretreatment, combining the wide linear range of electrochemical detection with the high sensitivity of electrochemiluminescence detection.

Description

Technical Field

[0001] This application relates to the field of biosensor technology, and in particular to a DNase I synergistic silicon channel dual-mode sensor, its construction method, and its application. Background Technology

[0002] Ovarian cancer is the fifth leading cause of cancer-related death in women and the leading cause of death among gynecological malignancies. Cancer antigen 125 (CA125), as the most common tumor marker in epithelial ovarian cancer, has high diagnostic sensitivity. The normal level of CA125 in healthy women is 0-35 U / mL, but serum CA125 levels may transiently increase in cases of endometriosis, peritonitis, and other malignancies. Currently, CA125 is widely used as a gold biomarker for diagnosing ovarian cancer, in early cancer diagnosis, pelvic mass identification, and treatment monitoring. Therefore, developing sensitive and accurate CA125 diagnostic methods is of great significance.

[0003] Electrogenerated chemiluminescence (ECL) combines the advantages of electrochemistry and chemiluminescence, offering benefits such as low background, high signal-to-noise ratio, high sensitivity, and miniaturization. Electrochemistry (EC) methods are widely accepted due to their high sensitivity, rapid response, and simple instrumentation. EC / ECL dual-mode detection, combining the advantages of both, has attracted considerable attention. Tris(2,2'-bipyridine)ruthenium (Ru(bpy)3) 2+ As a bifunctional signaling molecule with excellent EC and ECL responses, it can be used to construct EC / ECL dual-mode sensors.

[0004] Aptamer sensors, constructed based on the specific recognition of target molecules by aptamers, hold promise as an alternative to traditional immunodiagnostics. Aptamers are single-chain molecules composed of ribonucleotides or deoxyribonucleic acid that can specifically bind to target molecules, offering advantages such as high stability, low cost, and simple synthesis. Graphene oxide (GO), with its high specific surface area, excellent electronic conductivity, and biocompatibility, provides an ideal platform for aptamer attachment, enhancing detection sensitivity.

[0005] In recent years, signal amplification using nanomaterials has become an effective strategy for improving the sensitivity of electrochemical sensors. Vertically ordered mesoporous silica nanochannel films (VMSFs) have become an important method for fabricating high-performance electrochemical sensors. The unique pore size of 2-3 nm in VMSF nanochannels can exclude macromolecules through size effects, and their negatively charged surface can significantly enhance the electrostatic enrichment of cationic electrochemical probes, thereby achieving signal enhancement.

[0006] Currently, there is still a lack of homogeneous detection electrochemical / electrochemiluminescence sensors that use mesoporous silica films as signal amplifiers and DNase I as a co-assisted signal amplification. Summary of the Invention

[0007] The purpose of this application is to provide a DNase I synergistic silicon channel dual-mode sensor, its construction method, and its application, to achieve highly selective and sensitive detection of tumor markers.

[0008] To achieve the above objectives, this application provides a DNase I-coordinated silicon channel dual-mode sensor, comprising: a working electrode, a reference electrode, and a counter electrode; The working electrode is a vertically ordered mesoporous silica nanochannel thin film modified electrode. The vertically ordered mesoporous silica nanochannel film has a negatively charged array of mesoporous nanochannels; the pore size of the nanochannels is 2nm~3nm.

[0009] Preferably, the DNase I-coordinated silicon channel dual-mode sensor further includes: a composite probe; The composite probe is composed of tri(bipyridine)ruthenium(II), graphene oxide, and an aptamer; The aptamer is one of the following: cancer antigen 125 aptamer, carcinoembryonic antigen aptamer, alpha-fetoprotein aptamer, and cancer antigen 15-3 aptamer.

[0010] Preferably, the composite probe contains 10 μM to 30 μM of tri(bipyridine)ruthenium(II), 1 mg / mL to 5 mg / mL of graphene oxide, and 50 μM to 200 μM of aptamer.

[0011] This application provides a method for constructing a DNase I-coordinated silicon channel dual-mode sensor, which includes the following steps: S1. Mix ethanol, sodium nitrate solution and hexadecyltrimethylammonium bromide. After complete dissolution, add tetraethyl orthosilicate and stir at room temperature to pre-hydrolyze to obtain the precursor solution. S2. The electrode is placed in the precursor solution, and a constant current growth method is used to deposit the current. After deposition, the electrode is removed, rinsed, dried with nitrogen, and aged to obtain a silicon dioxide nanochannel modified electrode containing micelles. The electrode is one of indium tin oxide electrode, glassy carbon electrode, screen-printed electrode, gold electrode, fluorine-doped tin oxide electrode, graphite electrode, and carbon fiber electrode. S3. Immerse the silica nanochannel modified electrode containing micelles in an ethanol solution containing inorganic acid and stir for 1 min to 30 min to remove the micelles and obtain a vertically ordered mesoporous silica nanochannel thin film modified electrode. S4. Dissolve terpyridine ruthenium chloride in phosphate buffer solution, then add graphene oxide and aptamer, sonicate to combine, centrifuge and wash, remove supernatant, redisperse in buffer solution to obtain composite probe; S5. A vertically ordered mesoporous silica nanochannel film modified electrode is used as the working electrode, and a three-electrode system is formed with a reference electrode and a counter electrode. Combined with a composite probe, a DNase I synergistic silicon channel dual-mode sensor is obtained.

[0012] Preferably, the molar concentration of the sodium nitrate solution is 0.01 mol / L to 0.5 mol / L; and the concentration of the inorganic acid is 0.01 M to 0.5 M.

[0013] Preferably, the amount of hexadecyltrimethylammonium bromide added is 1g to 2g; and the amount of tetraethyl orthosilicate added is 1mL to 5mL.

[0014] Preferably, the concentration of the phosphate buffer solution is 0.005 mol / L to 0.5 mol / L, and the pH value is 6.5 to 7.5.

[0015] Preferably, the amount of ruthenium terpyridine chloride added is 1 mg to 10 mg; the volume ratio of the graphene oxide solution to the aptamer solution is 1 to 5:1; the concentration of the graphene oxide solution is 1 mg / mL to 5 mg / mL; and the concentration of the aptamer solution is 50 μM to 200 μM.

[0016] Preferably, the applied current density is -0.05 mA / cm². 2 ~-1mA / cm 2 The aging temperature is 100℃~150℃, and the time is 12h~24h.

[0017] This application also provides an application of a DNase I synergistic silicon channel dual-mode sensor in the detection of tumor markers for non-diagnostic purposes, which performs quantitative analysis of tumor markers by detecting the intensity of electrochemical signals or electrochemiluminescence signals; the tumor markers include one of cancer antigen 125, carcinoembryonic antigen, alpha-fetoprotein, cancer antigen 15-3, and cancer antigen 19-9.

[0018] In summary, the DNase I-coordinated silicon channel dual-mode sensor, its construction method, and its application provided in this application offer the following advantages compared to traditional technologies: (1) The DNase I synergistic silicon channel dual-mode sensor in this application uses a vertically ordered mesoporous silica nanochannel thin film to modify the electrode. It utilizes the size exclusion effect of the ultra-small nanochannel and the electrostatic enrichment effect of the surface negative charge on the cation probe to achieve efficient enrichment and detection of signal molecules without complicated sample pretreatment. It has both the wide linear range of electrochemical detection and the high sensitivity of electrochemiluminescence detection.

[0019] (2) This application combines tri(bipyridine)ruthenium(II) bifunctional signal molecules with graphene oxide and aptamers, and removes free signal molecules by centrifugation and washing, which significantly reduces the background signal; during detection, it can be combined with the cyclic shearing amplification effect of DNase I to achieve high selectivity and high sensitivity detection of tumor markers.

[0020] (3) The DNase I synergistic silicon channel dual-mode sensor in this application adopts a homogeneous recognition strategy, which does not require the immobilization and modification of biomolecules on the electrode surface, thus avoiding the complex modification steps and loss of bioactivity of traditional immune sensors. It is simple to operate, low in cost and has good reproducibility.

[0021] (4) The DNase I synergistic silicon channel dual-mode sensor in this application has good anti-interference ability, can effectively distinguish cancer antigen 125 from a variety of common interfering substances, and has been successfully applied to the spiked recovery detection of cancer antigen 125 in actual serum samples, demonstrating its application potential in the clinical diagnosis of tumor markers.

[0022] The technical solution of this application will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is a characterization diagram of the VMSF surface in Embodiment 1 of this application; Figure 1 (a) is a top view of the VMSF surface using a transmission electron microscope (TEM). Figure 1 (b) in the figure is a TEM cross-sectional view of the VMSF surface; Figure 1 (c) in the image is a cross-sectional view of the VMSF surface obtained by scanning electron microscopy (SEM). Figure 2 The figures show the cyclic voltammetry curves of the bare ITO electrode, SM@VMSF / ITO electrode, and VMSF / ITO electrode in different electrical probe solutions in Example 1 of this application. Figure 2 (a) in the text is Fe(CN)6 3-Cyclic voltammetry curves of bare ITO electrode, SM@VMSF / ITO electrode and VMSF / ITO electrode when used as probes; Figure 2 (b) in the example is Ru(NH3)6 3+ Cyclic voltammetry curves of bare ITO electrode, SM@VMSF / ITO electrode and VMSF / ITO electrode when used as probes; Figure 2 (c) shows the cyclic voltammetry curves of the bare ITO electrode, SM@VMSF / ITO electrode, and VMSF / ITO electrode when FcMeOH is used as the probe. Figure 3 This is a characterization diagram of GO in Embodiment 1 of this application; according to Figure 3 (a) in the image is an atomic force microscopy (AFM) image of GO; Figure 3 (b) in the image is the TEM image of GO; Figure 3 (c) in the figure is the Fourier Transform Infrared Spectroscopy (FT-IR) spectrum of GO; Figure 4 This is a feasibility analysis diagram of the DNase I synergistic silicon channel dual-mode sensor in Embodiment 1 of this application; Figure 4 (a) in the figure is the current-potential curve obtained by cyclic voltammetry. Figure 4 (b) in the figure is the current-potential curve of the differential pulse voltammetry (DPV) test; Figure 4 (c) in the figure is the electrochemiluminescence intensity-potential curve; Figure 4 (d) in the figure is the electrochemiluminescence intensity-time curve; Figure 5 This is an optimized diagram of the detection conditions in Embodiment 1 of this application; Figure 5 (a) represents Ru(bpy)3Cl2 in the ternary complex Ru(bpy)3 2+ Concentration optimization plot in / Apt&GO; Figure 5 (b) in the figure shows the signal optimization diagram for different shearing times; Figure 6 This is a performance graph of the DNase I-coordinated silicon channel dual-mode sensor in Embodiment 1 of this application; Figure 6 (a) shows the DPV curves of the DNase I synergistic silicon channel dual-mode sensor for different concentrations of CA125 (0.1 U / mL, 0.5 U / mL, 1 U / mL, 5 U / mL, 10 U / mL, 50 U / mL and 100 U / mL); Figure 6 (b) in the figure is the linear regression curve of the EC model; Figure 6(c) shows the ECL intensity-time response of the DNase I-co-silicon channel dual-mode sensor to different concentrations of CA125 (0.001 U / mL, 0.01 U / mL, 0.1 U / mL, 1 U / mL, 10 U / mL and 100 U / mL); Figure 6 (d) in the figure is the linear regression curve of the ECL model; Figure 7 The signal response difference diagram of EC and ECL in Embodiment 1 of this application; Figure 7 (a) in the figure is the EC signal response difference diagram; Figure 7 (b) in the figure is the ECL signal response difference diagram. Detailed Implementation

[0024] The technical methods of this application will be further described below with reference to the accompanying drawings and embodiments.

[0025] The following description of at least one exemplary embodiment is merely illustrative and is not intended to limit the scope of this application or its application or use.

[0026] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. All materials and reagents used in this application are commercially available products.

[0027] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by a person of ordinary skill in the art to which this application pertains.

[0028] Example 1 A DNase I-coordinated silicon channel dual-mode sensor includes: a working electrode, a reference electrode, a counter electrode, and a composite probe.

[0029] The working electrode is a vertically ordered mesoporous silica nanochannel film modified electrode. The vertically ordered mesoporous silica nanochannel film has a negatively charged array of mesoporous nanochannels with a pore size of 2.3 nm.

[0030] The composite probe consists of ruthenium(II) tris(bipyridine), graphene oxide, and the cancer antigen 125 (CA125) aptamer. The concentration of ruthenium(II) tris(bipyridine) is 20 μM, the concentration of graphene oxide is 2 mg / mL, and the concentration of the CA125 aptamer is 100 μM.

[0031] A method for constructing a DNase I-coordinated silicon channel dual-mode sensor includes the following steps: S1. Dissolve 1.585g of hexadecyltrimethylammonium bromide in a mixed solution of 20mL ethanol and 20mL NaNO3 solution (concentration of 0.1mol / L, pH value of 2.6). After stirring continuously until the hexadecyltrimethylammonium bromide is completely dissolved, add 3.050mL of tetraethyl orthosilicate and stir at room temperature for 2.5h for pre-hydrolysis to obtain a clear and transparent precursor solution.

[0032] S2. The indium tin oxide (ITO) electrode is placed in the precursor solution, and a constant current growth method is used, with a current density of -0.7 mA / cm². 2 A constant current was applied, and after deposition for 10 seconds, the electrode was removed, rinsed, dried with nitrogen, and aged at 120°C for 12 hours to obtain a silica nanochannel modified electrode containing micelles, named SM@VMSF / ITO electrode.

[0033] S3. Immerse the SM@VMSF / ITO electrode in an ethanol solution containing HCl (concentration of 0.01mol / L) and stir for 10 min. Remove the micelles to obtain a vertically ordered mesoporous silica nanochannel film modified electrode, named VMSF / ITO electrode.

[0034] S4. Weigh 7.5 mg of ruthenium tripyridine chloride (Ru(bpy)3Cl2) and dissolve it in 20 mL of 0.01 mol / L phosphate buffer (PBS) with a pH of 7.4 to obtain a stock solution. Then, take 685 μL of the stock solution and dilute it to 20 mL with PBS to obtain Ru(bpy)3Cl2 with a concentration of 20 μmol / L. 2+ The probe solution was then mixed with 25 μL of graphene oxide (GO) solution (2 mg / mL) and 10 μL of CA125 aptamer solution (100 μmol / L). The mixture was sonicated for 1 h, then centrifuged at 5000 rpm for 1 h, washed, and the supernatant was removed. The mixture was washed three times with PBS and then redispersed in PBS to obtain the composite probe Ru(bpy)3. 2+ / Apt&GO.

[0035] S5. Using VMSF / ITO as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode, a three-electrode system is formed, and then combined with the composite probe Ru(bpy)3. 2+ The / Apt&GO combination yields a DNase I synergistic silicon channel dual-mode sensor.

[0036] Characterization of VMSF / ITO electrodes, such as Figure 1 As shown.

[0037] Figure 1 (a) in the image is a TEM image of the VMSF surface. According to... Figure 1As shown in (a), the pores on the VMSF surface exhibit an ordered hexagonal arrangement and are uniformly distributed. ImageJ software analysis revealed that the pore size of the VMSF is approximately 2.3 nm. According to... Figure 1 As shown in (b), VMSF has vertical, ordered mass transfer channels, providing a pathway for the efficient and rapid transport of analytes. According to... Figure 1 As shown in (c), the VMSF / ITO electrode exhibits a clear interfacial delamination phenomenon, with the VMSF material, indium tin oxide and glass arranged from top to bottom, and the VMSF thickness being 96 nm.

[0038] Through negatively charged Fe(CN)6 3- and positively charged Ru(NH3)6 3+ Using two probes as analytes, and with bare ITO, SM@VMSF / ITO, and VMSF / ITO electrodes as working electrodes, respectively, VMSF was characterized using cyclic voltammetry. Figure 2 As shown. Figure 2 (a) and Figure 2 (b) in the figure represents Fe(CN)6 3- and Ru(NH3)6 3+ Potential-current curves were plotted on three electrodes (bare ITO electrode, SM@VMSF / ITO electrode, and VMSF / ITO electrode). No potential or current signals could be measured on the SM@VMSF / ITO electrode. This is because the presence of micelles hinders the detection of Fe(CN)6. 3- Or Ru(NH3)6 3+ The free diffusion of molecules from the solution to the electrode surface proves that the VMSF film is intact and crack-free. VMSF / ITO, compared to bare ITO electrodes, exhibits superior performance in the presence of negatively charged Fe(CN)6. 3- The signal decreases in the solution containing the positively charged Ru(NH3)6. 3+ The probe's signal is enhanced in solution due to the presence of silanol groups (p) in the VMSF channels. K a ~2) The negative charge results in different responses to probes with different charges. Simply put, negatively charged probes are repelled by the VMSF channels, while positively charged probes are attracted, indicating good charge selectivity of the VMSF / ITO electrode. For example... Figure 2In (c), when using the SM@VMSF / ITO electrode to test the electrically neutral probe FcMeOH, the hydrophobic interaction between the surfactant micelles (SM) and the probe molecules does not prevent the neutral probe molecules from reaching the electrode surface and generating a response. Compared to the bare ITO electrode and the VMSF / ITO electrode, the potential of the SM@VMSF / ITO electrode showed a significant shift to a higher potential when detecting FcMeOH, which is due to energy loss. The combined phenomena of these three electroactive probes indicate that the VMSF film was prepared intact and without cracks, and possesses excellent enrichment capabilities for positively charged probe molecules.

[0039] Characterization of composite probes, such as Figure 3 As shown. According to Figure 3 As shown in (a), atomic force microscopy (AFM) was used to clarify the lateral dimensions, interlayer thickness, and surface morphology of GO. Cross-sectional analysis of GO showed that the thickness of the monolayer material was 1.2 nm. Figure 3 As shown in (b), GO exhibits a good lamellar structure and rich surface wrinkle structure, which is typical of GO surface morphology. Figure 3 (c) in the figure is the Fourier Transform Infrared Spectroscopy (FT-IR) spectrum of GO, in which the OH stretching vibration (3416 cm⁻¹) is observed. -1 C=O stretching vibration (1731cm) -1 ), sp 2 Carbon (1625cm) -1 COC stretching vibration (1300cm) -1 ) and CO stretching vibration (1050cm) -1 Characteristic peaks can be observed at (), proving the presence of oxygen-containing groups on GO. Based on the above characterization results, graphene oxide material has been successfully prepared.

[0040] Sensor feasibility analysis, such as Figure 4 As shown. Figure 4 (a) and Figure 4(b) The feasibility of the DNase I-co-silicon channel dual-mode sensor construction strategy was analyzed using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The redispersed ternary complex of ruthenium(II), aptamer, and graphene oxide exhibited a low background signal of only 0.47 μA in solution (black line). After the addition of DNase I, the electrochemical signal remained low due to the effective protection of the aptamer CA125 by GO from cleavage; the signal change was only 0.3 μA compared to the solution without DNase I (blue line). Upon addition of the target substrate CA125, the aptamer complex desorbed from the GO sheets into the solution, releasing a large amount of probe molecules Ru(bpy)3 adsorbed by GO and the aptamer. 2+ The increased concentration of probe molecules in solution leads to the enrichment of positively charged probe molecules by the negatively charged pore electrode, resulting in a significant enhancement of the electrochemical signal response (red line). In electrochemiluminescence mode, such as... Figure 4 (c) and Figure 4 As shown in (d), the signal response enhancement mechanism is consistent with electrochemistry, and Ru(bpy)3 is a bifunctional cation probe with EC / ECL activity. 2+ In the presence of the co-reactant tripropylamine (TPA), ECL signals can be triggered under certain electrochemical conditions. The luminescence mechanism is as follows: Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ ; TPA−e - →TPA ·+ ; TPA ·+ -H + →TPA · ; TPA · +Ru(bpy)3 3+ →Ru(bpy)3 2+ ; Ru(bpy)3 2+ →Ru(bpy)3 2+ +hν(λ max (≈620nm); Among them, TPA ·+ It is a tripropylamine cationic free radical, TPA · It is a neutral free radical of tripropylamine, Ru(bpy)32+ For the excited state of tri(bipyridine)ruthenium(II), h is Planck's constant, ν is the frequency of light, and λ max This is the maximum emission wavelength.

[0041] During the positive scan of the potential, TPA and Ru(bpy)3 2+ It is oxidized by the electrode into TPA free radicals and Ru(bpy)3 3+ TPA free radicals and Ru(bpy)3 3+ A redox reaction occurs, resulting in the excited state of Ru(bpy)3. 2+ The light signal generated when the excited state returns to the ground state is finally recorded by a photomultiplier tube.

[0042] Optimization of detection conditions, such as Figure 5 As shown. Figure 5 (a) in the text refers to the signaling molecule Ru(bpy)3. 2+ The concentration was optimized, and Ru(bpy)3 was found to be... 2+ At a concentration of 5 μmol / L, graphene oxide has a certain effect on Ru(bpy)3. 2+ The strong adsorption force plays a dominant role, and even when the target analyte and aptamer are added to react, the probe molecules are difficult to desorb into the bulk solution. Although this results in a very low background signal, it also reduces the detection sensitivity of the analyte, thus affecting the detection performance. During the preparation of the complex, the Ru(bpy)3 group was gradually modulated... 2+ The concentration was such that the adsorption of the probe by graphene oxide and aptamer reached saturation, thus yielding the maximum change in electrochemiluminescence intensity Δ after the addition of the target compound CA125. I (△) I = I - I 0, where I The electrochemiluminescence signal intensity was measured after adding the target compound CA125 and DNase I. I 0 represents the background electrochemiluminescence signal intensity measured without the addition of the target compound CA125. In the solution without CA125, the system still exhibits a low ECL signal response after washing and separation. I 0), when Ru(bpy)3 2+ When the concentration increased to 20 μmol / L, with the specific recognition of the target substrate by the aptamer and the cyclic shearing effect of DNase I, the electrode exhibited a higher ECL signal response for the ternary composite system. I That is, to achieve GO and aptamer pair Ru(bpy)3 2+The adsorption capacity reached its maximum. Considering both low ECL background signal and the degree of absolute change in the ECL response, 20 μmol / L Ru(bpy)3 was selected for subsequent experiments. 2+ As the optimal incubation concentration. Figure 5 (b) of the study optimized the incubation time of DNase I with the aptamer. The results showed that extending the DNase I degradation reaction time was beneficial for the cyclic dissociation of the aptamer complex. After 60 min of reaction, the ECL intensity change tended to level off, reaching the reaction saturation point. To save testing time, a 60-min enzyme reaction time was selected as the optimal detection condition.

[0043] The dual-mode detection of CA125 in serum samples was performed using the DNase I-synergistic silicon channel dual-mode sensor described in Example 1. The process is as follows: First, a homogeneous reaction system was established, specifically by adding the composite probe Ru(bpy)3. 2+ / Apt&GO was mixed with CA125 standard solution at different concentrations (0.1 U / mL, 0.5 U / mL, 1 U / mL, 5 U / mL, 10 U / mL, 50 U / mL, and 100 U / mL) and 20 U of DNase I, and brought to a final volume with 0.01 mol / L PBS solution (pH 7.4). The mixture was incubated in a 37°C water bath for 60 min to obtain the post-reaction solution. The post-reaction solution was then subjected to electrochemical and electrochemiluminescence detection using the DNase I-co-silicon channel dual-mode sensor described in Example 1. Electrochemical detection was performed using differential pulse voltammetry at a scan rate of 50 mV / s. Electrochemiluminescence detection was performed in 0.3 mmol / L tripropylamine PBS solution, triggered by cyclic voltammetry with a potential range of 0 V to 1.4 V, a scan rate of 0.1 V / s, and a photomultiplier tube voltage of 400 V. Quantitative analysis of CA125 is based on the intensity of electrochemical and electrochemiluminescence signals, such as... Figure 6 As shown.

[0044] according to Figure 6 As shown in (a), the anodic peak current increases significantly with increasing CA125 concentration. According to... Figure 6 As shown in (b), EC intensity is related to logC. CA125 The linear regression curve, within the range of 0.1 U / mL-100 U / mL, has the following linear regression equation: I EC =1.42(±0.0511)logC CA125 +2.42 (±0.0439) (R) 2 The coefficient of determination is R, and R0 is the coefficient of determination. 2 =0.994), detection limit LOD = 91 mU / mL. The electrochemiluminescence intensity-time curve for CA125 in ECL mode is shown below. Figure 6 (c) and Figure 6 As shown in (d), the linear relationship of CA125 in the range of 0.001 U / mL-100 U / mL is I. ECL =1360(±48.7)logC CA125 +5319(±86.62)(R 2 =0.994), and the detection limit (LOD) calculated using three times the signal-to-noise ratio (S / N=3, where S is the signal and N is the noise) was determined to be 0.4 mU / mL.

[0045] Selective analysis such as Figure 7 As shown. Figure 7 (a) and Figure 7 As shown in (b), various other interfering agents (including CA199, CA15-3, CEA, AFP, Glu, and Lysine) were used to investigate the selectivity of the DNase I synergistic silicon channel dual-mode sensor. It was found that only CA125 induced a significant response in EC / ECL, while the other interfering agents showed almost no response, indicating that the DNase I synergistic silicon channel dual-mode sensor has excellent selectivity in CA125 determination.

[0046] The DNase I synergistic silicon channel dual-mode sensor of this application employs a three-electrode system, including a working electrode, a reference electrode, and a counter electrode. During detection, an electrolyte solution containing tripropylamine is used as the detection medium. The working electrode is an indium tin oxide electrode modified with a vertically ordered mesoporous silica film. A composite probe Ru(bpy)3 is used... 2+ / Apt&GO co-incubated with DNase I to achieve homogeneous detection of cancer antigen 125. This application utilizes the nanoconfining effect and electrostatic enrichment of VMSF to significantly enhance the cationic probe Ru(bpy)3. 2+ The electrochemical and electrochemiluminescence signals were detected. During the detection process, CA125 specifically recognized the aptamer to form a complex, inducing DNase I cyclic cleavage and releasing Ru(bpy)3. 2+ The molecules, after being efficiently enriched by VMSF, exhibit an enhanced EC / ECL response, thereby achieving highly sensitive dual-mode detection of CA125. The size exclusion and charge selectivity of VMSF effectively block the two-dimensional nanoprobe, reducing the background signal, while the synergistic cyclic shearing of DNase I further amplifies the detection signal. Furthermore, the DNase I-synergistic silicon channel dual-mode sensor of this application exhibits advantages such as a wide linear range, low detection limit, and high selectivity in CA125 detection, making it suitable for clinical diagnosis and bioanalysis of tumor markers such as ovarian cancer.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of this application, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of this application.

Claims

1. A DNase I-coordinated silicon channel dual-mode sensor, characterized in that, include: Working electrode, reference electrode, and counter electrode; The working electrode is a vertically ordered mesoporous silica nanochannel thin film modified electrode. The vertically ordered mesoporous silica nanochannel film has a negatively charged array of mesoporous nanochannels; the pore size of the nanochannels is 2nm~3nm.

2. The DNase I-coordinated silicon channel dual-mode sensor according to claim 1, characterized in that, The DNase I co-located silicon channel dual-mode sensor also includes: a composite probe; The composite probe is composed of tri(bipyridine)ruthenium(II), graphene oxide, and an aptamer; The aptamer is one of the following: cancer antigen 125 aptamer, carcinoembryonic antigen aptamer, alpha-fetoprotein aptamer, and cancer antigen 15-3 aptamer.

3. The DNase I synergistic silicon channel dual-mode sensor according to claim 2, characterized in that, The composite probe contains 10 μM to 30 μM of tri(bipyridine)ruthenium(II), 1 mg / mL to 5 mg / mL of graphene oxide, and 50 μM to 200 μM of aptamer.

4. A method for constructing a DNase I-coordinated silicon channel dual-mode sensor, characterized in that, To construct the DNase I co-silicon channel dual-mode sensor according to any one of claims 1 to 3, the following steps are included: S1. Mix ethanol, sodium nitrate solution and hexadecyltrimethylammonium bromide. After complete dissolution, add tetraethyl orthosilicate and stir at room temperature to pre-hydrolyze to obtain the precursor solution. S2. The electrode is placed in the precursor solution, and a constant current growth method is used to deposit the current. After deposition, the electrode is removed, rinsed, dried with nitrogen, and aged to obtain a silicon dioxide nanochannel modified electrode containing micelles. The electrode is one of indium tin oxide electrode, glassy carbon electrode, screen-printed electrode, gold electrode, fluorine-doped tin oxide electrode, graphite electrode, and carbon fiber electrode. S3. Immerse the silica nanochannel modified electrode containing micelles in an ethanol solution containing inorganic acid and stir for 1 min to 30 min to remove the micelles and obtain a vertically ordered mesoporous silica nanochannel thin film modified electrode. S4. Dissolve terpyridine ruthenium chloride in phosphate buffer solution, then add graphene oxide solution and aptamer solution, sonicate to combine, centrifuge and wash, remove supernatant, redisperse in buffer solution to obtain composite probe; S5. A vertically ordered mesoporous silica nanochannel film modified electrode is used as the working electrode, and a three-electrode system is formed with a reference electrode and a counter electrode. Combined with a composite probe, a DNase I synergistic silicon channel dual-mode sensor is obtained.

5. The method for constructing a DNase I-coordinated silicon channel dual-mode sensor according to claim 4, characterized in that, The molar concentration of the sodium nitrate solution is 0.01 mol / L to 0.5 mol / L; the concentration of the inorganic acid is 0.01 mol / L to 0.5 mol / L.

6. The method for constructing a DNase I-coordinated silicon channel dual-mode sensor according to claim 4, characterized in that, The amount of hexadecyltrimethylammonium bromide added is 1g to 2g; the amount of tetraethyl orthosilicate added is 1mL to 5mL.

7. The method for constructing a DNase I-coordinated silicon channel dual-mode sensor according to claim 4, characterized in that, The concentration of the phosphate buffer solution is 0.005 mol / L to 0.5 mol / L, and the pH value is 6.5 to 7.

5.

8. The method for constructing a DNase I-coordinated silicon channel dual-mode sensor according to claim 4, characterized in that, The amount of ruthenium chloride added is 1 mg to 10 mg; The volume ratio of the graphene oxide solution to the aptamer solution is 1~5:1; the concentration of the graphene oxide solution is 1mg / mL~5mg / mL; and the concentration of the aptamer solution is 50μM~200μM.

9. The method for constructing a DNase I-coordinated silicon channel dual-mode sensor according to claim 4, characterized in that, The applied current density is -0.05 mA / cm². 2 ~-1mA / cm 2 The aging temperature is 100℃~150℃, and the time is 12h~24h.

10. An application of a DNase I-coordinated silicon channel dual-mode sensor, characterized in that, The DNase I synergistic silicon channel dual-mode sensor according to any one of claims 1-3 is used for the detection of tumor markers for non-diagnostic purposes, specifically for the quantitative analysis of tumor markers by detecting the intensity of electrochemical signals or electrochemiluminescence signals. The tumor marker is one of cancer antigen 125, carcinoembryonic antigen, alpha-fetoprotein, cancer antigen 15-3, and cancer antigen 19-9.

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