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 sensor comprises a three-electrode system of an immune electrode, a reference electrode and a counter electrode, and when the electrochemical luminescence immunosensor is used, a detection solution comprises luminol and hydrogen peroxide; the immunoelectrode is an SNF modified electrode confined with metal palladium nanoparticles and nitrogen-doped graphene quantum dots, immunoglobulin A of immunospheres is covalently immobilized, then a primary antibody of a directionally immobilized tumor marker is constructed, and non-specific sites are closed, so that the immunoelectrode is obtained. The independent nano channel of the SNF film effectively avoids agglomeration of a confinement material, and the in-situ deposition mode is efficient and rapid; the good biocompatibility of the SNF provides a high-quality platform for the preparation of an immunosensing interface. The prepared immunosensor has the advantages of fast response speed, high ECL signal and good selective anti-interference capability, and has potential application prospects in tumor marker analysis and detection.

Description

Technical Field

[0001] The present invention relates to the 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] Pancreatic cancer poses a serious threat to human life and health due to its high mortality rate. To date, pancreatic cancer has become the third most common cancer and the second most deadly cancer in the world. Poor prognosis is one of the main reasons for the high mortality rate. Therefore, early detection of pancreatic cancer by detecting tumor biomarkers is particularly important. As a biomarker for diagnosing gastrointestinal malignancies, carbohydrate antigen 242 (CA242) has a high specificity for pancreatic cancer, even higher than CA199. Currently, there are more and more methods for immunodetection of CA242. For example, immunoradiometric assay (IRMA), immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), etc. These methods often face some problems such as long detection time, complex and expensive detection instrument operation, etc. To solve these problems, various electrochemical-related immunosensors have been developed and used.

[0003] The sensitivity and stability of immunosensors depend on many parameters, such as orientation, surface density, and antigen binding efficiency after antibody immobilization on the functionalized surface. Non-covalent physical adsorption or chemical adsorption is attractive due to its technical simplicity, but it can lead to random arrangement of antibodies on the surface, resulting in low sensor performance, leakage and instability problems. This is due to the fact that asymmetric antibody molecules may be immobilized through variable antigen binding sites (Fab), resulting in reduced or complete elimination of binding activity. The use of covalent immobilization techniques can improve the stability and density of immobilized antibodies, but these methods are not entirely ideal because they are usually not site-oriented. Therefore, directional immunosensing has received extensive attention and research, among which directional immobilization of antibodies through Fc-binding proteins, immunoglobulin A and protein G are widely used. The strong affinity of immunoglobulin A to the Fc end of the antibody effectively avoids the binding of the antibody Fab end to the electrode surface, exposing more antigen binding sites to improve sensor sensitivity.

[0004] Among the development and use of various immunosensors, electrochemiluminescence (ECL) immunoassay has been widely developed due to its advantages such as fast analysis speed, high sensitivity, low cost, and simple equipment. Luminophores and co-reactants are important components of the ECL system, and luminescence efficiency is one of the important indicators for evaluating their capabilities. The luminol-hydrogen peroxide (Luminol-H2O2) system is one of the most widely used luminescence systems. However, in order to obtain a more efficient ECL response, a more negative or positive potential is inevitably required to stimulate and promote the ECL response. Excessively wide scanning potentials will produce various electrochemical interferences, and may even destroy the biological activity of the target and reduce sensitivity and specificity. Therefore, constructing a high-performance ECL system with a low trigger potential is of great significance to broaden its application areas.

[0005] CN111208177A discloses a method for constructing a signal dual-amplification electrochemiluminescence sensor based on copper oxide and nano-platinum to enhance the luminol luminescence. In the present invention, luminol as a luminophore reacts with gold nanoparticles Au@BSA coated with bovine serum albumin to generate Luminol-Au@BSA, which is then combined with copper oxide nanospheres to generate Luminol-Au@BSA-CuO as a signal probe. Copper oxide and nano-platinum can catalyze H2O2 to obtain more O2 ·- , which greatly enhances the luminescence intensity of Luminol at low potential and realizes double amplification of the signal. Different concentrations of procalcitonin PCT can bind to different amounts of secondary antibody marker Ab2-Luminol-Au@BSA-CuO, thus causing changes in the luminescence intensity of the sensor and realizing quantitative detection of PCT.

[0006] Therefore, for ECL system biosensors, utilizing the electrocatalytic properties of nanomaterials is an effective means to construct a low-potential luminol ECL system, hoping to further improve its detection performance for tumor markers. Summary of the invention

[0007] In view of the problem of high activation potential of ECL system biosensors, the present invention provides an electrochemiluminescence immunosensor with a low trigger potential for detecting tumor markers. The electrochemiluminescence immunosensor takes a silicon dioxide nanochannel film as a matrix, and sequentially combines graphene quantum dots and palladium nanomaterials to obtain a high luminol-hydrogen peroxide ECL system response at a low trigger potential of -0.2V.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A low trigger potential electrochemiluminescent immunosensor, comprising a three-electrode system of an immunoelectrode, a reference electrode, and a counter electrode, wherein the detection solution of the electrochemiluminescent immunosensor comprises luminol and hydrogen peroxide when the electrochemiluminescent immunosensor is used;

[0010] The immunoelectrode is a SNF modified electrode of confined metal palladium nanoparticles and nitrogen-doped graphene quantum dots, which is obtained by covalently fixing immunoglobulin A, constructing a directional fixed tumor marker primary antibody and blocking non-specific sites.

[0011] The present invention uses a tumor marker primary antibody as a specific recognition element; uses luminol electrochemiluminescent signal molecules, uses hydrogen peroxide as a co-reactant of luminol, and combines the synergistic catalytic effect of a PdNPs / nitrogen-doped graphene quantum dot composite material confined in a vertically ordered mesoporous silica nanochannel film to construct an immunosensor; triggers the electrochemiluminescence of luminol-hydrogen peroxide at a low potential and improves the detection sensitivity.

[0012] The luminol-H2O2 system is the most common ECL system, but the triggering potential is often above 0.6 V. Taking the ITO electrode as an example, the bare ITO electrode or the SNF / ITO electrode has almost no ECL response in the luminol-H2O2 system under the potential scan of -0.2 to 0.3 V, because luminol oxidation or reactive oxygen radicals (ROS) generation almost does not occur at this potential.

[0013] As a precious metal nanomaterial, PdNPs can effectively promote the ECL response of the luminol-H2O2 system at low potential, and the addition of N-GQDs further improves the ECL signal. Pd nanomaterials can catalyze the luminol-H2O2 system to produce a strong ECL response at low potential, and the nanomaterials grown in situ in the nanopores have a higher specific surface area and higher catalytic activity than traditional large particle materials. N-GQDs have good pseudo-peroxidase activity. When N-GQDs and Pd are confined together in the nanochannels of SNF, the composite nanomaterials show stronger catalytic activity, catalyzing H2O2 or luminol to produce a large amount of ROS and luminol intermediates at an oxidative potential, thereby improving the ECL signal.

[0014] The immunoelectrode preparation specifically comprises the following steps:

[0015] Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode with confined N-GQDs;

[0016] Step 2, immersing the confined N-GQDs electrode in a Na2PdCl4 solution for electrodeposition to obtain a confined PdNPs / N-GQDs electrode;

[0017] Step 3, immersing the confined PdNPs / N-GQDs electrode in a siloxane solution containing epoxy groups to react, thereby obtaining an epoxy group-functionalized confined PdNPs / N-GQDs electrode;

[0018] Step 4, immersing the electrode of epoxy group functionalized confined PdNPs / N-GQDs in a buffer solution containing immunoglobulin A for incubation, and after rinsing, obtaining an electrode of immunoglobulin A modified confined PdNPs / N-GQDs;

[0019] Step 5, immersing the immunoglobulin A-modified confined PdNPs / N-GQDs electrode in a buffer solution containing a tumor marker primary antibody for incubation, and then immersing it in a bovine serum albumin solution for incubation after rinsing to obtain the immunoelectrode.

[0020] In the present invention, an immunosensor is successfully constructed on the electrode surface. First, a layer of SNF film is grown on the electrode surface to obtain a thin film electrode with open pores. The electrodes are placed in N-GQDs solution and Na2PdCl4 solution respectively, and N-GQDs and palladium nanomaterials are confined in nanochannels in steps by electrophoresis and electrodeposition respectively. Based on the abundant silanol groups on the surface of the SNF film, the electrode surface is functionalized with epoxy groups by modification with GPTMS. Immunoglobulin A is incubated on the electrode surface and covalently bound to the epoxy groups. Subsequently, tumor marker antibodies are incubated and non-specific sites are blocked with BSA. Immunoglobulin A dripped on the electrode surface is covalently bound to the epoxy functional group, and the strong affinity of immunoglobulin A to the Fc end of the antibody exposes the Fab end of the antibody to the outer surface of the electrode to achieve directional fixation. Subsequently, non-specific sites are blocked with BAS. The stability of nanomaterials can be significantly improved by confining PdNPs / N-GQDs using the nanochannel array of SNF. In addition, porous materials provide dispersion space, which can effectively increase the active sites of nanomaterials, thereby improving their catalytic activity and stability. In addition, the directed fixed antibodies provide more binding sites for subsequent specific binding to tumor markers.

[0021] Preferably, the nitrogen-doped graphene quantum dots are synthesized by a hydrothermal method in an ammonia solution using 1-aminopyrene as a carbon source;

[0022] Further preferably, the preparation of the nitrogen-doped graphene quantum dots specifically comprises the steps of: dispersing 1-aminopyrene in ammonia water and reacting at 170-200° C. for 2-10 hours, and purifying the product to obtain nitrogen-doped graphene quantum dots.

[0023] More preferably, the mass concentration of 1-aminopyrene in aqueous ammonia is 0.1 to 5 mg / mL.

[0024] The solvent used in the nitrogen-doped graphene quantum dot solution and the Na2PdCl4 solution is water, and the solvent used in the GPTMS solution is ethanol;

[0025] The electrode in the SNF modified electrode is 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;

[0026] The preparation method of the SNF modified electrode comprises the following steps: SNF is grown on the electrode by any of the following methods: solution growth 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.

[0027] Specifically, using The method for preparing SNF modified electrode by solution growth method comprises the following steps:

[0028] Sodium hexadecyl sulfonate (CTAB) was used as a template surfactant, dissolved in a water-ethanol solution, and ammonia and an organosiloxane precursor were added to obtain a precursor solution for growing SNF;

[0029] Inserting the electrode into the precursor solution for incubation, removing, washing and drying to obtain a modified electrode containing micelle (SM) template in the pores;

[0030] The micelles can be removed by immersing the electrode in a Cl-ethanol solution and stirring to obtain a SNF-modified electrode with open pores.

[0031] The method for preparing SNF modified electrode by EASA method comprises the following steps:

[0032] An organosiloxane precursor is added to a CTAB aqueous solution to obtain a precursor solution, an electrode is inserted into the precursor solution, a negative voltage is applied to the electrode to grow SNF, and the electrode is taken out, cleaned, and dried to obtain a modified electrode containing an SM template in the pores;

[0033] The micelles can be removed by immersing the electrode in a Cl-ethanol solution and stirring to obtain a SNF-modified electrode with open pores.

[0034] The nitrogen-doped graphene quantum dot solution in step 1 has a nitrogen-doped graphene quantum dot mass concentration of 0.05-3.0 mg / mL, an electrodeposition time of 1-120 s, and an electrodeposition voltage of +0.7 V to 0.9 V;

[0035] The molar concentration of Na2PdCl4 in the Na2PdCl4 solution of step 2 is 0.05-3.0mM, the electrodeposition time is 0.1-60s, and the electrodeposition voltage is -0.8V to -0.4V.

[0036] In step 3, the concentration of GPTMS in the GPTMS solution is 0.5-5 mM GPTMS, and the reaction is allowed to stand for 20-80 minutes at a temperature of 20-40° C.;

[0037] In step 4, the incubation is at 0-5°C for 0.5-4h;

[0038] In step 5, the incubation is performed at 0-5° C. for 0.5-4 h.

[0039] The concentration of immunoglobulin A in the buffer solution of step 4 is 1-20 mg / mL, the buffer solution is a phosphate buffer, the phosphate concentration is 0.005-0.3 mol / L, and the pH is 6.5-7.5;

[0040] The concentration of the tumor marker primary antibody in the buffer solution of step 5 is 1-20 mg / mL, the buffer solution is a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution, the solute concentration in the buffer solution is 0.005-0.5 mol / L, and the pH is 6-8.

[0041] The epoxy group siloxane includes any one or more of 3-aminopropyltriethoxysilane (GPTMS), (3-glycidylpropoxy)trimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane.

[0042] Preferably, each step is rinsed with water or buffer after completion to remove excess undeposited or unincubated solution;

[0043] The present invention also provides a construction method, comprising the steps of:

[0044] Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode with confined N-GQDs;

[0045] Step 2, immersing the confined N-GQDs electrode in a Na2PdCl4 solution for electrodeposition to obtain a confined PdNPs / N-GQDs electrode;

[0046] Step 3, immersing the confined PdNPs / N-GQDs electrode in a siloxane solution containing epoxy groups to react, thereby obtaining an epoxy group-functionalized confined PdNPs / N-GQDs electrode;

[0047] Step 4, immersing the electrode of epoxy group functionalized confined PdNPs / N-GQDs in a buffer solution containing immunoglobulin A for incubation, and after rinsing, obtaining an electrode of immunoglobulin A modified confined PdNPs / N-GQDs;

[0048] Step 5, immersing the electrode of immunoglobulin A modified confined PdNPs / N-GQDs in a buffer solution containing a tumor marker primary antibody for incubation, and then immersing it in a bovine serum albumin solution for incubation after washing to obtain the immunoelectrode;

[0049] Step 6: Using the three-electrode system of the immunoelectrode, 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.

[0050] The molar concentration of luminol in the detection solution is 5-300 μmol / L, and the concentration of hydrogen peroxide is 50-300 μmol / L.

[0051] The present invention also provides the electrochemiluminescent immunosensor with low trigger potential for detecting tumor markers for non-diagnostic purposes, comprising the steps of:

[0052] The immunoelectrode is incubated with a sample solution containing tumor markers and then rinsed to obtain an immune complex modified electrode;

[0053] The immune complex modified electrode is used as the modified electrode, and a three-electrode system is formed with the reference electrode and the counter electrode. The electrochemical luminescence signal is detected using an electrolyte solution containing luminol and hydrogen peroxide as the detection solution. The detection principle is as follows: When tumor markers are present in the serum sample, the primary antibody of the tumor marker binds to the tumor marker, and the larger antigen-antibody immune complex hinders the mass transfer of luminol and hydrogen peroxide, causing the ECL signal to change, thereby achieving quantitative detection of tumor markers. The N-GQDs and Pd composite nanomaterials trigger the ECL of the luminol-H2O2 system at a low potential, and have a very high improvement in the ECL emphasis, greatly improving the sensitivity of the sensor.

[0054] Preferably, the sample to be tested may contain one or more interfering compounds such as glucose, non-target tumor markers, inorganic salts, etc. Even in the presence of multiple interfering compounds, the sensor of the present invention still has a high sensitivity.

[0055] The electrochemiluminescent immunosensor can have a detection limit below 0.8 mU / mL.

[0056] The triggering potential of the electrochemiluminescence immunosensor is -0.2 V, which is lower than the triggering potential of the electrochemiluminescence immunosensor in the prior art.

[0057] 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 immune response, detect residual tumor markers after drug action, etc.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) The electrochemiluminescence immunosensor of the present invention is based on SNF confined palladium nanoparticles / nitrogen-doped graphene quantum dots (PdNPs / N-GQDs) composite nanomaterials, which serves as a high-efficiency catalyst for the luminol-H2O2 system, realizes low-potential ECL of the luminol-H2O2 system, enhances the ECL signal, and improves the detection sensitivity.

[0060] (2) Using electrophoresis and in-situ electrodeposition strategies, N-GQDs and PdNPs were confined in the nanochannels of SNF, respectively. The independent ultrafine nanochannel array effectively avoided the agglomeration of nanomaterials, giving the nanocatalysts high catalytic efficiency and stability. Among them, PdNPs can effectively catalyze H2O2 to produce a large amount of ROS at a low potential of -0.2 to 0.3V, realizing the low-potential electrochemiluminescence of luminol; the introduction of N-GQDs further sensitized the ECL response of luminol at this potential.

[0061] (3) The electrochemiluminescent immunosensor of the present invention adopts the strategy of immunoglobulin A directional fixation antibody modification to construct the immunosensor, which realizes the sensitive detection of tumor markers. Based on the good biocompatibility of SNF, the immunosensor interface is constructed, immunoglobulin A (PrA) is introduced on the outer surface of SNF, and the antibody is directional fixed based on the affinity between PrA and the Fc end of the antibody, so that more antigen recognition sites at the Fab end are exposed to the outer surface of the electrode, which can realize the specific recognition of more target tumor markers, thereby improving the detection sensitivity.

[0062] (4) When the immunosensor of the present invention is used for detection, the electrochemical signal can be measured after the immunoelectrode is incubated with the sample to be tested, without the need for a sandwich immunoassay mode, which makes the operation simpler and the cost lower.

[0063] (5) The present invention confines platinum nanoparticles / nitrogen-doped graphene quantum dots in nanochannels, and the two materials produce a synergistic effect, which significantly enhances the signal sensitivity of luminol-hydrogen peroxide compared with a single material, and achieves low-potential ECL.

[0064] (6) The detection method of the present invention has a wide linear range for tumor marker detection, high selectivity, high detection sensitivity and low detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1In Example 1, (A) is a top-view transmission electron microscope (TEM) photograph of an ITO electrode (SNF / ITO) with silica nanopores. (B) is a scanning electron microscope (SEM) image of a cross section of an SNF-modified ITO electrode (SNF / ITO). (C) is a SEM top view of SNF / ITO. (D) is a SEM top view of an ITO electrode (PdNPs / N-GQDs@SNF / ITO) modified with silica nanopores of confined palladium nanoparticles / nitrogen-doped graphene quantum dots. (E) is a SEM top view of a PdNPs / / N-GQDs@SNF / ITO electrode, with PdNPs deposited for 10s, and (F) is a Pd element mapping diagram in Figure (E).

[0066] Figure 2 In Example 1, (A) SEM top view of the electrode obtained by dissolving SNF in NaOH with PdNPs / N-GQDs@SNF / ITO electrode, where the PtNPs deposition time is 0.5s and the N-GQDs confinement is 1min, (B) is its enlarged view, and (C) is its corresponding elemental spectrum. (D) XPS spectrum measurement spectra of different electrodes, (E) high-resolution Pd 3d spectrum of PdNPs / N-GQDs@SNF / ITO. (F) Fluorescence intensity of SNF / ITO and N-GQDs@SNF / ITO, the inset is the fluorescence image of SNF / ITO (left) and N-GQDs@SNF / ITO (right).

[0067] Figure 3 (A) ECL signal diagram of different modified electrodes in Example 1. (B) ECL signal diagram of PdNPs / N-GQDs@SNF / ITO electrode after repeated rinsing and drying. (C) ECL-potential diagram of PdNPs / N-GQDs@SNF / ITO electrode. (D) Different free radical scavengers including hydroxyl radical (·OH) scavenger tert-butyl alcohol (TBA) and superoxide radical (O2 ·- ) Relative ratio of the ECL intensity of benzoquinone (BQ) in the absence (I0) or presence (I) of the capture agent.

[0068] Figure 4 (A) ECL signals on different electrodes during the preparation of the immunosensor in Example 1. (B) ECL signals of electrodes obtained with different immunoglobulin A incubation times (0-120 minutes). (C) ECL signals obtained from immunosensors prepared with different antibody incubation times (0-120 minutes). (D) ECL intensity of electrodes obtained with different incubation times of immunosensor and CA242.

[0069] Figure 5(A) ECL signals obtained on the prepared immunosensor after incubation with different concentrations of CA242 in Example 1. (B) Linear fitting curve of Ab / PrA / O-PdNPs / N-GQDs@SNF / ITO for detecting CA242. (C) Change rate of ECL signal in the absence (I0) or presence (I) of interfering substances in different species. Interfering substances include carcinoembryonic antigen CEA (1 ng mL -1 )、CA19-9(10mU mL -1 ) tumor markers and possible glucose (10 μM), K + (1μM), Na + (1μM). The ECL signal of the electrodes before and after the immunosensor was incubated with the above substances was measured, and the change rate was obtained by comparing the ECL signal change value with the initial ECL signal of the electrode before the action. (D) Signal response of five identical electrodes (detection of 0.1UmL -1 CA242). DETAILED DESCRIPTION

[0070] 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.

[0071] The raw materials used in the following specific embodiments are all purchased from the market.

[0072] Example 1

[0073] The process of using a silica nanopore modified electrode of a nanochannel confined PdNPs / N-GQDs composite material to covalently immobilize the tumor marker CA242 antibody on the outer surface to prepare an immunoelectrode and detecting CA242 in serum samples by electrochemiluminescence:

[0074] (1) SNF / ITO Solution growth method preparation

[0075] First, dissolve 0.1600 g of hexadecyl ammonium bromide (CTAB) in 70 mL of water and 30 mL of ethanol solution, and stir for 5 min until the CTAB is completely dissolved; then add 100 μL of 10% ammonia water and 80 μL of TEOS as a silicon source while stirring, and continue stirring for 5 min until the solution becomes clear and transparent without bubbles, and carefully transfer the solution to a staining jar.

[0076] Immerse clean ITO glass (2.5×5cm) in the precursor solution, seal the staining jar, and place it in a 60℃ water bath for 24h. After the growth is completed, take out the ITO electrode, rinse it with a large amount of deionized water, blow dry it with nitrogen, and age it overnight at 100℃ in an oven to obtain a modified electrode containing micelle (SM) templates in the pores, named SM@SNF / ITO. The prepared electrode is cut and its electrode area is controlled to 0.5×1cm with insulating glue as a working electrode. The electrode is immersed in 0.1M HCl-ethanol solution and stirred for 5min to remove the micelles, and the SNF / ITO electrode with open pores is obtained.

[0077] (2) N-GQDs were synthesized by hydrothermal method in aqueous ammonia solution using 1-aminopyrene as carbon source. The specific process is as follows: 1-aminopyrene (1 mg / mL) was added to aqueous ammonia (0.2 M), and after ultrasonic dispersion, the resulting mixture was added to the polytetrafluoroethylene liner of the reactor and reacted at 200 °C for 2 h. The resulting solution was first filtered with a 0.22 μm filter membrane to remove large-sized particles to obtain a reddish-brown solution, and then dialyzed for 2 days using a dialysis bag (molecular weight cutoff of 500 Da and 3500 Da, respectively) to remove unreacted small molecules and salts generated by the reaction. The dialyzate was freeze-dried to obtain N-GQDs solid.

[0078] (3) Preparation of PdNPs / N-GQDs@SNF / ITO

[0079] PdNPs / N-GQDs@SNF / ITO were prepared by confining N-GQDs and PdNPs in the nanopores of SNF / ITO by electrophoresis and in-situ electrodeposition. The specific methods are as follows:

[0080] The prepared SNF / ITO with open pores was immersed in an aqueous solution of N-GQDs (1 mg / mL), and a potential of +0.8 V was applied. The electrophoresis lasted for 1 min to obtain the N-GQDs@SNF / ITO electrode.

[0081] The solution for depositing PdNPs is Na2PdCl4 solution (1mM, 0.1M KNO3 solution). The prepared N-GQDs@SNF / ITO electrode is immersed in Na2PdCl4 solution, and a constant potential of -0.5V is applied for electrodeposition for 0.5s. The PdNPs structure is confined in the nanopores of SNF, and the obtained electrode is recorded as PdNPs / N-GQDs@SNF / ITO.

[0082] (4) Preparation of immunosensor

[0083] PdNPs / N-GQDs@SNF / ITO was used as the substrate electrode, and γ-glycidyloxypropyltrimethylsilane (GPTMS) was used as a crosslinker to covalently crosslink the antibody and SNF layers. GPTMS was incubated before removing the micelles to avoid a large amount of GPTMS modification on the inner wall of the pores, which would hinder mass transfer.

[0084] The specific experimental steps are as follows: After the PdNPs / N-GQD@SNF / ITO electrode is placed in a GPTMS solution (2.26 mM, ethanol) and incubated for 30 min, it is rinsed with a large amount of deionized water and blown dry with N2 to obtain an O-PdNPs / N-GQD@SNF / ITO electrode;

[0085] Immunoglobulin A (PrA) was used to fix the antibody. A stock solution of PrA (10 mg mL -1 ). Dilute the immunoglobulin A stock solution to 100 μg mL with 10 mM PBS (pH = 7.4). -1 For subsequent use. Take 50 μL of immunoglobulin A (100 μg mL -1 ) was drop-coated on the surface of the O-PdNPs / N-GQD@SNF / ITO electrode and incubated at 4°C to covalently bind to the epoxy group. The electrode surface was slowly rinsed with the same buffer solution to remove excess unbound immunoglobulin A, and the PrA / O-PdNPs / N-GQD@SNF / ITO electrode was obtained;

[0086] The CA242 stock solution was diluted to 20 μg mL with 10 mM PBS (pH = 7.4). -1 , 50 μL was applied to the electrode surface and evenly covered. Incubated at 4°C. The electrode surface was rinsed with 10 mM PBS (pH = 7.4) to remove unbound CA242 antibodies, and the final immunosensor Ab / PrA / O-PdNPs / N-GQD@SNF / ITO was obtained for subsequent experimental detection.

[0087] (5) CA242 electrochemiluminescence detection

[0088] In order to detect CA242, the Ab / PrA / O-PdNPs / N-GQD@SNF / ITO electrode was incubated with a series of known concentrations of CA242 antigen on the surface of the Ab / PrA / O-PdNPs / N-GQD@SNF / ITO electrode, and after the antigen-antibody specific binding reached saturation, the electrode surface was carefully rinsed with the same PBS buffer to remove the unbound CA242, and the obtained electrode was recorded as CA242 / Ab / PrA / O-PdNPs / N-GQD@SNF / ITO.

[0089] The obtained electrode was subjected to ECL detection, and the electrolyte solution used for the test was a PBS (0.01M, pH=7.4) solution containing 100 μM luminol and 100 μM H2O2. The ECL process was triggered by continuous CV scanning, and the CV scanning potential range was -1 to 0.8 V, and the scanning rate was 100 mV / s.

[0090] In step 1, Electron microscopy of the ITO electrode modified with silica nanopores (SNF / ITO) prepared by the solution growth method Figure 1 shown.

[0091] Figure 1 (A) TEM top view of the ITO electrode modified with silica nanopores (SNF / ITO), where the white bright spots are the pores. The TEM top view of SNF shows a uniform disordered distribution, with the pore size uniformly around 2-3 nm; the black part is silica, and it can be seen that the film as a whole has no large-area defects. Figure 1 (B) shows the SEM image of the SNF / ITO cross section, where three layers can be clearly seen, namely SNF film, ITO layer and glass layer from top to bottom. The thickness of SNF is 103nm. Figure 1 (C) shows the SEM image of the SNF / ITO surface, where it can be clearly seen that the surface is evenly distributed without cracks on the entire ITO surface.

[0092] To verify the successful confinement of PdNPs, SEM images of the electrode before and after the deposition of PdNPs in step 3 were taken. Figure 1 (D) shows the surface of the PdNPs / N-GQDs@SNF / ITO electrode obtained after 0.5s deposition. Compared with the SNF / ITO surface, there is almost no significant change on the surface, indicating that the confined material has almost no effect on the electrode surface. When the material electrodeposition time is extended, due to the limited space of the nanochannel, the material may overflow the pores and distribute on the electrode surface, affecting the electrode performance. Figure 1 (E) The PdNPs / N-GQDs@SNF / ITO electrode surface is obtained by deposition for 10s. The granular materials of uneven sizes are distributed on the surface. Through EDS spectrum element characterization, it can be seen that the main element of the material is Pd (such as Figure 1 (F)). In order to verify whether the nanopores of the PdNPs / N-GQDs@SNF / ITO electrode contain deposited materials, the thin film on the electrode surface was dissolved with NaOH, and the nanomaterials in the pores were exposed to form large aggregates and evenly distributed on the electrode surface. Figure 2(AC) shows that the deposition time of PtNPs is 0.5s, and the confinement time of N-GQDs is 1min. (B) is its enlarged image, and (C) is its corresponding elemental spectrum. (D) XPS spectrum measurement spectrum of different electrodes. The enlarged image and EDS element characterization prove that the main element of the material is Pd. The main reason for the appearance of larger aggregates may be that the precursor does not contain a protective agent in the one-step electrodeposition method of nanochannel confinement. When the film exists, the independent nanochannels can protect the nanomaterials from agglomeration. After the film is dissolved and destroyed, the nanomaterials agglomerate to form large aggregates.

[0093] The material was further characterized by XPS. Figure 2 (D) is the XPS total spectrum of different electrodes. Compared with SNF / ITO, N characteristic peaks can be observed from N-GQDs@SNF / ITO and PdNPs / N-GQDs@SNF / ITO, while obvious Pd characteristic peaks can be observed on PdNPs@SNF / ITO and PdNPs / N-GQDs@SNF / ITO, proving the existence of confined material PdNPs / N-GQDs. Figure 2 (E) is the Pd 3d spectrum corresponding to the Pd element, corresponding to the Pd 3d of metal Pd(0) 5 / 2 and Pd 3 / 2 Components. Figure 2 As shown in (F), N-GQDs@SNF / ITO has a significant fluorescence intensity compared with SNF / ITO, and the color of the fluorescence microscopy photograph in the inset is also consistent with the fluorescence of N-GQDs.

[0094] like Figure 3 As shown in (A), the SNF / ITO electrode has almost no ECL response in the luminol-H2O2 system under the potential scan of -0.2~0.3V, because almost no luminol oxidation or ROS generation occurs at this potential. N-GQDs have good pseudoperoxidase activity, but there is still no obvious low-potential ECL at this scanning potential. Pd nanomaterials can catalyze the luminol-H2O2 system to produce a strong ECL response at low potentials, and the nanomaterials grown in situ in the nanopores have a higher specific surface area than traditional large particle materials, and have higher catalytic activity. The PdNPs / N-GQDs@SNF / ITO electrode has an improvement of nearly 50% compared with the previous electrodes. This is because PdNPs / N-GQDs can act as a co-reactant promoter to catalyze H2O2 to produce a large amount of ROS to enhance the ECL signal of luminol, and the PdNPs / N-GQDs composite material grown in the SNF pores has a larger specific surface area, which further improves its catalytic activity. In addition, PdNPs / N-GQDs have a synergistic effect, producing a signal sensitization effect that is higher than the superposition of single materials.

[0095] The PdNPs / N-GQDs@SNF / ITO electrode was rinsed 10 times and the ECL signal was tested. Figure 3 As shown in (B), the electrode still maintains a stable ECL signal in 10 consecutive measurements, indicating that the composite material can be stably confined in the nanochannel and can maintain a stable ECL response even after multiple scanning washes.

[0096] like Figure 3 (C) shows the ECL potential diagram of the PdNPs / N-GQDs@SNF / ITO electrode. From the ECL potential diagram, it can be seen that the CV scan starts from +0.3V and scans negatively to -0.2V and then scans back. When the oxidation scan reaches -0.1V, a luminescence signal appears. Therefore, it is speculated that the possible luminescence mechanism of the ECL luminescence system with luminol as the luminophore, H2O2 as the co-reactant, and PdNPs / N-GQDs as the co-reactant promoter is: the composite material catalyzes H2O2 or luminol at the oxidation potential to produce a large amount of ROS and luminol intermediates, thereby increasing the ECL signal.

[0097] It is speculated that the possible luminescence mechanism of the ECL luminescence system with luminol as the luminophore, H2O2 as the co-reactant, and PdNPs / N-GQDs as the co-reactant promoter is: the composite material catalyzes H2O2 or luminol at an oxidative potential to produce a large amount of ROS and luminol intermediates, thereby increasing the ECL signal. The reactive oxygen species in the reaction process were explored through free radical capture experiments.

[0098] 0.5 mM of p-benzoquinone (BQ) or tert-butyl alcohol (TBA) was added to the luminol hydrogen peroxide solution. Figure 3 As shown in (D), superoxide radicals (O2 ·- ) scavenger benzoquinone (BQ), the ECL signal decreased significantly and almost no response. However, after adding hydroxyl radical (·OH) scavenger tert-butyl alcohol (TBA), the ECL signal remained almost unchanged. Therefore, it is proved that the active oxygen species in the system are mainly O2 ·- The ECL reaction mechanism reaction formula is as follows:

[0099]

[0100]

[0101] AP 2-* →AP 2- +hv

[0102] First, Pd nanoparticles are electrocatalytically oxidized by N-GQDs to Pd 2+; Then, due to Pd 2+ Due to the excellent catalytic reduction effect of N-GQDs, H2O2 will be reduced to superoxide anion radicals (O2 ·- ). Luminol loses protons in an alkaline environment to become luminol anion (LH - ), while the luminol anion radical (L ·- ) and superoxide anion radical (O2 ·- ) reacts to generate luminol excited state (AP 2-* ), which returns to the ground state 3-aminophthalate (AP 2- ) is accompanied by ECL luminescence (hv).

[0103] from Figure 4 (A) is the ECL response of the electrode during the construction of the CA242 immunosensor and the electrode obtained after the immunosensor was incubated with CA242. It can be seen that the ECL response gradually decreases. The more and more modifiers there are on the electrode surface, the greater the surface steric hindrance. When the CA242 antibody is covalently immobilized on the electrode surface, the ECL signal of the resulting immunosensor decreases, which may be due to the insulating properties of the immune. After the immunosensor was incubated with CA242, the ECL signal of the electrode decreased significantly. This is attributed to the fact that when CA242 specifically recognizes the immune and forms a complex at the interface, on the one hand, the interface resistance increases, and on the other hand, the large structure of the complex reduces the diffusion of luminol and the electrode support liquid to the surface of the supporting electrode, thereby reducing the ECL signal.

[0104] The incubation conditions for detecting CA242 in step 4, including the incubation time for immobilizing immunoglobulin A when preparing the immunoelectrode, the incubation time for immobilizing the antibody, and the incubation time for binding with the recognition antigen in step 5, were optimized to determine the final ECL response. Figure 4 As shown in (C) and (D), the incubation time of the antibody and antigen was optimized, and the A protein and antibody reached incubation saturation at 90 minutes, and the antigen reached incubation saturation at 120 minutes.

[0105] like Figure 5 As shown in (A) and (B), when the concentration of CA242 varies from 1 mU / mL to 100 U / mL, the ECL intensity has a linear relationship with the logarithmic value of the CA242 concentration (logCCA242) (IECL = -1062 (±49) logCCA242 + 7574 (±184), R2 = 0.992). The detection limit calculated based on the three-fold signal-to-noise ratio (S / N = 3) is 0.8 mU / mL.

[0106] When measuring the anti-interference ability of the immunosensor, the interference substances include carcinoembryonic antigen CEA (1ng mL -1)、CA19-9(10mUmL -1 ) tumor markers and possible glucose (10 μM), K + (1μM), Na + (1 μM). The ECL signal of the electrode before and after the immunosensor was incubated with the above substances was measured, and the change rate was obtained by comparing the ECL signal change value with the initial ECL signal of the electrode before the action. Figure 5 As shown in (C), when there is no CA242 in the test object and only contains interferences, the ECL signal of the sensor remains basically unchanged, while when the test object contains CA242 or contains CA242 and other interferences, the ECL signal response changes of the two are almost consistent. This fully demonstrates that the prepared sensor has specific recognition ability only for the target CA242 and is not easily affected by other coexisting substances, proving its high selectivity.

[0107] like Figure 5 As shown in (D), if five immunosensors were prepared in parallel, they were incubated with CA242 (0.1 U mL -1 ) After incubation and binding, the ECL signal of the electrode was measured, and it was found that the relative representation deviation (RSD) of the ECL signal of CA242 detected by the five sensors was 2.6%.

Claims

1. An electrochemiluminescent immunosensor with low trigger potential, It is characterized in that A three-electrode system comprising an immunoelectrode, a reference electrode and a counter electrode, wherein the detection solution of the electrochemiluminescent immunosensor comprises luminol and hydrogen peroxide when the electrochemiluminescent immunosensor is used; The immunoelectrode is a SNF modified electrode of confined metal palladium nanoparticles and nitrogen-doped graphene quantum dots, which is obtained by covalently fixing immunoglobulin A, constructing a directional fixed tumor marker primary antibody and blocking non-specific sites.

2. The electrochemiluminescent immunosensor with low trigger potential according to claim 1, It is characterized in that The immunoelectrode preparation specifically comprises the following steps: Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode with confined N-GQDs; Step 2: Immerse the electrode of confined N-GQDs in Na 2 PdCl 4 Electrodeposition in solution to obtain a confined PdNPs / N-GQDs electrode; Step 3, immersing the confined PdNPs / N-GQDs electrode in a siloxane solution containing epoxy groups to react, thereby obtaining an epoxy group-functionalized confined PdNPs / N-GQDs electrode; Step 4, immersing the electrode of epoxy group functionalized confined PdNPs / N-GQDs in a buffer solution containing immunoglobulin A for incubation, and after rinsing, obtaining an electrode of immunoglobulin A modified confined PdNPs / N-GQDs; Step 5, immersing the immunoglobulin A-modified confined PdNPs / N-GQDs electrode in a buffer solution containing a tumor marker primary antibody for incubation, and then immersing it in a bovine serum albumin solution for incubation after rinsing to obtain the immunoelectrode.

3. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, It is characterized in that The nitrogen-doped graphene quantum dots are synthesized by hydrothermal method in an ammonia solution using 1-aminopyrene as a carbon source; the nitrogen-doped graphene quantum dot solution, Na 2 PdCl 4 The solvent used for the solution was water, and the solvent used for the GPTMS-containing solution was ethanol; The electrode in the SNF modified electrode is 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; The preparation method of the SNF modified electrode comprises the following steps: SNF is grown on the electrode by any of the following methods: solution growth 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.

4. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, It is characterized in that The mass concentration of nitrogen-doped graphene quantum dots in the nitrogen-doped graphene quantum dot solution of step 1 is 0.05-3.0 mg / mL, the electrodeposition time is 1-120 s, and the electrodeposition voltage is +0.7V to 0.9V.

5. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, It is characterized in that Step 2 Na 2 PdCl 4 Na in solution 2 PdCl 4 The molar concentration is 0.05-3.0 mM, the electrodeposition time is 0.1-60 s, and the electrodeposition voltage is -0.8 V to -0.4 V.

6. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, It is characterized in that In step 3, the concentration of GPTMS in the epoxy group-containing siloxane solution is 0.5-5 mM GPTMS, and the reaction is allowed to stand for 20-80 minutes at a temperature of 20-40° C.; In step 4, the incubation is at 0-5°C for 0.5-4h; In step 5, the incubation is performed at 0-5° C. for 0.5-4 h.

7. The electrochemiluminescent immunosensor with low trigger potential according to claim 2, It is characterized in that The concentration of immunoglobulin A in the buffer solution of step 4 is 1-20 mg / mL, the buffer solution is a phosphate buffer, the phosphate concentration is 0.005-0.3 mol / L, and the pH is 6.5-7.5; The concentration of the tumor marker primary antibody in the buffer solution of step 5 is 1-20 mg / mL, the buffer solution is a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution, the solute concentration in the buffer solution is 0.005-0.5 mol / L, and the pH is 6-8; The epoxy group siloxane includes any one or more of 3-aminopropyltriethoxysilane, (3-glycidylpropoxy)trimethoxysilane, 3-(2,3-epoxypropoxy)propyltrimethoxysilane, and 2-(3,4-epoxycyclohexyl)ethyltriethoxysilane.

8. The electrochemiluminescent immunosensor with low trigger potential according to claim 1, It is characterized in that The molar concentration of luminol in the detection solution is 5-300 μmol / L, and the concentration of hydrogen peroxide is 50-300 μmol / L.

9. A method for constructing an electrochemiluminescent immunosensor with low trigger potential. It is characterized in that Includes steps: Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode with confined N-GQDs; Step 2: Immerse the electrode of confined N-GQDs in Na 2 PdCl 4 Electrodeposition in solution to obtain a confined PdNPs / N-GQDs electrode; Step 3, immersing the confined PdNPs / N-GQDs electrode in a siloxane solution containing epoxy groups to react, thereby obtaining an epoxy group-functionalized confined PdNPs / N-GQDs electrode; Step 4, immersing the electrode of epoxy group functionalized confined PdNPs / N-GQDs in a buffer solution containing immunoglobulin A for incubation, and after rinsing, obtaining an electrode of immunoglobulin A modified confined PdNPs / N-GQDs; Step 5, immersing the electrode of immunoglobulin A modified confined PdNPs / N-GQDs in a buffer solution containing a tumor marker primary antibody for incubation, and then immersing it in a bovine serum albumin solution for incubation after washing to obtain the immunoelectrode; Step 6: Using the three-electrode system of the immunoelectrode, 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.

10. Use of the electrochemiluminescent immunosensor with low trigger potential according to any one of claims 1 to 7 in the detection of tumor markers for non-diagnostic purposes, It is characterized in that Includes steps: The immunoelectrode is incubated with a sample solution containing tumor markers and then rinsed to obtain an immune complex modified electrode; The immune complex modified electrode was used as the modified electrode, and a three-electrode system was formed with a reference electrode and a counter electrode. An electrolyte solution containing luminol and hydrogen peroxide was used as the detection solution for electrochemiluminescence signal detection.

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