CN115060772A - CA50 antigen electrochemical detection method based on functionalized nucleopore membrane - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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Abstract
The invention discloses a CA50 antigen electrochemical detection method based on a functionalized nuclear pore membrane, which is characterized in that an anti-CA 50 monoclonal antibody (C-50) which is coupled by copolymerization on the inner wall of a nuclear pore membrane pore channel is combined with a CA50 antigen, and the CA50 antigen is detected by detecting the change of micro-current passing through a nuclear pore membrane pore channel through an electrochemical workstation. The preparation method has the characteristics of mild reaction, easily controlled process, simple and convenient operation, low cost, high sensitivity, high speed, high specificity and simple and convenient operation. Has very important theoretical and practical significance for the diagnosis and monitoring of tumors and the identification of benign and malignant tumors.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane.
Background
Glycolipids or glycoproteins on the cell surface play an important role in the transmission, growth, and differentiation of information in cells. When cells are malignant, some invertases which tend to be stationary during maturation due to inactivation of glycosyltransferases or activity of certain embryonic stages are activated, thereby causing changes in carbohydrates on the cell surface. The CA (carbohydrate antigen) class of antigens are believed to be the result of this change, which appears on the cell as gangliosides or bound to glycoproteins, and the determination of these tumor bound antigens is a useful indicator for the diagnosis of malignant lesions. The monoclonal antibody prepared by the hybridoma technique is a specific immunological reagent, and has the capacity of recognizing tumor antigens. With the development of hybridoma and monoclonal technology, French scholars obtained monoclonal antibody C-50 from colon adenocarcinoma cell line Colo205 by the hybridoma technology and verified that it recognizes two different sugar chains-sialylated Lewis-alpha and sialylated tetralactose. The carbohydrate antigen CA50 is found in gastrointestinal tumors such as pancreatic cancer, gastric cancer, colon cancer and liver cancer, and uterine cancer. The CA50 antigen enters blood from tumor, and the immunoassay technology based on the anti-CA 50 monoclonal antibody can specifically determine the content of the CA50 in serum, and has great application value for the diagnosis and monitoring of various tumors and the identification of benign and malignant tumors.
The nuclear pore membrane is a track etching membrane which utilizes heavy ions accelerated by an accelerator or thermal neutrons in a reactor to enable 235U to be fissioned, fragments generated by fission are injected into the organic polymer plastic film through a collimator and penetrate through the organic polymer plastic film, and a narrow irradiation channel is reserved on a path through which particles pass. The tracks on the film can be etched into cylindrical or double-cone micropores by using a proper chemical reagent, and the nuclear pore membranes with different pore diameters and different pore densities can be obtained by controlling the irradiation condition and the etching condition.
The nuclear pore membrane with pore channels is rapidly developed due to the characteristics of mechanical and chemical stability, controllable channel shape and chemical modification of the channel surface, and the characteristics enable the nuclear pore membrane to be widely used for constructing high-performance biosensors. The nature of biosensing is to convert molecular recognition into a measurable current. The solid channel is modified by various methods, such as solution chemical covalent modification, electrostatic self-assembly, plasma modification and the like to obtain different functionalized channels, when the functional probe is combined with a target, the effective aperture, the inner surface charge and/or the inner wall wettability of the channel can be changed, so that the change of ion current is caused, and the change of the ion current before and after the action is monitored by electrochemical detection equipment. And the iron terpyridine complex is covalently fixed on the inner wall of the PET nano channel by the aid of the Ali and the like, and is used for detecting lactoferrin, and the detection sensitivity can reach 1 pM. Li and the like fix the 4-carboxyphenylboronic acid on the inner wall of the nano-channel, and when glucose exists, the ion transmission of the nano-channel is switched between a non-rectification state and a rectification state, so that the response to the glucose is realized. The current detection method commonly used for detecting CA50 is chemiluminescence. The method has the advantages that the detection signal value is attenuated quickly, and the attenuation speeds of high-concentration and low-concentration signals are inconsistent, so that the low-end detection sensitivity, stability and repeatability of the method in the immune quantitative analysis are limited. Therefore, the development of a detection method with high detection speed, high sensitivity, simple operation and low cost has very important theoretical and practical significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane.
The technical scheme of the invention is summarized as follows:
a CA50 antigen electrochemical detection method based on a functionalized nuclear pore membrane is characterized in that an anti-CA 50 antibody is coupled on the inner wall of a pore channel of the nuclear pore membrane in a copolymerization mode.
Preferably, the thickness of the nuclear pore membrane is 0.1-100 mu m, the pore diameter of the pore canal of the nuclear pore membrane is 0.01-10 mu m, and the distribution density of the pore canal of the nuclear pore membrane is 5 multiplied by 10 4 ~2×10 12 Per cm 2 。
Preferably, the material of the nuclear pore membrane is polyethylene terephthalate, polycarbonate or polyimide.
A CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane, wherein the preparation process of the functionalized nucleopore membrane comprises the following steps: soaking a nuclear pore membrane in a mixed solution containing EDC and NHS, reacting for 1-2 h to activate carboxyl on the inner surface of a pore passage of the nuclear pore membrane into a reactive amine ester molecule, taking out, soaking in 1-15 mu M CA50 antibody aqueous solution, incubating and reacting for 1-2 h at room temperature, taking out, washing for 2-3 times with 0.1-0.3M MES buffer solution, and airing to obtain a functional nuclear pore membrane for CA-50 antigen detection;
EDC concentration in the mixed solution is 10-15mg/ml, NHS concentration is 1-5mg/ml, and solvent is 0.1-0.3M MES buffer solution;
EDC is an abbreviation for N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride;
the NHS is an abbreviation for N-hydroxysulfosuccinimide.
A CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane comprises the step of incubating the functionalized nucleopore membrane in 0.1-1 nM CA50 antigen water solution for 2 h.
A CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane comprises the following steps: and (3) placing the functionalized nuclear pore membrane between the electrolytic cell clamps, injecting electrolyte with a certain concentration into the electrolytic cell, inserting electrodes into two ends of the electrolytic cell, applying pulse voltage by using an electrochemical workstation, and detecting the micro-current change of the nuclear pore membrane after capturing the CA50 antigen.
Preferably, the electrolyte is a 0.5 x PBS phosphate buffer standard solution.
Preferably, the electrode is an Ag/AgCl electrode.
Preferably, the pulse voltage applied by the electrochemical workstation is-2V, and each pulse voltage lasts for 5 s.
The invention has the advantages that:
the preparation method has the advantages of mild reaction, easily controlled process, simple and convenient operation, high covalent grafting rate and low cost. The detection of the CA50 antigen is realized by detecting the change of micro-current passing through the nuclear pore membrane pore channel through an electrochemical workstation, has the characteristics of high sensitivity, rapidness, high specificity and simple and convenient operation, and has very important theoretical and practical significance for the diagnosis and monitoring of tumors and the identification of benign and malignant tumors.
Drawings
FIG. 1 is a schematic view of an electrochemical detection apparatus used in the present invention.
FIG. 2 is a micro-current curve of electrochemical detection of functionalized nucleopore membrane after incubation with different concentrations of CA50 antigen.
Detailed Description
The nucleopore membranes used in the present invention are commercially available. EDC is an abbreviation for N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride; NHS is an abbreviation for N-hydroxysulfosuccinimide.
The present invention will be further described with reference to the following specific examples.
Example 1
A CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane, the preparation process of the functionalized nucleopore membrane comprises the following steps: soaking a polyethylene terephthalate (PET) nuclear pore membrane in a mixed solution containing EDC and NHS, reacting for 1h to activate carboxyl groups (-COOH) on the inner surface of a nuclear pore membrane pore passage into reactive amine ester molecules, taking out, soaking in 10 mu M CA50 antibody aqueous solution, incubating at room temperature for 2h, taking out, washing for 2 times by using 0.1M MES buffer solution, and airing to obtain a functional nuclear pore membrane;
EDC concentration in the mixed solution is 10mg/ml, NHS concentration is 1mg/ml, and the solvent is 0.1M MES buffer solution;
the thickness of the nuclear pore membrane is 15 μm, the pore diameter of the nuclear pore membrane pore canal is 2 μm, and the distribution density of the nuclear pore membrane pore canal is 2 multiplied by 10 11 Per cm 2 。
The nuclear pore membrane is made of polyethylene terephthalate (PET).
The micro-current detection method comprises the following steps:
(1) the functionalized nucleopore membranes prepared in example 1 are respectively immersed in CA50 antigen aqueous solutions with the concentrations of 0.1, 0.5 and 1nM for incubation for 2h, and then taken out:
(2) and (2) putting the functionalized nucleopore membrane obtained in the step (1) between electrolytic cell clamps of an electrochemical detection device (shown in figure 1), injecting 0.5 multiplied by PBS phosphate buffer solution into the electrolytic cell to serve as electrolyte, inserting Ag/AgCl electrodes at two ends of the electrolytic cell, applying pulse voltage to-2V by using an electrochemical workstation, and detecting the micro-current change of the nucleopore membrane after capturing CA50 antigen. The duration of the pulse voltage was 5 seconds and the corresponding microcurrent values were automatically recorded by the electrochemical workstation on the connected computer (see figure 2).
Comparative example 1
The comparative example is the functionalized nucleopore membrane prepared in example 1 without being immersed in the aqueous CA50 antigen solution, step (2) is the same as step (2) of the microcurrent detection method (see fig. 2).
From the microcurrent curves of example 1 and comparative example 1, it can be seen that when the functionalized nucleopore membrane captures the CA50 antigen, the absolute value of the current passing through the pore channels of the nucleopore membrane is reduced under the same applied voltage, and the absolute value of the generated current is smaller when the nucleopore membrane is incubated with the CA50 antigen aqueous solution with higher concentration. In fig. 2 it can be produced that the difference between the starting current value (-current at 2V) and the ending current value (current at 2V) of the three curves in example 1 is significantly reduced compared to the curves of comparative example 1. By utilizing the characteristic, whether the solution incubated with the functionalized nucleopore membrane contains the CA50 antigen or not and the concentration range of the CA50 antigen can be judged.
Claims (9)
1. A CA50 antigen electrochemical detection method based on a functionalized nucleopore membrane is characterized in that the inner wall of a nucleopore membrane pore channel is coupled with an anti-CA 50 antibody in a copolymerization mode, and after the antibody is combined with the antigen, the electrochemical workstation detects the change of micro-current passing through the nucleopore membrane pore channel to realize the detection of the antigen.
2. The functionalized nucleopore membrane-based electrochemical detection method for the CA50 antigen according to claim 1, wherein the nucleopore membrane has a thickness of 0.1-100 μm, the pore diameter of the nucleopore membrane pore channel is 0.01-10 μm, and the distribution density of the nucleopore membrane pore channel is 5 x 10 4 ~2×10 12 Per cm 2 。
3. The method for electrochemical detection of the CA50 antigen based on the functionalized nucleopore membrane of claim 1, wherein the nucleopore membrane is made of polyethylene terephthalate, polycarbonate or polyimide.
4. The method for the electrochemical detection of the CA50 antigen based on the functionalized nucleopore membrane as claimed in claim 1, wherein the preparation process of the functionalized nucleopore membrane comprises the following steps: soaking a nuclear pore membrane in a mixed solution containing EDC and NHS, reacting for 1-2 h to activate carboxyl on the inner surface of a pore passage of the nuclear pore membrane into a reactive amine ester molecule, taking out, soaking in 1-15 mu M anti-CA 50 antibody aqueous solution, incubating and reacting for 1-2 h at room temperature, taking out, washing for 2-3 times with 0.1-0.3M MES buffer solution, and airing to obtain a functional nuclear pore membrane for CA50 antigen detection;
EDC concentration in the mixed solution is 10-15mg/ml, NHS concentration is 1-5mg/ml, and the solvent is 0.1-0.3M MES buffer solution;
EDC is an abbreviation for N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride;
the NHS is an abbreviation for N-hydroxysulfosuccinimide.
5. The method for the electrochemical detection of the CA50 antigen based on the functionalized nucleopore membrane of claim 1, wherein the functionalized nucleopore membrane is incubated for 2h in an aqueous solution of the CA50 antigen with a concentration of 0.1-1 nM.
6. The electrochemical detection method of the CA50 antigen based on the functionalized nucleopore membrane as claimed in claim 1, characterized in that the electrochemical detection method comprises the following steps: and (3) placing the functionalized nuclear pore membrane between the electrolytic cell clamps, injecting electrolyte with a certain concentration into the electrolytic cell, inserting electrodes into two ends of the electrolytic cell, applying pulse voltage by using an electrochemical workstation, and detecting the micro-current change of the nuclear pore membrane after capturing the CA50 antigen.
7. The electrochemical detection method of claim 6, wherein the electrolyte is a 0.5 x PBS phosphate buffer standard solution.
8. The electrochemical detection method according to claim 6, wherein the electrode is an Ag/AgCl electrode.
9. The electrochemical detection method according to claim 6, wherein the electrochemical workstation applies a pulse voltage of-2 to 2V for 5s per pulse voltage.
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