Electrochemiluminescence sensor based on antibody modified nano-channel and construction method and use method of electrochemical luminescence sensor

Abstract

The invention relates to the field of biological detection, in particular to an electrochemical luminescence sensor based on an antibody modified nano-channel as well as a construction method and a use method of the electrochemical luminescence sensor. The electrochemical luminescence sensor based on the antibody modified nano-channel comprises an anodic aluminum oxide film with the nano-channel penetrating through the surface, hydrogel is attached to the surface of one side of the anodic aluminum oxide film, the problem that the anodic aluminum oxide film is prone to fragmentation due to direct contact operation can be solved, the hydrogel layer is completely modified with modifiers, and the electrochemical luminescence sensor is high in electrochemical luminescence efficiency. The modifiers and the nano channel are separated, the problem that the nano channel of the anodic aluminum oxide film modified by the antibody is easy to block is solved, in addition, all the modifiers can be removed through boiling, and the non-directional utilization rate of the anodic aluminum oxide film is improved; a construction method of the electrochemical luminescence sensor based on the antibody modified nano channel can achieve a good modification effect in a short time, and the electrochemical luminescence sensor is used in collagenous fiber acidic protein analysis, so that the clinical operation efficiency is improved, and the effect of rapid, high-sensitivity and high-accuracy complete quantitative detection is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an electrochemiluminescence sensor based on an antibody-modified nanochannel and a construction method and a use method thereof. Background Art

[0002] Electrochemiluminescence (ECL) is light emission triggered by an electrochemical reaction, and has unique advantages such as low background noise, high sensitivity, and strong operability. Thanks to the advantages of ECL and the specificity of the immune reaction, ECL immunoassay (ECLIA) has become a powerful analytical tool that helps overcome the limitations of enzyme-linked immunosorbent assay.

[0003] Anodic aluminum oxide (AAO) membrane is an anodic aluminum oxide nanostructure membrane with a pore size of 10-400 nm formed by electrochemical anodization of aluminum in an acidic solution at a voltage of 20-200 V. The pore size of the AAO membrane can be adjusted and the distribution can be regularized by specific regulation of the anodization conditions. Based on the characteristics of the AAO membrane, researchers have applied it in the fields of synthesizing nano-ordered structure templates, preparing biosensors, preparing nanoelectronic devices, and nano-optoelectronic devices.

[0004] Agarose is a polysaccharide with an average molecular weight of 120 kDa, composed of 1,3-linked β-D-galactofuranose and 1,4-linked 3,6-anhydro α-L-galactofuranose. It has special gel properties, strong elasticity, high stability and good biological affinity, and is an ideal polymer for fixing proteins. At present, the application of agarose hydrogel in immunoelectrophoresis is very complete, and it is a protein carrier with excellent performance.

[0005] Glial fibrillary acidic protein (GFAP) is a brain-specific astrocyte intermediate filament protein that only exists in the central nervous system. It is released into the human blood when the brain is damaged. It is a biomarker for a series of neurological diseases. It has high sensitivity and high specificity physiological characteristics, and has significant advantages in early diagnosis and identification of different types of neurological damage diseases. Therefore, the high-sensitivity detection of GFAP is of great significance for clinical diagnosis.

[0006] At present, there are many effective results in the research of using ECLIA to detect and analyze atopic antigens. For example, the patent document with publication number CN115825044A discloses a method for constructing a reusable nanopore-gated electrochemiluminescent sensor and its application in detecting T-2 toxin. Luminol nano-gold luminescent composite material is prepared and adsorbed on the surface of AAO membrane. Then, T-2 toxin aptamer is modified on the nano-gold membrane. After encapsulation, an electrochemiluminescent sensor specifically modified with T-2 toxin aptamer is obtained to achieve quantitative detection of T-2 toxin. In addition, the used sensor is immersed in ultrapure water to unlock the thiol group, and then it can be reused after drying.

[0007] However, although the above-mentioned patents have successfully modified specific antibodies on the AAO membrane and achieved high-sensitivity quantitative detection of antigens by utilizing the ordered and adjustable porous structure on the AAO membrane, they have not solved the problem that the AAO membrane itself is prone to clogging and breakage. How to avoid clogging of AAO nanochannels and breakage of AAO membranes when using antibody-modified AAO membrane nanochannels is still a problem difficult for technicians in this field to solve. Summary of the invention

[0008] In order to solve the problem of how to solve the blockage of AAO nanochannels and the fragmentation of AAO membranes when using antibody-modified AAO membrane nanochannels mentioned in the above background technology, an embodiment of the present invention provides an electrochemiluminescence sensor based on antibody-modified nanochannels, including an anodized aluminum film, characterized in that: The anodized aluminum film is provided with nanochannels penetrating the surface thereof, and a hydrogel is attached to one side of the surface of the anodized aluminum film. The interior of the hydrogel is amino-modified with an amino-modification solution, and then the antibody is coupled with an antibody coupling solution; The amino modification solution includes 3-aminopropyltriethoxysilane; The antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0009] On the basis of the above embodiment, further, it also includes a light emitting system, an electrolyte solution, an H-type electrolytic cell, a three-electrode system and an electrochemical workstation; The luminescent system includes phenanthroline ruthenium and tri-n-propylamine; The electrolyte solution includes 1× phosphate buffer and 0.1 mmol / L potassium chloride solution; The three-electrode system includes a working electrode glassy carbon electrode, a reference electrode silver chloride electrode and a counter electrode platinum electrode.

[0010] On the basis of the above embodiment, further, the nanochannel is an anodic aluminum nanochannel with a pore size of 10-400 nm, which is arranged in an array and penetrates the anodic aluminum oxide film with a pore spacing of 100-200 nm.

[0011] On the basis of the above embodiment, further, the hydrogel is a 1% agarose gel solution prepared with physiological saline as solvent; The thickness of the hydrogel attached to the anodized aluminum film is 1 to 2 mm.

[0012] On the basis of the above embodiment, further, the amino-modified solution is a 5%-10% 3-aminopropyltriethoxysilane solution prepared with anhydrous ethanol as a solvent.

[0013] On the basis of the above embodiment, further, the antibody coupling solution is prepared by the following steps: Dissolve the antibody in 1× phosphate buffer as a solvent to prepare an antibody solution with a concentration of 20-30 μg / mL; Using 1× phosphate buffer as solvent to dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide to prepare a crosslinker solution with a concentration of 5-8 mmol / L; The antibody solution and the cross-linking agent solution are mixed to obtain an antibody coupling solution.

[0014] The present invention also provides a method for constructing an electrochemiluminescence sensor based on an antibody-modified nanochannel as described in any one of the above items, comprising the following steps: S100, immersing the anodized aluminum film having nanochannels in anhydrous ethanol, ultrasonically applying it for 5 to 8 min, and then immersing it in ultrapure water, ultrasonically applying it for 5 to 8 min; S101, taking 10-20 μL of the hydrogel, dropping it onto one side of the anodized aluminum film, and letting it stand at room temperature for 10 seconds to form a gel, thereby obtaining a hydrogel nanochannel; S102, immersing the hydrogel nanochannel in the amino-modified solution for 2 to 3 hours, then immersing it in anhydrous ethanol for 5 to 8 minutes, and then immersing it in ultrapure water for 5 to 8 minutes to obtain an amino-modified nanochannel; S103, taking 5-10 μL of the antibody coupling solution, dropping it onto the hydrogel surface of the amino-modified nanochannel, incubating at 37° C. for 2-3 h, and then immersing it in ultrapure water for 5-8 min to obtain the antibody-modified nanochannel; S104, placing the antibody-modified nanochannel clamp inside the central circular channel of the H-type electrolytic cell, placing the three-electrode system in the electrolytic chamber of the H-type electrolytic cell, and adding an electrolyte solution.

[0015] On the basis of the above embodiment, further, the modification effect verification step is included: Place one of the silver chloride electrodes in the central circular channel of the H-type electrolytic cell, and place another silver chloride electrode in the electrolytic chamber of the H-type electrolytic cell, so that the antibody-modified nanochannel is located between the two silver chloride electrodes; The 0.1 mmol / L potassium chloride solution was used as the electrolyte and the electrochemical workstation was used to perform IV testing on the antibody-modified nanochannel.

[0016] On the basis of the above embodiment, the antibody-modified nanochannel is further placed in water and boiled to remove the modification, thereby obtaining an anodized aluminum film with clean nanochannels.

[0017] The present invention also provides a method for using the electrochemiluminescence sensor construction method based on antibody-modified nanochannels as described in any of the above items in the analysis of collagen fibrillary acidic protein, comprising the following steps: Using 1× phosphate buffer as solvent, prepare collagen fibrillary acid protein solutions of different concentrations; The collagen fibrillary acid protein solution with the lowest concentration was dripped onto the surface of the hydrogel of the antibody-modified nanochannel and allowed to stand at room temperature for 1 h; Washing the antibody-modified nanochannel to which the collagen fibrillary acid protein solution has been dropped with ultrapure water; The antibody-modified nanochannel with collagen fibrillary acid protein solution dripped on it is placed inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The glassy carbon electrode and the silver chloride electrode are placed in the left chamber of the H-type electrolytic cell, and the platinum electrode is placed in the right chamber of the H-type electrolytic cell; Using 1× phosphate buffer as the electrolyte solution, the luminescent system is added into the left chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; The antibody-modified nanochannel after the test was taken out, and the collagen fibrillary acid protein solution of the next concentration step was dripped onto the hydrogel surface of the antibody-modified nanochannel, and after standing at room temperature for 1 hour, it was washed with ultrapure water; The antibody-modified nanochannel is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; Repeat the operation to test the change degree of the electrochemiluminescent signal under different concentrations of collagen fibrillary acidic protein solution, and draw a working curve by plotting the change degree of the electrochemiluminescent signal and the concentration of the collagen fibrillary acidic protein solution; The antibody-modified nanochannel incubated with the collagen fibrillary acid protein solution of the to-be-tested concentration is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The electrochemical workstation is used to capture the electrochemical luminescence intensity of the collagen fibrillary acidic protein solution with a concentration to be measured, and the concentration of the collagen fibrillary acidic protein solution is calculated according to the working curve.

[0018] The present invention has the following beneficial effects: The electrochemiluminescent sensor based on antibody-modified nanochannels provided by the present invention comprises an anodized aluminum oxide membrane (AAO membrane) and a hydrogel layer. The AAO membrane has nanochannels arranged in an array pattern throughout the surface, and a hydrogel is attached to one side of the surface, which can reduce direct contact operations on the AAO membrane, thereby solving the problems of easy breakage and difficult operation during use, and effectively improving the use efficiency of the AAO membrane.

[0019] The electrochemiluminescent sensor based on antibody-modified nanochannels provided by the present invention modifies amino groups and antibodies inside the hydrogel to avoid direct modification of the AAO membrane, separates the modifier from the AAO membrane, and solves the problem that the nanochannels are easily blocked when the AAO membrane is used.

[0020] The method for constructing an electrochemiluminescent sensor based on antibody-modified nanochannels provided by the present invention modifies all the modifiers inside the hydrogel without causing direct modification changes to the AAO membrane. Based on the property that the hydrogel can be dissolved in water after boiling at 100°C and thus completely removed, the used antibody-modified nanochannels are placed in water and boiled to regain the AAO membrane with clean nanochannels, thereby realizing multiple uses of the AAO membrane without limiting the purpose, greatly improving the utilization rate of the AAO membrane, and effectively saving costs.

[0021] The method for constructing an electrochemiluminescent sensor based on an antibody-modified nanochannel provided by the present invention can complete amino modification within 2 hours, while the in-situ modification method takes 12 hours to complete amino modification. Under the same modification time, the modification effect of the antibody-modified nanochannel provided by the present invention is significantly better than that of the in-situ modified nanochannel, and the modification time can be significantly shortened and the modification efficiency can be improved. At the same time, the method does not need to perform acid treatment on the AAO membrane in advance during the experiment, reduces the use of toxic chemicals, and retains the activity of the antibody and the safety of the operation to the greatest extent.

[0022] The method for constructing an electrochemiluminescent sensor based on an antibody-modified nanochannel provided by the present invention particularly adopts an H-type electrolytic cell, utilizes the structural features of the H-type electrolytic cell, a double electrolytic cell, and a narrow exchange channel, and further combines the barrier effect of the AAO film itself to separate the luminescent pool from the antigen-antibody reaction site, thereby ensuring the activity of the antigen-antibody and the structural stability of the antigen-antibody complex, and protecting the luminescent substance and the luminescent electrode from biological contamination, thereby avoiding a reduction in luminescent efficiency.

[0023] The present invention also provides a method for constructing an electrochemiluminescence sensor based on an antibody-modified nanochannel and using it in the analysis of collagen fibrillary acidic protein. A working curve is prepared by using a GFAP concentration step and an electrochemiluminescence light intensity. The method has good linearity within a concentration range of 0.01 to 100 ng / mL, and the electrochemiluminescence light intensity at different concentrations is significantly different, and the sensitivity is high. In addition, the theoretical detection limit can reach 1 pg / mL, which improves the shortcomings of traditional immunoelectrophoresis experiments, immunodiffusion experiments, etc., which can only be qualitative or semi-quantitative, and achieves a completely quantitative technical effect of protein detection and analysis.

[0024] The present invention provides a method for using the electrochemiluminescent sensor construction method based on antibody-modified nanochannels in the analysis of collagen fibrillary acidic protein. The whole process IV test monitoring is performed on different pieces of antibody-modified nanochannels. The test results have high correlation and good repeatability, and comprehensively realize rapid, high-sensitivity, and high-accuracy quantitative detection of GFAP.

[0025] Other features and beneficial effects of the present invention will be described in the following description, and some of the technical features and beneficial effects can be obviously derived from the description or understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, some of the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is an electron microscope scanning image of the nanochannels of the anodized aluminum film provided in Example 1 of the present invention; Figure 2 This is an IV test diagram for verifying the modification effect of the antibody-modified nanochannel provided in Example 2 of the present invention; Figure 3 This is a feasibility analysis diagram of the application of antibody-modified nanochannels provided in Example 3 of the present invention in GFAP analysis; Figure 4This is a schematic diagram of a working curve of the antibody-modified nanochannel provided in Example 3 of the present invention applied to GFAP analysis; Figure 5 This is a IV test diagram of the whole process of applying the antibody-modified nanochannel to GFAP analysis provided in Example 3 of the present invention; Figure 6 This is a IV test diagram of the whole process of GFAP analysis using different antibody-modified nanochannels provided in Example 3 of the present invention; Figure 7 This is an IV test diagram comparing the modification effects of the antibody-modified nanochannel and the in-situ modified nanochannel provided in Example 2 and Comparative Example 1 of the present invention at the same modification time; Figure 8 This is an IV test diagram of the full process of GFAP analysis using the in-situ modified nanochannel provided in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Example 1 Embodiment 1 of the present invention provides an electrochemiluminescent sensor based on antibody-modified nanochannels, comprising an anodized aluminum film; The anodized aluminum film is provided with nanochannels penetrating the surface thereof, and a hydrogel is attached to one side of the surface of the anodized aluminum film. The interior of the hydrogel is amino-modified with an amino-modification solution, and then the antibody is coupled with an antibody coupling solution; The amino modification solution includes 3-aminopropyltriethoxysilane; The antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0030] When used specifically, it also includes: a luminescence system, an electrolyte solution, an H-type electrolytic cell, a three-electrode system and an electrochemical workstation; The luminescent system includes phenanthroline ruthenium and tri-n-propylamine; The electrolyte solution includes 1× phosphate buffer and 0.1 mmol / L potassium chloride solution; The three-electrode system includes a working electrode glassy carbon electrode, a reference electrode silver chloride electrode and a counter electrode platinum electrode; In the specific operation, 1× phosphate buffer is prepared according to the following steps: weigh 8 g NaCl, 0.2 g KCl, 1.44 g NaH2PO4, and 0.245 g K2HPO4, fully dissolve them with 800 mL distilled water, and adjust the pH of the mixed solution to 7.4 to obtain 1× phosphate buffer.

[0031] For specific use, refer to Figure 1 As shown, the nanochannel is an anodic aluminum nanochannel with a pore size of 10-400 nm, which is arranged in an array and penetrates the anodic aluminum oxide membrane with a pore spacing of 100-200 nm.

[0032] When used specifically, the hydrogel is a 1% agarose gel solution prepared with physiological saline as a solvent; The thickness of the hydrogel attached to the anodized aluminum film is 1 to 2 mm; In the specific operation, 10 mg of agarose powder was weighed into a glass container, 1 mL of 9% saline was added, and the mixture was heated to 100°C to completely dissolve the agarose powder. The mixture was cooled to 55-65°C and then kept warm to maintain a liquid state.

[0033] When used specifically, the amino-modified solution is a 5% to 10% 3-aminopropyltriethoxysilane solution prepared with anhydrous ethanol as a solvent.

[0034] In specific operation, the antibody coupling solution is prepared by the following steps: Dissolve the antibody in 1× phosphate buffer as a solvent to prepare an antibody solution with a concentration of 20-30 μg / mL; Using 1× phosphate buffer as solvent to dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide to prepare a crosslinker solution with a concentration of 5-8 mmol / L; The antibody solution and the cross-linking agent solution are mixed to obtain an antibody coupling solution.

[0035] In this embodiment, preferably, the nanochannel has a pore size of 90 nm, is arranged in an array throughout the AAO membrane, and has a pore spacing of 150 nm.

[0036] In this embodiment, preferably, the concentration of the 1% agarose gel solution is maintained at 57° C. to maintain a liquid state.

[0037] In this embodiment, preferably, the concentration of the 3-aminopropyltriethoxysilane (APTES) solution is 5%.

[0038] In this embodiment, preferably, the concentration of the antibody solution is 20 μg / mL.

[0039] In this embodiment, preferably, the concentration of the cross-linking agent solution is 5 mmol / L, wherein the content of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is 5 mmol / L, and the content of N-hydroxysuccinimide (NHS) is 5 mmol / L.

[0040] In this embodiment, by using hydrogel to attach to the surface of one side of the AAO membrane, mechanical support is provided for the AAO membrane, thereby solving the technical problem of the AAO membrane being easily broken during use; in addition, since the amino group and antibody modifiers are both modified inside the hydrogel and do not directly contact the AAO membrane, the problem of clogging the AAO membrane nanochannels is avoided.

[0041] Example 2 Based on Example 1, Example 2 of the present invention provides a method for constructing an electrochemiluminescence sensor based on an antibody-modified nanochannel, comprising the following steps: S100, immersing the anodized aluminum film having nanochannels in anhydrous ethanol, ultrasonically applying it for 5 to 8 min, and then immersing it in ultrapure water, ultrasonically applying it for 5 to 8 min; S101, taking 10-20 μL of the hydrogel, dropping it onto one side of the anodized aluminum film, and letting it stand at room temperature for 10 seconds to form a gel, thereby obtaining a hydrogel nanochannel; S102, immersing the hydrogel nanochannel in the amino-modified solution for 2 to 3 hours, then immersing it in anhydrous ethanol for 5 to 8 minutes, and then immersing it in ultrapure water for 5 to 8 minutes to obtain an amino-modified nanochannel; S103, taking 5-10 μL of the antibody coupling solution, dropping it onto the hydrogel surface of the amino-modified nanochannel, incubating at 37° C. for 2-3 h, and then immersing it in ultrapure water for 5-8 min to obtain the antibody-modified nanochannel; S104, placing the antibody-modified nanochannel clamp inside the central circular channel of the H-type electrolytic cell, placing the three-electrode system in the electrolytic chamber of the H-type electrolytic cell, and adding an electrolyte solution; Among them, the APTES in the amino modification solution will modify the amino group inside the agarose gel through covalent bond connection. Furthermore, the antibody is coupled with the amino group with the help of EDC / NHS in the cross-linker solution, thereby realizing the modification of the antibody inside the hydrogel and indirectly completing the modification of the nanochannel of the AAO membrane.

[0042] The specific operation also includes the modification effect verification step: The antibody-modified nanochannel is sandwiched inside the central circular channel of the H-type electrolytic cell; Place one of the silver chloride electrodes in the central circular channel of the H-type electrolytic cell, and place another silver chloride electrode in the electrolytic chamber of the H-type electrolytic cell, so that the antibody-modified nanochannel is located between the two silver chloride electrodes; Using the 0.1 mmol / L potassium chloride solution as the electrolyte solution, using the electrochemical workstation to perform an IV test on the antibody-modified nanochannel; Specifically, when conducting the modification effect verification test, the CV mode of the electrochemical workstation was used, the scanning voltage was -1~1 V, and the scanning rate was 0.05 V / s; specifically, the IV test results of the AAO membrane nanochannel, the hydrogel nanochannel and the antibody modified nanochannel were referenced Figure 2 As shown, compared with the AAO membrane nanochannel, the current change amplitude of the hydrogel nanochannel and the antibody-modified nanochannel is around 3 μA, which is significantly different from the AAO membrane nanochannel, and the modification effect is good.

[0043] When used specifically, the antibody-modified nanochannel is placed in water and boiled to remove the modification, thereby obtaining an anodized aluminum film with clean nanochannels; In the specific operation, the agarose gel attached to the surface of the AAO membrane will transform from solid to liquid at 100°C and dissolve in water. Since each modification step is located inside the agarose gel and does not directly act on the AAO membrane, all modifications can be removed by removing the agarose gel to obtain an AAO membrane with clean nanochannels.

[0044] In this embodiment, preferably, the ultrasonic time is 5 min.

[0045] In this embodiment, preferably, the amount of the hydrogel is 10 μL.

[0046] In this embodiment, preferably, the hydrogel nanochannel is immersed in the amino modification solution for 2 h for amino modification, then immersed in anhydrous ethanol for 5 min to wash away unmodified APTES, and then immersed in ultrapure water for 5 min to wash away residual ethanol.

[0047] In this embodiment, preferably, the antibody coupling solution is used in an amount of 5 μL, which is dropped onto the hydrogel surface of the amino-modified nanochannel, incubated at 37° C. for 2 h for antibody modification, and then immersed in ultrapure water for 5 min to remove the fixed antibodies.

[0048] In this embodiment, the advantages of agarose gel rapid gelation and good mechanical strength are utilized to quickly attach to the surface of the AAO membrane. Further, APTES is used to covalently link the modified amino group and EDC / NHS coupled amino group with the antibody to achieve indirect modification of the antibody modification on the AAO membrane, obtain the antibody modified nanochannel, and sandwich it in the H-type electrolytic cell. In conjunction with the three-electrode system and the electrochemical workstation, an electrochemiluminescent sensor based on the antibody modified nanochannel is constructed. In addition, the antibody modified nanochannel is subjected to a modification effect IV scanning verification test. The test results show that the nanochannels modified by hydrogel and antibody are significantly different from the unmodified AAO membrane nanochannels, and the modification effect is good. While retaining the advantages of high spatiotemporal resolution and sensitivity of the AAO membrane nanochannel, this embodiment effectively solves the problem of easy breakage of the AAO membrane and easy clogging of the nanochannel during use.

[0049] In this embodiment, by boiling the antibody-modified nanochannel in water, all the modifications inside the agarose gel can be removed at once by utilizing the property of agarose gel that it will transform from solid to liquid and dissolve in water at 100°C, and the AAO membrane with clean nanochannels can be obtained again, thereby realizing the multiple uses of the AAO membrane without limitation.

[0050] Example 3 On the basis of Examples 1 and 2, Example 3 of the present invention provides a method for using an electrochemiluminescence sensor construction method based on antibody-modified nanochannels in the analysis of collagen fibrillary acidic protein, comprising the following steps: Using 1× phosphate buffer as solvent, prepare collagen fibrillary acid protein solutions of different concentrations; The collagen fibrillary acid protein solution with the lowest concentration was dripped onto the surface of the hydrogel of the antibody-modified nanochannel and allowed to stand at room temperature for 1 h; Washing the antibody-modified nanochannel to which the collagen fibrillary acid protein solution has been dropped with ultrapure water; The antibody-modified nanochannel with collagen fibrillary acid protein solution dripped on it is placed inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The glassy carbon electrode and the silver chloride electrode are placed in the left chamber of the H-type electrolytic cell, and the platinum electrode is placed in the right chamber of the H-type electrolytic cell; Using 1× phosphate buffer as the electrolyte solution, the luminescent system is added into the left chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; The antibody-modified nanochannel after the test was taken out, and the collagen fibrillary acid protein solution of the next concentration step was dripped onto the hydrogel surface of the antibody-modified nanochannel, and after standing at room temperature for 1 hour, it was washed with ultrapure water; The antibody-modified nanochannel is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; Repeat the operation to test the change degree of the electrochemiluminescent signal under different concentrations of collagen fibrillary acidic protein solution, and draw a working curve by plotting the change degree of the electrochemiluminescent signal and the concentration of the collagen fibrillary acidic protein solution; The antibody-modified nanochannel incubated with the collagen fibrillary acid protein solution of the to-be-tested concentration is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The electrochemical workstation is used to capture the electrochemical luminescence intensity of the collagen fibrillary acidic protein solution with a concentration to be measured, and the concentration of the collagen fibrillary acidic protein solution is calculated according to the working curve.

[0051] In this embodiment, preferably, the luminescent system is 10 μL of 99% tri-n-propylamine solution and 2.5 μmol / L of phenanthroline ruthenium solution.

[0052] In this embodiment, specifically, the collagen fibrillary acid protein (GFAP) antibody has a specific recognition and binding effect on the GFAP antigen. When the two combine with each other to form an antigen-antibody complex, it will cause changes in the charge density and electroosmotic flow inside the hydrogel nanochannel, resulting in changes in ion current and ion concentration, further affecting the luminescence intensity, thereby realizing the detection of GFAP.

[0053] In this embodiment, specifically, before applying the electrochemiluminescence sensor construction method based on antibody-modified nanochannels to the analysis of collagen fibrillary acidic protein (GFAP), a feasibility analysis was performed. The feasibility analysis results were referenced to Figure 3 As shown, the electrochemiluminescence intensity of GFAP solutions at concentrations of 100 pg / mL and 1 ng / mL was significantly different from that of the blank control group, and there was also an obvious difference between the 100 pg / mL GFAP solution group and the 1 ng / mL GFAP solution group, which was sufficient for capture and differentiation.

[0054] In this embodiment, when the test is performed, the scanning voltage is 0.4-1.6 V and the scanning rate is 0.1 V / s; The concentrations of the collagen fibrillary acidic protein (GFAP) solution are 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL and 100 ng / mL, and the working curve is referenced Figure 4 As shown in the figure, the method has good linearity in the GFAP solution concentration range of 0.01~100 ng / mL, and the theoretical detection limit can reach 1 pg / mL.

[0055] In this example, the antibody-modified nanochannel used in GFAP analysis was further subjected to full-process IV test monitoring, with a scanning voltage of -1 to 1 V and a scanning rate of 0.05 V / s, which was consistent with Example 2. The test results were referenced to Figure 5 As shown, the current change after binding to the antigen varies by about 0.5 μA, which is clearly different from the current change of the antibody-modified nanochannel without binding to the antigen.

[0056] In this embodiment, further, the IV test monitoring is performed on different AAO membrane nanochannels at different modification or use stages used in GFAP analysis, with a scanning voltage of -1 to 1 V and a scanning rate of 0.05 V / s, which is consistent with Example 2. The linearity of the test results is shown in Figure 2. Figure 6 As shown, at the same time, the current data in the test results is used as a comparison object to perform correlation analysis, and the analysis results are shown in Table 1.

[0057] Table 1 Correlation analysis of IV tests of different nanochannels at different stages in GFAP analysis

[0058] It can be seen that at the same process stage, such as the antibody modification stage, the antibody modified nanochannels in different slices have good correlation, and the correlation results are within 10 -4 The order of magnitude is consistent and close to 1, that is, the limit is close to perfect correlation. Figure 6 It is reflected in the high linear overlap rate of each group. It can be seen that the electrochemiluminescence sensor construction method based on antibody-modified nanochannels provided by the present invention has good repeatability in each stage of GFAP analysis and small differences between slices.

[0059] In this embodiment, by using the electrochemiluminescence sensor construction method based on antibody-modified nanochannels for GFAP analysis, the dual advantages of high spatiotemporal resolution and high sensitivity of AAO membrane nanochannels and high mechanical strength and good molecular carrier performance of hydrogels are fully combined. This not only solves the problems of AAO nanochannel blockage and AAO membrane fragmentation when using antibody-modified AAO membrane nanochannels, but also optimizes the disadvantages of traditional detection methods such as the inability to accurately quantify, time-consuming and complicated operation, and large fluctuations in repeatability, thereby achieving the technical effect of sensitive, rapid and easy-to-operate GFAP analysis and detection.

[0060] Comparative Example 1 Comparative Example 1 provides an application of a nanochannel in situ modification method in GFAP analysis. Except for the different nanochannel modification methods, other steps and specific parameters are consistent with the corresponding steps and parameters of Example 2 and Example 3, and the nanochannel in situ modification specifically includes the following steps: S200, immersing the AAO membrane nanochannel in anhydrous ethanol, ultrasonicating for 10 min, then immersing it in ultrapure water, ultrasonicating for 10 min, and then drying it in a nitrogen atmosphere; S201, immersing the AAO membrane nanochannel in a 5% HCl solution for 1 min, then rinsing the HCl solution on the surface of the AAO membrane nanochannel with ultrapure water, then immersing it in ultrapure water for 5 min, and then drying it in a nitrogen atmosphere; S202, immersing the AAO membrane nanochannel in a 5% APTES solution prepared with anhydrous ethanol as solvent, and incubating in the dark for 12 h; S203, rinsing the AAO membrane nanochannels incubated in S202 with anhydrous ethanol, and immersing them in anhydrous ethanol for 1 hour, and then placing them in an oven and drying them at 120° C. for 2 hours; S204. Pipette 5 μL of GFAP antibody solution containing 5 mmol / L EDC / NHS and drop it onto the surface of the AAO membrane nanochannel. After standing for 2 h, rinse off the residual antibody with ultrapure water to obtain the in situ modified nanochannel.

[0061] In this comparative example, the in situ modified nanochannel was subjected to IV test, with a scanning voltage of -1 to 1 V and a scanning rate of 0.05 V / s. The test results are shown in Table 1. Figure 7 As shown, after 2 h of modification, the amplitude of the change in the amino current of the AAO membrane nanochannel in situ modified with amino groups was only around 0.1 μA, while after 2 h of amino modification of the AAO membrane nanochannel with a hydrogel modified layer provided in Example 2 of the present invention, the amplitude of the change in the current was around 2 μA. Its modification efficiency is significantly higher than the in situ modification method provided in Comparative Example 1, which can greatly shorten the modification time.

[0062] In this comparative example, the in situ modified nanochannel used in GFAP analysis was further subjected to full-process IV test monitoring, with a scanning voltage of -1 to 1 V and a scanning rate of 0.05 V / s. The test results are shown in Table 1. Figure 8 As shown, it can be seen that the difference in the amplitude of the current change after the in situ modified nanochannel binds to the antigen is less than 0.05 μA compared with when it is not bound to the antigen, and no significant distinction can be formed, that is, the response to changes in antigen concentration is not sensitive.

[0063] In this comparative example, an in situ modified nanochannel electrochemiluminescence sensor was constructed and applied to GFAP analysis, and its modification effect and the change in ion flux after binding to the antigen were monitored and analyzed by IV testing. From the analysis results, it can be seen that the electrochemiluminescence sensor based on antibody-modified nanochannels containing a hydrogel-modified carrier layer provided by the present invention is significantly superior to comparative example 1 in terms of modification effect, modification efficiency and sensitivity when used in GFAP analysis.

[0064] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present invention can be improved in only one or several aspects, without having to solve all the technical problems listed in the prior art or background technology at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be used as a limitation on the claim.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An electrochemiluminescent sensor based on antibody-modified nanochannels, comprising an anodized aluminum film, characterized in that: The anodized aluminum film is provided with nanochannels penetrating the surface thereof, and a hydrogel is attached to one side of the surface of the anodized aluminum film. The interior of the hydrogel is amino-modified with an amino-modification solution, and then the antibody is coupled with an antibody coupling solution; The amino modification solution includes 3-aminopropyltriethoxysilane; The antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The electrochemiluminescence sensor based on antibody-modified nanochannel according to claim 1, characterized in that: Also includes: Luminescence system, electrolyte solution, H-type electrolytic cell, three-electrode system and electrochemical workstation; The luminescent system includes phenanthroline ruthenium and tri-n-propylamine; The electrolyte solution includes 1× phosphate buffer and 0.1 mmol / L potassium chloride solution; The three-electrode system includes a working electrode glassy carbon electrode, a reference electrode silver chloride electrode and a counter electrode platinum electrode.

3. The electrochemiluminescence sensor based on antibody-modified nanochannel according to claim 1, characterized in that: The nanochannel is an anodic aluminum nanochannel with a pore size of 10-400 nm, which is arranged in an array and penetrates the anodic aluminum oxide film with a pore spacing of 100-200 nm.

4. The electrochemiluminescence sensor based on antibody-modified nanochannel according to claim 1, characterized in that: The hydrogel is a 1% agarose gel solution prepared with physiological saline as solvent; The thickness of the hydrogel attached to the anodized aluminum film is 1 to 2 mm.

5. The electrochemiluminescence sensor based on antibody-modified nanochannel according to claim 1, characterized in that: The amino modification solution is a 5% to 10% 3-aminopropyltriethoxysilane solution prepared with anhydrous ethanol as a solvent.

6. The electrochemiluminescence sensor based on antibody-modified nanochannel according to claim 1, characterized in that: The antibody coupling solution is prepared by the following steps: Dissolve the antibody using 1× phosphate buffer as a solvent to prepare an antibody solution with a concentration of 20-30 μg / mL; Using 1× phosphate buffer as solvent to dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide to prepare a crosslinker solution with a concentration of 5-8 mmol / L; The antibody solution and the cross-linking agent solution are mixed to obtain an antibody coupling solution.

7. A method for constructing an electrochemiluminescence sensor based on antibody-modified nanochannels according to any one of claims 1 to 6, characterized in that: The following steps are involved: S100, immersing the anodized aluminum film having nanochannels in anhydrous ethanol, ultrasonically applying it for 5 to 8 min, and then immersing it in ultrapure water, ultrasonically applying it for 5 to 8 min; S101, taking 10-20 μL of the hydrogel, dropping it onto one side of the anodized aluminum film, and letting it stand at room temperature for 10 s to form a gel, thereby obtaining a hydrogel nanochannel; S102, immersing the hydrogel nanochannel in the amino-modified solution for 2 to 3 hours, then immersing it in anhydrous ethanol for 5 to 8 minutes, and then immersing it in ultrapure water for 5 to 8 minutes to obtain an amino-modified nanochannel; S103, taking 5-10 μL of the antibody coupling solution, dropping it onto the hydrogel surface of the amino-modified nanochannel, incubating at 37° C. for 2-3 h, and then immersing it in ultrapure water for 5-8 min to obtain the antibody-modified nanochannel; S104, placing the antibody-modified nanochannel clamp inside the central circular channel of the H-type electrolytic cell, placing the three-electrode system in the electrolytic chamber of the H-type electrolytic cell, and adding an electrolyte solution.

8. The method for constructing an electrochemiluminescence sensor based on antibody-modified nanochannels according to claim 7, characterized in that: It also includes the modification effect verification step: Place one of the silver chloride electrodes in the central circular channel of the H-type electrolytic cell, and place another silver chloride electrode in the electrolytic chamber of the H-type electrolytic cell, so that the antibody-modified nanochannel is located between the two silver chloride electrodes; The 0.1 mmol / L potassium chloride solution was used as the electrolyte solution, and the electrochemical workstation was used to perform IV testing on the antibody-modified nanochannel.

9. The method for constructing an electrochemiluminescence sensor based on antibody-modified nanochannels according to claim 7, characterized in that: The antibody-modified nanochannel is placed in water and boiled to remove the modification, thereby obtaining an anodized aluminum membrane with clean nanochannels.

10. A method for using the electrochemiluminescence sensor construction method based on antibody-modified nanochannels as claimed in any one of claims 7 to 9 in the analysis of collagen fibrillary acidic protein, comprising the following steps: Using 1× phosphate buffer as solvent, prepare collagen fibrillary acid protein solutions of different concentrations; The collagen fibrillary acid protein solution with the lowest concentration was dripped onto the surface of the hydrogel of the antibody-modified nanochannel and allowed to stand at room temperature for 1 h; Washing the antibody-modified nanochannel to which the collagen fibrillary acid protein solution has been dropped with ultrapure water; The antibody-modified nanochannel with collagen fibrillary acid protein solution dripped on it is placed inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The glassy carbon electrode and the silver chloride electrode are placed in the left chamber of the H-type electrolytic cell, and the platinum electrode is placed in the right chamber of the H-type electrolytic cell; Using 1× phosphate buffer as the electrolyte solution, the luminescent system is added into the left chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; The antibody-modified nanochannel after the test was taken out, and the collagen fibrillary acid protein solution of the next concentration step was dripped onto the hydrogel surface of the antibody-modified nanochannel, and after standing at room temperature for 1 hour, it was washed with ultrapure water; The antibody-modified nanochannel is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; Using the electrochemical workstation to perform an electrochemiluminescence test and capture the electrochemiluminescence signal intensity; Repeat the operation to test the change degree of the electrochemiluminescent signal under different concentrations of collagen fibrillary acidic protein solution, and draw a working curve by plotting the change degree of the electrochemiluminescent signal and the concentration of the collagen fibrillary acidic protein solution; The antibody-modified nanochannel incubated with the collagen fibrillary acid protein solution of the to-be-tested concentration is clamped inside the central circular channel of the H-type electrolytic cell, wherein the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell; The electrochemical workstation is used to capture the electrochemical luminescence intensity of the collagen fibrillary acidic protein solution with a concentration to be measured, and the concentration of the collagen fibrillary acidic protein solution is calculated according to the working curve.

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