Antibody-modified nanochannel electrogenerated chemiluminescence sensor and methods of construction and use thereof

Abstract

The present application relates to the field of biological detection, in particular to an electrochemiluminescence sensor based on antibody modified nanochannel and a construction method and use method thereof. The electrochemiluminescence sensor based on antibody modified nanochannel comprises an anodic aluminum oxide film with a nanochannel penetrating through the surface, and a hydrogel is attached to one side surface of the anodic aluminum oxide film, which can reduce the problem of easy fragmentation of the anodic aluminum oxide film caused by direct contact operation, and the modifier is completely modified on the hydrogel layer, so that the modifier is separated from the nanochannel, and the problem of easy blockage of the anodic aluminum oxide film nanochannel modified by the antibody is solved. In addition, all the modifiers can be removed by boiling, and the non-directional utilization rate of the anodic aluminum oxide film is improved. The construction method of the electrochemiluminescence sensor based on antibody modified nanochannel can achieve good modification effect in a short time and improve the efficiency. When used in collagen fiber acid protein analysis, the clinical operation efficiency is improved, and rapid, high sensitivity, high accuracy and complete quantitative detection of collagen fiber acid protein are realized.

Description

TECHNICAL FIELD

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

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

[0003] Anodic aluminum oxide (AAO) film is a kind of anodic aluminum nano-structured film with pore size of 10-400 nm formed by electrochemical anodization of aluminum in acidic solution by applying 20-200 V voltage, and by specific regulation of anodization conditions, the pore size of AAO film can be adjusted and distributed regularly. Based on the characteristics of AAO film, researchers have applied it in the fields of synthesis of nano-ordered structure template, preparation of biosensor, preparation of nano-electronic devices and nano-optoelectronic devices, etc.

[0004] Agarose is a polysaccharide with an average molecular weight of 120 kDa, which is composed of 1,3-linked β-D-galactofuranose and 1,4-linked 3,6-anhydro-α-L-galactofuranose, and has special gel properties, strong elasticity, high stability and good biological affinity. It is an ideal high molecular polymer for immobilizing 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 intermediate filament protein that only exists in central nervous system astrocytes, which is released into the human blood when the brain is damaged, and is a biomarker for a series of nervous system diseases. It has high sensitivity and high specificity of physiological characteristics, and has significant advantages in early diagnosis and identification of different types of nervous system damage diseases. Therefore, the high sensitivity detection of GFAP has important significance for clinical diagnosis.

[0006] At present, there are many effective results in the research of detecting and analyzing specific antigens by using ECLIA. For example, the patent document with publication number CN115825044A discloses a reusable nanopore gate electrochemiluminescence sensor construction method and its application in detecting T-2 toxin. A luminol nanogold luminescent composite material is prepared, adsorbed on the surface of an AAO membrane, and then a T-2 toxin aptamer is modified on the nanogold membrane. After packaging, a T-2 toxin aptamer specific modified electrochemiluminescence sensor is obtained, realizing quantitative detection of T-2 toxin. In addition, the used sensor is immersed in ultrapure water for mercapto unlocking, and then dried to be reusable.

[0007] However, although the above-mentioned patent successfully modifies specific antibodies on the AAO membrane, utilizes the ordered and adjustable porous structure on the AAO membrane to realize high-sensitivity quantitative detection of antigens, it does not solve the problem of easy clogging and easy fragmentation of the AAO membrane. How to avoid AAO nanopore clogging and AAO membrane fragmentation when using antibody modified AAO nanopore is still a problem difficult to solve for those skilled in the art. SUMMARY

[0008] To solve the problem of AAO nanopore clogging and AAO membrane fragmentation when using antibody modified AAO nanopore mentioned in the background art, an embodiment of the present application provides an electrochemiluminescence sensor based on antibody modified nanopore, comprising an anodic aluminum oxide membrane, characterized in that:

[0009] The anodic aluminum oxide membrane is provided with nanopores penetrating through its surface, and one side surface of the anodic aluminum oxide membrane is attached with a hydrogel, the inside of which is modified with an amino modification solution and then coupled with an antibody using an antibody coupling solution;

[0010] The amino modification solution comprises 3-aminopropyltriethoxysilane;

[0011] The antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide.

[0012] On the basis of the above-mentioned embodiment, further comprising a luminescence system, an electrolyte solution, an H-type electrolytic cell, a three-electrode system, and an electrochemical workstation;

[0013] The luminescence system comprises phenanthroline ruthenium and tri-n-propylamine;

[0014] The electrolyte solution comprises 1× phosphate buffer and 0.1 mmol / L potassium chloride solution;

[0015] The three-electrode system comprises a working electrode glassy carbon electrode, a reference electrode silver chloride electrode, and a counter electrode platinum electrode.

[0016] Further, based on the above-mentioned embodiments, the nanochannel is an anodic aluminum nanochannel, the pore size is 10-400 nm, the array is arranged through the anodic aluminum oxide film, and the hole distance is 100-200 nm.

[0017] Further, based on the above-mentioned embodiments, the hydrogel is a 1% agarose gel solution configured with physiological saline as a solvent.

[0018] The thickness of the hydrogel attached to the anodic aluminum oxide film is 1-2 mm.

[0019] Further, based on the above-mentioned embodiments, the amino modification solution is a 5%-10% 3-aminopropyltriethoxysilane solution configured with anhydrous ethanol as a solvent.

[0020] Further, based on the above-mentioned embodiments, the antibody coupling solution is prepared by the following steps:

[0021] The antibody is dissolved with 1× phosphate buffer as a solvent to obtain an antibody solution with a concentration of 20-30 μg / mL;

[0022] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide is dissolved with 1× phosphate buffer as a solvent to obtain a crosslinking agent solution with a concentration of 5-8 mmol / L;

[0023] The antibody solution and the crosslinking agent solution are mixed to obtain the antibody coupling solution.

[0024] The application also provides a construction method of the antibody-modified nanochannel-based electrochemiluminescence sensor, comprising the following steps:

[0025] S100, the anodic aluminum oxide film with nanochannels is immersed in anhydrous ethanol for 5-8 min, and then immersed in ultrapure water for 5-8 min;

[0026] S101, 10-20 μL of the hydrogel is taken and added dropwise to one side surface of the anodic aluminum oxide film, and gelled at room temperature for 10 s to obtain a hydrogel nanochannel;

[0027] S102, the hydrogel nanochannel is immersed in the amino modification solution for 2-3 h, then immersed in anhydrous ethanol for 5-8 min, and then immersed in ultrapure water for 5-8 min to obtain an amino-modified nanochannel;

[0028] S103, 5~10 μL of the antibody conjugated solution is dropped to the hydrogel surface of the amino-modified nanochannel, and is incubated at 37℃ for 2~3 h, and then is immersed in ultrapure water for 5~8 min to obtain an antibody-modified nanochannel;

[0029] S104, the antibody-modified nanochannel is sandwiched in the central circular channel of the H-type electrolytic cell, the three-electrode system is placed in the electrolytic chamber of the H-type electrolytic cell, and an electrolyte solution is added.

[0030] On the basis of the above embodiment, further comprising a modification effect verification step:

[0031] One of the silver chloride electrodes is placed in the central circular channel of the H-type electrolytic cell, and another one of the silver chloride electrodes is placed in the electrolytic chamber of the H-type electrolytic cell, so that the antibody-modified nanochannel is between the two silver chloride electrodes;

[0032] The 0.1 mmol / L potassium chloride solution is used as the electrolyte, and the I-V test of the equipped antibody-modified nanochannel is performed by using the electrochemical workstation.

[0033] On the basis of the above embodiment, further, the antibody-modified nanochannel is placed in water and boiled to remove the modifier, and thus a clean nanochannel anodic aluminum membrane is obtained.

[0034] The application also provides a use method of the antibody-modified nanochannel-based electrochemiluminescence sensor construction method in collagen fiber acidic protein analysis, comprising the following steps:

[0035] The 1× phosphate buffer is used as a solvent to configure collagen fiber acidic protein solutions with different concentrations.

[0036] The collagen fiber acidic protein solution with the lowest concentration is dropped on the hydrogel surface of the antibody-modified nanochannel, and is left at room temperature for 1 h.

[0037] The antibody-modified nanochannel to which the collagen fiber acidic protein solution is dropped is washed with ultrapure water.

[0038] The antibody-modified nanochannel to which the collagen fiber acidic protein solution is dropped is sandwiched in the central circular channel of the H-type electrolytic cell, and the side with the attached hydrogel faces the right chamber of the H-type electrolytic cell.

[0039] 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.

[0040] The 1× phosphate buffer is used as an electrolyte solution, and the luminescence system is added to the left chamber of the H-type electrolytic cell.

[0041] Carrying out electrochemiluminescence test by using the electrochemical workstation, and capturing electrochemiluminescence signal intensity;

[0042] After the test, the antibody-modified nanochannel is taken out, and a collagen fiber acid protein solution of a next concentration step is added dropwise on the hydrogel surface of the antibody-modified nanochannel, and after being placed at room temperature for 1 h, ultrapure water is used for cleaning;

[0043] The antibody-modified nanochannel is clamped in the central circular channel of the H-type electrolytic cell, and the side with the attached hydrogel faces the right chamber of the H-type electrolytic cell;

[0044] Carrying out electrochemiluminescence test by using the electrochemical workstation, and capturing electrochemiluminescence signal intensity;

[0045] The operation is repeated, the degree of change of the electrochemiluminescence signal under different concentrations of collagen fiber acid protein solution is tested, and a working curve is drawn by plotting the degree of change of the electrochemiluminescence signal and the concentration of the collagen fiber acid protein solution;

[0046] The antibody-modified nanochannel incubated with the collagen fiber acid protein solution of the to-be-tested concentration is clamped in the central circular channel of the H-type electrolytic cell, and the side with the attached hydrogel faces the right chamber of the H-type electrolytic cell;

[0047] The electrochemiluminescence light intensity of the collagen fiber acid protein solution of the to-be-tested concentration is captured by using the electrochemical workstation, and the concentration of the collagen fiber acid protein solution is calculated according to the working curve.

[0048] The application has the following beneficial effects:

[0049] The electrochemiluminescence sensor based on the antibody-modified nanochannel provided by the application comprises an anodic aluminum oxide film (AAO film) and a hydrogel layer, the AAO film has nanochannels arranged in an array through a surface, and one side surface is attached with the hydrogel, so that direct contact operation on the AAO film can be reduced, and the problem that the AAO film is easy to break and difficult to operate during use is solved, and the use efficiency of the AAO film is effectively improved.

[0050] The electrochemiluminescence sensor based on the antibody-modified nanochannel provided by the application modifies amino groups and antibodies in the hydrogel, avoids direct modification on the AAO film, separates the modification from the AAO film, and solves the problem that the nanochannels of the AAO film are easy to block during use.

[0051] The application provides a construction method of an electrochemiluminescence sensor based on an antibody modified nanochannel, all modifiers are modified in a hydrogel, no direct modification change is generated to an AAO film, and based on the characteristics that the hydrogel can be dissolved in water after boiling at 100 DEG C and is removed completely, the antibody modified nanochannel after use is placed in water to boil, and the AAO film with clean nanochannels can be obtained again, so that the AAO film can be used repeatedly without limitation, the utilization rate of the AAO film is greatly improved, and the cost is effectively saved.

[0052] The construction method of the electrochemiluminescence sensor based on the antibody modified nanochannel can complete amino modification within 2 h, while in-situ modification needs to consume 12 h to complete amino modification, and under the same modification time, the modification effect of the antibody modified nanochannel is significantly better than that of the in-situ modified nanochannel, the modification time can be significantly shortened, the modification efficiency is improved, meanwhile, the method does not need to perform acid treatment on the AAO film in advance in the experimental process, the use of toxic chemicals is reduced, and the activity of the antibody and the safety of operation are maximally reserved.

[0053] The construction method of the electrochemiluminescence sensor based on the antibody modified nanochannel particularly adopts an H-type electrolytic cell, utilizes the structural characteristics of the double electrolytic cell and the narrow exchange channel of the H-type electrolytic cell, and further combines the blocking effect of the AAO film, so that the light-emitting cell and the antigen-antibody reaction position can be separated from each other, the activity of the antigen-antibody and the structural stability of the antigen-antibody complex are ensured, and the light-emitting substance and the light-emitting electrode can be protected from biological pollution and the reduction of light-emitting efficiency.

[0054] The application further provides a use method of the construction method of the electrochemiluminescence sensor based on the antibody modified nanochannel in collagen fiber acid protein analysis, a working curve is made by taking the GFAP concentration ladder and the electrochemiluminescence light intensity, good linearity is obtained in the concentration range of 0.01-100 ng / mL, the electrochemiluminescence light intensity is obviously different under different concentrations, the sensitivity is high, in addition, the theoretical detection limit can reach 1 pg / mL, the traditional immunoelectrophoresis experiment and immunodiffusion experiment and the like are improved, and the qualitative or semi-quantitative deficiency is overcome, and the technical effect of completely quantitative protein detection analysis is realized.

[0055] The use method of the construction method of the electrochemiluminescence sensor based on the antibody modified nanochannel in collagen fiber acid protein analysis also performs whole-process I-V test monitoring on different piece antibodies modified nanochannels, the test result has high correlation and good repeatability, and rapid, high-sensitivity and high-accuracy quantitative detection of GFAP is comprehensively realized.

[0056] Other features and advantages of the present application will be set forth in the following description, and in part will be apparent from the description, or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings required to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, part of the drawings in the following description is some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0058] Figure 1 is an anodic aluminum oxide film nanochannel electron microscope scanning graph provided by the embodiment 1 of the present application;

[0059] Figure 2 is an I-V test graph for verifying the modification effect of the antibody modified nanochannel provided by the embodiment 2 of the present application;

[0060] Figure 3 is a feasibility analysis graph of the antibody modified nanochannel applied to GFAP analysis provided by the embodiment 3 of the present application;

[0061] Figure 4 is a working curve schematic graph of the antibody modified nanochannel applied to GFAP analysis provided by the embodiment 3 of the present application;

[0062] Figure 5 is a full-process I-V test graph of the antibody modified nanochannel applied to GFAP analysis provided by the embodiment 3 of the present application;

[0063] Figure 6 is a full-process I-V test graph of the antibody modified nanochannel applied to GFAP analysis provided by the embodiment 3 of the present application;

[0064] Figure 7 is an I-V test graph for comparing the modification effects of the antibody modified nanochannel and the in-situ modified nanochannel under the same modification time provided by the embodiment 2 and the comparative example 1 of the present application;

[0065] Figure 8 is a full-process I-V test graph of the in-situ modified nanochannel applied to GFAP analysis provided by the comparative example 1 of the present application. DETAILED DESCRIPTION

[0066] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some but not all of the embodiments of the present application; as long as there is no conflict, the technical features in the different embodiments of the present application can be combined with each other; based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0067] Embodiment 1

[0068] The embodiment 1 of the present application provides an electrochemiluminescence sensor based on antibody modified nanochannel, comprising an anodic aluminum oxide film;

[0069] The anodic aluminum oxide film is provided with nanochannels penetrating through the surface thereof, and one side surface of the anodic aluminum oxide film is attached with a hydrogel, the inside of the hydrogel is modified with an amino modification solution, and then the antibody is coupled with an antibody coupling solution;

[0070] The amino modification solution comprises 3-aminopropyltriethoxysilane;

[0071] The antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0072] In specific use, it further comprises a luminescence system, an electrolyte solution, an H-type electrolytic cell, a three-electrode system and an electrochemical workstation;

[0073] The luminescence system comprises phenanthroline ruthenium and tri-n-propylamine;

[0074] The electrolyte solution comprises 1× phosphate buffer and 0.1 mmol / L potassium chloride solution;

[0075] The three-electrode system comprises a working electrode glassy carbon electrode, a reference electrode silver chloride electrode and a counter electrode platinum electrode;

[0076] In specific operation, the 1× phosphate buffer is prepared according to the following steps: 8 g of NaCl, 0.2 g of KCl, 1.44 g of NaH2PO4 and 0.245 g of K2HPO4 are weighed, and then dissolved in 800 mL of distilled water, and the pH of the mixed solution is adjusted to 7.4 to obtain the 1× phosphate buffer.

[0077] In specific use, reference is made to FIG. 1, Figure 1 As shown in the figure, the nanochannel is an anodic aluminum nanochannel, the pore size is 10-400 nm, and the nanochannels are arranged in an array and penetrate through the anodic aluminum oxide film, and the hole distance is 100-200 nm.

[0078] In particular, the hydrogel is a 1% agarose gel solution prepared with physiological saline as solvent.

[0079] The thickness of the hydrogel attached to the anodic aluminum oxide film is 1-2 mm.

[0080] In particular, 10 mg of agarose powder is weighed in a glass container, 1 mL of 9% physiological saline is added, and the agarose powder is completely dissolved by heating to 100°C. The solution is cooled to 55-65°C and maintained in a liquid state.

[0081] In particular, the amino-modified solution is a 5%-10% 3-aminopropyltriethoxysilane solution prepared with anhydrous ethanol as solvent.

[0082] In particular, the antibody coupling solution is prepared by the following steps:

[0083] The antibody is dissolved in 1× phosphate buffer to prepare an antibody solution with a concentration of 20-30 μg / mL;

[0084] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide is dissolved in 1× phosphate buffer to prepare a cross-linking agent solution with a concentration of 5-8 mmol / L;

[0085] The antibody solution and the cross-linking agent solution are mixed to obtain the antibody coupling solution.

[0086] In this embodiment, preferably, the nanochannel aperture is 90 nm, and the array is arranged through the AAO film with a hole distance of 150 nm.

[0087] In this embodiment, preferably, the 1% agarose gel solution has a concentration maintained in a liquid state at 57°C.

[0088] In this embodiment, preferably, the 3-aminopropyltriethoxysilane (APTES) solution has a concentration of 5%.

[0089] In this embodiment, preferably, the antibody solution has a concentration of 20 μg / mL.

[0090] In this embodiment, preferably, the cross-linking agent solution has a concentration of 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.

[0091] In the embodiment, the attachment of the hydrogel to one side surface of the AAO film provides mechanical support for the AAO film, and solves the technical problem of easy fragmentation of the AAO film during use; in addition, since the amino group and the antibody modifier are both modified inside the hydrogel and do not directly contact the AAO film, the problem of blocking the nanochannel of the AAO film is avoided.

[0092] Embodiment 2

[0093] Based on the embodiment 1, the embodiment 2 of the application provides a construction method of an electrochemiluminescence sensor based on an antibody modified nanochannel, comprising the following steps:

[0094] S100, immersing the anodic aluminum oxide film with a nanochannel in anhydrous ethanol for ultrasonic treatment for 5-8 min, then immersing in ultrapure water for ultrasonic treatment for 5-8 min;

[0095] S101, taking 10-20 μL of the hydrogel and dropping it on one side surface of the anodic aluminum oxide film, and then standing for 10 s at room temperature to form a gel, to obtain a hydrogel nanochannel;

[0096] S102, immersing the hydrogel nanochannel in the amino modification solution for 2-3 h, then immersing in anhydrous ethanol for 5-8 min, and then immersing in ultrapure water for 5-8 min, to obtain an amino modified nanochannel;

[0097] S103, taking 5-10 μL of the antibody coupling solution and dropping it on the hydrogel surface of the amino modified nanochannel, and then incubating at 37℃ for 2-3 h, and then immersing in ultrapure water for 5-8 min, to obtain an antibody modified nanochannel;

[0098] S104, sandwiching the antibody modified nanochannel in 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;

[0099] In the amino modification solution, the APTES is connected to the agarose gel inside by a covalent bond to modify the amino group inside the agarose gel, and further, the antibody is coupled with the amino group by means of the EDC / NHS in the crosslinking agent solution, so as to modify the antibody inside the hydrogel and indirectly modify the nanochannel of the AAO film.

[0100] In the specific operation, a modification effect verification step is further included:

[0101] Said antibody modified nanochannel is sandwiched in the central circular channel of the H-type electrolytic cell;

[0102] Put one of the silver chloride electrodes inside the central circular channel of the H-shaped electrolytic cell, and put another one of the silver chloride electrodes in the electrolytic chamber of the H-shaped electrolytic cell, so that the antibody-modified nanochannel is between the two silver chloride electrodes;

[0103] Use the electrochemical workstation to perform I-V testing on the antibody-modified nanochannel equipped with the 0.1 mmol / L potassium chloride solution as the electrolyte solution.

[0104] Specifically, when performing the modification effect verification test, use the CV mode of the electrochemical workstation, the scanning voltage is-1~1 V, and the scanning rate is 0.05 V / s. Specifically, the I-V test results of the AAO membrane nanochannel, the hydrogel nanochannel, and the antibody-modified nanochannel are as shown in the following table. Figure 2 As shown in the table, compared with the AAO membrane nanochannel, the current change of the hydrogel nanochannel and the antibody-modified nanochannel is about 3 μA, which is significantly different from the AAO membrane nanochannel, and the modification effect is good.

[0105] Specifically, when in use, the antibody-modified nanochannel is boiled in water to remove the modification, and an anodic aluminum oxide membrane with clean nanochannels is obtained.

[0106] Specifically, when in operation, the agarose gel attached to the surface of the AAO membrane will be converted from a solid state to a liquid state at 100℃, and dissolved in water. Since each modification step is located inside the agarose gel and does not directly act on the AAO membrane, the agarose gel can be removed to remove all modification, and an AAO membrane with clean nanochannels is obtained.

[0107] In this embodiment, preferably, the ultrasonic time is 5 min.

[0108] In this embodiment, preferably, the amount of the hydrogel is 10 μL.

[0109] 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 clean the unmodified APTES, and then immersed in ultrapure water for 5 min to clean the residual ethanol.

[0110] In this embodiment, preferably, the amount of the antibody coupling solution is 5 μL, which is added to the surface of the amino-modified nanochannel of the hydrogel, and incubated at 37℃ for 2 h for antibody modification, and then immersed in ultrapure water for 5 min to remove the fixed antibody.

[0111] In the embodiment, the advantages of rapid gelation and good mechanical strength of agarose gel are utilized to quickly adhere to the surface of AAO membrane. Further, the modification of amino group by APTES covalent bond and the coupling of amino group with antibody by EDC / NHS are utilized to realize the indirect modification of antibody on AAO membrane to obtain antibody modified nanochannel. The antibody modified nanochannel is sandwiched in the H-type electrolytic cell, and the three-electrode system and electrochemical workstation are used to construct the electrochemiluminescence sensor based on the antibody modified nanochannel. In addition, the modification effect I-V scanning verification test is specially performed on the antibody modified nanochannel. The test results show that the nanochannel modified by hydrogel and antibody is significantly different from the unmodified AAO membrane nanochannel, and the modification effect is good. The embodiment retains the advantages of high temporal and spatial resolution and sensitivity of AAO membrane nanochannel, and effectively solves the problems of easy fragmentation of AAO membrane and easy blockage of nanochannel.

[0112] In the embodiment, by placing the antibody modified nanochannel in water and boiling, the characteristics of agarose gel that will be converted from solid state to liquid state and dissolved in water at 100℃ are utilized to remove all the modifiers modified in the agarose gel and obtain AAO membrane with clean nanochannel, so that the AAO membrane can be used multiple times without limitation.

[0113] Embodiment 3

[0114] Based on the embodiments 1 and 2, the embodiment 3 of the present application provides a use method of the electrochemiluminescence sensor construction method based on antibody modified nanochannel in collagen fiber acid protein analysis, which comprises the following steps:

[0115] 1× phosphate buffer solution is used as a solvent to configure collagen fiber acid protein solutions with different concentrations.

[0116] The collagen fiber acid protein solution with the lowest concentration is added dropwise on the surface of the hydrogel of the antibody modified nanochannel, and is placed at room temperature for 1 h.

[0117] The antibody modified nanochannel with the added collagen fiber acid protein solution is washed with ultrapure water.

[0118] The antibody modified nanochannel with the added collagen fiber acid protein solution is sandwiched in the central circular channel of the H-type electrolytic cell, and the side with the attached hydrogel faces the right chamber of the H-type electrolytic cell.

[0119] 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.

[0120] 1× phosphate buffer solution is used as an electrolyte solution, and the luminescence system is added to the left chamber of the H-type electrolytic cell.

[0121] Perform electrochemiluminescence test by using the electrochemical workstation to capture electrochemiluminescence signal intensity;

[0122] After the test, the antibody-modified nanochannel is taken out, and the next concentration step of the collagen fiber acid protein solution is added to the hydrogel surface of the antibody-modified nanochannel. After standing at room temperature for 1 hour, ultrapure water is used for cleaning.

[0123] The antibody-modified nanochannel is clamped in the central circular channel of the H-type electrolytic cell, and the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell.

[0124] Perform electrochemiluminescence test by using the electrochemical workstation to capture electrochemiluminescence signal intensity;

[0125] Repeat the operation to test the degree of change in electrochemiluminescence signal under different concentrations of collagen fiber acid protein solution, and plot the working curve of the degree of change in electrochemiluminescence signal and the concentration of collagen fiber acid protein solution.

[0126] The antibody-modified nanochannel incubated with the collagen fiber acid protein solution to be tested is clamped in the central circular channel of the H-type electrolytic cell, and the side with the hydrogel attached faces the right chamber of the H-type electrolytic cell.

[0127] Capture the electrochemiluminescence light intensity of the collagen fiber acid protein solution to be tested by using the electrochemical workstation, and calculate the concentration of the collagen fiber acid protein solution according to the working curve.

[0128] In this embodiment, the preferred luminescence system is 10 μL of 99% tri-n-propylamine solution and 2.5 μmol / L of phenanthroline ruthenium solution.

[0129] In this embodiment, specifically, the collagen fiber acid protein (GFAP) antibody has specific recognition and binding effect on the GFAP antigen. When the two form an antigen-antibody complex, it will cause changes in the charge density and electroosmotic flow effect inside the hydrogel nanochannel, leading to changes in ionic current and ion concentration, further affecting the luminescence intensity, thereby realizing the detection of GFAP.

[0130] In this embodiment, specifically, before applying the antibody-modified nanochannel-based electrochemiluminescence sensor construction method to collagen fiber acid protein (GFAP) analysis, a feasibility analysis is performed, and the feasibility analysis results are referred to Figure 3As shown, the electrochemiluminescence light intensity of the GFAP solution at the concentration of 100 pg / mL and 1 ng / mL is significantly different from the blank control group, and there is also a significant difference between the 100 pg / mL GFAP solution group and the 1 ng / mL GFAP solution group, which is sufficient for capture and distinction.

[0131] In this embodiment, when the test is specifically performed, the scanning voltage is 0.4-1.6 V, and the scanning rate is 0.1 V / s.

[0132] The collagen fiber acidic protein (GFAP) solution has a concentration of 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL and 100 ng / mL, and the working curve is prepared Figure 4 As shown, it 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.

[0133] In this embodiment, further, the antibody-modified nanochannel applied to the GFAP analysis is further subjected to whole-process I-V test monitoring, the scanning voltage is-1-1 V, and the scanning rate is 0.05 V / s, which is consistent with embodiment 2, and the test result is referenced Figure 5 As shown, the current change amount after binding the antigen has a change amplitude of about 0.5 μA, which is obviously distinguished from the current change amount of the antibody-modified nanochannel without binding the antigen.

[0134] In this embodiment, further, the different pieces of AAO film nanochannels applied to the GFAP analysis in different modification or use stages are subjected to I-V test monitoring, the scanning voltage is-1-1 V, and the scanning rate is 0.05 V / s, which is consistent with embodiment 2. The linearity of the test result is referenced Figure 6 As shown, the current data in the test result is taken as a comparison object to perform correlation analysis, and the analysis result is referenced in table 1.

[0135] Table 1: I-V test correlation analysis of different pieces of nanochannels applied to different stages of GFAP analysis

[0136]

[0137] It can be known that, under the same process stage, for example, the antibody modification stage, the correlation between different pieces of the antibody-modified nanochannels is good, and the correlation result is consistent within 10 -4 orders of magnitude, and approaches to 1, that is, the limit approaches to complete correlation. The linear analysis graph is as follows: Figure 6The linear coincidence rate of each group is high, and it can be seen that the antibody-modified nanochannel-based electrochemiluminescence sensor construction method provided by the application has good repeatability and small inter-piece difference in each stage of GFAP analysis.

[0138] In the embodiment, the antibody-modified nanochannel-based electrochemiluminescence sensor construction method is used in GFAP analysis, and the high temporal and spatial resolution of the AAO membrane nanochannel, the high sensitivity, the high mechanical strength of the hydrogel, and the good molecular carrier performance are combined, which not only solves the problems of AAO nanochannel blockage and AAO membrane fragmentation when the antibody-modified AAO membrane nanochannel is used, but also optimizes the disadvantages of inaccurate quantification, time-consuming and complex operation, and large fluctuation of repeatability in the traditional detection method, and realizes the technical effect of sensitive, rapid and easy operation of GFAP analysis and detection.

[0139] Comparative Example 1

[0140] Comparative Example 1 provides an application of a nanochannel in-situ modification method in GFAP analysis. Except that the nanochannel modification method is different, the 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:

[0141] S200, immerse the AAO membrane nanochannel in anhydrous ethanol, ultrasonic for 10 min, then immerse in ultrapure water, ultrasonic for 10 min, and then dry in a nitrogen atmosphere;

[0142] S201, immerse the AAO membrane nanochannel in a 5% HCl solution for 1 min, then rinse the HCl solution on the surface of the AAO membrane nanochannel with ultrapure water, then immerse in ultrapure water for 5 min, and then dry in a nitrogen atmosphere;

[0143] S202, immerse the AAO membrane nanochannel in a 5% APTES solution prepared with anhydrous ethanol as a solvent, and incubate in the dark for 12 h;

[0144] S203, rinse the AAO membrane nanochannel incubated in S202 with anhydrous ethanol, immerse in anhydrous ethanol for 1 h, and then dry in an oven at 120°C for 2 h;

[0145] S204, drop 5 μL of GFAP antibody solution containing 5 mmol / L EDC / NHS onto the surface of the AAO membrane nanochannel, and after standing for 2 h, rinse the residual antibody with ultrapure water, to obtain the in-situ modified nanochannel.

[0146] In the present comparative example, the in-situ modified nanochannel is tested by I-V, the scanning voltage is-1~1 V, the scanning rate is 0.05 V / s, and the test results are shown in the following table Figure 7 As shown in the table, after modification for 2 h, the current change of the AAO membrane nanochannel modified in-situ is only about 0.1 μA, while the current change of the AAO membrane nanochannel provided by the embodiment 2 of the present application and having a hydrogel modification layer is about 2 μA after modification for 2 h, and the modification efficiency is significantly higher than that of the in-situ modification method provided by the comparative example 1, which can greatly shorten the modification time.

[0147] In the present comparative example, further, the in-situ modified nanochannel applied in GFAP analysis is tested by I-V, the scanning voltage is-1~1 V, the scanning rate is 0.05 V / s, and the test results are shown in the following table Figure 8 As shown in the table, the current change of the in-situ modified nanochannel combined with the antigen is not more than 0.05 μA, which cannot be significantly distinguished from the current change without antigen, and the reaction to the change of antigen concentration is not sensitive.

[0148] In the present comparative example, the in-situ modified nanochannel is tested by I-V, the scanning voltage is-1~1 V, the scanning rate is 0.05 V / s, and the test results are shown in the following table

[0149] 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 application can only be improved in one or several aspects, and it is not necessary to solve all the technical problems listed in the prior art or background art at the same time. Those skilled in the art should understand that what is not mentioned in a claim should not be regarded as a limitation to the claim.

[0150] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An electrochemiluminescence sensor based on antibody modified nanochannel, comprising an anodic aluminum oxide film, characterized in that: the anodic aluminum oxide film is provided with nanochannels penetrating through its surface, and one side surface of the anodic aluminum oxide film is attached with a hydrogel, the interior of the hydrogel is modified with an amino modification solution, and then an antibody is coupled with an antibody coupling solution; the amino modification solution comprises 3-aminopropyltriethoxysilane; the antibody coupling solution comprises an antibody, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; further comprising a luminescence system, an electrolyte solution, an H-type electrolytic cell, a three-electrode system and an electrochemical workstation; the luminescence system comprises phenanthroline ruthenium and tri-n-propylamine; the electrolyte solution comprises 1× phosphate buffer and 0.1 mmol / L potassium chloride solution; the three-electrode system comprises a working electrode glassy carbon electrode, a reference electrode silver chloride electrode and a counter electrode platinum electrode; the nanochannel is an anodic aluminum nanochannel with a pore size of 10-400 nm, which is arranged in an array and penetrates through the anodic aluminum oxide film, and the pore distance is 100-200 nm; the hydrogel is a 1% agarose gel solution prepared with physiological saline as a solvent; and the thickness of the hydrogel attached to the anodic aluminum oxide film is 1-2 mm; the amino modification solution is a 5%-10% 3-aminopropyltriethoxysilane solution prepared with anhydrous ethanol as a solvent; the antibody coupling solution is prepared by the following steps: dissolving an antibody with 1× phosphate buffer as a solvent to obtain an antibody solution with a concentration of 20-30 μg / mL; dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide with 1× phosphate buffer as a solvent to obtain a crosslinking agent solution with a concentration of 5-8 mmol / L; and mixing the antibody solution and the crosslinking agent solution to obtain the antibody coupling solution; and comprising the following steps: S100, immersing the anodic aluminum oxide film with nanochannels in anhydrous ethanol for ultrasonic treatment for 5-8 min, then immersing in ultrapure water for ultrasonic treatment for 5-8 min; S101, taking 10-20 μL of the hydrogel, dropping on one side surface of the anodic aluminum oxide film, and standing at room temperature for 10 s to form a gel, obtaining a hydrogel nanochannel; S102, immersing the hydrogel nanochannel in the amino modification solution for 2-3 h, then immersing in anhydrous ethanol for 5-8 min, and then immersing in ultrapure water for 5-8 min, obtaining an amino modified nanochannel; S103, taking 5-10 μL of the antibody coupling solution, dropping on the surface of the hydrogel of the amino modified nanochannel, incubating at 37°C for 2-3 h, and then immersing in ultrapure water for 5-8 min, obtaining an antibody modified nanochannel; S104, sandwiching the antibody modified nanochannel in the central circular channel of the H-type electrolytic cell, placing the three-electrode system in the electrolysis chamber of the H-type electrolytic cell, and adding the electrolyte solution; and further comprising a modification effect verification step. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 2. The antibody-modified nanochannel-based electrochemiluminescence sensor of claim 1, wherein: ​ 3. The antibody-modified nanochannel-based electrochemiluminescence sensor of claim 1, wherein, ​ ​ ​ ​ 4. A method for constructing an antibody-modified nanochannel-based electrochemiluminescent sensor according to any one of claims 1 to 3, characterized in that, ​ ​ ​ ​ ​ ​ 5. The method of constructing an antibody-modified nanochannel-based electrochemiluminescent sensor according to claim 4, wherein, ​ Put one of the silver chloride electrodes in the central circular channel of the H-shaped electrolytic cell, and put another one of the silver chloride electrodes in the electrolytic chamber of the H-shaped electrolytic cell, so that the antibody-modified nanochannel is between the two silver chloride electrodes; Use the electrochemical workstation to perform I-V test on the antibody-modified nanochannel equipped with the 0.1 mmol / L potassium chloride solution as the electrolyte solution.

6. The method of constructing an antibody-modified nanochannel-based electrochemiluminescent sensor according to claim 4, wherein: Put the antibody-modified nanochannel in water and boil to remove the modification, thereby obtaining an anodic aluminum oxide membrane with clean nanochannels.

7. A method for using the antibody-modified nanochannel-based electrochemiluminescence sensor construction method according to claim 5 or 6 in collagen fiber acidic protein analysis, comprising the following steps: Prepare collagen fiber acidic protein solutions with different concentrations using 1× phosphate buffer as the solvent; Drop the collagen fiber acidic protein solution with the lowest concentration on the surface of the hydrogel of the antibody-modified nanochannel, and stand at room temperature for 1 h; Wash the antibody-modified nanochannel to which the collagen fiber acidic protein solution is added with ultrapure water; Put the antibody-modified nanochannel to which the collagen fiber acidic protein solution is added in the central circular channel of the H-shaped electrolytic cell, with the side with the attached hydrogel facing the right chamber of the H-shaped electrolytic cell; Put the glassy carbon electrode and the silver chloride electrode in the left chamber of the H-shaped electrolytic cell, and put the platinum electrode in the right chamber of the H-shaped electrolytic cell; Add the luminescence system to the left chamber of the H-shaped electrolytic cell using 1× phosphate buffer as the electrolyte solution; Use the electrochemical workstation to perform electrochemiluminescence test and capture the electrochemiluminescence signal intensity; Take out the antibody-modified nanochannel after the test, drop the collagen fiber acidic protein solution with the next concentration step on the surface of the hydrogel of the antibody-modified nanochannel, stand at room temperature for 1 h, and then wash with ultrapure water; Put the antibody-modified nanochannel in the central circular channel of the H-shaped electrolytic cell, with the side with the attached hydrogel facing the right chamber of the H-shaped electrolytic cell; Use the electrochemical workstation to perform electrochemiluminescence test and capture the electrochemiluminescence signal intensity; Repeat the operation to test the degree of change of the electrochemiluminescence signal under different concentrations of collagen fiber acidic protein solution, and plot the working curve of the degree of change of the electrochemiluminescence signal and the concentration of the collagen fiber acidic protein solution; Put the antibody-modified nanochannel incubated with the collagen fiber acidic protein solution with the to-be-tested concentration in the central circular channel of the H-shaped electrolytic cell, with the side with the attached hydrogel facing the right chamber of the H-shaped electrolytic cell; Use the electrochemical workstation to capture the electrochemiluminescence light intensity of the collagen fiber acidic protein solution with the to-be-tested concentration, and calculate the concentration of the collagen fiber acidic protein solution according to the working curve.

Images