CN102268119B - Preparation method of molecular imprinted polymer for detecting lung cancer tumor markers - Google Patents
Preparation method of molecular imprinted polymer for detecting lung cancer tumor markers Download PDFInfo
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Abstract
The invention discloses a preparation method of a molecular imprinted polymer for detecting lung cancer tumor markers. The method comprises the following basic process of: dissolving template molecules (ferrocenecarboxylic acid-tumor marker), a functional monomer and a cross-linking agent in an organic solvent (pore-foaming agent); then transferring the solution into water, stirring and emulsifying; adding an initiator for cross-linking, performing ultraviolet polymerization to obtain spherical molecular imprinted substances with relatively uniform particle sizes; and washing by ethanol, and centrifuging to remove the template molecules to obtain the molecular imprinted polymer with specificity to the lung cancer tumor markers. The molecular imprinted polymer is also used as an identifying element of a biosensor. The invention provides a new tumor diagnosis method.
Description
Technical field
The present invention relates to materials chemistry and electrochemical field, be specifically related to a kind of preparation method of the molecularly imprinted polymer for detection of the lung cancer tumor markers.
Background technology
The generation of tumour, propagation are processes that develops gradually, in this progressive formation, by the malignant cell abnormal secretion or come off, or occur and breed closely-related material to be called tumor markers (Tumor marker with tumour in the entered blood that is produced because of tumor response by body or the body fluid, TM), rough segmentation is the six large classes such as carcinomebryonic antigen class, enzyme, hormones, glycoprotein analog, Oncogene and cell surface tumour antigen class.For dynamic surveillance and the mensuration of this class material, the evaluation that can be auxiliary diagnosis, differential diagnosis, observation of curative effect, state of illness monitoring and the prognosis of tumour provides reliable foundation.The immunologic detection method of present clinical blood serum tumor markers mainly contains immune shooting method, euzymelinked immunosorbent assay (ELISA), chemiluminescence immunoassay method, immunofluorescence technique, time-resolved fluoroimmunoassay of radioimmunology, traget antibody etc., the advantages such as that although these methods have is highly sensitive, high specificity, but still can't avoid radiocontamination, consuming time, can't be in shortcomings such as community hospital popularize.
Biosensor is comprised of recognition component and signal converter, recognition component is fixed on the surface of transmodulator by rights, when testing molecule when recognition component is combined, produce a physics or chemical signal, transmodulator with this signal convert to one can be quantitative output signal realize the real time measure to testing molecule by monitoring output signal.The biomolecules that consists of biosensor has enzyme, antibody, microorganism, tissue even complete organ; Transmodulator has electrode, field-effect transistor, optical fiber, thermistor and piezoquartz etc.Biosensor is because its highly sensitive and specificity are subject to extensive concern.Immunosensor is to be coupled the bio-sensitive film that contains the antigen/antibody molecule and a kind of Novel Biosensor of signal converter, have highly sensitive, high specific, easy to use, be suitable for the advantage popularized in community hospital, become the effective ways that tumor markers detects.But the defective that biomolecules is intrinsic is namely had relatively high expectations to environment for use, is difficult to prolonged preservation etc., biosensor is run in actual applications much be difficult for the obstacle that overcomes.Simultaneously, biomolecules derives from living organisms, because preparation and purifying are loaded down with trivial details, expensive, therefore, it also is an important factor of restriction biosensor development that recognition component is difficult to.Obtaining cheap, stable recognition component, is one of key of further developing of biosensor.
By research and the understanding to antigen-antibody, enzyme and substrate reactions principle, scientist has courageously proposed the imagination by the synthetic analog antibody of chemical reaction, started a brand-new technology---molecular imprinting (molecular imprinting technique, MIT).By synthetic molecularly imprinted polymer (the molecularly imprinted polymers of MIT, MIPs) be called as " plastics antibody ", therefore scientist has realized not relying on the long-cherished wish that living organisms obtains " antibody ", MIPs compares with biological identification element (such as enzyme, antibody, nucleic acid etc.) has lot of advantages: the 1. good stability of MIP, and can Reusability more than 50 times; In the drying at room temperature environment, can preserve the several years, can not affect its character more than 4 weeks of preservation in the water; Can not reduce its evident characteristics during with acid, alkalescence, metal ion and other multiple solution-treated; Can tolerate certain physical strength, high temperature and high pressure; 2. MIP has very high selectivity, is " making to measure " for marking molecule, and any one molecule all can prepare corresponding MIP in theory; 3. biological identification element comes from biogenic, often will use animal in process of production, and molecular imprinting is the chemosynthesis process fully; 4. the biomolecules recognition system is difficult for setting up mass production processes, so expensive, and the MIP expense is low, and for some micromolecular compound such as medicine, it is quite simple and save time to prepare its MIP, sets up easily scale operation.Replace biomolecules as the study hotspot that becomes gradually sensor field after the nineties in 20th century that is operated in of recognition component development molecular imprinting Biomimic sensor (MIPs biosensor) with this " plastics antibody ".
The preparation of MIPs comprises 3 steps: (1) or/and non covalent bond makes template or microsphere (being testing molecule) and function monomer form the mixture of functional group and space structure complementation, used function monomer must be with the function base that can have an effect with microsphere by covalent linkage.Function monomer commonly used has vinylformic acid, methacrylic acid, trifluoromethyl acrylate and vinylbenzene etc.; (2) add linking agent, around microsphere-function monomer mixture, produce polyreaction, the form of functional group and space structure complementation is fixed in the polymkeric substance.Linking agent commonly used has: ethyleneglycol dimethyacrylate, pentaerythritol triacrylate and trimethoxy propane trimethyl acrylic ester etc.; (3) remove template from polymkeric substance, forming can specific recognition, in conjunction with the hole of template, and this polymkeric substance is MIPs.
MIPs meets with testing molecule in the similar solution when synthetic to it, by the cooperation of functional group and spatial form, and selectivity recombine testing molecule.The advantage that MIPs has makes it as recognition component, is particularly suitable for sensor technology.Electrochemical immunosensor combines electrochemical analysis technology and immunological technique, is a comparatively ripe para-immunity sensor of present most study, is widely used in the detection of tumor markers.Yet, replace natural antibody to realize accurately detecting in a lot of small molecules (VITAMIN with MIPs this " artificial antibody ", amino acid, aldehydes, coenzyme, sterols, medicine morphine etc.) succeed in the detection, but study fewerly to macromolecular molecular imprintings such as protein, the used substrate maximum of MIPs of so far scientist's research can only reach polypeptide rank (3kD is following), does not reach protein rank (more than the 20kD).How protein is prepared MIPs as marking molecule and become the key that realizes the MIPs-biosensor.
Summary of the invention
The objective of the invention is to overcome the problem of macromolecular substance trace difficulty, provide a kind of can selectivity and specific recognition lung cancer tumor markers, can be used for the molecularly imprinted polymer that the lung cancer tumor markers detects.For achieving the above object, the invention provides a kind of simply for detection of the preparation method of the molecularly imprinted polymer of lung cancer tumor markers.
The present invention is take the state of conflict current sensor as the basis.Because the selectivity of MIPs depends primarily on cooperating of its functional group and spatial form and substrate, there are some researches show, for biomacromolecule, molecularly imprinted polymer is not only because in the imprinted polymer binding site that can carry out with substrate exclusive reaction is arranged selectivity and the avidity of substrate, the what is more important function monomer has formed the structure of high-sequential at imprinted polymer, the structure of this high-sequential with ion or (with) hydrophobic interaction power plays a major role to selectivity and the affinity of imprinted polymer.So focusing on to make up, the present invention has on the MIPs of high-sequential structure.Some is far-reaching factor in the small molecules trace, such as polarity, specific inductivity, protonation and the complexing action etc. of solvent, in macromolecular trace, as the influence factor of next.
The present invention selects lung cancer tumor markers carcinomebryonic antigen (CEA), neuronspecific enolase (NSE), cytokeratin 19 fragment antigen (CYFRA21-1) to be template, adopt diffusion/dispersion copolymerization method, prepare the molecularly imprinted polymer of specific tumor markers.As microsphere, its MIPs is as the singularity of sensor recognition component according to macromole.
The preparation method of a kind of molecularly imprinted polymer for detection of the lung cancer tumor markers of the present invention, concrete scheme comprises the steps:
(1) Electrochemical Modification of template molecule;
Select electroactive substance formic acid ferrocene 3-5mg, be abbreviated as FER, be dissolved in 500-1000 μ L, 0.15M, pH are 7.3 hydroxyethyl piperazine second thiosulfonic acid damping fluid, solution is through 0.22 μ m filtering with microporous membrane, add 8-15mg 1-ethyl-3-[3-dimethylaminopropylamine] carbodiimide-hydrochloric acid and 80-120 μ L, the tumor markers of 500 μ g/mL, and under room temperature, hatched 3-5 hour, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate; Make template composite in 4 ℃ of preservations; Described tumor markers is that carcinomebryonic antigen is abbreviated as CEA, neuronspecific enolase is abbreviated as NSE or cytokeratin 19 fragment antigen is abbreviated as CYFRA21-1;
(2) preparation of molecularly imprinted polymer;
Be formic acid ferrocene-tumor markers with template molecule, function monomer, linking agent is dissolved in pore-creating agent, and solution is moved in the water, stirs emulsification; Then it is crosslinked to add initiator, uv photopolymerization, get particle diameter than the spherical molecular imprinting material of homogeneous, wherein the amount ratio of template, function monomer, linking agent, pore-creating agent, initiator can be selected mmol:mmol:mmol:mL:mg=1:(8-10): (40-50): (90-100): (3-40); By the centrifugal template molecule of removing of washing with alcohol; Described template is FER-CEA or FER-NSE or FER-CYFRA21-1; Described function monomer is dihydroxymethyl ethyl propenoate, methyl methacrylate, methylene-succinic acid, to Ethenylbenzene formic acid or diacrylamine-2-methyl isophthalic acid-propanesulfonic acid; Described linking agent is N, N-two acryloyl piperazines, N, N '-methylene diacrylamine, 3,5-dibenzoic acid or 3,5-diacrylamine phenylformic acid; Described pore-creating agent is tetrahydrofuran (THF), methyl alcohol, ethanol or propyl alcohol; Described initiator is that Diisopropyl azodicarboxylate is abbreviated as AIBN or 2,2'-Azobis(2,4-dimethylvaleronitrile) is abbreviated as AIHN.
The concise and to the point process of the preparation of molecularly imprinted polymer such as Fig. 1.
Wherein in the Electrochemical Modification part of template molecule:
Biomacromolecule itself does not have electroactive, can not conduction current, therefore, before preparation MIPs, the present invention utilizes electric active matter confrontation template molecule to modify, and the Electrochemical Modification of existing biomolecules adopts ferrocene more, thionine, chitosans etc. adopt the formic acid ferrocene as electroactive substance as electroactive substance in this experiment, it possesses electroactive wetting ability with being applicable to this experimental situation simultaneously.Electroactive substance after the modification-template molecule mixture is re-used as template molecule and prepares its MIPs.
Wherein in the preparation part of molecularly imprinted polymer:
Because template molecule is biomacromolecule, adopt diffusion/dispersion copolymerization method, utilize at normal temperatures the UV-irradiation polymerization.The method generally is used for the preparation of the micromolecular MIPs of thermolability, technology comparative maturity in the preparation of micromolecular MIPs, primary process is: with template molecule (formic acid ferrocene-tumor markers), function monomer, linking agent is dissolved in organic solvent (pore-creating agent), then solution is moved in the water, stir emulsification.Then it is crosslinked to add initiator, and uv photopolymerization gets particle diameter than the spherical molecular imprinting material of homogeneous.Then by the centrifugal template molecule of removing of washing with alcohol, concise and to the point process as shown in Figure 1.
Improving velocity of diffusion, shorten the time of response, mainly is by reducing dosage of crosslinking agent, increasing the pore-creating agent consumption to increase the space between polymer chain.Protein is the template molecule with definite shape, so the consumption of linking agent will hang down a bit in right amount.The consumption of template molecule can not surpass 5%.In the method, key factor is the ratio of masterplate molecule, function monomer, the ratio of linking agent, pore-creating agent, ultraviolet lighting time three factors, the amount of other solvents is relative secondary cause, what of selected amount are the amount of initiator can come according to the size of container of preparation, and are its major effect reaction times, little to the preparation influential effect.
Remove the template molecule in the imprinted polymer, need to add in the molecule that organic solvent with water immiscible phase can make protein and intermolecular hydrogen bond changes and makes protein condenses, make with the drug release of protein bound out.Water-miscible organic solvent commonly used has: acetonitrile, methyl alcohol, ethanol, propyl alcohol, acetone, tetrahydrofuran (THF) etc.The volume ratio of serum and water-miscible organic solvent is 1:(1～3) time, just the protein more than 90% can be removed, wash protein precipitation and formic acid ferrocene off.
Molecularly imprinted polymer among the present invention is made as current sensor in order to using, and screen printing technique is adopted in concrete operations.
Molecularly imprinted polymer of the present invention, find after testing, the biosensor of this molecularly imprinted polymer preparation is used for the detection of tumor markers, detection to CEA is limited to 0.22ng/mL, linearity range is 0.50-25ng/mL, and relation conefficient is 0.9981, and the time of response is 35min, with other proteinaceous substances no cross reactions, CEA concentration-current curve as shown in Figure 2 below 112ng/mL; Detection to NSE is limited to 0.29ng/mL, and linearity range is 0.53-29ng/mL, and relation conefficient is 0.9990, and the time of response is 28min, and with other proteinaceous substances no cross reactions, NSE concentration-current curve as shown in Figure 3 below 231ng/mL; Detection to CYFRA21-1 is limited to 0.41ng/mL, linearity range 0.63-27ng/mL, and relation conefficient is 0.9986, time of response is 32min, below 89ng/mL with other proteinaceous substancess without obvious cross reaction, CYFRA21-1 concentration-current curve as shown in Figure 4.
Compared with prior art, the present invention has following effect:
(1) the present invention's preparation is the molecularly imprinted polymer of biomacromolecule protein, and corresponding substrate molecule is had higher specificity, has reserved simultaneously the binding site of electroactive substance.
(2) the present invention adopts water miscible two acryloyl piperazines (diacrylylpiperazine) as linking agent, the MIPs that has synthesized in water and can use in hydrophilic environment (10%～100% water).
(3) competitive electric current testing is adopted in detection of the present invention, uses the electroactive substance ferrocene and makees probe and replace labeled substrate in traditional immunosensor.
(4) suspension/dispersion copolymerization method of the present invention, and uv-light polymerization are more commonly used, simple to operate, and combine with screen printing technique and Flow Injection Technique, both realized the characteristics of the disposable practicality of chip, again so that easy to detect effective fast.
Description of drawings
Fig. 1 is the concise and to the point process of the preparation of molecularly imprinted polymer.
Fig. 2 is CEA concentration-current curve diagram.
Fig. 3 is NSE concentration-current curve diagram.
Fig. 4 is CYFRA21-1 concentration-current curve diagram.
Fig. 5 changes linking agent, pore-creating agent, CEA concentration-current curve diagram after the ratio of initiator.
Fig. 6 changes ultraviolet polymerization CEA concentration-current curve diagram after the time
Embodiment
The invention will be further described below in conjunction with specific embodiment, but specific embodiment is not done any restriction to the present invention.
Embodiment 1 is for detection of the molecularly imprinted polymer of CEA
(1) Electrochemical Modification of CEA
Get formic acid ferrocene (FER) 4mg, be dissolved in 800 μ L Na-HEPES damping fluid (0.15M, pH 7.3), solution adds 10mg(1-ethyl-3-[3-dimethylaminopropylamine through 0.22 μ m filtering with microporous membrane] carbodiimide-hydrochloric acid) (EDC) with 90 μ L's
Template molecule CEA and under room temperature, hatched 4 hours, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate.Make template composite in 4 ℃ of preservations.
(2) molecularly imprinted polymer is synthetic
Described molecularly imprinted polymer adopts the free radical polymerisation process that is caused by UV-light to be prepared from.Template molecule is resulting template molecule in (1), and function monomer is dihydroxymethyl ethyl propenoate (EGDMA), and linking agent is two acryloyl piperazines, and pore-creating agent is methyl alcohol, and initiator is Diisopropyl azodicarboxylate (AIBN).Its process is: get that the initiator A IBN of prepared template composite solution 0.1mmol and 3mg is dissolved in the 8mL pore-creating agent methyl alcohol in (1), the monomer EGDMA1mmol that adds 3mL linking agent two acryloyl piperazine 4.5mmol and 400 μ L, concussion mixing 10-30 minute, form homogeneous solution, specking is on the working electrode of screen printing electrode, nitrogen was got rid of oxygen in logical 2 minutes, and irradiation is 2 hours under ultraviolet lamp, 90%-95% washing with alcohol 2-6 time.Dry resulting polymers, 4 ℃ of Refrigerator stores.
(3) molecularly imprinted polymer performance measurement
Prepared MIPs-sensor chip is connected with the signal collection system of current sensor, inject the CEA(0.1ng/mL of different concns, 1ng/mL, 5ng/mL, 8ng/mL, 12ng/mL, 18ng/mL, 25ng/mL), and then inject respectively the formic acid ferrocene solution of equivalent, the record current pulse signal.Make the typical curve of CEA concentration-current signal, as shown in Figure 2, reading of data foundation during as test sample.
(1) Electrochemical Modification of NSE
Get formic acid ferrocene (FER) 4mg, be dissolved in 800 μ LNa-HEPES damping fluid (0.15M, pH 7.3), solution adds 10mg(1-ethyl-3-[3-dimethylaminopropylamine through 0.22 μ m filtering with microporous membrane] carbodiimide-hydrochloric acid) (EDC) with 90 μ L's
Template molecule NSE and under room temperature, hatched 4 hours, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate.Make template composite in 4 ℃ of preservations.
(2) molecularly imprinted polymer is synthetic
Described molecularly imprinted polymer adopts the free radical polymerisation process that is caused by UV-light to be prepared from.Template molecule is resulting template molecule in (1), and function monomer is dihydroxymethyl ethyl propenoate (EGDMA), and linking agent is two acryloyl piperazines, and pore-creating agent is methyl alcohol, and initiator is Diisopropyl azodicarboxylate (AIBN).Its process is: get that the initiator A IBN of prepared template composite solution (0.1mmol) and 3mg is dissolved in the 8mL pore-creating agent methyl alcohol in (1), the monomer EGDMA(1mmol that adds 3mL linking agent two acryloyl piperazines (4.5mmol) and 400 μ L), concussion mixing 10-30 minute, form homogeneous solution, specking is on the working electrode of screen printing electrode, nitrogen was got rid of oxygen in logical 2 minutes, and irradiation is 2 hours under ultraviolet lamp, 90%-95% washing with alcohol 2-6 time.Dry resulting polymers, 4 ℃ of Refrigerator stores.
(3) molecularly imprinted polymer performance measurement
Prepared MIPs-sensor chip is connected with the signal collection system of current sensor, inject the NSE(0.1ng/mL of different concns, 1ng/mL, 5ng/mL, 8ng/mL, 12ng/mL, 18ng/mL, 25ng/mL), and then inject respectively the formic acid ferrocene solution of equivalent, the record current pulse signal.Make the typical curve of NSE concentration-current signal, as shown in Figure 3, reading of data foundation during as test sample.
Embodiment 3 is for detection of the molecularly imprinted polymer of CYFRA21-1
(1) Electrochemical Modification of CYFRA21-1
Get formic acid ferrocene (FER) 4mg, be dissolved in 800 μ L Na-HEPES damping fluid (0.15M, pH 7.3), solution adds 10mg(1-ethyl-3-[3-dimethylaminopropylamine through 0.22 μ m filtering with microporous membrane] carbodiimide-hydrochloric acid) (EDC) with 90 μ L's
Template molecule CYFRA21-1 and under room temperature, hatched 4 hours, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate.Make template composite in 4 ℃ of preservations.
(2) molecularly imprinted polymer is synthetic
Described molecularly imprinted polymer adopts the free radical polymerisation process that is caused by UV-light to be prepared from.Template molecule is resulting template molecule in (1), and function monomer is dihydroxymethyl ethyl propenoate (EGDMA), and linking agent is two acryloyl piperazines, and pore-creating agent is methyl alcohol, and initiator is Diisopropyl azodicarboxylate (AIBN).Its process is: get that the initiator A IBN of prepared template composite solution (0.1mmol) and 3mg is dissolved in the 8mL pore-creating agent methyl alcohol in (1), the monomer EGDMA(1mmol that adds 3mL linking agent two acryloyl piperazines (4.5mmol) and 400 μ L), concussion mixing 10-30 minute, form homogeneous solution, specking is on the working electrode of screen printing electrode, nitrogen was got rid of oxygen in logical 2 minutes, and irradiation is 2 hours under ultraviolet lamp, 90%-95% washing with alcohol 2-6 time.Dry resulting polymers, 4 ℃ of Refrigerator stores.
(3) molecularly imprinted polymer performance measurement
Prepared MIPs-sensor chip is connected with the signal collection system of current sensor, inject the CYFRA21-1(0.1ng/mL of different concns, 1ng/mL, 5ng/mL, 8ng/mL, 12ng/mL, 18ng/mL, 25ng/mL), and then inject respectively the formic acid ferrocene solution of equivalent, the record current pulse signal.Make the typical curve of CYFRA21-1 concentration-current signal, as shown in Figure 4, reading of data foundation during as test sample.
Embodiment 4 is for detection of the molecularly imprinted polymer of CEA
(1) Electrochemical Modification of CEA
Get formic acid ferrocene (FER) 4mg, be dissolved in 800 μ L Na-HEPES damping fluid (0.15M, pH 7.3), solution adds 10mg(1-ethyl-3-[3-dimethylaminopropylamine through 0.22 μ m filtering with microporous membrane] carbodiimide-hydrochloric acid) (EDC) with 90 μ L's
Template molecule CEA and under room temperature, hatched 4 hours, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate.Make template composite in 4 ℃ of preservations.
(2) molecularly imprinted polymer is synthetic
Described molecularly imprinted polymer adopts the free radical polymerisation process that is caused by UV-light to be prepared from.Template molecule is resulting template molecule in (1), and function monomer is dihydroxymethyl ethyl propenoate (EGDMA), and linking agent is two acryloyl piperazines, and pore-creating agent is methyl alcohol, and initiator is Diisopropyl azodicarboxylate (AIBN).Its process is: get that the initiator A IBN of prepared template composite solution (0.1mmol) and 2mg is dissolved in the 10mL pore-creating agent methyl alcohol in (1), the monomer EGDMA(1mmol that adds 2mL linking agent two acryloyl piperazines (3mmol) and 400 μ L), concussion mixing 10-30 minute, form homogeneous solution, specking is on the working electrode of screen printing electrode, nitrogen was got rid of oxygen in logical 2 minutes, and irradiation is 2 hours under ultraviolet lamp, 90%-95% washing with alcohol 2-6 time.Dry resulting polymers, 4 ℃ of Refrigerator stores.
(3) molecularly imprinted polymer performance measurement
Prepared MIPs-sensor chip is connected with the signal collection system of current sensor, inject the CEA(0.1ng/mL of different concns, 1ng/mL, 5ng/mL, 8ng/mL, 12ng/mL, 18ng/mL, 25ng/mL), and then inject respectively the formic acid ferrocene solution of equivalent, the record current pulse signal.Make the typical curve of CEA concentration-current signal, as shown in Figure 5.
(1) Electrochemical Modification of CEA
Get formic acid ferrocene (FER) 4mg, be dissolved in 800 μ L Na-HEPES damping fluid (0.15M, pH 7.3), solution adds 10mg(1-ethyl-3-[3-dimethylaminopropylamine through 0.22 μ m filtering with microporous membrane] carbodiimide-hydrochloric acid) (EDC) with 90 μ L's
Template molecule CEA and under room temperature, hatched 4 hours, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate.Make template composite in 4 ℃ of preservations.
(2) molecularly imprinted polymer is synthetic
Described molecularly imprinted polymer adopts the free radical polymerisation process that is caused by UV-light to be prepared from.Template molecule is resulting template molecule in (1), and function monomer is dihydroxymethyl ethyl propenoate (EGDMA), and linking agent is two acryloyl piperazines, and pore-creating agent is methyl alcohol, and initiator is Diisopropyl azodicarboxylate (AIBN).Its process is: get that the initiator A IBN of prepared template composite solution (0.1mmol) and 3mg is dissolved in the 8mL pore-creating agent methyl alcohol in (1), the monomer EGDMA(1mmol that adds 3mL linking agent two acryloyl piperazines (4.5mmol) and 400 μ L), concussion mixing 10-30 minute, form homogeneous solution, specking is on the working electrode of screen printing electrode, nitrogen was got rid of oxygen in logical 2 minutes, and irradiation is 4 hours under ultraviolet lamp, 90%-95% washing with alcohol 2-6 time.Dry resulting polymers, 4 ℃ of Refrigerator stores.
(3) molecularly imprinted polymer performance measurement
Prepared MIPs-sensor chip is connected with the signal collection system of current sensor, inject the CEA(0.1ng/mL of different concns, 1ng/mL, 5ng/mL, 8ng/mL, 12ng/mL, 18ng/mL, 25ng/mL), and then inject respectively the formic acid ferrocene solution of equivalent, the record current pulse signal.Make the typical curve of CEA concentration-current signal, as shown in Figure 6.
Reference
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Claims (1)
1. the preparation method for detection of the molecularly imprinted polymer of lung cancer tumor markers is characterized in that, comprises step:
(1) Electrochemical Modification of template molecule;
Select electroactive substance formic acid ferrocene 3-5mg, be abbreviated as FER, be dissolved in 500-1000 μ L, 0.15M, pH are 7.3 hydroxyethyl piperazine second thiosulfonic acid damping fluid, solution is through 0.22 μ m filtering with microporous membrane, add 8-15mg 1-ethyl-3-[3-dimethylaminopropylamine] carbodiimide-hydrochloric acid and 80-120 μ L, the tumor markers of 500 μ g/mL, and under room temperature, hatched 3-5 hour, unconjugated formic acid ferrocene is removed in ultrafiltration, until no longer contain the formic acid ferrocene in the filtrate; Make template composite in 4 ℃ of preservations; Described tumor markers is that carcinomebryonic antigen is abbreviated as CEA, neuronspecific enolase is abbreviated as NSE or cytokeratin 19 fragment antigen is abbreviated as CYFRA21-1;
(2) preparation of molecularly imprinted polymer;
Be formic acid ferrocene-tumor markers with template molecule, function monomer, linking agent is dissolved in pore-creating agent, and solution is moved in the water, stirs emulsification; Then it is crosslinked to add initiator, uv photopolymerization, get particle diameter than the spherical molecular imprinting material of homogeneous, wherein the amount ratio of template, function monomer, linking agent, pore-creating agent, initiator can be selected mmol:mmol:mmol:mL:mg=1:(8-10): (40-50): (90-100): (3-40); By the centrifugal template molecule of removing of washing with alcohol; Described template is FER-CEA or FER-NSE or FER-CYFRA21-1; Described function monomer is dihydroxymethyl ethyl propenoate, methyl methacrylate, methylene-succinic acid, to Ethenylbenzene formic acid or diacrylamine-2-methyl isophthalic acid-propanesulfonic acid; Described linking agent is N, N-two acryloyl piperazines, N, N '-methylene diacrylamine, 3,5-dibenzoic acid or 3,5-diacrylamine phenylformic acid; Described pore-creating agent is tetrahydrofuran (THF), methyl alcohol, ethanol or propyl alcohol; Described initiator is that Diisopropyl azodicarboxylate is abbreviated as AIBN or 2,2'-Azobis(2,4-dimethylvaleronitrile) is abbreviated as AIHN.
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| CN109959791B (en) * | 2017-12-14 | 2021-09-24 | 中国科学院大连化学物理研究所 | Multiple-action imprinting material for specifically recognizing tumor cells and preparation and application thereof |
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