Nanochannel-confined aptamer sensor for detecting tumor marker by electrochemiluminescence as well as construction method and application of nanochannel-confined aptamer sensor

Abstract

The invention discloses an aptamer sensor for detecting a tumor marker through nanochannel confinement electrochemical luminescence and a construction method and application of the aptamer sensor. The aptamer sensor comprises a three-electrode system of an aptamer electrode, a reference electrode and a counter electrode, and an electrolyte solution containing luminol is used as a detection solution; the aptamer electrode is an SNF modified electrode of confined nitrogen-doped graphene quantum dot nano-enzyme, and is obtained by covalently immobilizing a tumor marker aptamer and closing a non-specific site. According to the invention, the gated ECL aptamer sensor of the endogenous co-reactant is constructed based on the SNF confinement N-GQDs nano-enzyme, the ECL signal of a luminol-O2 system is greatly sensitized, and the sensor has the advantages of low detection limit, high sensitivity, wide detection range and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to an aptamer sensor for detecting tumor markers by electrochemiluminescence in a nanochannel confinement, and a construction method and application thereof. Background Art

[0002] There is an increasing demand for the detection of disease-related proteins, especially tumor biomarkers. Carcinoembryonic antigen (CEA) is a glycoprotein produced by tumor cells and is often used as a broad-spectrum tumor marker. The specific and highly sensitive determination of CEA is of great significance for the clinical diagnosis and efficacy evaluation of tumors. At present, there are several methods for detecting CEA in serum, such as amperometry, fluorescence, electrochemistry, photoelectrochemistry, enzyme-linked immunosorbent assay, and capillary electrophoresis. In these methods, antibodies are mainly used as recognition elements. However, antibodies have problems such as easy inactivation and high cost. Aptamers have molecular recognition characteristics similar to antibodies, and compared with antibodies, they have advantages such as low cost, simple synthesis, and easy modification. At present, aptamer biosensors have shown great potential in tumor marker detection. For example, researchers have constructed a CEA nucleic acid aptamer sensor based on fluorescence resonance energy transfer, covalently labeled the amino-modified CEA aptamer on water-soluble PAA-modified upconversion nanoparticles (PAA-UCPs) as an energy donor, and achieved high-sensitivity detection of CEA.

[0003] Electrochemiluminescence (ECL) is an energy relaxation process based on high-energy electron transfer triggered by electrochemical stimulation. This analytical technique combines the advantages of electrochemistry and optics, and has the advantages of temporal and spatial controllability, high sensitivity and accuracy, and a wide linear range. In the field of ECL sensing, luminol-based electrochemiluminescence systems are favored by researchers due to their excellent luminescence properties and low excitation potential. However, the traditional luminol-H2O2 system is usually restricted by problems such as H2O2 self-decomposition and easy oxidation, which limits its application in bioanalysis. Therefore, it is of great significance to develop a new ECL signal enhancement method that is stable and does not require external co-reactants.

[0004] Graphene quantum dots (GQDs) are graphene-based materials with a size of less than 100nm and composed of a single layer or a few graphene layers. They have high stability, large specific surface area and excellent catalytic activity, and are widely used in sensing, bioimaging and other fields. Nanozymes are an emerging multifunctional nanomaterial that can simulate the various catalytic properties of natural enzymes. However, nanozymes in a free dispersed state in a homogeneous solution often have problems such as poor stability and low catalytic activity. Confining nanozymes in a closed environment with concentrated substrates can minimize the decomposition of active intermediates and enhance their catalytic activity.

[0005] The oxidation of luminol and the generation of reactive oxygen species (ROS) are the key to triggering the luminol ECL system. Nanozymes are a class of nanomaterials with catalytic activity and enzymatic reaction kinetics similar to natural enzymes, with the advantages of simple preparation, adjustable catalytic activity, and high stability. Among them, pseudoperoxidase nanozymes can catalyze H2O2 to produce a large amount of ROS, which is beneficial to improving the ECL signal of the luminol system. At present, the use of nanozymes to improve the ECL signal of the luminol system has become one of the hot research areas. However, in a homogeneous system, the performance of nanozymes alone cannot meet the requirements of signal amplification. Dispersed free nanozymes lead to a decrease in the local concentration of nanozymes and their intermediates (ROS), thereby limiting the overall effectiveness of the pseudoenzyme catalytic process. By fixing nanozymes on a carrier or confining them in a confined space, the stability and catalytic performance of the enzyme can be improved by constructing a nanocatalytic reactor. Because this restriction can minimize the decomposition of active intermediates, it also avoids the influence of the reaction medium on the nanozymes.

[0006] Mesoporous silica nanochannel film (SNF, also known as vertical ordered mesoporous silica film VMSF) is a type of nanomaterial with high pore density, uniform pore size, easy modification, and good mechanical and chemical stability. The high pore density and specific surface area give it excellent mass transfer ability, laying the foundation for the construction of gated sensors. Good mechanical and chemical stability is a prerequisite for SNF confined nanozymes. SNF pores are rich in reactive silanol groups, which can be used as covalent modification sites, making it possible to construct a biorecognition interface on the electrode surface. In addition, SNF's good anti-fouling and anti-interference capabilities are expected to achieve anti-fouling detection of targets in complex samples.

[0007] Therefore, SNF can be used as an excellent material to confine nanozymes, and its outer surface can be modified to construct a biorecognition interface, which is expected to construct an efficient ECL sensor for tumor marker detection. Summary of the invention

[0008] In view of the problems that the efficiency of luminol ECL is low and the detection sensitivity of immunosensors needs to be further improved, the present invention provides an aptamer sensor for detecting tumor markers by electrochemiluminescence in a nanochannel confinement, and constructs a gated ECL aptamer sensor of endogenous co-reactants based on SNF confined N-GQDs nanozymes, which greatly sensitizes the ECL signal of the luminol-O2 system and has the advantages of low detection limit and wide detection range.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] An aptamer sensor for detecting tumor markers by electrochemiluminescence in a nanochannel confinement, the aptamer sensor comprising a three-electrode system of an aptamer electrode, a reference electrode, and a counter electrode, with an electrolyte solution containing luminol as a detection liquid;

[0011] The aptamer electrode is a SNF modified electrode of confined nitrogen-doped graphene quantum dot nanozyme, which is obtained by covalently fixing the tumor marker aptamer and blocking the non-specific sites.

[0012] Based on SNF confined N-GQDs nanozymes, a gated ECL aptamer sensor with endogenous co-reactants is constructed to detect tumor markers. In the traditional luminol-H2O2 ECL system, the co-reactant H2O2 has poor stability and is prone to decomposition at room temperature. The ECL system is constructed by using luminol as a luminophore and dissolved oxygen as an endogenous co-reactant. The present invention prepares carbon-based nanozymes-nitrogen-doped graphene quantum dots (N-GQDs) with pseudo-peroxidase activity by a hydrothermal method, and then confines N-GQDs in SNF modified electrodes by electrophoretic deposition. The confined N-GQDs nanozyme has good stability and catalytic activity, plays an electrocatalytic role in the oxidation of luminol, and plays a dual role of enzyme catalysis and electrocatalysis in the reduction of O2, which greatly sensitizes the ECL signal of the luminol-O2 system. After the outer surface of SNF is modified with epoxy groups, the aptamer is covalently fixed to obtain the ECL aptamer sensor, which improves the convenience of detection, reduces the detection cost, significantly sensitizes the ECL signal, and improves the detection sensitivity. It is used for high-sensitivity detection of tumor markers and has the advantages of low detection limit and detection range. In addition, the detection does not require complicated pretreatment and separation processes, which reduces the detection cost, is simple to operate, and is suitable for large-scale applications.

[0013] Preferably, the preparation of the aptamer electrode specifically comprises the steps of:

[0014] Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode of confined N-GQDs nanozyme;

[0015] Step 2, immersing the electrode of the confined N-GQDs nanozyme in a (3-epoxypropyl)trimethoxysilane (GPTMS) solution and allowing it to react to obtain an electrode of epoxy-functionalized confined N-GQDs;

[0016] Step 3, after rinsing the electrode in step 2 with deionized water, immersing the electrode of epoxy-functionalized confined N-GQDs in a buffer solution containing a tumor marker aptamer for incubation, and then immersing it in a bovine serum albumin solution for incubation to obtain the aptamer electrode.

[0017] In the present invention, N-GQDs are confined in the porous material of SNF, and the porous material is used to confine the nanomaterial, which can significantly improve the stability of the nanomaterial. In addition, the porous material provides a dispersion space, which can effectively increase the active sites of the nanomaterial, thereby improving its catalytic activity and stability.

[0018] The nitrogen-doped graphene quantum dots are synthesized by hydrothermal method in ammonia solution with 1-aminopyrene as carbon source; graphene quantum dots (GQDs) are a new type of graphene material with a size less than 100nm, composed of a single layer or a few graphene layers, with high stability, large specific surface area, π-π conjugated structure and low biological toxicity, and have wide applications in sensing, bioimaging and other fields. Nanozyme is an emerging multifunctional nanomaterial that can simulate various catalytic properties of natural enzymes. By introducing doping atoms such as nitrogen into GQDs, it can be made to have nanozyme activity.

[0019] Preferably, the specific preparation of the nitrogen-doped graphene quantum dots comprises the following steps:

[0020] 1-aminopyrene is dispersed in ammonia water and reacted at 180-200° C. for 2-10 hours. The product is purified to obtain nitrogen-doped graphene quantum dots.

[0021] Furthermore, the specific preparation of the nitrogen-doped graphene quantum dots includes the following steps:

[0022] 1-Aminopyrene (0.5-5 mg / mL) was added to aqueous ammonia (0.05-0.4 M). After ultrasonic dispersion, the resulting mixture was added to the polytetrafluoroethylene liner of the reactor and reacted at 180-200°C under high pressure for 2-10 hours. The product was filtered, dialyzed and freeze-dried to obtain N-GQDs solid.

[0023] The solvent used in the nitrogen-doped graphene quantum dot solution or the (3-epoxypropyl)trimethoxysilane solution is water;

[0024] 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;

[0025] The preparation method of the SNF modified electrode comprises the following steps: A silicon dioxide nanoarray film is grown on the electrode by any one of 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.

[0026] Specifically, using The method for preparing SNF modified electrode by solution growth method comprises the steps of: using sodium hexadecyl sulfonate (CTAB) as a template surfactant, dissolving it in a water-ethanol solution, adding ammonia water, and then adding tetraethoxysiloxane (TEOS), an organosiloxane precursor, to obtain a precursor solution for growing SNF. Inserting the electrode into the precursor solution to grow a silicon dioxide nanochannel film. After the growth is completed, taking out the electrode, rinsing it with a large amount of deionized water, drying it with nitrogen, and aging it in an oven at 100°C overnight to obtain a modified electrode containing micelle (SM) templates in the pores. The micelles can be removed by immersing the electrode in a 0.1M HCl-ethanol solution and stirring it for 5 minutes to obtain an SNF modified electrode with open pores. The method for preparing SNF modified electrode by EASA method comprises the steps of: using CTAB as a template surfactant, dissolving it in an aqueous solution, adjusting the pH to 3, and then adding the organosiloxane precursor TEOS to obtain a precursor solution for growing SNF. The electrode was inserted into the precursor solution, and a negative voltage was applied to the electrode to electrolyze water, thereby obtaining an in-situ pH gradient on the electrode surface and growing a silica nanochannel film. After the growth was completed, the electrode was taken out, rinsed with a large amount of deionized water, dried with nitrogen, and aged overnight at 100°C in an oven to obtain a modified electrode containing an SM template in the pores. The electrode was immersed in a 0.1M HCl-ethanol solution to remove the micelles, and a SNF modified electrode with open pores was obtained.

[0027] The mass concentration of nitrogen-doped graphene quantum dots in the nitrogen-doped graphene quantum dot solution of step 1 is 0.1-2.0 mg / mL, the electrodeposition time is 3-12 min, and the electrodeposition voltage is +0.7V-+0.9V.

[0028] Preferably, the deposition time is 4 to 10 minutes, and more preferably, the deposition time is 5 to 10 minutes; further preferably, the electrodeposition time is 5 to 6 minutes, and the electrodeposition voltage is +0.8V.

[0029] In step 2, the molar concentration of GPTMS in the GPTMS solution is 2 mM to 3 mM, and the reaction is allowed to stand for 30 to 60 minutes; the reaction temperature is 20 to 40°C.

[0030] The incubation condition in step 3 is 0-5° C. for 30-90 min.

[0031] In step 3, the molar concentration of the tumor marker aptamer in the buffer solution containing the tumor marker aptamer is 0.1-0.2 μM, and 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.

[0032] The molar concentration of luminol in the detection solution is 30-80 μmol / L. When the concentration of luminol is too low, the ECL signal measured by the electrode is low. Dissolved oxygen is an endogenous co-reactant. Dissolved oxygen is commonly present in biological samples, and under constant temperature and atmospheric pressure, the dissolved oxygen concentration is basically constant. In addition, electrochemiluminescence is an electrode interface reaction, and only the dissolved oxygen on the electrode surface is consumed. Relative to the bulk solution, the dissolved oxygen in the bulk solution is excessive. Therefore, using dissolved oxygen as an endogenous co-reactant can ensure the stability of the electrochemiluminescence signal.

[0033] Preferably, each step is rinsed with water or buffer after completion to remove excess undeposited or unincubated solution;

[0034] The present invention also provides a method for constructing the nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers, comprising the steps of:

[0035] Step 1, immersing the SNF modified electrode in a nitrogen-doped graphene quantum dot solution, and electrodepositing to obtain an electrode of confined N-GQDs nanozyme;

[0036] Step 2, immersing the electrode of the confined N-GQDs nanozyme in a (3-epoxypropyl)trimethoxysilane (GPTMS) solution and allowing it to react to obtain an electrode of epoxy-functionalized confined N-GQDs;

[0037] Step 3, after rinsing the electrode in step 2 with deionized water, immersing the electrode of epoxy-functionalized confined N-GQDs in a buffer solution containing a tumor marker aptamer for incubation, and then immersing it in a bovine serum albumin solution for incubation to obtain the aptamer electrode;

[0038] Step 4: construct the aptamer sensor using a three-electrode system of the aptamer electrode, the reference electrode and the counter electrode and an electrolyte solution containing luminol as a detection solution.

[0039] The present invention also provides the use of the nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers in non-diagnostic tumor marker detection, comprising the steps of:

[0040] The aptamer electrode is incubated with a sample solution containing tumor markers and then rinsed to obtain an aptamer complex modified electrode; the aptamer complex modified electrode is used as a modified electrode to form a three-electrode system with a reference electrode and a counter electrode, and an electrolyte solution containing luminol is used as a detection solution for electrochemiluminescence signal detection.

[0041] The detection principle is as follows: when tumor markers are present in the serum sample, the tumor marker aptamer binds to the tumor marker, and the large tumor marker aptamer bound to the electrode interface will produce a significant steric hindrance effect on the diffusion mass transfer of luminol, thereby changing the ECL signal of the electrode. Therefore, according to the quenching degree of the electrochemiluminescence signal, the electrochemiluminescence detection of tumor markers can be achieved.

[0042] The sample solution to be tested includes a standard solution containing a tumor marker or a serum sample.

[0043] The sample to be tested contains glucose, non-target tumor markers, and one or more interfering compounds in inorganic salts.

[0044] The aptamer sensor for detecting tumor markers by electrochemiluminescence confined by the nanochannel has a minimum detection limit of 0.44 pg mL in tumor marker detection for non-diagnostic purposes. -1 .

[0045] 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.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) The present invention constructs a gated ECL sensor based on SNF confined N-GQDs nanozyme to enhance the ECL signal of the luminol-O2 system to achieve the detection of CEA, improves the stability of the nanozyme while enhancing its catalytic activity, can electrocatalytically oxidize luminol, and electrocatalytically reduce O2, thereby sensitizing the ECL signal of the luminol-O2 system. At the same time, the abundant silanol groups on the surface of the nanochannel give it the characteristic of easy modification, and after epoxy modification, a biorecognition interface can be constructed.

[0048] (2) In the present invention, the N-GQDs are directly fixed on the electrode, and will agglomerate on the electrode surface after a period of time, presenting a uniformly dispersed spindle-shaped structure, while the N-GQDs confined in the nanochannel are well protected in the channel due to the size effect. Therefore, the confined N-GQDs have strong stability and still have excellent detection sensitivity after multiple washings and reuses during detection.

[0049] (3) The present invention constructs an ECL aptamer sensor for highly sensitive detection of tumor marker CEA based on the catalytic amplification of ECL signals by nanochannel-confined N-GQDs and the gating effect caused by the binding of the detected object to the aptamer sensing interface. Inside the nanochannel, the confined space can provide a higher surface area, and the confined N-GQDs have more active sites and surface reaction activity, showing significant catalytic activity, thereby sensitizing the ECL signal of the luminol system. On the outer surface of the nanochannel, the silanol groups on the outer surface are used as active sites to couple epoxy groups, and the ring-opening reaction between the epoxy groups and the amino-functionalized aptamers is used to construct the aptamer recognition interface. When CEA specifically binds to the recognition interface, the formed complex reduces the diffusion of luminol to the substrate electrode, and the gating effect generated by biorecognition achieves highly sensitive detection of CEA. The constructed aptamer sensor has the advantages of simple manufacturing and the like.

[0050] (4) The sensor of the present invention is used for tumor marker detection for non-therapeutic or diagnostic purposes and has the advantages of wide linear range, simple manufacturing, high detection sensitivity, and low detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 (A) Transmission electron microscope (TEM) image of N-GQDs in Example 1. Inset is a high-resolution TEM image and particle size distribution of N-GQDs. (B) Ultraviolet (UV) spectrum of N-GQDs. Inset is a photo of N-GQDs under 365nm ultraviolet light. (C) XPS measurement spectrum of N-GQDs. High-resolution C1s spectrum (D) O 1s spectrum (E) and N1s spectrum (F) of N-GQDs.

[0052] Figure 2 CV graphs of ITO, SNF / ITO, and N-GQDs@SNF / ITO in 50 mM KHP solution containing 0.5 mM K3[Fe(CN)6] (A) and [Ru(NH3)6]Cl3 (B) in Example 1. (C) Fluorescence intensity of SNF / ITO and N-GQDs@SNF / ITO. Insets are fluorescence images of SNF / ITO (left) and N-GQDs@SNF / ITO (right). (D) XPS spectra of SNF / ITO and N-GQDs@SNF / ITO.

[0053] Figure 3The electrochemiluminescence IT images of (A) ITO, (B) N-GQDs@ITO (electrophoresis), (C) N-GQDs@ITO (drop coating), (D) SNF / ITO, (E) N-GQDs@SNF / ITO (electrophoresis), and (F) N-GQDs@SNF / ITO (drop coating) in Example 1 are shown in the insets. The concentrations of luminol and N-GQDs are 100 μM and 0.5 mg / mL, respectively. The electrophoretic deposition time is 5 minutes, and the volume of drop-coated N-GQDs is 40 μL. PMT = 600 V.

[0054] Figure 4 ECL signal diagram of SNF / ITO and N-GQDs@SNF / ITO in different atmospheres in the presence of 100 μM luminol in Example 1 (A). PMT = 600 V. Scan rate 100 mV s -1 CV curves of SNF / ITO and N-GQDs@SNF / ITO in (B) PBS (0.01 M, pH = 7.4) and (C) luminol (100 μM). Scan rate was 100 mV s -1 (D) ECL response of N-GQDs@SNF / ITO in 0.01 M PBS (pH = 7.4) buffer solution with different free radical scavengers added. I and I0 are the ECL signals in the presence or absence of free radical scavengers, respectively.

[0055] Figure 5 The ECL response diagram of (A) N-GQDs@SNF / ITO under different initial potential excitation in Example 1. I represents the signal of each sample, and I0 represents the maximum signal value measured in a series of samples. The inset is a photo of N-GQDs@SNF / ITO after scanning for 5 minutes at different initial potentials. Different electrodes in the presence of 2.5mM Fe(CN)6 3- / 4- CV (B) and EIS (C) graphs of aptamer in 0.1 M KCl solution. The concentration of aptamer is 0.1 μM. The concentration of CEA is 10 ng / mL. The scan rate is 50 mV s -1 (D) ECL signals of different electrodes in 50 μM luminol solution. PMT = 600 V. Scan rate: 100 mV s -1 .

[0056] Figure 6 The effects of (A) electrophoretic deposition potential and (B) electrophoretic deposition time on the ECL signal of the composite electrode in Example 1.

[0057] Figure 7(A) ECL signal diagram of BSA / Apt / N-GQDs@O-SNF / ITO electrode incubated with different concentrations of CEA in Example 1. (B) Linear calibration curve for detecting CEA.

[0058] Figure 8 The anti-interference ability of the ECL aptamer sensor (A) in Example 1 to different species. The glucose concentration was 5 μM, and the concentrations of NGAL, S100, and AFP were 10 ng mL -1 , CEA concentration was 1 ng mL -1 (B) Reproducibility of 10 pg / mL CEA / Aptamer / N-GQDs@O-SNF / ITO. DETAILED DESCRIPTION

[0059] 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.

[0060] The raw materials used in the following specific embodiments are all purchased from the market.

[0061] Example 1

[0062] The process of using a silica nanopore modified electrode with nanochannel-confined N-GQDs to covalently immobilize the tumor marker CEA aptamer on the outer surface to prepare an aptamer electrode and detecting CEA in serum samples by electrochemiluminescence:

[0063] (1) SNF / ITO Solution growth method preparation

[0064] First, dissolve 0.1600 g of hexadecyltrimethylammonium 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 tetraethoxysilane (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.

[0065] 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.

[0066] (2) Synthesis of N-GQDs by hydrothermal method under alkaline conditions with 1-aminopyrene as carbon source. Nitrogen-doped graphene quantum dots were synthesized by hydrothermal method under alkaline conditions with 1-aminopyrene as carbon source. The specific process is as follows: 1-aminopyrene (1 mg / mL) was added to ammonia water (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.

[0067] (3) Preparation of N-GQDs@SNF / ITO

[0068] N-GQDs@SNF / ITO was prepared by in-situ electrodeposition of N-GQDs in the nanopores of SNF / ITO by constant potential method. The specific method is as follows: the prepared SNF / ITO with open pores was immersed in an aqueous solution of N-GQDs (0.5 mg / mL) and a potential of +0.8 V was applied for 5 min. The N-GQDs@SNF / ITO electrode was obtained.

[0069] (4) Preparation of aptamer sensors

[0070] N-GQDs@SNF / ITO was used as the substrate electrode, and γ-glycidyloxypropyltrimethylsilane (GPTMS) was used as a crosslinker to covalently immobilize the amino-modified aptamer and SNF layer. 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.

[0071] The specific experimental steps are as follows: immerse the N-GQDs@SNF / ITO electrode in an ethanol solution containing 2.26 mM GPTMS and react at room temperature for 1 hour. Modification of GPTMS on the SNF surface is achieved through silanization reaction. The electrode is washed with deionized water to obtain a N-GQDs@O-SNF / ITO electrode.

[0072] Secondly, 40 μL CEA aptamer (0.1 μM) was drop-coated on the surface of N-GQDs@O-SNF / ITO electrode, incubated at 4°C for 60 min, fully washed with PBS (0.01 M, pH = 7.4) to remove unbound aptamer, and blown dry to obtain Apt / N-GQDs@O-SNF / ITO electrode. Finally, Apt / N-GQDs@O-SNF / ITO electrode was incubated with 40 μL bovine serum albumin (BSA) solution (1%, w / w) at room temperature for 15 min, fully washed with PBS (0.01 M, pH = 7.4), and blown dry to obtain BSA / Apt / N-GQDs@O-SNF / ITO electrode.

[0073] (5)CEA electrochemiluminescence detection

[0074] To detect CEA, the BSA / Ab / N-GQDs@O-SNF / ITO electrode was incubated with 50 μL of CEA samples with different concentrations at 4 °C for 60 min, and the electrode was slowly rinsed with PBS (0.01 M, pH = 7.4) solution to wash away unbound CEA.

[0075] The obtained electrode was subjected to ECL detection, and the electrolyte solution used for the test was an electrolyte PBS (0.01M, pH=7.4) solution containing 50 μM luminol. 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 0.1 V / s.

[0076] Figure 1 (A) Transmission electron microscope (TEM) image of N-GQDs in Example 1. Inset is a high-resolution TEM image and particle size distribution of N-GQDs. (B) Ultraviolet (UV) spectrum of N-GQDs. Inset is a photo of N-GQDs under 365nm ultraviolet light. (C) XPS measurement spectrum of N-GQDs. High-resolution C1s spectrum (D) O 1s spectrum (E) and N1s spectrum (F) of N-GQDs.

[0077] like Figure 1 As shown in (A), the size and distribution of N-GQDs were characterized by TEM. N-GQDs are evenly distributed with an average size of 2.5nm. Clear lattice fringes can be observed in the high-resolution TEM image, and the lattice parameter is 0.21nm, which corresponds to the (100) crystal plane of graphene.

[0078] like Figure 1As shown in (B), compared with two-dimensional graphene materials, zero-dimensional graphene quantum dots have an open energy band, thus having a photoluminescence phenomenon. Under the excitation of ultraviolet light at a wavelength of 365nm, N-GQDs exhibit green fluorescence. In addition, N-GQDs exhibit obvious ultraviolet characteristic absorption near 350nm, which is due to the n→π of C=O * Caused by transition.

[0079] like Figure 1 As shown in (C), compared with two-dimensional graphene materials, zero-dimensional graphene quantum dots have open energy bands, thus exhibiting photoluminescence. N-GQDs are mainly composed of three elements: C, N, and O, with corresponding atomic percentages of 72.13%, 1.28%, and 17.31%.

[0080] like Figure 1 As shown in (D), the C1s high-resolution spectrum of N-GQDs, in which the signal peak at the binding energy of 285.9 eV is the graphite C (CC=C, sp2 carbon) structure, and the peaks at the binding energies of 286.3 eV and 288.5 eV correspond to the CN bond and CO bond, respectively. Figure 1 (E) is the high-resolution O1s spectrum of N-GQDs. The peaks at binding energies of 531.4 eV and 532.0 eV are CO bonds and C=O bonds, respectively. The excellent water dispersibility of N-GQDs may be attributed to the presence of oxygen-containing groups such as hydroxyl groups. Figure 1 In the N1s high-resolution spectrum shown in (F), the peaks at binding energies of 398.4 eV, 399.5 eV, 401.0 eV, and 402.1 eV correspond to pyridinic N, pyrrolic N, graphitic N, and amino N, respectively. The above experiments confirmed the successful synthesis of N-GQDs.

[0081] Figure 2 CV graphs of ITO, SNF / ITO, and N-GQDs@SNF / ITO in 50 mM KHP solution containing 0.5 mM K3[Fe(CN)6] (A) and [Ru(NH3)6]Cl3 (B) in Example 1. (C) Fluorescence intensity of SNF / ITO and N-GQDs@SNF / ITO. Insets are fluorescence images of SNF / ITO (left) and N-GQDs@SNF / ITO (right). (D) XPS spectra of SNF / ITO and N-GQDs@SNF / ITO.

[0082] like Figure 2As shown in (AB), SNF / ITO shows an electrochemical signal lower than that of the bare ITO electrode in the negatively charged K3[Fe(CN)6] probe solution, indicating that the probe is repelled, while it shows a peak current higher than that of the bare electrode in the positively charged [Ru(NH3)6]Cl3 probe, showing significant charge selective permeability. After confining N-GQDs, the electrode shows stronger charge selective permeability because the surface of N-GQDs is negatively charged. These electrochemical behaviors indirectly prove the successful confinement of N-GQDs.

[0083] like Figure 2 As shown in (C), compared with SNF / ITO, SNF / ITO with confined N-GQDs exhibits obvious green fluorescence, and the color of the fluorescence microscope photo in the inset is also consistent with the fluorescence of N-GQDs. The elemental composition of N-GQDs@SNF / ITO was studied by XPS, such as Figure 2 As shown in (D), compared with SNF / ITO, the C content of the electrode confined with N-GQDs increased from 47.4% to 73.2%, and the presence of N was observed from the spectrum. The above experiments prove that N-GQDs are successfully confined in the silicon dioxide nanochannel.

[0084] In order to prove that confinement can achieve stable immobilization of N-GQDs, two blank electrodes were prepared by the method of drip coating of N-GQDs. The confinement method of N-GQDs in step 3 was drip coating, and the specific method was: 20μL N-GQDs (0.5mg / mL) solution was drip coated on the SNF / ITO electrode to obtain N-GQDs SNF / @ITO (drip coating) prepared by the drip coating method. In addition, 20μL N-GQDs (0.5mg / mL) solution was drip coated on the ITO electrode to obtain the N-GQDs@ITO (drip coating) electrode.

[0085] Figure 3 These are the electrochemiluminescence IT images of (A) ITO, (B) N-GQDs@ITO (electrophoresis), (C) N-GQDs@ITO (drop coating), (D) SNF / ITO, (E) N-GQDs@SNF / ITO (electrophoresis), and (F) N-GQDs@SNF / ITO (drop coating) in Example 1, and the insets are the corresponding SEM images.

[0086] like Figure 3 As shown, compared with ITO ( Figure 3 A), SNF / ITO( Figure 3 D), the ECL signals of the electrodes modified with N-GQDs were all improved to a certain extent, but only the N-GQDs@SNF / ITO prepared by electrophoretic deposition had a stable ECL signal response ( Figure 3E). This may be because N-GQDs can be fixed in the nanochannels through the size effect, while direct electrophoretic deposition or drop coating will make the water-dispersible N-GQDs free into the homogeneous solution. Figure 3 As can be seen from the illustrations C and F, N-GQDs will agglomerate on the electrode surface after a period of time, presenting a uniformly dispersed spindle-shaped structure, while the N-GQDs confined in the nanochannel are well protected in the pores due to the size effect.

[0087] Figure 4 ECL signal diagram of SNF / ITO and N-GQDs@SNF / ITO in different atmospheres in the presence of 100 μM luminol in Example 1 (A). PMT = 600 V. Scan rate 100 mV s -1 CV curves of SNF / ITO and N-GQDs@SNF / ITO in (B) PBS (0.01 M, pH = 7.4) and (C) luminol (100 μM). Scan rate was 100 mV s -1 (D) ECL response of N-GQDs@SNF / ITO in 0.01 M PBS (pH = 7.4) buffer solution with different free radical scavengers added. I and I0 are the ECL signals in the presence or absence of free radical scavengers, respectively.

[0088] like Figure 4 As shown in (A), N-GQDs have a significant sensitization effect on the ECL signal of the luminol-O2 system, and its electrochemiluminescence mechanism was further explored. Under deoxygenated conditions, N-GQDs@SNF / ITO only has an electrochemiluminescence signal of 733. Under aerobic conditions, the electrochemiluminescence signal increased to 12752, a 17.4-fold increase in the signal. SNF / ITO without confined N-GQDs had no electrochemiluminescence signal response under aerobic and anaerobic conditions. We speculate that O2 plays the role of an endogenous co-reactant, while N-GQDs act as a co-reaction promoter, which can promote the production of more co-reaction intermediates, resulting in a significant amplification of the ECL signal.

[0089] The electrochemical behavior of N-GQDs towards luminol and O2 was further investigated. Figure 4 As shown in (B), under aerobic conditions, the reduction peak potential of O2 in N-GQDs@SNF / ITO is earlier than that in SNF / ITO, and the reduction peak current is larger, indicating that N-GQDs plays an electrocatalytic role in the reduction of O2. On this basis, 100 μM luminol was added, as shown in Figure 4As shown in (C), compared with the blank PBS solution, after adding luminol to the system, the Faraday current of N-GQDs@SNF / ITO at 0.8V increased from 2.63μA to 9.98μA. The increase of 7.35μA was caused by the electrocatalytic oxidation of luminol by N-GQDs. In addition, we measured the types of ROS generated by electrocatalytic O2 reduction, using tert-butyl alcohol as ·OH scavenger and p-benzoquinone as O2 ·- Scavengers, such as Figure 4 As shown in (D), it can be observed that the ROS generated by the electrochemiluminescence process in the luminol-O2 system is in the form of O2 ·- In summary, it is proved that N-GQDs can electrocatalyze the oxidation of luminol to produce more luminol -· At the same time, it can electrocatalytically reduce O2 to produce O2 at low potential ·- , the two react to generate excited AP 2-* , and then releases energy to emit blue light. Therefore, the more intermediate reactants are generated, the higher the ECL efficiency.

[0090] Figure 5 The ECL response diagram of (A) N-GQDs@SNF / ITO under different initial potential excitation in Example 1. I represents the signal of each sample, and I0 represents the maximum signal value measured in a series of samples. The inset is a photo of N-GQDs@SNF / ITO after scanning for 5 minutes at different initial potentials. Different electrodes in the presence of 2.5mM Fe(CN)6 3- / 4- CV (B) and EIS (C) graphs of aptamer in 0.1 M KCl solution. The concentration of aptamer is 0.1 μM. The concentration of CEA is 10 ng / mL. The scan rate is 50 mV s -1 (D) ECL signals of different electrodes in 50 μM luminol solution. PMT = 600 V. Scan rate: 100 mV s -1 .

[0091] The effects of different initial negative potential scans on the ECL signal of the luminol-O2 system were investigated. Figure 5 As shown in (A), as the initial potential shifts negatively, the ECL signal increases first and then decreases. The increase in the ECL signal is because, on the one hand, it is more conducive to the electrocatalytic reduction of O2 by N-GQDs to produce ROS at a negative potential, and on the other hand, the negative shift in potential is more conducive to the accumulation of ROS. The decrease in the ECL signal is because an overly negative potential will affect the ITO (indium tin oxide) electrode ( Figure 5A illustration). At the same time, an ECL signal response is generated at an initial potential of -0.3 V, which is the same as the potential of N-GQDs electrocatalytic reduction of O2, which further verifies the reaction mechanism of N-GQDs as a co-reaction accelerator.

[0092] like Figure 5 As shown in (B), with the gradual modification of SNF, the steric hindrance effect caused by the complex formed on the SNF surface gradually increases, hindering the diffusion of the probe and reducing the electrochemical signal. Figure 5 A similar phenomenon can be observed in the EIS data shown in (C): R ct The lowest (1497Ω), after epoxy modification, R ct After further modification of the aptamer and blocking of the specific site, R ct Increased to 2416Ω, and finally, after the target specifically binds to the aptamer, R ct Increased to 4721Ω. Similarly, the same trend is also seen in the ECL signal response, such as Figure 5 (D) As shown. In summary, the ECL aptasensor we developed is feasible for CEA determination.

[0093] The potential and time of electrodeposition in step 3 were changed separately, and the ECL signal of the corresponding N-GQDs@SNF / ITO electrode was detected to obtain the optimal experimental conditions. The results are as follows: Figure 6 As shown in (A, B), the potential and time of N-GQDs@SNF / ITO electrophoretic deposition were optimized, and the optimal experimental conditions were electrophoretic deposition potential +0.8V and deposition time of 5min.

[0094] like Figure 7 (A) is the ECL signal graph measured after incubating different concentrations of CEA (1pg / mL to 10ng / mL) with the aptamer sensor under optimal conditions. As the concentration of CEA increases, more complexes are formed on the outer surface of SNF, the steric hindrance increases, the hindrance to the probe becomes greater, and the ECL signal gradually decreases. Figure 7 (B) The CEA assay calibration curve shown in Figure 2 shows that the ECL signal (IECL) and the logarithm of CEA concentration (logC CEA ) has a good linear range, and the regression equation is ECL =–815logC CEA (ng / mL)+3923(R 2 =0.998), and the LOD was 0.44 pg / mL.

[0095] The anti-interference ability of the sensor was investigated. The interference substances included glucose (Glu), carcinoembryonic antigen neutrophil gelatinase-associated lipocalin (NGAL), calcium-binding protein β (S100), and alpha-fetoprotein (AFP). At the same time, the signal changes in the presence of the test substance carcinoembryonic antigen (AFP) were measured. Among them, the glucose concentration was 5 μM, and the NGAL, S100, and AFP concentrations were 10 ng mL -1 , CEA concentration was 1 ng mL -1 The ECL signals of the electrodes before and after the immunosensor was incubated with the above substances were measured, and the ECL signal change value was compared with the initial ECL signal of the electrode before the action to obtain the ECL signal change rate.

[0096] The results are as follows Figure 8 (A) As shown. Except for CEA and its mixture, the aptamer sensor incubated with other interferents did not produce obvious signal changes, showing good selectivity. The aptamer sensor was incubated with CEA and its mixture, respectively, and the signal changes produced by the two were similar, which means that the constructed aptamer sensor has good anti-interference ability. In order to evaluate the reproducibility of the developed aptamer sensor, five BSA / Apt / N-GQDs@O-SNF / ITO electrodes were prepared and used to detect 10pg / mL CEA.

[0097] like Figure 8 As shown in (B), if five aptamer sensors were prepared in parallel, they were incubated with CEA (10 pg / mL) and then the ECL signals of the electrodes were measured. It was found that the relative standard deviation (RSD) of the ECL signal values ​​of the five sensors detecting CEA was 1.3%, indicating that the constructed electrochemiluminescent aptamer sensor had good reproducibility.

Claims

1. A nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers, characterized in that: The aptamer sensor comprises a three-electrode system of an aptamer electrode, a reference electrode, and a counter electrode, and uses an electrolyte solution containing luminol as a detection liquid; The aptamer electrode is a SNF modified electrode of confined nitrogen-doped graphene quantum dot nanozyme, which is obtained by covalently fixing the tumor marker aptamer and blocking the non-specific sites.

2. The nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 1, characterized in that: The preparation of the aptamer electrode 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 of confined N-GQDs nanozyme; Step 2, immersing the electrode of the confined N-GQDs nanozyme in a (3-epoxypropyl)trimethoxysilane solution and allowing it to react to obtain an electrode of epoxy-functionalized confined N-GQDs; Step 3, after rinsing the electrode in step 2 with deionized water, immersing the electrode of epoxy-functionalized confined N-GQDs in a buffer solution containing a tumor marker aptamer for incubation, and then immersing it in a bovine serum albumin solution for incubation to obtain the aptamer electrode.

3. The nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 2, characterized in that: The nitrogen-doped graphene quantum dots are synthesized by a hydrothermal method in an ammonia solution using 1-aminopyrene as a carbon source; The solvent used in the nitrogen-doped graphene quantum dot solution or the (3-epoxypropyl)trimethoxysilane solution is water; 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: A silicon dioxide nanoarray film is grown on the electrode by any one of 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 nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 2, characterized in that: The mass concentration of nitrogen-doped graphene quantum dots in the nitrogen-doped graphene quantum dot solution of step 1 is 0.1-2.0 mg / mL, the electrodeposition time is 3-12 min, and the electrodeposition voltage is +0.7V-+0.9V.

5. The nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 2, characterized in that: In step 2, the molar concentration of GPTMS in the GPTMS solution is 2 mM to 3 mM, and the reaction is allowed to stand for 30 to 60 minutes; the reaction temperature is 20 to 40°C.

6. The nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 2, characterized in that: In step 3, the incubation condition is 0-5°C for 30-90 min; In step 3, the molar concentration of the tumor marker aptamer in the buffer solution containing the tumor marker aptamer is 0.1-0.2 μM, and 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.

7. The nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 1, characterized in that: The molar concentration of luminol in the detection solution is 30-80 μmol / L.

8. The method for constructing an aptamer sensor for detecting tumor markers by electrochemiluminescence through nanochannel confinement according to any one of claims 1 to 7, 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 of confined N-GQDs nanozyme; Step 2, immersing the electrode of the confined N-GQDs nanozyme in a (3-epoxypropyl)trimethoxysilane solution and allowing it to react to obtain an electrode of epoxy-functionalized confined N-GQDs; Step 3, after rinsing the electrode in step 2 with deionized water, immersing the electrode of epoxy-functionalized confined N-GQDs in a buffer solution containing a tumor marker aptamer for incubation, and then immersing it in a bovine serum albumin solution for incubation to obtain the aptamer electrode; Step 4: construct the aptamer sensor using a three-electrode system of the aptamer electrode, the reference electrode and the counter electrode and an electrolyte solution containing luminol as a detection solution.

9. Use of the nanochannel-confined electrochemiluminescence aptasensor for detecting tumor markers according to any one of claims 1 to 7 in detecting tumor markers for non-diagnostic purposes, characterized in that: Includes steps: The aptamer electrode is incubated with a sample solution containing tumor markers and then rinsed to obtain an aptamer complex modified electrode; the aptamer complex modified electrode is used as a modified electrode to form a three-electrode system with a reference electrode and a counter electrode, and an electrolyte solution containing luminol is used as a detection solution for electrochemiluminescence signal detection.

10. Use of the nanochannel-confined electrochemiluminescence aptamer sensor for detecting tumor markers according to claim 9 in detecting tumor markers for non-diagnostic purposes, characterized in that: The sample to be tested may contain one or more interfering compounds such as glucose, non-target tumor markers, and inorganic salts.

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