Low-trigger-potential electrochemiluminescence aptamer sensor based on porous silicon as well as preparation method and application of low-trigger-potential electrochemiluminescence aptamer sensor

Abstract

The invention relates to the technical field of biological detection, and discloses a low-trigger-potential electrochemiluminescence aptamer sensor based on porous silicon as well as a preparation method and application of the low-trigger-potential electrochemiluminescence aptamer sensor. Wherein the working electrode is obtained by modifying an electrode with a porous silicon confinement platinum nanoparticle material, then covalently immobilizing an MMP-9 aptamer and closing a non-specific site; when the sensor is used for detection, a solution containing luminol and hydrogen peroxide is used as an electrolyte. According to the invention, PSi is adopted to limit PdNPs, so that a low-trigger-potential ECL signal of a luminol-hydrogen peroxide system is improved, ultra-sensitive rapid detection of trace MMP-9 is realized, and the method plays an important role in research on various physiological and pathological processes of pregnancy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a low trigger potential electrochemiluminescence aptamer sensor based on porous silicon and a preparation method and application thereof. BACKGROUND

[0002] Matrix metalloproteinases (MMPs) play a key role in many physiological and pathological processes of pregnancy, especially in extracellular matrix remodeling. Therefore, MMPs detection has important clinical significance in the field of obstetrics. For example, matrix metalloproteinase-9 (MMP-9) detection provides an objective molecular diagnostic tool for obstetrics, which has significant value in rapidly and accurately diagnosing premature rupture of membranes and identifying pregnant women at high risk of infection-related preterm birth, and helps to optimize clinical decision-making and improve pregnancy outcomes. Specifically, detecting MMP-9 in cervical vaginal secretions is a high-sensitivity and specific biomarker for diagnosing rupture of membranes (especially high rupture of membranes that are difficult to diagnose or atypical cases with symptoms), which is more accurate than traditional methods (such as pH test paper or fern-shaped crystal test), can effectively distinguish between ruptured membranes and normal secretions, and guide timely intervention. In addition, significantly increased MMP-9 levels in the serum, cervical vaginal secretions or amniotic fluid of pregnant women in the middle and late stages of pregnancy are key indicators for predicting preterm birth (especially those caused by intrauterine infection / inflammation), which helps to identify high-risk groups and guide preventive treatments such as anti-inflammatory, anti-infection or fetal lung maturation promotion. MMP-9 is also a key enzyme that mediates cervical collagen degradation and promotes cervical maturation, and its elevated levels can reflect the onset of cervical softening and dilation, which helps to assess the success of induced labor and predict imminent delivery (especially for those with preterm labor symptoms to determine whether they are true labor). Therefore, it is of great significance to develop convenient and efficient MMP-9 detection technology.

[0003] Electrochemiluminescence (ECL) technology combines the precise control of electrochemistry with the high sensitivity of chemiluminescence. By applying an electric potential on the electrode surface to excite the luminescence reaction, it has the advantages of low background noise, wide dynamic range and good controllability, and is an ideal platform for trace biomarker analysis.

[0004] Among the many ECL luminescence systems, the luminol-hydrogen peroxide (H2O2) system is widely used due to its excellent performance: luminol has a very high luminescence quantum yield (>5%), and its luminescence efficiency is significantly better than that of isoluminol and other similar substances; its oxidized state intermediate is stable, and the luminescence reaction has good reversibility, which is beneficial to signal repeatability and stability; the reaction can be efficiently carried out at a mild near-neutral physiological pH (7.0-8.5), which is compatible with biological samples; the raw materials are low in cost and easy to modify chemically, which is convenient for the construction of sensing interfaces. These characteristics make luminol one of the most practical ECL luminophores in biomedical detection.

[0005] Low-triggered ECL technology (triggering potential usually in the range of -0.3 V to +0.3 V) has attracted much attention in recent years. The core of this strategy is to use efficient catalysts to generate strong ROS at low potentials to drive the ECL process. This strategy can maximize the inhibition of electrode side reactions and biological matrix interference, obtain ultra-low background, significantly reduce energy consumption, improve device portability, reduce electrode corrosion, enhance sensor stability and service life, and is particularly suitable for super-sensitive and anti-interference detection in complex biological samples, providing new tools for precision medicine.

[0006] The introduction of nano-catalysts is an effective way to achieve high efficiency and stable ECL emission of the luminol-H2O2 system at low potentials. However, the traditional catalyst synthesis methods (such as hydrothermal method, chemical reduction method) often have limited performance due to particle agglomeration and active site masking, and the low stability of their drop-coating onto the electrode surface easily leads to catalytic activity decay. The use of porous materials to confine the synthesis of nano-catalysts can significantly drive / sensitize the low-potential ECL of luminol by improving the activity and stability of nano-catalysts through confinement effect and interface interaction.

[0007] Among the porous materials, Porous silicon (PSi) has attracted much attention due to its unique structure and semiconductor properties. PSi can be fabricated on monocrystalline silicon wafers by electrochemical anodization (electrochemical etching) at low cost and in batches. By adjusting the etching parameters, the mesoporous channel diameter, specific surface area and porosity (40%-90%) of PSi can be precisely controlled. Combined with its surface properties, PSi nanochannel films can simultaneously construct high-performance confined nanocatalyst systems and low-biofouling interfaces. The core advantages include: (1) It can improve the electrode interface micro zone concentration: When the co-reactants, ROS or active intermediates in PSi undergo one-dimensional restricted diffusion, a high micro zone concentration can be formed at the electrode interface, which will obviously lead to higher detection sensitivity. (2) Confined synthesis of small size / high dispersion nanocatalysts, and interface interaction to improve catalytic activity and stability: The mesoporous nanochannel has a high specific surface area, and the physical constraints of the confined space can regulate the nucleation and growth kinetics of nanoparticles, allowing the preparation of small size, high dispersion, high activity nanocatalysts. In addition, the semiconductor matrix and noble metal nanocatalysts can form strong electronic metal-support interaction (EMSI) to improve charge transfer efficiency and improve the activity of nanocatalysts. The confinement effect can also prevent the shedding of noble metal nanocatalysts and other materials during electrochemical cycling, significantly improving the stability of nanocatalysts. (3) Spontaneous reducibility and rich surface modifiability: The freshly etched PSi surface is rich in Si-Si-H bonds, which can spontaneously reduce noble metal ions without the need for external chemical reducing agents to directly prepare highly dispersed noble metal nanocatalysts. In addition, after chemical modification, PSi can be used to immobilize biological probes such as aptamers and antibodies to construct biological recognition interfaces. (4) Constructing an "physically blocked" anti-biofouling interface: By adjusting the electrochemical etching parameters, the size of the PSi nanochannel can be precisely controlled, and its mesoporous structure can act as a "molecular sieve" to selectively size-exclude large molecule interferents such as serum proteins.

[0008] CN120334320A discloses an electrochemical sensor based on a porous silicon platinum nanoflower DNA composite material, a preparation method and application thereof. The invention prepares a porous silicon layer on the polished surface of a silicon wafer, then modifies a platinum nanoflower and a T-rich thiolated DNA probe, and plates a metal film on the other side of the silicon wafer to obtain an electrochemical sensor, which is applied to the detection of melamine. The DNA probe can adsorb melamine on the surface of the material through hydrogen bonds and produce an electric current response linearly related to the concentration of melamine; the hydroxyl groups on the surface of the porous silicon can enhance the adsorption effect of melamine; the flower-shaped platinum nanomaterial can enhance the sensitivity of the sensor; in addition, the negative charge carried by the surface of the porous silicon has an electrostatic repulsion effect with common coexisting substances of melamine, making the sensor have good selectivity. This high-performance nanosensor provides a new practical method for the on-site rapid detection of melamine.

[0009] However, there is no related ECL detection report for the ultra-sensitive, anti-interference and rapid detection of trace matrix metalloproteinase-9 in serum. Such detection technology is expected to significantly improve the accuracy and timeliness of preterm birth prediction and risk stratification and premature rupture of membranes. SUMMARY

[0010] The present application aims at the problems of low detection sensitivity, great detection difficulty and high or low potential detection interference in the detection of matrix metalloproteinase-9 in biological samples in the prior art, and provides a porous silicon confined palladium nanoparticle, realizes low potential triggered luminol-hydrogen peroxide system ECL signal, realizes trace and rapid ECL detection effect of MMP-9, and plays an important role in the research of many physiological and pathological processes of pregnancy.

[0011] To achieve the above object, the technical scheme adopted by the present application is: A low trigger potential electrochemical luminescence aptamer sensor based on porous silicon, comprising a three-electrode system, wherein the working electrode is obtained by covalently fixing MMP-9 aptamer and blocking non-specific sites after modifying porous silicon confined platinum nanoparticle material on the electrode; The sensor detects a solution containing luminol and hydrogen peroxide as an electrolyte.

[0012] The present application is based on in-situ reduction of PdNPs by PSi, and the material of PSi confined PdNPs is prepared to enhance the ECL signal of luminol-H2O2 at low trigger potential. PSi has high specific surface area, good biocompatibility and adjustable pore structure, and is considered as an ideal nanocarrier material. In-situ reduction of Pd is realized by using the rich Si-H bonds on the surface of PSi, and a material with uniform size and small size is obtained, which effectively enhances the catalytic activity and further enhances the ECL signal of luminol-H2O2 system. PSi is a semiconductor material with unique structure. The PSi layer is prepared on a single crystal silicon wafer at low cost by electrochemical anodic oxidation method (electrochemical etching), and the PSi layer is detached from the surface of the silicon wafer through electrochemical polishing process, and becomes particles after ultrasonic crushing, which can be batch prepared.

[0013] The PSi used in the present application is an electrochemically etched PSi nanoparticle, so that the PSi has a small pore size, which is conducive to the synthesis of small size, high dispersibility and high activity PdNPs. In addition, the semiconductor matrix and the PdNPs can form a strong metal-support electron interaction (EMSI) to improve the charge transfer efficiency and improve the activity of the PdNPs. The freshly etched PSi surface is rich in Si-Si-H bonds, which can spontaneously reduce noble metal ions, and high-dispersible PdNPs can be directly prepared without the need for an external chemical reducing agent. In the nanochannel of the PSi, the free radicals generated by the PdNPs catalysis can react with luminol, which increases the collision probability of the reactants and intermediates while forming a high micro-zone concentration, thereby facilitating the generation of more excited states and increasing the ECL intensity.

[0014] In addition, PSi generates a small amount of silicon hydroxyl groups during use, which can be covalently connected to antibodies through chemical bonding to construct an efficient biosensing interface. In addition, the size of the PSi nanochannel can play a "molecular sieve" effect to selectively size-exclude macromolecular interferents such as serum proteins. An anti-biofouling interface is constructed. When matrix metalloproteinase-9 is present in the solution, the aptamer on the interface can specifically capture the matrix metalloproteinase-9 to form a complex, which causes a steric hindrance effect, reduces the diffusion of luminol and H2O2 to the electrode surface, and thus reduces the ECL signal. Based on this specific recognition, ECL detection of matrix metalloproteinase-9 in human samples can be achieved.

[0015] The working electrode preparation comprises the following steps: Step 1: mixing the porous silicon dispersion liquid with the platinum salt solution to obtain a porous silicon confined platinum nanoparticle material by in-situ reduction; modifying the porous silicon confined platinum nanoparticle material on the surface of the electrode, and obtaining a porous silicon confined platinum nanoparticle material modified electrode by aging treatment; Step 2: immersing the porous silicon confined platinum nanoparticle material modified electrode in an epoxy silane solution and standing for reaction, then immersing it in a solution containing MMP-9 aptamer and a solution containing bovine serum albumin for incubation, to obtain the working electrode.

[0016] In the present application, PdNPs are confined in the porous material of PSi. The confinement of the porous material can significantly improve the stability of PdNPs. In addition, the porous material provides a dispersion space, which can effectively increase the active sites of PdNPs, and thus improve the catalytic activity and stability of PdNPs. In addition to the effect of the confinement space on the nucleation kinetics of nanomaterials, the semiconductor matrix of PSi and PdNPs can also form a strong metal-support electron interaction (EMSI) to improve the activity of PdNPs.

[0017] The mass concentration of the porous silicon in the porous silicon dispersion liquid is 0.1-3 mg / mL. The platinum salt includes [Pd(NH3)4]SO4, and the platinum salt has a more obvious effect.

[0018] The molar concentration of the platinum salt in the platinum salt solution is 1-12 mM; preferably, the molar concentration of the platinum salt is 5-10 mM; further preferably, the molar concentration of the platinum salt is 5-8 mM. The in-situ reduction in step 1 is a shaking reaction at room temperature for 2-60 min; preferably, the shaking reaction is performed for 10-20 min.

[0019] The process of modifying the porous silicon confined platinum nanoparticle material on the electrode surface specifically includes: dropping the porous silicon confined platinum nanoparticle material on the electrode surface, naturally air-drying, and then obtaining a stable PdNPs@PSi modification layer on the electrode through aging treatment. A small amount of silicon hydroxyl groups is generated in the use process of PSi, and In-O-Si and Sn-O-Si bonds are formed on the ITO electrode surface in the aging process, so that a stable PdNPs@PSi modification layer is obtained. The reason for adopting vacuum aging is to avoid the oxidation of PdNPs in air at high temperature.

[0020] The aging treatment is vacuum aging at 120-150℃ for 8-12 h.

[0021] The epoxy silane includes γ-glycidoxypropyltrimethylsilane.

[0022] The molar solution concentration of the solute in the epoxy silane-containing solution is 2.0 mM-3.0 mM. The standing reaction in step 2 is performed for 30-60 min, and the standing reaction temperature is 20-40℃.

[0023] The solution containing MMP-9 aptamer is a buffer solution with a molar concentration of MMP-9 aptamer of 0.1-0.5 μM; the buffer solution is one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution, or a phosphate buffer solution, and the pH is 6.5-7.5.

[0024] The mass concentration of the bovine serum albumin in the bovine serum albumin-containing solution is 0.05-2 mg / ml.

[0025] The incubation in step 2 is incubation in a temperature environment of 4-6℃ for 10-120 min.

[0026] Preferably, after each incubation, a buffer solution is used for washing to remove the excess raw materials that are not connected; The solvent of the porous silicon dispersion is water, the solvent of the platinum salt solution is water, the solvent of the solution containing bovine serum albumin is one or more buffers such as phosphate buffer, HEPES buffer, etc. with a pH of 6.5-7.5; the solvent of the epoxysilane solution includes ethanol or isopropanol.

[0027] The molar concentration of luminol in the electrolyte is 5-300 μmol / L, and the molar concentration of hydrogen peroxide is 10-1000 μmol / L.

[0028] Preferably, the triggering luminescence potential of the sensor is -0.1 V to 0 V and 0.2 V to 0.3 V. This low triggering point can minimize electrode side reactions and biological matrix interference, thereby improving detection sensitivity and stability.

[0029] The present invention also provides a method for preparing the porous silicon-based low trigger potential electrochemiluminescence aptamer sensor, comprising the steps of: Step 1: mixing a porous silicon dispersion with a platinum salt solution, and performing in-situ reduction to obtain a porous silicon-confined platinum nanoparticle material; modifying the porous silicon-confined platinum nanoparticle material on an electrode surface, and performing an aging treatment to obtain a porous silicon-confined platinum nanoparticle material-modified electrode; Step 2: immersing the porous silicon confined platinum nanoparticle material modified electrode in a solution containing epoxysilane and allowing it to react. After taking it out, immersing it in a solution containing an MMP-9 aptamer and a solution containing bovine serum albumin in sequence to incubate to obtain a working electrode; Step 3: A three-electrode system is formed by using a working electrode, a counter electrode, and a reference electrode to obtain the sensor. During detection, the sensor uses a solution containing luminol and hydrogen peroxide as an electrolyte.

[0030] The present invention also provides the use of the low trigger potential electrochemiluminescent aptamer sensor in detecting MMP-9 for non-diagnostic or non-therapeutic purposes, such as in research work on detecting MMP-9 in serum for premature birth prediction, risk stratification, and premature rupture of membranes prediction.

[0031] The present invention also provides a method for detecting MMP-9 by low trigger potential electrochemiluminescence, comprising the steps of: incubating the working electrode with a test solution and then rinsing to obtain a test electrode; using the test electrode as the working electrode, a reference electrode, and a counter electrode to form a three-electrode system, and performing electrochemiluminescence signal detection using an electrolyte solution containing luminol and hydrogen peroxide as the detection solution.

[0032] The detection principle is as follows: when there is matrix metalloproteinase-9 in the serum sample, the aptamer is combined with the matrix metalloproteinase-9, and the large complex combined on the electrode interface will produce a significant steric hindrance effect on the diffusion mass transfer of luminol and hydrogen peroxide, thereby changing the ECL signal of the electrode. Therefore, according to the quenching degree of the electrochemiluminescence signal, the electrochemiluminescence detection of matrix metalloproteinase-9 can be realized.

[0033] The concentration of MMP-9 in the to-be-detected solution is below 100 ng / mL; The to-be-detected solution comprises one or more interference compounds such as glucose, inorganic salt, cytokine and tumor marker; and the total molar concentration of the interference compounds is below 0.5 M.

[0034] Compared with the prior art, the present application has the following beneficial effects: (1) The present application based on PSi confined PdNPs enhances the low trigger potential ECL signal of luminol-H2O2 system. PSi has adjustable and uniform nanochannel array, and confines PdNPs in the pore of PSi, thereby improving the stability. In addition, the simultaneously confined PdNPs have high peroxidase-like activity. The confinement effect of PSi and the metal-carrier electron interaction of the semiconductor significantly improve the catalytic activity of PdNPs, and the performance of the ECL signal of the luminol-H2O2 system sensitized by PSi confined PdNPs is significantly higher than that of non-PSi confined PdNPs.

[0035] (2) The present application drops PdNPs@PSi on the surface of an ITO electrode, and obtains a stable PdNPs@PSi modified layer on the electrode through vacuum high-temperature aging treatment. In the aging process, PdNPs@PSi forms In-O-Si and Sn-O-Si bonds with the surface of the ITO electrode, thereby obtaining a stable PdNPs@PSi modified layer. Vacuum aging can avoid the oxidation of PdNPs in air at high temperature and prevent the catalytic performance of PdNPs from being reduced.

[0036] (3) The outer surface of PSi in the present application is easy to modify, and after modification by an epoxy group, the antibody can be covalently fixed, and an immune recognition interface is prepared. When there is a detection substance MMP-9 in the solution, the aptamer on the immune recognition interface can specifically capture MMP-9 to form a complex. The steric hindrance effect caused by the complex will reduce the diffusion of luminol and H2O2 to the electrode surface, thereby reducing the ECL signal. Based on this specific recognition, the ECL detection of MMP-9 can be realized. When the immune sensor of the present application is used for detection, only the working electrode needs to be incubated with the to-be-detected sample, and then the electrochemical signal can be measured, without the need for a sandwich-type immune analysis mode, so that the detection is fast and the cost is low. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 SEM images of the micro-morphology and structure characterization of various electrodes prepared in Example 1, Figure 1 (A) is the SEM image of the porous silicon (PSi) modified ITO electrode (PSi / ITO); Figure 1 (B) is the SEM image of the PdNPs@PSi modified ITO electrode (PdNPs@PSi / ITO) after in-situ reduction of PdNPs on PSi; Figure 1 (C) is the TEM image of the porous silicon (PSi); Figure 1 (D) is the TEM image of PdNPs@PSi synthesized with Pd source of PdCl2; (E) is the TEM image of PdNPs@PSi synthesized with Pd source of [Pd(NH3)4]SO4; Figure 1 (F) and Figure 1 (G) is the elemental analysis image of PdNPs@PSi; Figure 1 (H) is the top-view TEM image of PdNPs@PSi; Figure 1 (I) is the lattice structure image of PdNPs.

[0038] Figure 2 XPS spectra of PdNPs@PSi aged in different environments in Example 1, Figure 2 (A) is the comparison of XPS spectra of PdNPs@PSi aged in vacuum, in air and without aging; Figure 2 (B) is the Pd3d spectrum of PdNPs@PSi aged in vacuum; Figure 2 (C) is the Pd3d spectrum of PdNPs@PSi without aging; Figure 2 (D) is the Pd3d spectrum of PdNPs@PSi aged in air.

[0039] Figure 3 ECL signal graphs of PdNPs@PSi / ITO continuous scanning obtained from PdNPs@PSi aged in different environments and different Pd precursors in Example 1, Figure 3 (A) is aged in vacuum; Figure 3 (B) is aged in air; Figure 3 (C) is without aging; Figure 3 (D) is the ECL signal graph of PdNPs@PSi / ITO continuous scanning obtained from PdNPs@PSi synthesized with Pd source of PdCl2.

[0040] Figure 4 Electrochemical detection related data graphs of different electrodes prepared in Example 1, Figure 4 (A) is the ECL signal of different electrodes in phosphate buffer solution (0.01 M, pH = 7.4) containing 100 μM luminol and 1 mM hydrogen peroxide; Figure 4Middle (B) shows the ECL-potential curves of different electrodes (ITO, PSi / ITO and PdNPs@PSi / ITO); Figure 4 Middle (C) shows the CV signals of different electrodes in phosphate buffer solution (0.01 M, pH = 7.4) containing 100 μM luminol and 1 mM hydrogen peroxide; Figure 4 Middle (D) shows the CV curves of different electrodes (ITO, PSi / ITO, PdNPs@PSi / ITO, and PdNPs / ITO) in 1MH2SO4 at a scan rate of 50 mV / s. Figure 4 Middle (E) shows electrodes with different modifications in phosphate buffer solution (0.01 M, pH = 7.4) containing luminol (100 μM) and H2O2 (1 mM). PMT = 700 V, scan rate 100 mV / s, scanning range –0.2 V to 0.3 V. Figure 4 Middle (F) shows the ECL intensity of PdNPs@PSi / ITO after adding luminol to the test solution without hydrogen peroxide at –0.2 V without electrolysis (left) and electrolysis (right); Figure 4 Middle (G) shows the ECL intensity of PdNPs@PSi / ITO in the presence of hydrogen peroxide, after 120 s without electrolysis (left) and electrolysis (right) at –0.2 V, and then the electrolysis was stopped and luminol was added, PMT = 700 V; Figure 4 The middle (H) is a parallel test of the parallelism of the three electrodes; by changing the voltage from –0.2V to 0.3V ( Figure 4 (I)) and from 0.3V to –0.2V ( Figure 4 Middle (J) Step pulse, ECL transient of PdNPs@PSi / ITO, the yellow curve indicates the applied potential step, PMT = 700 V.

[0041] Figure 4 CV curves of different electrodes in Example 1 and the linear relationship between CV peak current and the square root of scan rate, Figure 5 Middle (A) is the CV curve of ITO electrode; Figure 5 Middle (B) is the CV curve of PSi / ITO electrode; Figure 5 Middle (C) is the CV curve of PdNPs@PSi / ITO electrode; Figure 5 Middle (D) is the linear relationship between the CV peak current of the ITO electrode and the square root of the scan rate; Figure 5 Middle (E) is the linear relationship between the CV peak current of the PSi / ITO electrode and the square root of the scan rate; Figure 5 (F) in the middle is the linear relationship between the CV peak current of the PdNPs@PSi / ITO electrode and the square root of the scan rate.

[0042] Figure 5CV signals of electrodes with different modification in different atmospheres in PBS for Example 1, Figure 6 CV signals of electrodes with different modification in different atmospheres in PBS for Example 1, Figure 6 CV signals of electrodes with different modification in different atmospheres in PBS for Example 1, Figure 6 ECL signals of electrodes in different atmospheres for Example 1, Figure 6 UV-Vis absorption spectra of different electrodes (test condition: 0.01M NaAc buffer solution containing 1 mM TMB and 1 mM H2O2, pH=4) for Example 1, Figure 6 ECL response of PdNPs@PSi / ITO in PBS (0.01M, pH=7.4) containing luminol (100 μM) and H2O2 (1 mM) with the addition of TBA (1 mM), BQ (100 μM) and Thiourea (1 mM) for Example 1, where I and I0are the ECL signals with or without ROS scavengers, respectively, Figure 6 Mechanism summary of PdNPs@PSi promoting low trigger potential electrochemiluminescence of luminol for Example 1.

[0043] Figure 6 Effect of different precursor concentrations and oscillation times on ECL signals of PdNPs@PSi / ITO electrodes for Example 1, Figure 7 Optimization of [Pd(NH3)4]SO4 precursor concentration for Example 1, where the oscillation time is fixed at 20 min, Figure 7 Time optimization of [Pd(NH3)4]SO4 oscillation reaction with PSi for Example 1, where the detection solution is phosphate buffer solution (0.01M, pH=7.4) containing luminol (100 μM) and H2O2 (1 mM), PMT=700 V, scan rate is 100 mV / s, and the scan range is –0.2 V~0.3 V.

[0044] Figure 7 Electrochemical data graphs of different electrodes in different electrolytes for Example 1, Figure 8 CV curves of different electrodes in 0.1M KCl containing 2.5 mM [Fe(CN)6] 3– / 4– for Example 1, Figure 8 EIS graphs of different electrodes in 0.1M KCl containing 2.5 mM [Fe(CN)6] 3– / 4– for Example 1, Figure 8 ECL signal graphs of different electrodes in PBS solution (0.01M, pH 7.4) containing 100 μM luminol and 1 mM H2O2 for Example 1.

[0045] Figure 8Electrochemical signals of the electrode in Example 1 under different aptamer concentrations, aptamer incubation time and antigen binding time, Figure 9 (A) is the optimization of aptamer concentration; Figure 9 (B) is the optimization of aptamer incubation time; Figure 9 (C) is the optimization of antigen binding time.

[0046] Figure 9 ECL signals of the BSA / Apt / PdNPs@O-PSi / ITO electrode in Example 1 and PBS incubated with different concentrations of MMP-9 Figure 10 (A) and the calibration curve Figure 10 (B) after incubation, PMT = 700 V, sweep rate is 100 mV / s, and the scanning range is -0.2 V ~ 0.3 V.

[0047] Figure 10 Anti-interference and stability of the BSA / Apt / PdNPs@O-PSi / ITO electrode in Example 1, Figure 11 (A) is the anti-interference ability of the BSA / Apt / PdNPs@O-PSi / ITO electrode to different species; Figure 11 (B) is the reproducibility of the electrode incubated with MMP-9; Figure 11 (C) is the long-term stability of the electrode incubated with MMP-9, PMT = 700 V, sweep rate is 100 mV / s, and the scanning range is -0.2 V ~ 0.3 V. DETAILED DESCRIPTION

[0048] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. Any modification or equivalent replacement made by those skilled in the art based on the technical solutions of the present application without departing from the spirit and scope of the present application should be covered within the protection scope of the present application.

[0049] The raw materials used in the following specific embodiments are all purchased from the market.

[0050] Example 1 The PdNPs@PSi modified electrode prepared by in-situ reduction of PdNPs on porous silicon covalently fixes the MMP-9 aptamer as the detection object on the outer surface to prepare a working electrode, and the process of detecting MMP-9 in a serum sample by electrochemiluminescence method is as follows: (1) Preparation of porous silicon by electrochemical etching: Freshly etched porous silicon is obtained by the method of electrochemical etching, specifically as follows: p ++PSi was prepared by anodic oxidation of boron-doped Si wafers in an electrolyte consisting of hydrofluoric acid (HF) and ethanol (95%, v:v=4:1). 2 The silicon wafer was etched at a constant current density of 100 nm for 600 seconds, followed by etching with a 3.3% HF-ethanol solution for 180 seconds to remove the PSi layer from the silicon substrate. After drying the material in a vacuum oven for two hours, a certain amount of PSi powder was weighed and dispersed in 95% ethanol to obtain a 2 mg / mL PSi ethanol dispersion. The porous silicon layer was then ultrasonically disrupted at 500W for 30 minutes to obtain a dispersion of nanoscale PSi.

[0051] (2) Preparation of PdNPs@PSi / ITO The above 2 mg / mL PSi ethanol dispersion was mixed with 5 mM [Pd(NH3)4]SO4 solution, and after shaking for 15 minutes in the dark, a 1 mg / mL PdNPs@PSi suspension was obtained. 20 μL was drop-coated on the surface of the indium tin oxide (ITO) electrode twice. After it was naturally air-dried, it was aged at 150°C overnight for 12 hours to obtain a PdNPs@PSi modified electrode.

[0052] (3) Preparation of PdNPs@O-PSi / ITO: PdNPs@O-PSi / ITO was used as the substrate electrode, and γ-glycidyloxypropyltrimethylsilane (GPTMS) was used as the cross-linking agent to covalently cross-link the antibody and PSi layer.

[0053] The specific experimental steps are as follows: immerse the PdNPs@PSi / ITO electrode in an ethanol solution of GPTMS (2.26 mM), let it react at room temperature for 1 hour, modify the PSi surface with GPTMS through silanization reaction, and wash the electrode with deionized water to obtain a PdNPs@O-PSi / ITO electrode.

[0054] (4) Preparation of sensors To prepare the recognition interface, a PdNPs@O-PSi / ITO electrode was immersed in a phosphate buffer solution (pH 7.4) containing the MMP-9 aptamer (0.3 μM, 40 μL) and incubated at 4°C for 90 minutes. The electrode was then washed thoroughly with 0.1 M PBS, pH 7.4. The electrode with the antibody covalently immobilized was designated Apt / PdNPs@O-PSi / ITO. Subsequently, the Apt / PdNPs@O-PSi / ITO electrode was placed in a 1 wt% bovine serum albumin (BSA) solution (0.01 M PBS, pH 7.4) and incubated at room temperature for 15 minutes to block nonspecific binding sites, resulting in the immunosensor (BSA / Apt / PdNPs@O-PSi / ITO).

[0055] (5) MMP-9 electrochemiluminescence detection To detect MMP-9, the BSA / Apt / PdNPs@O-PSi / ITO electrode was incubated with 40 μL of MMP-9 at different concentrations at 4 °C for 60 min, and then the electrode was slowly rinsed with PBS (0.01 M, pH = 7.4) solution to wash off the unbound MMP-9, and the obtained electrode was recorded as MMP-9 / BSA / Apt / PdNPs@O-PSi / ITO.

[0056] The obtained electrode was subjected to ECL detection, and the electrolyte solution used for testing was PBS (0.01 M, pH = 7.4) solution containing 100 μM luminol and 1 mM H2O2. The ECL process was triggered by continuous CV scanning, and the CV scanning potential scanning range was -0.2-0.3 V, and the scanning rate was 0.1 V / s. The voltage of the photomultiplier tube (PMT) was set to 700 V.

[0057] Figure 11 The micro-morphology and structure characterization diagrams of various electrodes prepared in Example 1 are shown in FIG. 1, Figure 1 FIG. 1(A) is a SEM diagram of the porous silicon (PSi) modified ITO electrode (PSi / ITO); Figure 1 FIG. 1(B) is a SEM diagram of the PdNPs@PSi modified ITO electrode (PdNPs@PSi / ITO) obtained after in-situ reduction of PdNPs on the porous silicon; Figure 1 FIG. 1(C) is a TEM diagram of the porous silicon (PSi); Figure 1 FIG. 1(D) is a TEM diagram of the PdNPs@PSi synthesized using PdCl2 as the Pd source; and FIG. 1(D) is a TEM diagram of the PdNPs@PSi synthesized using [Pd(NH3)4]SO4 as the Pd source; Figure 1 FIG. 1(F) and Figure 1 FIG. 1(G) is an elemental analysis diagram of the PdNPs@PSi; Figure 1 FIG. 1(H) is a top-view TEM diagram of the PdNPs@PSi; Figure 1 FIG. 1(I) is a lattice structure diagram of the PdNPs.

[0058] As shown in Figure 1 FIG. 1(A) and Figure 1 FIG. 1(B) are a scanning electron microscope diagram of the porous silicon (PSi) modified ITO electrode (PSi / ITO) and a scanning electron microscope diagram of the PdNPs@PSi modified ITO electrode (PdNPs@PSi / ITO) obtained after in-situ reduction of PdNPs on the porous silicon, and it can be seen that the PSi and the PdNPs@PSi are both successfully modified on the ITO surface and the dispersed phases are relatively uniform. Figure 1 FIG. 1(C) is a transmission electron microscope diagram of the prepared porous silicon (PSi) particles, and it can be seen that the internal part contains long strip-shaped pore structures. Figure 1The PdNPs@PSi prepared by using PdCl2 as the palladium source in the middle (D) can be seen to have a larger size and uneven distribution of PdNPs. In comparison Figure 1 The PdNPs@PSi prepared by using [Pd(NH3)4]SO4 as the palladium source in the middle (E) has a small size and uniform distribution. The reason is that, in the deposition process using PdCl2 as the palladium source, the insulating oxide formed on the surface of silicon inhibits uniform nucleation, while [Pd(NH3)4] 2+ The release of ammonia molecules during reduction locally produces a high pH value, thereby significantly accelerating the dissolution of the oxide, exposing fresh silicon on the surface for reduction and nucleation of more Pd, and the accumulation of the oxide on the surface of porous Si is limited, making the deposition more uniform. Figure 1 The middle (F) and Figure 1 The middle (G) is the element mapping characterization, which can be seen that the Si, O and Pd elements are uniformly distributed in the material, and the Figure 1 The middle (H) and Figure 1 The middle (I) is a high-resolution TEM image of PdNPs@PSi, which can be seen that the PdNPs are uniformly distributed, and the 0.22 nm lattice spacing corresponds to the Pd(111) crystal face, further proving the successful preparation of PdNPs@PSi.

[0059] The reaction equation of PdNPs@PSi prepared by using [Pd(NH3)4]SO4 as the palladium source is as follows: Si + 2H2O→SiO2+ 4H + + 4e ‒ [Pd(NH3)4] 2+ + 2e ‒ → Pd + 4NH3 SiO2+ 2H2O→Si(OH) 4(aq) Figure 1 The XPS spectra of PdNPs@PSi aged in different environments in Example 1 are shown in Figure 2 The middle (A) is a comparison of the XPS spectra of PdNPs@PSi obtained by not aging, air aging and vacuum aging. Figure 2 The middle (B) is the Pd3d spectrum of PdNPs@PSi aged in vacuum. Figure 2 The middle (C) is the Pd3d spectrum of PdNPs@PSi not aged. Figure 2 The middle (D) is the Pd3d spectrum of PdNPs@PSi aged in air.

[0060] As shown in Figure 2 The middle (A) compares the XPS spectra of PdNPs@PSi obtained by not aging, air aging and vacuum aging. Figure 2Figure 1 shows the ECL signals of the PdNPs@PSi / ITO electrodes prepared in Example 1 under different conditions, wherein (A) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by vacuum aging; Figure 2 Figure 1 (C) shows that there are two peaks at 339.5 eV and 334.2 eV in the spectra of the vacuum-aged and unaged XPSPd2p, which correspond to Pd(0) 3d5 / 2 and 3d3 / 2, respectively, and there are smaller peaks at 337 eV and 342.1 eV, which belong to Pd(II), proving that vacuum aging can effectively prevent PdNPs from being oxidized by oxygen in the air, thereby affecting the catalytic activity thereof. However, as shown in Figure 1 (B), after air aging, in addition to the Pd(0) peaks, there are obvious Pd(II) peaks, and the peak height is obviously higher than that of Pd(0), proving the generation of a large amount of Pd oxide, which proves that the generation of Pd oxide is one of the reasons affecting the catalytic activity thereof. Figure 2

[0061] Figure 1 (D) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by replacing [Pd(NH3)4]SO4 with PdCl2 as the Pd source to synthesize PdNPs@PSi. Figure 2 Figure 2 shows the ECL signals of the PdNPs@PSi / ITO electrodes prepared in Example 1 under different conditions, wherein (A) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by vacuum aging; Figure 3 Figure 2 (B) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by air aging; Figure 3 Figure 2 (C) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by unaging; Figure 3 Figure 2 (D) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by replacing [Pd(NH3)4]SO4 with PdCl2 as the Pd source to synthesize PdNPs@PSi. Figure 3 Figure 3 shows the ECL signals of the PdNPs@PSi / ITO electrodes prepared in Example 1 under different conditions, wherein (A) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by vacuum aging; Figure 3 (B) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by air aging;

[0062] Figure 3 (C) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by unaging; Figure 3 Figure 3 (D) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by replacing [Pd(NH3)4]SO4 with PdCl2 as the Pd source to synthesize PdNPs@PSi. Figure 3 Figure 4 shows the ECL signals of the PdNPs@PSi / ITO electrodes prepared in Example 1 under different conditions, wherein (A) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by vacuum aging; Figure 3 Figure 4 (D) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by replacing [Pd(NH3)4]SO4 with PdCl2 as the Pd source to synthesize PdNPs@PSi. Figure 5 shows the ECL signals of the PdNPs@PSi / ITO electrodes prepared in Example 1 under different conditions, wherein (A) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by vacuum aging;

[0063] Figure 5 (B) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by air aging; Figure 3 Figure 5 (C) is the ECL signal of the PdNPs@PSi / ITO electrode prepared by unaging; Figure 4(A) shows the ECL signals of different electrodes in phosphate buffer solution (0.01 M, pH = 7.4) containing 100 μM luminol and 1 mM hydrogen peroxide; Figure 4 Middle (B) shows the ECL-potential curves of different electrodes (ITO, PSi / ITO and PdNPs@PSi / ITO); Figure 4 Middle (C) shows the CV signals of different electrodes in phosphate buffer solution (0.01 M, pH = 7.4) containing 100 μM luminol and 1 mM hydrogen peroxide; Figure 4 Middle (D) shows the CV curves of different electrodes (ITO, PSi / ITO, PdNPs@PSi / ITO, and PdNPs / ITO) in 1MH2SO4 at a scan rate of 50 mV / s. Figure 4 Middle (E) shows electrodes with different modifications in phosphate buffer solution (0.01 M, pH = 7.4) containing luminol (100 μM) and H2O2 (1 mM). PMT = 700 V, scan rate 100 mV / s, scanning range –0.2 V to 0.3 V. Figure 4 Middle (F) shows the ECL intensity of PdNPs@PSi / ITO after adding luminol to the test solution without hydrogen peroxide at –0.2 V without electrolysis (left) and electrolysis (right); Figure 4 (G) shows the ECL intensity of the test solution at -0.2V without electrolysis (left) and electrolysis (right) for 120s in the presence of hydrogen peroxide, and then the electrolysis was stopped and luminol was added to PdNPs@PSi / ITO, PMT = 700V; by increasing the temperature from -0.2V to 0.3V ( Figure 4 Medium (H) and from 0.3V to –0.2V ( Figure 4 Step pulse in (I), ECL transient of PdNPs@PSi / ITO, the yellow curve indicates the applied potential step, PMT = 700 V.

[0064] like Figure 4 As shown in (AC), it can be seen that in the potential range of -0.2V~0.3V, only PdNPs@PSi / ITO has a large ECL signal, while ITO and PSi / ITO have no ECL signal and CV signal. Figure 4 In (B), it can be seen that the peak potential when it is stable is -0.1V~0V and 0.2V~0.3V, with double peaks. Figure 4 As shown in (D), it can be seen that only PdNPs@PSi / ITO and PdNPs / ITO have obvious hydrogen adsorption and desorption curves and Pd oxidation and reduction peaks in 1MH2SO4, while ITO and PSi / ITO have no obvious peaks. Figure 4As shown in (E), compared with Figure 4 In (D), the ECL signals of PdNPs@PSi / ITO and PdNPs / ITO electrodes with similar peak shapes are continuously scanned. It can be seen that the electrochemical peak currents of PdNPs are similar, and thus the amount of Pd on the electrodes is similar. After PSi confines PdNPs, the ECL signal obtained is higher and more stable, proving the role of PSi confinement in enhancing the ECL signal. This is attributed to the confined synthesis and the EMSI effect of PSi and PdNPs.

[0065] In order to verify the ROS-dominated ECL reaction, the luminescence process was studied by adding luminol alone. Figure 4 The ECL signals of (F, G) before and after electrolysis of PBS and addition of luminol, and before and after electrolysis of PBS and H2O2 mixed solution and addition of luminol are compared. It can be seen that Figure 4 In (F), there is no obvious ECL signal when luminol is injected without electrolysis or after electrolysis without H2O2. Figure 4 Middle (G), after electrolysis of H2O2 at –0.2 V for 120 s, a stronger ECL intensity was detected with the addition of luminol, indicating that the generation of ROS is a key step in ECL.

[0066] Further investigation was carried out by parallel testing of electrodes from the same batch to explore the parallelism of the electrodes. Figure 4 As shown in (H), there is no significant difference in the ECL signal values ​​of the three electrodes prepared in parallel, and the RSD value is 0.98%, which proves that the prepared electrodes have excellent parallelism.

[0067] In addition, step-pulse tests were further performed to investigate the proposed ROS-dominated ECL mechanism. Figure 4 As shown in (I), after accumulating at an initial step potential of –0.2 V, luminol shows a strong ECL transient, but when the potential is stepped to a positive potential, the ECL is almost gradually quenched. Figure 4 As shown in (J), when the initial step potential is set to 0.3 V and then switched to a negative potential, there is no obvious emission in all the first pulse cycles due to the absence of ROS, indicating that the ECL reaction is mainly dominated by the generation of ROS, and luminol anions are mainly produced by ROS oxidation rather than electrochemical oxidation.

[0068] Figure 4 CV curves of different electrodes in Example 1 and the linear relationship between CV peak current and the square root of scan rate, Figure 5 Middle (A) is the CV curve of ITO electrode; Figure 5 Middle (B) is the CV curve of PSi / ITO electrode; Figure 5Figure 6. CV curves of PdNPs@PSi / ITO electrode (C); Figure 5 Figure 7. Linear relationship between the peak current and the square root of scan rate of ITO electrode (D); Figure 5 Figure 8. Linear relationship between the peak current and the square root of scan rate of PSi / ITO electrode (E); Figure 5 Figure 9. Linear relationship between the peak current and the square root of scan rate of PdNPs@PSi / ITO electrode (F).

[0069] As Figure 5 Figure 6. CV curves of PdNPs@PSi / ITO electrode (C); 2− / 3− The electroactive areas of ITO, PSi / ITO and PdNPs@PSi / ITO were compared as shown in Figures 6-9. The experiments were carried out in 0.1 M KCl (pH=4) solution containing 5 mM [Fe(CN)6]3-4- at a scan rate ranging from 30 mV / s to 100 mV / s (30 mV / s, 40 mV / s, 50 mV / s, 60 mV / s, 70 mV / s, 80 mV / s, 90 mV / s and 100 mV / s) by cyclic voltammetry (CV).

[0070] By analyzing the changes of the oxidation peak potential and the reduction peak potential, it was found that the oxidation peak potential and the reduction peak potential of the three electrodes all showed a good linear relationship with the scan rate. The linear regression equation of the anodic oxidation peak current and the square root of the scan rate of ITO was Figure 5 1 / 2 +83 R 2 = 0.995), the linear equation of the cathodic reduction peak current and the square root of the scan rate was I pc =−26v 1 / 2 −84 R 2 =0.996), the linear regression equation of the anodic oxidation peak current and the square root of the scan rate of PSi / ITO was I pa = 8.1v 1 / 2 +60 R 2 =0.987), the linear equation of the cathodic reduction peak current and the square root of the scan rate was I pc =−7.4v 1 / 2 −67 R 2 ​​​​= 0.994), the linear regression equation of the anodic oxidation peak current versus the square root of the scan rate for PdNPs@PSi / ITO was I pa =9.3v 1 / 2 +63 ( R 2 = 0.990), the linear equation of the cathodic reduction peak current versus the square root of the scan rate was I I pc =−9.6v 1 / 2 −54 ( R 2 = 0.991), according to I p Ipa = 28 v 1 / 2 n 3 / 2 v 1 / 2 C (A (cm 2 ): effective area; D (cm / s): diffusion coefficient; n: number of transferred electrons; v (V / s): scan rate; C (mmol / L): molar volume concentration) The calculation results show that the electroactive areas of the electrodes modified with PSi and PdNPs@PSi are decreased, and the significant increase in the ECL intensity can be attributed to the electrocatalytic effect of PdNPs rather than the significant increase in the electrode active area.

[0071] = 2.69 x 105 AD Figure 1 shows the electrochemical signals of the electrodes in different environments in Example 1, Figure 6 Figure 1(A) shows the CV signals of the electrodes in different modification conditions in different atmospheres in PBS; Figure 6 Figure 1(B) shows the CV signals of the electrodes in different modification conditions in different atmospheres in PBS+H2O2; Figure 6 Figure 1(C) shows the ECL signals of the electrodes in different atmospheres; Figure 6 Figure 1(D) shows the UV-Vis absorption spectra of different electrodes (test conditions: 0.01M NaAc buffer containing 1 mM TMB and 1 mM H2O2, pH=4); Figure 6 Figure 1(E) shows the ECL response of PdNPs@PSi / ITO in PBS (0.01M, pH=7.4) containing luminol (100 μM) and H2O2 (1 mM) when TBA (1 mM), BQ (100 μM) and Thiourea (1 mM) are added, wherein I and I0 are the ECL signals with or without ROS scavengers, respectively; Figure 6 Figure 1(F) shows the mechanism summary of PdNPs@PSi promoting the low trigger potential electrochemiluminescence of luminol.

[0072] AsFigure 6 As shown in (A, B), the electrochemical signals under different atmospheres and different solution media are compared. It can be seen that the electrochemical signal increased by 10.7μA when H2O2 was added in the air atmosphere, proving that PdNPs@PSi has an obvious electrocatalytic effect on H2O2. The trends in different atmospheres are similar. After nitrogen and oxygen are introduced at the same time, the electrochemical signal is reduced and improved to a certain extent. Figure 6 (C) Comparison of the ECL signals of PdNPs@PSi / ITO under different atmospheres shows that although there are changes in the electrochemical signal, there is no obvious difference in the ECL signal, proving that H2O2 is the main source of ROS in low trigger potential electrochemiluminescence.

[0073] like Figure 6 As shown in (D), using 3,3',5,5'-tetramethylbenzidine (TMB) as a colorimetric substrate, the peroxidase-like activity of different catalytic systems was compared in the presence of H2O2. It can be seen that the electrode modified with PdNPs has good peroxidase-like activity. Figure 6 As shown in (E), hydroxyl radical (OH·) scavenger tert-butyl alcohol (TBA) and superoxide radical (O2 ·− ) scavenger p-benzoquinone (BQ), and broad-spectrum free radical scavenger thiourea (Thiourea) were used to observe their effects on ECL signals. After adding TBA and p-benzoquinone, the ECL signals were reduced to a certain extent. This result proves that OH· and O2 ·− Free radicals play a key role in the reaction. Figure 6 As shown in (F), the ECL luminescence mechanism is summarized as follows:

[0074] The effects of changing the concentration of the [Pd(NH3)4]SO4 precursor reacting with PSi and the oscillation time of the [Pd(NH3)4]SO4 precursor in step 1 on the ECL signal of the PdNPs@PSi / ITO electrode were observed. Figure 6 As shown, Figure 7 The effects of different precursor concentrations and oscillation times on the ECL signal of PdNPs@PSi / ITO electrode are shown in Figure 2. Figure 7 Middle (A) shows the optimization of [Pd(NH3)4]SO4 precursor concentration, where the oscillation time is fixed at 20 min; Figure 7 (B) shows the optimized oscillation time for the reaction between [Pd(NH3)4]SO4 and PSi. The detection solution is a phosphate buffer solution (0.01M, pH=7.4) containing luminol (100μM) and H2O2 (1mM). The PMT is 700V, the scan rate is 100mV / s, and the scan range is –0.2V~0.3V.

[0075] like Figure 7 As shown in (A), the concentration of the [Pd(NH3)4]SO4 precursor reacting with PSi was optimized, and the ECL signals obtained from materials prepared with [Pd(NH3)4]SO4 precursor concentrations of 1mM, 3mM, 5mM, 7mM, 10mM, and 12mM were compared. The results show that with increasing [Pd(NH3)4]SO4 precursor concentration, the ECL intensity of the electrode initially increases and then decreases. This is because increasing [Pd(NH3)4]SO4 precursor concentration increases the amount of NH3 generated in the transient reaction, leading to significant local pH changes. At excessively high concentrations, the locally high alkalinity destroys the PSi structure, hindering the deposition of PdNPs. Therefore, to achieve the highest ECL signal, the electrode prepared with a [Pd(NH3)4]SO4 precursor concentration of 5mM was selected for subsequent experiments.

[0076] like Figure 7 As shown in (B), the reaction oscillation time with the [Pd(NH3)4]SO4 precursor was optimized, and the ECL signals of reactions of 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, and 60 min were compared. The results show that as the reaction oscillation time increases, the ECL intensity of the electrode shows a trend of first increasing and then decreasing. The main reason for this is that as the reaction time increases, the amount of PdNPs increases, causing pore blockage or deposition outside the pores, which will reduce the diffusion of the ECL luminophore and co-reactant within the nanochannel. Therefore, in order to achieve the highest ECL signal, the electrode obtained by oscillating the reaction with the [Pd(NH3)4]SO4 precursor for 15 minutes was selected for subsequent experiments.

[0077] In the presence of 2.5 mMFe(CN)6 3- / 4- In 0.1M KCl solution, multiple electrodes PdNPs@PSi / ITO, PdNPs@O-PSi / ITO, Apt / PdNPs@O-PSi / ITO (0.3 μM), BSA / Apt / PdNPs@O-PSi / ITO and MMP-9 / BSA / Apt / PdNPs@O-PSi / ITO (10 ng mL -1 ) Test CV graph as follows Figure 7 (A) and EIS diagram (such as Figure 8 (B)). Scan rate is 50mVs -1 In the presence of 100μM luminol and 1mM H2O2, the electrodes with different modification conditions were tested. Figure 8 Middle (C) ECL signal. PMT = 700 V. Scan rate = 100 mVs. -1 .

[0078] As Figure 8 The electrodes at different modification stages were characterized by CV and EIS. The CV curves of the electrodes at different modification stages in 5 mM [Fe(CN)6] 3− / 4− With the stepwise modification of Apt, BSA and MMP-9 on the electrode, the steric hindrance on the PSi surface gradually increased, the conductivity decreased, and the redox reaction activity of the probe gradually decreased.

[0079] As Figure 8 As shown in (B) of the same figure, similar phenomena can be observed in the EIS data. The EIS spectra are composed of a semicircle at high frequency and a linear part at low frequency. The right inset in the figure is the equivalent circuit diagram, which includes solution resistance (Rs), double-layer capacitance (Cdl), Warburg impedance (Zw) and apparent charge transfer resistance (Rct). Rct is equal to the equivalent diameter of the semicircle at high frequency. The Rct of PdNPs@PSi / ITO electrode is 396 Ω. After modification with epoxy groups, the Rct increases to 1214 Ω. The covalent immobilization of Apt makes the Rct increase to 1347 Ω, and the Rct further increases to 2097 Ω after BSA blocking. When MMP-9 binds to the aptamer, the Rct sharply increases to 2508 Ω, which proves the feasibility of the constructed sensor.

[0080] As Figure 8 As shown in (C) of the same figure, the PdNPs@PSi / ITO electrode exhibits a significant ECL signal at a low trigger potential, which is due to the catalytic effect of PdNPs on H2O2. However, the signal gradually decreases after modification with epoxy groups, covalent immobilization of Apt, and BSA blocking. This phenomenon can be attributed to the insulating nature of the protein layer, which leads to an increase in interfacial resistance, thereby limiting electron transfer and the diffusion of reactants. When MMP-9 binds to the aptamer, the ECL signal intensity decreases significantly. The above results prove the feasibility of the construction of the sensor and the detection of MMP-9.

[0081] The effects of the concentration of Apt in step 3, the incubation time of Apt, and the antigen binding time on the ECL signal of the PdNPs@PSi / ITO electrode were observed. The detection solution was phosphate buffer solution (0.01 M, pH = 7.4) containing luminol (100 μM) and H2O2 (1 mM). PMT = 700 V, the scan rate was 100 mV / s, the scan range was –0.2 V ~ 0.3 V, and the results Figure 8 are shown in the figure.Figure 9 (A) is the optimization of aptamer concentration; Figure 9 (B) is the optimization of aptamer incubation time; Figure 9 (C) is the optimization of antigen binding time. As shown in Figure 9 (A), the ECL intensity of the Apt / PdNPs@O-PSi / ITO electrode obtained at different aptamer concentrations was compared. The results showed that with the increase of the aptamer concentration, the ECL intensity of the electrode showed a trend of first decreasing and then tending to be flat. This is because with the increase of the aptamer concentration, the amount of aptamer covalently combined with the epoxy group increases, which gradually reduces the ECL signal. When the aptamer concentration reaches 0.3 μM, the loaded aptamer on the electrode surface reaches saturation, so the subsequent ECL signal remains stable. Considering the cost factor, the electrode obtained at 0.3 μM of the aptamer concentration is selected for subsequent experiments.

[0082] As shown in Figure 9 (B-C), the incubation time of the aptamer and the binding time of MMP-9 were optimized, considering the time and cost factors. Finally, the incubation time of the antibody was selected as 90 min, and the binding time of MMP-9 was selected as 60 min.

[0083] Figure 9 The ECL signal of the BSA / Apt / PdNPs@O-PSi / ITO electrode and the PBS incubated with different concentrations of MMP-9 in Example 1 (A) and the calibration curve (B) are shown. Figure 10 (A) and the calibration curve (B) are shown. Figure 10 (A) and the calibration curve (B) are shown.

[0084] As shown in Figure 10 (A), the ECL signal of the electrode was measured in the luminol-H2O2 system after the constructed sensor was incubated with different concentrations of MMP-9 for 60 minutes. With the increase of the concentration of MMP-9, the ECL signal of the electrode decreased. This is due to the gradual increase of the electron transfer resistance after the formation of the complex, and the hindering effect of the diffusion of the ECL emitter and the co-reactant is enhanced. Figure 10 (B) is the corresponding cathode ECL linear fitting curve. The results show that in the range of 100 fg / mL to 100 ng / mL, the ECL signal (A) shows a good linear relationship with the logarithm (log I ECL ) of the concentration of MMP-9. The cathode regression equation is C MMP-9 I ECL =–834± Figure 10 MMP-9 (18) log C ​2 =0.997) , LOD was 39fg / mL.

[0085] (ng / mL) + 50 15 ± (45) (R The anti-interference and stability of the BSA / Apt / PdNPs@O-PSi / ITO electrode in Example 1, Figure 11 Middle (A) shows the anti-interference ability of BSA / Apt / PdNPs@O-PSi / ITO electrode against different species; Figure 11 Middle (B) shows the reproducibility of electrodes incubated with MMP-9; Figure 11 Middle (C) shows the long-term stability of the electrode incubated with MMP-9, PMT=700V, scan rate of 100mV / s, and scan range of –0.2V~0.3V.

[0086] like Figure 11 As shown in (A), some tumor markers, cytokines and common substances in serum including Na + , K + 、NO3 ‒ , Cl ‒ , Glu, CEA, CA-153 and IL-6. Among them, Na + , K + , NO3 ‒ , Cl ‒ The concentration of Glu was 10 μM, the concentration of CEA, IL-6 and MMP-9 was 1 ng mL ‒1 The concentration of CA-125 was 10 U / mL. After incubation with these interfering substances in step 4, the ECL signal was measured. None of the interfering substances caused a significant change in the electrode ECL signal. However, when the target MMP-9 was added, the ECL signal changed significantly. Furthermore, after mixing the interfering substances, the magnitude of the ECL signal change was similar to that of the single target MMP-9, demonstrating its good selectivity and anti-interference properties.

[0087] like Figure 11 Middle (B) Five BSA / Apt / PdNPs@O-PSi / ITO electrodes were prepared in parallel to detect 1 ng mL ‒1 ECL signal diagram of MMP-9. Figure 11 Middle (C) Electrode detection of MMP-9 long-term stability of the ECL signal.

[0088] The reproducibility of the immunosensor was studied, e.g. Figure 11 As shown in (B), the RSD value of 5 electrodes prepared in the same batch was 3.1%. The long-term stability of the prepared sensor was investigated over five consecutive days ( Figure 11 Figure 11 The relative standard deviation (RSD) value was 2.8%.

Claims

1. A porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor, characterized in that, The three-electrode system comprises a working electrode, wherein the working electrode is obtained by covalently fixing MMP-9 aptamer and blocking non-specific sites after modifying porous silicon confined platinum nanoparticle material on the electrode; The sensor detects a solution containing luminol and hydrogen peroxide as an electrolyte.

2. The porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to claim 1, characterized in that, The preparation of the working electrode comprises the following steps: Step 1: mixing a porous silicon dispersion liquid with a platinum salt solution, and obtaining porous silicon confined platinum nanoparticle material through in-situ reduction; modifying the porous silicon confined platinum nanoparticle material on the surface of an electrode, and obtaining a porous silicon confined platinum nanoparticle material modified electrode through aging treatment; Step 2: immersing the porous silicon confined platinum nanoparticle material modified electrode in an epoxy silane solution for static reaction, and then immersing the electrode in a solution containing MMP-9 aptamer and a solution containing bovine serum albumin for incubation, to obtain the working electrode.

3. The porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The mass concentration of the porous silicon in the porous silicon dispersion liquid is 0.1-3 mg / mL; The platinum salt comprises [Pd(NH3)4]SO4; The molar concentration of the solute in the platinum salt solution is 1-12 mM.

4. The porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The in-situ reduction in step 1 is a shaking reaction at room temperature for 2-60 min; The aging treatment is vacuum aging at 120-150 ℃ for 8-12 h.

5. The porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The epoxy silane comprises γ-glycidoxypropyltrimethylsilane; The molar concentration of the solute in the epoxy silane solution is 2.0-3.0 mM; The static reaction in step 2 is performed for 30-60 min at a temperature of 20-40 ℃; The solution containing MMP-9 aptamer is a buffer solution with a molar concentration of MMP-9 aptamer of 0.1-0.5 μM; the buffer solution is one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution, and the pH is 6.5-7.5; The mass concentration of bovine serum albumin in the solution containing bovine serum albumin is 0.05-2 mg / ml; The incubation in step 2 is performed for 10-120 min in an environment at a temperature of 4-6 ℃; The solvent of the porous silicon dispersion liquid is water, the solvent of the platinum salt solution is water, the solvent of the solution containing bovine serum albumin is one or more of a phosphate buffer solution with a pH of 6.5-7.5 and a HEPES buffer solution, and the solvent of the epoxy silane solution comprises ethanol or isopropanol.

6. The porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to claim 1, wherein, The molar concentration of luminol in the electrolyte is 5-300 μmol / L, and the molar concentration of hydrogen peroxide is 10-1000 μmol / L.

7. The method for the preparation of a porous silicon-based low-trigger potential electrochemiluminescence aptamer sensor according to any one of claims 1-6, characterized in that, The three-electrode system comprises a working electrode, wherein the working electrode is obtained by covalently fixing MMP-9 aptamer and blocking non-specific sites after modifying porous silicon confined platinum nanoparticle material on the electrode; Step 2: immersing the porous silicon confined platinum nanoparticle material modified electrode in an epoxy silane solution for static reaction, and then immersing the electrode in a solution containing MMP-9 aptamer and a solution containing bovine serum albumin for incubation, to obtain the working electrode. ​ Step 3: the sensor is obtained by forming a three-electrode system with the working electrode, the counter electrode and the reference electrode, and the sensor detects a solution containing luminol and hydrogen peroxide as an electrolyte.

8. Use of the low trigger potential electrochemiluminescence aptamer sensor according to any one of claims 1-6 in detecting MMP-9 for non-diagnostic or non-therapeutic purposes.

9. A method for detecting MMP-9 by low trigger potential electrochemiluminescence, characterized in that, The method comprises the steps of: incubating the working electrode of any one of claims 1-6 with a to-be-detected solution, and then washing to obtain a to-be-detected electrode; using the to-be-detected electrode as a working electrode, and combining a reference electrode and a counter electrode to form a three-electrode system, and using an electrolyte solution containing luminol and hydrogen peroxide as a detection solution to detect an electrochemiluminescence signal.

10. The method for detecting MMP-9 by low trigger potential electrochemiluminescence according to claim 9, wherein, The concentration of MMP-9 in the to-be-detected solution is less than 100 ng / mL. And / or, the to-be-detected solution comprises one or more interference compounds such as glucose, inorganic salt, cytokine, and tumor marker; and the total molar concentration of the interference compounds is less than 0.5 M. And / or, the to-be-detected solution comprises one or more interference compounds such as glucose, inorganic salt, cytokine, and tumor marker; and the total molar concentration of the interference compounds is less than 0.5 M.

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