Electrochemical luminescence immunosensor with low trigger potential and construction method and application thereof

Abstract

The invention discloses an electrochemical luminescence immunosensor with low trigger potential and a construction method and application thereof, the immunosensor comprises a working electrode, a reference electrode and a counter electrode, and a liquid to be detected contains luminol and hydrogen peroxide; the working electrode is a VMSF modified electrode of confined gold-palladium nanoparticles, and is obtained by covalently immobilizing an antibody and closing non-specific active sites. Luminol and an antibody are respectively used as an electrochemical luminescence signal molecule and a specific recognition element; in combination with the anti-pollution effect of the VMSF and the efficient catalytic effect of the gold-palladium nanoparticles on a low-trigger-potential electrochemical luminescence system, the sensor has the characteristics of high sensitivity, wide detection linear range and low detection limit, and can be applied to detection of the content of tumor markers, cell surface factors and bacterial or virus surface factors in biological samples. The method has great application potential in the fields of clinical diagnosis and monitoring and basic medical research.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to an electrochemiluminescent immunosensor with a low trigger potential and a construction method and application thereof. Background Art

[0002] Electrochemiluminescence (ECL) is a luminescence process triggered by an electrochemical reaction on the electrode surface. In this process, substances form excited states through electron transfer reactions, and then release light energy when these excited state substances fall back to the ground state, thereby realizing the conversion of electrical energy into radiation energy. Luminol is an efficient ECL probe with low toxicity, high quantum yield and excellent water solubility. It has been widely used in many fields such as clinical analysis and food safety monitoring. Current research mainly focuses on the anodic ECL characteristics of luminol, while the exploration of its cathodic ECL behavior is relatively small. This is mainly because the efficient cathodic ECL of luminol needs to meet more stringent conditions, such as a more negative luminescence potential, which often brings various electrochemical interferences or causes irreversible damage to many biorecognition molecules, thereby reducing the sensitivity of detection. Therefore, constructing a high-performance luminol ECL system with a low trigger potential is of great significance to broaden its application field.

[0003] Researchers have combined carbonized polydopamine nanotubes (ACS Sustainable Chemistry & Engineering, 2022, 10, 10361), palladium nanoclusters (Analytical chemistry, 2022, 94, 12845), and single-atom iron (Analytical chemistry, 2022, 94, 9459) to achieve low-potential ECL in the luminol-hydrogen peroxide system. So far, no one has used gold-palladium nanoparticles to explore low-potential ECL in the luminol-hydrogen peroxide system.

[0004] Vertically-ordered mesoporous silica films (VMSF) are composed of many cylindrical nanochannels, with the characteristics of uniform and adjustable pore size (2-1.8nm), pores perpendicular to the electrode substrate, and high porosity. The nanochannels of VMSF can be used as micro-nanoreactors to effectively limit the diffusion and aggregation of metals and precursors, thereby avoiding the formation of larger particles. The unique confinement effect of VMSF can produce ultra-small and uniform nanoparticles. In addition, the size of nanoparticles can be further precisely controlled by regulating the pore size of VMSF.

[0005] At present, VMSF, as an ideal hard template for confined synthesis of nanoclusters or nanoparticles, has been used to synthesize single metal nanoparticles such as gold (Chem.—Eur.J.2014,20,12777-12780), silver (Analytical Chemistry,2024,96,10264-10273), platinum (ChemElectroChem,2020,7,2081-2086), copper (Molecules,2023,28,7515), and nickel (Journal of Materials Chemistry B,2020,8,3616-3622). However, whether VMSF can be used as a nanoreactor to synthesize gold-palladium alloy nanoparticles (AuPd NPs) to achieve low trigger potential ECL of luminol-hydrogen peroxide system and applied to the design of ECL immunosensors has been studied and tested. Summary of the invention

[0006] In order to solve the problems of low detection sensitivity and great detection difficulty of target analytes in biological samples, the present invention provides a low trigger potential electrochemiluminescence immunosensor, which is used to detect tumor markers, cell surface factors, and bacterial or viral surface factors in biological samples. It has the advantages of high detection sensitivity and specificity, good selectivity, and low cost, and the detection process does not require a complicated pretreatment process.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] An electrochemiluminescent immunosensor with a low trigger potential, the electrochemiluminescent immunosensor comprising a working electrode, a reference electrode and a counter electrode, and the liquid to be detected contains luminol and hydrogen peroxide;

[0009] The working electrode is a VMSF-modified electrode of confined gold-palladium nanoparticles, which is obtained by covalently fixing antibodies and blocking nonspecific active sites.

[0010] The present invention adopts luminol as the electrochemiluminescent signal molecule and antibody as the specific recognition element; combined with the reduction of ECL trigger potential and signal enhancement effect of gold palladium nanoparticles synthesized by VMSF confinement on the luminol-hydrogen peroxide system, an immunosensor is constructed for high-sensitivity detection of tumor markers in biological samples, with low detection limit and wide detection range, and detection does not require complicated pretreatment and separation process, thus reducing detection cost, simple operation, and being suitable for large-scale application.

[0011] The working electrode preparation method comprises the steps of:

[0012] Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode;

[0013] Step 2, 3-glycidyloxypropylmethyldiethoxysilane (GPTMS) was drop-coated on the surface of the confined AuPd NPs@VMSF electrode, and the electrode was incubated at room temperature away from light to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode;

[0014] Step 3, drop-coat the surface of the epoxy-functionalized confined AuPd NPs@VMSF electrode in step 2 with a buffer solution containing an antibody and incubate; then drop-coat the surface with a bovine serum albumin solution and incubate to obtain the working electrode.

[0015] The VMSF modified electrode is Growing a silicon dioxide nanoarray film with pores perpendicular to the substrate and orderly arranged on the electrode by one or more of the following methods: solution growth method, electrochemical assisted self-assembly method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent induced self-assembly method;

[0016] The electrode in the VMSF modified electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode.

[0017] The gold precursor is any one of chloroauric acid, gold nitrate, gold cyanide, and a gold complex; the palladium precursor is any one of sodium tetrachloropalladate, palladium chloride, palladium nitrate, an aminopalladium compound, and a palladium complex;

[0018] The solution containing the gold precursor and the palladium precursor has a gold precursor concentration of 0.01-1 mmol / L and a palladium precursor concentration of 0.05-2 mmol / L;

[0019] The molar ratio of gold to palladium in the solution containing the gold precursor and the palladium precursor is 1:5 to 1:1; preferably, the molar ratio of gold to palladium is 1:4 to 1:2; more preferably, the molar ratio of gold to palladium is 1:3;

[0020] The solvent used in the solution containing the gold precursor and the palladium precursor includes any one or more of a potassium nitrate aqueous solution, a sodium chloride aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution.

[0021] In step 1, the electrodeposition potential is -1.0 to 0V, the electrodeposition time is 1 to 10s, and the temperature is room temperature; when the deposition time is short, the amount of deposited gold palladium nanoparticles is small, resulting in a weak electrochemical luminescence intensity of the luminol-hydrogen peroxide system, thereby affecting the detection sensitivity. If the deposition time is too long, the amount of deposited gold palladium nanoparticles is too much, thereby blocking the pores and affecting the mass transfer process, thereby causing the electrochemical luminescence signal of the luminol-hydrogen peroxide system to decrease. Preferably, the electrodeposition time is 1-8s, and more preferably 2-5s.

[0022] The solvent of the GPTMS solution in step 2 includes any one or more of ethanol, water and ether; the volume proportion of GPTMS in the GPTMS solution is about 0.02-0.5%; and epoxy groups are introduced by modifying GPTMS for subsequent antibody fixation. The amount of epoxy groups introduced at this ratio can effectively take into account both the amount of antibodies and the mass transfer effect at the electrode interface, and has good economic benefits.

[0023] In step 2, the incubation time is 0.2-2h and the temperature is 5-40°C.

[0024] In step 3, the mass concentration of the antibody in the antibody-containing buffer solution is 1 to 20 μg / mL; preferably, the mass concentration of the antibody is 5 to 15 μg / mL; the solvent used includes any one or more of STE buffer solution and TE buffer solution;

[0025] In step 3, the incubation condition is 0-5°C for 0.5-2h;

[0026] In step 3, the mass concentration of the aqueous solution of bovine serum albumin is 0.2-2%;

[0027] The molar concentration of luminol in the detection solution is 10-300 μmol / L. Within this concentration range, the ECL signal is ensured to remain within a suitable absolute value range, making the detection technology more sensitive and economical. The hydrogen peroxide concentration is 0.2-5 mmol / L. Hydrogen peroxide is a co-reactant of luminol, which can produce active oxygen to enhance the electrochemiluminescence signal of the luminol molecule. Too low a concentration of hydrogen peroxide will result in a decrease in the electrochemiluminescence signal.

[0028] The present invention also provides a method for constructing the electrochemiluminescent immunosensor with a low trigger potential, comprising the steps of:

[0029] Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode;

[0030] Step 2, drop-coat GPTMS on the surface of the confined AuPd NPs@VMSF electrode, incubate at room temperature away from light to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode;

[0031] Step 3, drop-coating the surface of the epoxy-functionalized confined AuPd NPs@VMSF electrode in step 2 with a buffer solution containing an antibody and incubating; then drop-coating a bovine serum albumin solution and incubating to obtain the working electrode;

[0032] Step 4: Using the three-electrode system of the working electrode, the reference electrode and the counter electrode, and the electrolyte solution containing luminol and hydrogen peroxide as the detection solution, the electrochemiluminescent immunosensor is constructed.

[0033] The reference electrode includes any one or more of an Ag / AgCl electrode (saturated KCl), a saturated calomel electrode, and a Hg / HgO electrode, and the counter electrode includes any one or more of a platinum wire and a platinum sheet.

[0034] The present invention also provides the use of the electrochemiluminescent immunosensor with low trigger potential in detecting biological antigens for non-therapeutic or diagnostic purposes, comprising the steps of:

[0035] The working electrode is incubated with a test solution containing biological antigens to obtain a test electrode, and a three-electrode system is formed by the test electrode, a reference electrode and a counter electrode. Electrochemical luminescence signal detection is performed in an electrolyte solution containing luminol and hydrogen peroxide as the test solution.

[0036] The detection principle is as follows: vertical ordered mesoporous silica film is used as a nanoreactor to in situ synthesize gold palladium nanoparticles to achieve low trigger potential electrochemiluminescence of the luminol-hydrogen peroxide system and generate an enhanced signal, and then the antibody is further fixed as the working electrode. When the target analyte antigen is present in the serum, the antigen-antibody specifically binds to form an immune complex, which hinders the diffusion of luminol and hydrogen peroxide to the electrode substrate, resulting in a decrease in the electrochemiluminescence signal. The decrease in the electrochemiluminescence signal is associated with the amount of the target analyte antigen in the biological sample. Therefore, according to the degree of reduction in the electrochemiluminescence signal, the electrochemiluminescence quantitative detection of the target analyte antigen can be achieved.

[0037] This detection method is used to detect tumor markers for non-diagnostic purposes, and can be used to study the relevant mechanisms of anti-tumor aptamer response, detect residual tumor markers after drug action, etc.

[0038] The incubation time in the liquid to be detected is 0.5-2h, and the incubation temperature is 0-5°C; the biological antigen includes one of a tumor marker, a cell surface factor, a bacterial or viral surface factor, and its concentration is above 5mU / mL; preferably, the concentration of the target biological antigen in the liquid to be detected is 5mU / mL-500U / mL. In the actual detection process, due to the problem of high concentration of the target analyte antigen (or many interfering substances) in the biological sample, during the detection, an electrolyte solution can be used to dilute it, such as diluting it 1-100 times, to achieve a reduction in the concentration of the target analyte antigen.

[0039] The pH of the liquid to be tested is 6-8. The pH of the liquid to be tested can avoid inhibiting the deprotonation of luminol, and has high electrochemical luminescence intensity and sensitivity and high detection stability. Preferably, the pH of the liquid to be tested is 6.5-8, and more preferably, the pH is 6.5-7.5.

[0040] The tumor markers include one or more of carbohydrate antigen 15-3, carbohydrate antigen 19-9, carbohydrate antigen 125, carcinoembryonic antigen, alpha-fetoprotein, prostate-specific antigen or ferritin;

[0041] The test solution also includes interfering substances other than the target biological antigen, and the interfering substances include one or more of CA19-9, AFP, and CEA, and the concentration of each interfering protein is less than 50U / mL. Since the immunosensor in the present invention involves antigen-antibody specific binding, it has high selectivity, is not easily affected by other interfering substances, has high detection stability, and has a wide range of applications.

[0042] The logarithmic value of the detection concentration of the target analyte antigen by the sensor in the present invention is linear in the range of 5mU / mL-500U / mL, and the detection limit is 3.3mU / mL. The detection method has a wide linear detection range, high selectivity, high detection sensitivity, and low detection limit. In addition, the detection does not require complex pretreatment and separation processes, which reduces the detection cost, is easy to operate, and is suitable for large-scale applications.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The low trigger potential electrochemiluminescent immunosensor of the present invention uses a vertical ordered mesoporous silica film modified electrode to synthesize gold palladium nanoparticles. The gold palladium precursor is confined inside the pores. Under the action of negative potential, the gold palladium nanoparticles are synthesized in situ to obtain stable gold palladium nanoparticles. The nanochannel can protect the gold palladium nanoparticles inside the pores from interference from other substances in the actual sample, maintaining a high catalytic activity.

[0045] (2) The present invention uses vertical ordered mesoporous silica membranes to in-situ confine gold-palladium nanoparticles to achieve efficient cathode and anode electrochemical luminescence in the luminol-hydrogen peroxide system at low trigger potentials. The lower trigger potential can reduce damage to biorecognition molecules; gold-palladium nanoparticles can enhance the electrochemical luminescence intensity of the luminol-hydrogen peroxide system.

[0046] (3) The silica nanoporous membrane of the present invention has ultra-small nanochannels (2-3 nm), the surface of the pores is rich in silanol groups, and its pK a The film is about 2. Ionization in conventional solutions makes the film negatively charged. It can hinder the influence of large-sized negatively charged pollutants (such as cells, proteins and other biological macromolecules) in complex samples on the substrate electrode through electrostatic repulsion, hydrophilic effect and size screening effect, and has good anti-fouling and anti-interference capabilities.

[0047] (4) The low trigger potential electrochemiluminescence immunosensor of the present invention has strong specificity and high sensitivity, and is expected to open up a new way to detect a series of important biomarkers related to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the structure of the VMSF-modified indium tin oxide (ITO) electrode prepared in Example 1 with CA15-3 antibody fixed on the outer surface and AuPd NPs confinedly grown in the pores after incubation with antigen.

[0049] Figure 2 This is a scanning electron microscope cross-sectional image (A) of the VMSF-modified ITO electrode (VMSF / ITO) in Example 1, as well as a transmission electron microscope top view (B) and cross-sectional image (C) of VMSF.

[0050] Figure 3 This is the scanning electron microscope cross-sectional image (A) of the AuPd NPs@VMSF modified ITO electrode (AuPd NPs@VMSF / ITO) in Example 1, the transmission electron microscope top view (B) and cross-sectional image (C) of AuPd NPs@VMSF, as well as the scanning electron microscope cross-sectional image (D) of the AuPd NPs@VMSF / ITO electrode and the corresponding elemental distribution diagrams of Pd (E) and Au (F).

[0051] Figure 4 The XPS total spectrum of AuPd NPs@VMSF / ITO prepared in Example 1 (A) and the high-resolution XPS deconvolution images of O1s, Au 4f, and Pd3d (BD).

[0052] Figure 5(A) is a high-resolution XPS deconvolution image of Pd3d of VMSF-confined platinum nanoparticles modified ITO electrode (Pd NPs@VMSF / ITO) in Example 1. (B) is a high-resolution XPS energy spectrum of Au 4f of AuPd NPs@VMSF / ITO and VMSF-confined gold nanoparticles modified ITO electrode (Au NPs@VMSF / ITO). (C) is a high-resolution XPS energy spectrum of Pd 3d of AuPd NPs@VMSF / ITO and Pd NPs@VMSF / ITO.

[0053] Figure 6 CV curves of Pd NPs@VMSF / ITO and AuPd NPs@VMSF / ITO electrodes in sulfuric acid (0.1 M) in Example 1. The scan rate was 50 mV / s.

[0054] Figure 7 (A) is the ECL potential curve of AuPd NPs@VMSF / ITO electrode in Application Example 1. (B) is the ECL signal of different electrodes in the low trigger potential range. The detection solution contains luminol (100 μM) and H 2 O 2 (1 mM) phosphate buffer solution (0.01 M, pH = 7.4).

[0055] Figure 8 (A) is the CV curves of different electrodes in pure phosphate buffer solution (0.01M, pH=7.4) in Application Example 1. (B) is the CV curves of different electrodes in H 2 O 2 CV curves obtained in phosphate buffer solution (0.01 M, pH = 7.4) with a scan rate of 50 mV / s.

[0056] Fig. 9 The AuPd NPs@VMSF / ITO in Application Example 1 was prepared in the presence of luminol (100 μM) and H 2 O 2 The ECL response was obtained in phosphate buffered saline (0.01 M, pH = 7.4) with 100 μM tert-butyl alcohol (TBA, 100 μM) and p-benzoquinone (BQ, 100 μM) added as reactive oxygen species scavengers, where I is and I 0 ECL signals in the presence or absence of reactive oxygen species scavengers, respectively.

[0057] Fig.10 In Application Example 2, each electrode contains luminol (100 μM) and H 2 O 2The ECL signals (A) obtained in PBS (0.01 M, pH = 7.4) (1 mM) and the ECL signals of each electrode in the presence of 2.5 mM (1:1) [Fe(CN) 6 ] 3- / 4- Impedance spectrum obtained in KCl (0.1 M) solution (B). The concentration of CA15-3 was 100 U / mL.

[0058] Fig.11 (A) is the optimized ratio diagram of the deposition precursor solution in Application Example 3. (B) is the optimized diagram of the CA15-3 antibody concentration. The incubation time is 1.5h. (CD) is the optimized diagram of the incubation time of CA15-3 antibody and CA15-3. The concentration of CA15-3 antibody is 10μg / mL, and the concentration of CA15-3 is 5U / mL.

[0059] Fig.12 The Ab / AuPd NPs@O-VMSF / ITO immunosensor in Application Example 4 was used to detect different concentrations of CA15-3 (0 U / mL, 0.005 U / mL, 0.05 U / mL, 0.5 U / mL, 5 U / mL, 50 U / mL and 500 U / mL) in an aqueous solution containing luminol (100 μM) and H 2 O 2 (A) ECL response obtained in PBS (0.01M, pH=7.4) with 1 mM (C) (B) Calibration curve of cathode ECL intensity and logarithm of CA15-3 concentration, with inset as calibration curve of cathode ECL intensity and logarithm of CA15-3 concentration. (C) Calibration curve of anodic ECL intensity and logarithm of CA15-3 concentration, with inset as calibration curve of anodic ECL intensity and logarithm of CA15-3 concentration.

[0060] Fig.13 (A) is the selectivity of the immunosensor in Application Example 5, where the concentrations of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are 10 ng / mL, the concentration of CA19-9 is 50 U / mL, and the concentration of CA15-3 is 5 U / mL. (B) is the reproducibility of the immunosensor, where the concentration of CA15-3 is 0.5 U / mL. (CD) is the signal stability of AuPd NPs@VMSF / ITO and the immunosensor, where the concentration of CA15-3 is 0.05 U / mL. The electrolyte contains 100 μM luminol and 1 mM H 2 O 2 PBS (0.01 M, pH = 7.4), PMT = 700 V. Error bars indicate the standard deviation of three measurements under the same experimental conditions. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0062] Unless otherwise specified, the raw materials used in the following specific embodiments were purchased from commercial sources and used directly without special treatment. The main raw materials used in the examples are as follows:

[0063] Cetyltrimethylammonium bromide (CTAB): Shanghai Aladdin Biotechnology Co., Ltd.;

[0064] Tetraethoxysilane (TEOS): Shanghai Aladdin Biotechnology Co., Ltd.;

[0065] Sodium dihydrogen phosphate hydrate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0066] Potassium hydrogen phthalate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0067] 3-Glycidyloxypropyltrimethoxysilane (GPMTS): Shanghai Aladdin Biotechnology Co., Ltd.;

[0068] Luminol: Shanghai Aladdin Biotechnology Co., Ltd.;

[0069] Hydrogen peroxide (H 2 O 2 ): Shanghai Aladdin Biochemical Technology Co., Ltd.

[0070] Sodium hydroxide: Shanghai MacLean Biochemical Co., Ltd.;

[0071] Sodium hydrogen phosphate hydrate: Shanghai MacLean Biochemical Co., Ltd.;

[0072] Hexaammineruthenium trichloride: Shanghai MacLean Biochemical Co., Ltd.;

[0073] CA15-3: Beijing Keyue Biotechnology Co., Ltd.;

[0074] CA15-3 antibody: Beijing Keyue Biotechnology Co., Ltd.;

[0075] Carcinoembryonic antigen (CEA): Beijing Keyue Zhongkai Biotechnology Co., Ltd.;

[0076] Alpha-fetoprotein (AFP): Beijing Keyue Zhongkai Biotechnology Co., Ltd.;

[0077] Carbohydrate antigen 19-9 (CA19-9): Beijing Keyue Zhongkai Biotechnology Co., Ltd.;

[0078] Hydrochloric acid: Hangzhou Shuanglin Chemical Reagent Co., Ltd.;

[0079] Potassium nitrate: Tianjin Komiou Chemical Reagent Co., Ltd.;

[0080] Ethanol: Sinopharm Chemical Reagent Co., Ltd.

[0081] Example 1

[0082] The construction process of the low trigger potential electrochemiluminescent immunosensor for CA15-3 antigen detection includes the following steps:

[0083] (1) Adoption Solution growth method to grow VMSF on the electrode surface: configuration H 2 O (70mL), ethanol (30mL), CTAB (160mg), ammonia (100μL) and TEOS (80μL) to form a precursor solution. Immerse the cleaned ITO electrode in the above precursor solution and incubate it in a constant temperature water bath at 60℃ for 24h. After the growth is completed, take the electrode out of the precursor solution and quickly rinse it with a large amount of clean water. After rinsing, blow dry the electrode with nitrogen and age it at 120℃ for 12h. The obtained electrode is called SM@VMSF / ITO electrode;

[0084] (2) Immerse the SM@VMSF / ITO electrode obtained in step (1) in a 0.1M hydrochloric acid ethanol solution and stir for 5 minutes to remove the residual micelles in the pores. Obtain a VMSF / ITO electrode with open pores. Immerse the VMSF / ITO electrode in a solution containing a gold precursor and a palladium precursor (containing 0.25mM chloroauric acid and 0.75mM sodium tetrachloropalladate, and the supporting electrolyte is 0.1M potassium nitrate), and apply a constant voltage of -0.2V for 2s. Take out the electrode, wash it with a large amount of flowing ultrapure water, and blow it dry with nitrogen to obtain a VMSF / ITO electrode with confined gold palladium nanoparticles (AuPd NPs@VMSF / ITO);

[0085] By changing the precious metal precursor solution in step 2 separately, separately preparing precursor solutions containing 0.25 mM chloroauric acid and 0.75 mM sodium tetrachloropalladate, and keeping other steps unchanged, electrode NPs@VMSF / ITO and PdNPs@VMSF / ITO loaded with gold or palladium separately were obtained.

[0086] (3) The AuPd NPs@VMSF / ITO electrode obtained in step (2) was drop-coated with GPMTS as a crosslinker and incubated in the dark at room temperature for 1 h. After the reaction was completed, the unbound GPMTS was washed away with phosphate buffer solution to obtain the AuPd NPs@O-VMSF / ITO electrode. CA15-3 antibody (10 μg / mL, 50 μL) was then drop-coated on the electrode surface and incubated at 4°C for 1.5 h to finally obtain a VMSF / ITO electrode (Ab / AuPd NPs@O-VMSF / ITO) with CA15-3 antibody (Ab) fixed on the outer surface and gold palladium nanoparticles confined in the pores.

[0087] (4) Bovine serum albumin (BSA) was drop-coated on the Ab / AuPd NPs@O-VMSF / ITO electrode obtained in step (3) and incubated at 4°C for 15 minutes to block nonspecific sites. After the reaction, the unbound BSA was washed away with phosphate buffer solution to obtain a working electrode BSA / Ab / AuPd NPs@O-VMSF / ITO electrode.

[0088] (5) The working electrode BSA / Ab / AuPd NPs@O-VMSF / ITO electrode and the silver / silver chloride electrode (the solution is saturated potassium chloride) are obtained by step 4, and the counter electrode is a platinum wire to form a three-electrode system. The electrolyte solution containing luminol and hydrogen peroxide is used as the detection solution to construct the electrochemiluminescent immunosensor.

[0089] Application Example 1

[0090] The process of detecting CA15-3 in serum by electrochemiluminescence method using the vertical ordered silica film modified working electrode BSA / Ab / AuPd NPs@O-VMSF / ITO with CA15-3 antibody fixed on the surface and gold palladium nanoparticles confined in the pores of Example 1:

[0091] The serum was diluted 50 times with phosphate buffer (0.01 mol / L, pH = 7.4), and a certain amount of CA15-3 antigen was added in proportion. The final CA15-3 antigen concentrations were 0.005U / mL, 0.05U / mL, 0.5U / mL, 5U / mL, 50U / mL and 500U / mL. CA15-3 antigen was drop-coated on the BSA / Ab / AuPd NPs@O-VMSF / ITO electrode and incubated at 4°C for 1h. Unbound CA15-3 was rinsed with phosphate buffer (0.01 mol / L, pH = 7.4) to obtain the electrode to be tested. The schematic diagram of its internal structure is shown in Figure 1 shown.

[0092] The electrode to be tested was used as the working electrode, the reference electrode was a silver / silver chloride electrode (the solution was saturated potassium chloride), and the counter electrode was a platinum wire. A simple three-electrode system was used to carry out electrochemiluminescence testing in a detection solution containing luminol (100 μM) and hydrogen peroxide (1 mM) in a phosphate buffer solution, with a potential range of -0.2 to 0.3 V.

[0093] Performance observation test

[0094] 1. Morphology observation and electrochemical characterization

[0095] The electron microscopy of the VMSF / ITO electrode obtained in step (2) of Example 1 is as follows: Figure 2 shown.

[0096] Figure 2 (A) is a cross-sectional scanning electron microscope image of the VMSF / ITO electrode. It can be observed that the typical three-layer structure of the VMSF / ITO electrode consists of VMSF, ITO layer and glass substrate, where the thickness of VMSF is about 102 nm. Figure 2 The top view and cross-section of the transmission electron microscopy image (BC) reveal a complete film structure without cracks, with a pore size of about 2 to 3 nm. The thickness of the cross-section image is consistent with the data obtained from the cross-section image in the scanning electron microscopy image.

[0097] 2. Characterization of gold-palladium nanoparticles

[0098] Figure 3 (AC) shows the scanning electron microscopy cross-section of AuPd NPs@VMSF / ITO electrode and the top view and cross-section of TEM image. From the scanning electron microscopy cross-section, it can be observed that the thickness of the VMSF layer in the AuPd NPs@VMSF / ITO electrode is different from that of the VMSF / ITO electrode ( Figure 2 (A)) compared to the VMSF layer, the thickness remains the same, and no large nanoparticles appear on the VMSF layer, proving that the gold-palladium alloy nanoparticles are deposited in the pores and do not grow outside the pores. Observing the top view and cross-section of the transmission electron microscopy image of the AuPd NPs@VMSF / ITO electrode, it can be seen from the figure that the nanoparticle morphology is obvious and the particle size is small. However, due to the indispensable ultrasonic step in the transmission electron microscopy sample preparation process, the particles have a certain agglomeration phenomenon. In order to better characterize the confinement of AuPd NPs inside the VMSF nanochannel, energy dispersive X-ray spectroscopy (EDS) was used to characterize the AuPd NPs@VMSF / ITO electrode. Figure 3 (DF) are the cross-sectional SEM images of AuPd NPs@VMSF / ITO electrode and EDS images of palladium and gold elements. It can be seen that Pd and Au elements are distributed in the VMSF layer, proving that the synthesized AuPd NPs exist inside the VMSF nanochannel.

[0099] Figure 4 The XPS overall spectrum of the prepared AuPd NPs@VMSF / ITO (A) and the high-resolution XPS deconvolution images of O1s, Au4f, and Pd 3d (BD). Figure 4 (A) shows the presence of O, Si, Pd, and Au elements in the AuPd NPs@VMSF / ITO sample. The high-resolution X-ray photoelectron spectra of O1s, Au 4f, and Pd 3d were deconvoluted. Figure 4 In (B), a main peak (532.77 eV) and two shoulder peaks (530.67 eV, 535.30 eV) can be observed. According to the literature, they are attributed to SiO 2 , metal oxides (in subsequent studies, found to be palladium oxides) and Pd 3p 3 / 2 .like Figure 4 (C) As shown, Au 4f 7 / 2 (Au 4f 5 / 2 ) peak is located at a binding energy (BE) of 83.31 eV (86.98 eV), and the spin-orbit splitting is measured to be 3.67 eV, which is consistent with the spin-orbit splitting of gold. 5 / 2 (Pd 3d 3 / 2 ) peak is located at the binding energy of 334.7eV (339.9eV), and the spin-orbit splitting is measured to be 5.2±0.1eV, which is consistent with the spin-orbit splitting of metal palladium. Secondly, two other peaks were observed at 335.7eV and 336.9eV ( Figure 4 (D)), referring to some previously published reports, the component with the highest binding energy (about 336.9 eV) was attributed to palladium oxide (PdO), while the component with the medium binding energy value (about 335.7 eV) was attributed to the surface oxide or PdO in contact with metallic palladium, recorded as PdOx. At the same time, the XPS peak of Au 4d was also observed at 334.7 eV.

[0100] Figure 5 Taking Au NPs@VMSF / ITO and Pd NPs@VMSF / ITO as comparison samples, the electronic structure of AuPd NPs@VMSF / ITO was analyzed by XPS. Figure 5 (A)) and AuPd NPs@VMSF / ITO( Figure 4From the deconvolution peak of Pd 3d in (D), it can be found that the PdO content in the AuPd NPs@VMSF / ITO sample decreases and the PdOx content increases, which is consistent with the literature reports, that is, the introduction of gold will reduce a certain amount of oxidized palladium.

[0101] Further comparison of the Au 4f and Pd 3d high-resolution XPS patterns of Au NPs@VMSF / ITO, Pd NPs@VMSF / ITO, and AuPd NPs@VMSF / ITO ( Figure 5 (BC)), it was found that the binding energy of both in the AuPd NPs@VMSF / ITO sample moved toward the low binding energy direction, which is consistent with the results of the gold-palladium alloy research reported in the literature. This change in binding energy can be attributed to the re-hybridization and redistribution of charges inside the metal. Au lost its d charge and gained almost the same number of sp-like electrons, while Pd gained d charge and lost sp charge. This redistribution of charge led to a significant change in the binding energy between the two metal atoms, thereby affecting the electronic structure and chemical properties of the material. It further shows that the interior of the VMSF pores is alloyed AuPd NPs.

[0102] Figure 6 The cyclic voltammetry curves of AuPd NPs@VMSF / ITO and Pd NPs@VMSF / ITO modified electrodes in sulfuric acid (0.1M) are shown. There are obvious peaks (–0.4 to 0V) derived from hydrogen desorption / adsorption on both AuPd NPs@VMSF / ITO and Pd NPs@VMSF / ITO modified electrodes. Redox peaks at around 0.46V, 0.64V and 1.1V were observed on the AuPd NPs@VMSF / ITO electrode, which were attributed to the reduction peak of palladium oxide, the oxidation peak of palladium and the oxidation peak of gold, respectively. Compared with the PdNPs@VMSF / ITO modified electrode, the reduction peak of palladium oxide in the AuPd NPs@VMSF / ITO modified electrode moved to the positive potential, which is also the result of alloying. The positive shift of the reduction potential of palladium oxide in the AuPd NPs@VMSF / ITO modified electrode (80 mV vs Pd NPs@VMSF / ITO) is conducive to the adsorption and desorption of hydroxyl groups and has better catalytic performance.

[0103] In summary, it can be demonstrated that AuPd NPs were successfully confined inside the nanochannels of VMSF.

[0104] 3. Mechanism exploration

[0105] The mechanism of low trigger potential electrochemiluminescence of luminol-hydrogen peroxide system catalyzed by gold-palladium nanoparticles was investigated. Figure 7As shown in A, in the ultra-low potential range (–0.2-0.3 V), strong cathode emission and anode emission were observed in the luminol-hydrogen peroxide electrochemiluminescence system of the AuPd NPs@O-VMSF / ITO electrode. Compared with the bare ITO electrode, VMSF / ITO electrode, Au NPs@VMSF / ITO electrode and Pd NPs@VMSF / ITO electrode, in such a low potential range, only the AuPd NPs@VMSF / ITO electrode and Pd NPs@VMSF / ITO electrode showed a certain ECL signal, and the ECL signal of the AuPd NPs@VMSF / ITO electrode was higher ( Figure 7 (B)). This may be due to the synergistic effect between the gold and palladium alloys.

[0106] Figure 8 Considering that the oxidation potential of luminol is far from being reached in such a low potential range, we used electrochemical methods to verify the source of the strong ECL. Figure 8 The results of different electrodes in air atmosphere without (A) and with (B) H 2 O 2 The cyclic voltammetry curves were obtained in phosphate buffer solution (1 mM). After observation, we noticed that both Pd NPs and AuPd NPs can react with oxygen and H 2 O 2 However, they are not very effective in catalyzing H 2 O 2 The current response of AuPd NPs is more significant. Therefore, we can infer that AuPd NPs mainly catalyze H 2 O 2 The ECL intensity of luminol was significantly enhanced by generating a large amount of reactive oxygen species, rather than dissolved oxygen. 2 O 2 We found that AuPdNPs not only showed more prominent H 2 O 2 catalytic effect, but also retains similar properties to Pd NPs, that is, it is more conducive to H 2 O 2 This phenomenon has also been reported previously. Therefore, AuPd NPs can be used as co-reactant promoters to achieve luminol-H reduction at ultra-low potential. 2 O 2 The system has strong cathodic and anodic ECL.

[0107] p-Benzoquinone (BQ) and tert-butyl alcohol (TBA) are two commonly used reactive oxygen species scavengers, which can specifically capture hydroxyl radicals (OH · ) and superoxide anion radicals (O2 ·- ), thereby affecting the intensity of the ECL signal. The specific steps are to 2 O 2 The capture agent was added to the ECL detection solution (1 mM) and the change in signal compared with the substrate signal was observed. Fig. 9 (AB) The free radical capture experiments were carried out using these two active oxygen scavengers. It was observed that after adding TBA and BQ, both the anode ECL and the cathode ECL decreased, proving that OH · and O 2 ·- All participate in low trigger potential ECL.

[0108] Application Example 2 Optimization of detection conditions

[0109] The ratio of the gold-palladium nanoparticle electrodeposition precursor solution, the concentration of CA15-3 antibody, the incubation time of CA15-3 antibody and the optimization of the incubation time of CA15-3 were investigated respectively. Step (2) of Example 1 was changed to adjust the molar ratio of gold to palladium in the solution containing the gold precursor and the palladium precursor to Au:Pd of 0:1, 1:5, 1:3, 1:1, 3:1 and 1:0 respectively, to obtain working electrodes with different gold-palladium ratios, and the detection was carried out according to Application Example 1. The electrochemical luminescence intensity results are shown in FIG. Fig.10 As shown in (A), it can be seen that the optimal ratio of gold to palladium in the precursor solution is 1:3.

[0110] The concentration of CA15-3 antibody in step (3) of Example 1 was changed to 0 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, and 20 μg / mL, respectively. The obtained working electrode was tested according to Application Example 1. The electrochemical luminescence intensity results were as follows: Fig.10 As shown in (B), the optimal concentration condition was 10 μg / mL.

[0111] The incubation time of CA15-3 antibody and electrode in step (3) of Example 1 and the incubation time of CA15-3 antigen and working electrode in step (5) were changed to 0 min, 30 min, 60 min, 90 min, 120 min and 0 min, 20 min, 40 min, 60 min, 90 min, respectively. The obtained working electrode was tested according to Application Example 1. The electrochemical luminescence intensity test results were as follows: Fig.10 (C) and Fig.10 As shown in (D), it can be seen that the optimal incubation times for the two are 1-2h and 0.5-1h, and the optimal incubation times are 1.5h and 1h, respectively.

[0112] Application Example 3: Low Trigger Potential Electrochemiluminescence Immunosensor for Detection of CA15-3

[0113] The different electrodes prepared in Example 1 (AuPd NPs@O-VMSF / ITO, Ab / AuPd NPs@O-VMSF / ITO, BSA / Ab / AuPd NPs@O-VMSF / ITO, Ag / BSA / Ab / AuPd NPs@O-VMSF / ITO) were placed in a buffer system containing 100 μM luminol and 1 mM hydrogen peroxide to compare the ECL responses. 2 O 2 The strong catalytic effect of ECL makes ECL have strong ECL intensity in a narrow low trigger potential range. Then, GPTMS and CA15-3 antibodies are gradually modified on the electrode, and the binding target CA15-3 is found to decrease the ECL intensity ( Fig.11 This may be attributed to the steric hindrance that hinders the H 2 O 2 Reach the electrode surface to participate in the ECL reaction. The trend of electrochemical impedance also further proves the successful preparation of the immunosensor ( Fig.11 (B)).

[0114] Under the optimal experimental conditions, the Ab / AuPd NPs@O-VMSF / ITO electrode prepared in Example 1 was incubated with different concentrations of antigen and placed in a solution containing 100 μM luminol and 1 mM H 2 O 2 in a buffer system to evaluate its detection performance.

[0115] like Fig.12 As shown in (A), as the concentration of CA15-3 increases, the ECL signal obtained by the prepared low trigger potential ECL immunosensor also decreases. In the concentration range of 5mU / mL-500U / mL, the cathode ECL intensity and the anode ECL intensity show a good linear relationship with the logarithm of CA15-3 concentration. The cathode ECL linear regression equation is I ECL-cathode =–1307lgC CA15-3 (U / mL)+5179, the correlation coefficient is 0.999, and the detection limit (LOD) is calculated to be 3.3mU / mL ( Fig.12 (B)). The linear regression equation of anode ECL is: ECL-anode =–637lgC CA15-3 (U / mL)+2717, the correlation coefficient is 0.996, and the LOD is calculated to be 0.16mU / mL ( Fig.12(C)). Compared with other sensors for detecting CA15-3, the low trigger potential ECL immunosensor we constructed has a wider detection range, lower detection limit, simple electrode materials, and is easy to prepare. It can generate stable cathode and anode ECL signals in a smaller potential range.

[0116] Application Example 4: Sensor Selectivity

[0117] Fig.13 Using cancer markers such as AFP, CA199, CEA and their mixtures as interfering substances, interfering substances AFP (10 ng / mL), CEA (10 ng / mL), and CA19-9 (50 U / mL) were added to the test solution in Application Example 1 to explore the selectivity of the designed Ab / AuPd NPs@O-VMSF / ITO immunosensor. Fig.13 As shown in (A), when only AFP, CA199, and CEA were present, no obvious ECL signal changes were shown. When the target CA15-3 was present, it would cause obvious ECL signal changes. All of the above cancer markers or mixtures with CA15-3 would not affect the detection of CA15-3, proving the excellent selectivity of the proposed low trigger potential ECL immunosensor for CA15-3. In order to further evaluate the reproducibility of the designed Ab / AuPd NPs@O-VMSF / ITO immunosensor, five prepared electrodes were selected to detect 0.5U / mL CA15-3. There was no obvious fluctuation in the ECL signal (relative standard deviation (RSD) = 1.2%), which shows that the developed immunosensor has good reproducibility ( Fig.13 (B)). In addition, AuPd NPs@VMSF / ITO electrode and Ag / BSA / Ab / AuPd NPs@O-VMSF / ITO electrode were used for continuous scanning on the ECL detection instrument. It was found that both the anode and cathode ECL signals were very stable without obvious fluctuations (RSD < 4.1%), which proved that the constructed low-potential ECL immunosensor remained stable during the detection process and was not affected by H 2 O 2 Impact Fig.13 (CD)).

[0118] The results showed that higher concentrations of interfering substances did not cause significant changes in the electrochemiluminescence signal. Only CA15-3 or a mixture of CA15-3 and other interfering species could induce significant changes in the ECL signal, and the amplitude of their ECL signal changes was comparable. This was attributed to the excellent specificity of the CA15-3 antibody, which can only specifically recognize the CA15-3 antigen. As well as the size exclusion effect of the ultra-small and uniform VMSF on macromolecular substances, the prepared sensor has good selectivity and anti-interference properties. In addition, it also has good reproducibility and stability.

[0119] The above analysis results prove that the anti-fouling effect of vertically ordered mesoporous silica film and the efficient enhancement effect of gold palladium nanoparticles on the luminol-hydrogen peroxide electrochemiluminescence system can achieve specific high-sensitivity and high-selectivity detection of CA15-3 in serum.

Claims

1. An electrochemiluminescent immunosensor with low trigger potential, characterized in that: The electrochemiluminescent immunosensor comprises a working electrode, a reference electrode and a counter electrode, and the liquid to be detected contains luminol and hydrogen peroxide; The working electrode is a VMSF-modified electrode of confined gold-palladium nanoparticles, which is obtained by covalently fixing antibodies and blocking nonspecific active sites.

2. The electrochemiluminescent immunosensor with low trigger potential according to claim 1, characterized in that: The working electrode preparation method comprises the steps of: Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode; Step 2, drop-coat 3-glycidyloxypropylmethyldiethoxysilane on the surface of the confined AuPd NPs@VMSF electrode, incubate at room temperature away from light, and obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode; Step 3, drop-coat the surface of the epoxy-functionalized confined AuPd NPs@VMSF electrode in step 2 with a buffer solution containing an antibody and incubate; then drop-coat the surface with a bovine serum albumin solution and incubate to obtain the working electrode.

3. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, characterized in that: The VMSF modified electrode is Growing a silicon dioxide nanoarray film on an electrode by one or more of the following methods: solution growth method, electrochemical assisted self-assembly method, two-phase layered growth method, evaporation induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent induced self-assembly method; The electrode in the VMSF modified electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode.

4. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, characterized in that: The gold precursor is any one of chloroauric acid, gold nitrate, gold cyanide, and a gold complex; the palladium precursor is any one of sodium tetrachloropalladate, palladium chloride, palladium nitrate, an aminopalladium compound, and a palladium complex; The solution containing the gold precursor and the palladium precursor has a gold precursor concentration of 0.01-1 mmol / L and a palladium precursor concentration of 0.05-2 mmol / L; The molar ratio of gold to palladium in the solution containing the gold precursor and the palladium precursor is 1:5 to 1:1; The solvent used in the solution containing the gold precursor and the palladium precursor includes any one or more of a potassium nitrate aqueous solution, a sodium chloride aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution.

5. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, characterized in that: In step 1, the electrodeposition potential is -1.0 to 0 V, the electrodeposition time is 1 to 10 s, and the temperature is room temperature; and / or, the solvent of the GPTMS solution in step 2 comprises any one or more of ethanol, water and ether; And / or, the volume proportion of GPTMS in the GPTMS solution is about 0.02-0.5%; And / or, in step 2, the incubation time is 0.2-2h and the temperature is 5-40°C.

6. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, characterized in that: In step 3, the mass concentration of the antibody in the antibody-containing buffer solution is 1 to 20 μg / mL; the solvent used includes any one or more of STE buffer solution and TE buffer solution; And / or, the incubation condition in step 3 is 0-5°C for 0.5-2h; And / or, in step 3, the mass concentration of the aqueous solution of bovine serum albumin is 0.2-2%.

7. The electrochemiluminescent immunosensor with low trigger potential according to claim 1, characterized in that: The molar concentration of luminol in the detection solution is 10-300 μmol / L, and the concentration of hydrogen peroxide is 0.2-5 mmol / L.

8. The method for constructing an electrochemiluminescent immunosensor with a low trigger potential according to any one of claims 1 to 7, characterized in that: Includes steps: Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode; Step 2, drop-coat GPTMS on the surface of the confined AuPd NPs@VMSF electrode, incubate at room temperature away from light to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode; Step 3, drop-coat the surface of the epoxy-functionalized confined AuPd NPs@VMSF electrode in step 2 with a buffer solution containing an antibody and incubate; Then, drop-coating a bovine serum albumin solution and incubating to obtain the working electrode; Step 4: Using the three-electrode system of the working electrode, the reference electrode and the counter electrode, and the electrolyte solution containing luminol and hydrogen peroxide as the detection solution, the electrochemiluminescent immunosensor is constructed.

9. Use of the electrochemiluminescent immunosensor with low trigger potential according to any one of claims 1 to 7 in detecting biological antigens for non-therapeutic or diagnostic purposes, characterized in that: Includes steps: The working electrode is incubated with a test solution containing biological antigens to obtain a test electrode, and a three-electrode system is formed by the test electrode, a reference electrode and a counter electrode. Electrochemical luminescence signal detection is performed in an electrolyte solution containing luminol and hydrogen peroxide as the test solution.

10. The use according to claim 9, characterized in that: The incubation time in the test solution is 0.5-2h, the incubation temperature is 0-5°C, and the pH of the test solution is 6-8; And / or, the biological antigen includes one of a target tumor marker, a cell surface factor, a bacterial or viral surface factor, and its concentration is above 5 mU / mL.

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