CN107607498B - Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof - Google Patents
Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof Download PDFInfo
- Publication number
- CN107607498B CN107607498B CN201710592816.1A CN201710592816A CN107607498B CN 107607498 B CN107607498 B CN 107607498B CN 201710592816 A CN201710592816 A CN 201710592816A CN 107607498 B CN107607498 B CN 107607498B
- Authority
- CN
- China
- Prior art keywords
- solution
- sensor
- molecular imprinting
- reaction
- freeze
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Weighing carbon dot powder, ultrasonically dispersing the carbon dot powder in borate buffer solution, sequentially adding 1-ethyl- (3- (dimethylamino) propyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring for reaction at room temperature in a dark place, adding 4-vinylaniline, continuing stirring for reaction, dialyzing the obtained product in water, and freeze-drying to obtain carbon dots with surface double bonds functionalized; adding template molecules and two different functional monomers into a pore-foaming agent, ultrasonically dissolving, and stirring at room temperature for prepolymerization to obtain a preassembled solution A; ultrasonically dispersing carbon points with surface double bond functionalized in a pore-foaming agent to obtain a solution B; uniformly mixing the solution A and the solution B, adding a cross-linking agent and an initiator, introducing nitrogen, stirring, centrifuging, collecting precipitate, and washing with distilled water; and finally, eluting the template protein by HAc-SDS, and freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
Description
Technical Field
The invention belongs to the field of fluorescent nano molecular imprinting bionic sensors, and particularly relates to a fluorescent nano molecular imprinting bionic sensor of a liver cancer tumor marker sensitive to pH and temperature, and a preparation method and application thereof.
Background
Liver cancer, which is a malignant tumor of the liver, is one of the most common malignant tumors in the world, with the fifth highest morbidity and the third highest mortality. The main reason for the extremely high mortality rate of liver cancer is that the liver cancer is diagnosed in a near late stage in clinic, so that the early detection and early diagnosis of liver cancer are very important for the successful cure of cancer and the improvement of the survival rate of patients. Currently, Alpha Fetoprotein (AFP), as a specific indicator of liver cancer, is an extremely important marker for finding primary liver cancer. AFP is a glycoprotein with a molecular weight of 70kDa, and in early liver cancer, the content of AFP is very low (ng/mL) and glycosylation sites and structures are complex, so that the quantitative determination of the AFP is very challenging. At present, the clinical detection of serum tumor markers mainly comprises an enzyme-linked immunosorbent assay, a radioimmunoassay and the like, but has the defects of complex operation, long time consumption, high cost, large instrument volume, huge radioactive pollution, incapability of being popularized in community hospitals and the like. Therefore, there is a need to find a simple and rapid detection method with high specificity, high sensitivity and accuracy for AFP at low cost.
As a new carbon nano functional material, the carbon quantum dot has the advantages of good photostability, photobleaching resistance, small particle size, good water solubility, easy functionalization, no toxicity, good biocompatibility and the like, and is widely used as a signal conduction unit of a fluorescent nano sensor at present. Although the carbon dot fluorescence sensor has the advantages of high sensitivity, high response speed, simple sample pretreatment and the like, due to the fact that bioactive components such as antibodies, enzymes, microorganisms and the like used as molecular recognition units are extremely easily inactivated, the types of available biomolecules are limited, the manufacturing cost of the sensor is high, the stability is poor and the like, and the large-scale popularization and application of the carbon dot fluorescence sensor are limited. Therefore, obtaining an identification unit that has strong adaptability, low manufacturing cost, long life span, and reusability is one of the keys for further development of biosensors.
Molecular Imprinting Technology (MIT) is a technology for artificially preparing a polymer having selectivity and affinity for a specific target molecule (template molecule, imprinted molecule) based on the principle of specific molecular recognition between an antigen and an antibody in nature. Molecularly Imprinted Polymers (MIPs) synthesized by MIT are called "artificial antibodies", fulfilling the desire to obtain "antibodies" independent of living organisms. Because the molecular imprinting material has good selective recognition capability on the target protein, the selective recognition behavior of the carbon quantum dots can be improved by modifying the molecular imprinting material on the surfaces of the carbon quantum dots. Based on the consideration, the high selective recognition capability of the molecular imprinting technology is combined with the fluorescence property of the quantum dots, a novel protein imprinting material with fluorescence property is developed, and then the AFP is sensitively, efficiently and quickly detected. The literature search method in the prior art finds that Liu Ji and the like adopt the traditional method to prepare the molecularly imprinted polymer specifically binding the designated glycoprotein, but the polymer prepared by the method has the defects of slow mass transfer rate, low selectivity, low sensitivity, difficulty in synthesis and identification in water and the like (see Liu Ji, Li 28583, a molecularly imprinted polymer specifically binding the designated glycoprotein and a preparation method and application thereof, CN 201110416198.8. The molecular imprinting gel prepared by Karfa, Tan and the like has specific selectivity for AFP, but the modification of the carrier surface by silicon dioxide is easy to cause protein configuration change, template removal difficulty and poor contraction effect (see P. Karfa, E.Roy, S.Patra, et al, biosens. Bioelectron,2016,78, 454-containing 463; L.Tan, K.C.Chen, C.Huang, et al, Microchim Acta,2015,182, 2615-containing 2622). At present, a method for detecting the AFP content in serum of a liver cancer patient by combining a molecular imprinting technology and a carbon quantum dot is not reported.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a fluorescent nano molecular imprinting bionic sensor and a preparation method and application thereof, aiming at the problems of clinical rapid, economical, convenient, high-sensitivity and high-specificity detection difficulty of tumor marker alpha fetoprotein. The invention takes carbon points with surface double bond functionalization as a carrier substrate, takes temperature-sensitive and pH-sensitive intelligent materials as functional monomers, and adopts a surface printing method to create a convenient, low-cost, high-sensitivity and high-specificity eco-friendly molecular imprinting bionic sensor for specifically identifying and sensitively detecting AFP in the serum of a liver cancer patient.
The technical scheme is as follows: a preparation method of a fluorescent nano molecular imprinting bionic sensor comprises the following preparation steps: weighing carbon dot powder, ultrasonically dispersing the carbon dot powder in borate buffer solution with the pH value of 4-6, sequentially adding 1-ethyl- (3- (dimethylamino) propyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring the mixture at room temperature in a dark place for reaction, adding 4-vinylaniline, continuously stirring the mixture for reaction, dialyzing the obtained product in water, and freeze-drying the dialyzed product to obtain carbon dots with surface double bonds functionalized; adding template molecules and two different functional monomers into a pore-foaming agent, ultrasonically dissolving, and stirring at room temperature for prepolymerization to obtain a preassembled solution A; ultrasonically dispersing carbon dots with surface double-bond functionalization in a pore-foaming agent to obtain a solution B; uniformly mixing the solution A and the solution B, adding a cross-linking agent and an initiator, introducing nitrogen to remove oxygen for 15min, stirring and reacting for 3-6 h at 60 ℃, centrifuging, collecting precipitate, and washing with distilled water for 5-10 times; finally, eluting the template protein by using 10% (v/v) HAc-10% (w/v, g/mL) SDS, and freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor; the molar ratio of the two functional monomers is 2: 4-4: 2; the molar ratio of the functional monomer to the cross-linking agent is 2 (1-4); the using amount of the initiator is 5 percent of the total mass of the template molecules, the functional monomers and the cross-linking agent; the mass ratio of the carbon points functionalized by the surface double bonds to the template molecules is (50-100): 1, the mass ratio of the functional monomers to the template molecules is 1 (353-1201), and the mass-to-volume ratio (w/v, mg/mL) of the carbon points functionalized by the surface double bonds to the total volume of the pore-foaming agent is (1-6): 6.
The mass ratio of the carbon dot powder to the 1-ethyl- (3- (dimethylamino) propyl) carbodiimide hydrochloride is 5 (3-10), the molar ratio of the 1-ethyl- (3- (dimethylamino) propyl) carbodiimide hydrochloride to the N-hydroxysuccinimide hydrochloride is 1: 1-1: 4, and the mass ratio of the carbon dot powder to the 4-vinylaniline is 20 (2-7).
The preparation method of the carbon dots comprises the following steps: adding 1g of citric acid and 335 mu L of ethylenediamine into 20mL of ultrapure water, and stirring for dissolving; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing for 48h, and finally freezing and freeze-drying for 12h to obtain the carbon dot powder.
The template molecules are alpha fetoprotein, and the functional monomers are any two of 4-vinylphenylboronic acid, 3-acrylamidophenylboronic acid, 4- (3-butenylsulfonyl) phenylboronic acid, 4-ethylmercapto phenylboronic acid, m-aminobenzoic acid, p-hydroxybenzeneboronic acid, N-isopropylacrylamide, N-diethylacrylamide, ethylene glycol oligomer, N-ethylacrylamide and N-N-propylacrylamide; the cross-linking agent is N, N-methylene bisacrylamide, ethylene glycol dimethacrylate, methyl orthosilicate, 3-isocyanatopropyl triethoxysilane or 1, 6-hexanediamine; the initiator is potassium persulfate or azodiisobutyronitrile; the pore-forming agent is at least one of pure water, pH 8.5 buffer solution, polyethylene glycol and dimethyl sulfoxide.
The fluorescent nano molecular imprinting bionic sensor prepared by the preparation method.
The fluorescent nano molecular imprinting bionic sensor is applied to the preparation of a liver cancer diagnosis kit.
The fluorescent nano molecular imprinting bionic sensor is applied to screening of liver cancer drugs.
Has the advantages that: (1) the invention combines the high sensitivity of the carbon quantum dots and the high selectivity of the molecular imprinting, and the molecular imprinting layer is modified on the surface of the modified carbon dots to obtain the protein fluorescence nanometer molecular imprinting bionic sensor with specific recognition capability and fluorescence response, thereby solving the problem of difficult mass transfer of biomacromolecules to a certain extent and shortening the time for recognizing specific imprinting sites by proteins. (2) Biological receptors (such as antibodies, enzymes and the like) have good molecular recognition capability, excellent stimulus response performance to external stimuli (such as temperature, illumination, pH and the like) and the like. In the experiment, an intelligent response type high polymer material sensitive to pH and temperature is used as a bifunctional monomer, so that the fluorescent nano molecular imprinting bionic sensor with high identification and high selectivity is successfully prepared. (3) The synthesis and identification of the imprinting sensor are carried out in a water phase, and the bifunctional monomer with the synergistic effect of covalent bonds and non-covalent bonds is used, so that the effect of weakening hydrogen bonds by surrounding water molecules is overcome, the selective identification capability of the imprinting sensor is improved to a certain extent, and the application of molecular imprinting in practical samples is widened. (4) The pH and temperature sensitive fluorescent nano molecular imprinting bionic sensor successfully prepared by the invention is used for detecting AFP in serum, and has the advantages of less serum use amount, short analysis time, low cost, high accuracy and precision, simple and convenient operation, good repeatability and the like. The kit is suitable for clinical AFP detection in community hospitals and township hospitals, and has wide application prospect in diagnosing and monitoring the AFP level in the serum of liver cancer patients.
Drawings
FIG. 1 is a schematic diagram of a preparation process of a fluorescent nano-molecular imprinting biomimetic sensor.
Fig. 2 is a transmission electron microscope image of a fluorescent nano-molecular imprinted biomimetic sensor (FMIP) and a fluorescent nano-non-molecular imprinted biomimetic sensor (FNIP) prepared by surface printing.
FIG. 3 is a fluorescence property evaluation diagram of a fluorescent nano molecular imprinting bionic sensor, wherein, A and C are fluorescence emission spectra of FMIP and FNIP combined with different AFP concentrations, and B and D are Stern-Volmer graphs of FMIP and FNIP.
Fig. 4 is a kinetic adsorption curve diagram of the fluorescent nano-molecular imprinting biomimetic sensor.
FIG. 5 is a selective diagram of a fluorescent nano-molecular imprinting biomimetic sensor.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to limit the present invention in any way.
1-Ethyl- (3- (dimethylamino) propyl) carbodiimide hydrochloride (EDC)
N-hydroxysuccinimide (NHS)
4-Vinylaniline (4-VPA)
N-isopropyl acrylamide (NIPAAm)
4-vinyl phenyl boron (4-VPBA)
Para hydroxybenzene boric acid (APBA)
Ethylene glycol Oligomer (OEG)
N, N-Methylene Bisacrylamide (MBA)
Potassium persulfate (APS)
Azobisisobutyronitrile (AIBN)
Ethylene Glycol Dimethacrylate (EGDMA)
Example 1:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing in pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 20mL of phosphate buffer solution with the pH value of 5, sequentially adding 100 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to perform covalent condensation reaction, and the reaction was magnetically stirred for 4 hours. After the reaction, the reaction solution was dialyzed against water for 48 hours to remove unreacted 4-VPA, other reactants and ions. Freeze drying for 12h to obtain carbon dots with double-bonded modified surface for later use.
(3) Adding 160 mu L of AFP (alpha-fetoprotein) with the concentration of 1.25mg/mL, NIPAAm with the concentration of 0.5mmol and 4-VPBA into 15mL of pure water solution, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 40 min; dispersing 20mg of carbon dot powder with surface double-bonding modification in 15mL of pure water; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol MBA and 13.25mg AIBN, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction for 4h at 60 ℃. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally eluting the template proteins with 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the FMIP and FNIP fluorescence are nearly identical; and freeze-drying the obtained product to obtain the fluorescent nano-molecular imprinting bionic sensor.
The FNIP preparation process is the same as FMIP preparation process except that alpha-fetoprotein is not added during the synthesis process.
Example 2:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 20mL of phosphate buffer solution with the pH value of 5, sequentially adding 200 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to perform covalent condensation reaction, and the reaction was magnetically stirred for 4 hours. After the reaction was completed, the reaction solution was dialyzed against water for 48 hours to remove unreacted 4-VPA, other reactants and ions. Freeze drying for 12h to obtain carbon dots with double-bonded modified surface for later use.
(3) Adding 320 mu L of 1.25mg/mL AFP, 0.5mmol NIPAAm and 4-VPBA into 15mL pure water solution, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 40 min; dispersing 20mg of carbon dot powder with surface double-bonding modification in 15mL of pure water; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol MBA and 13.25mg APS, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction at 60 ℃ for 4 h. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally eluting the template proteins with 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the FMIP and FNIP fluorescence are nearly identical; and freeze-drying the obtained product to obtain the fluorescent nano-molecular imprinting bionic sensor.
The FNIP preparation process is the same as FMIP preparation process except that alpha-fetoprotein is not added during the synthesis process.
Example 3:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 20mL of phosphate buffer solution with the pH value of 5, sequentially adding 200 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to perform covalent condensation reaction, and the reaction was magnetically stirred for 4 hours. After the reaction was completed, the reaction solution was dialyzed against water for 48 hours to remove unreacted 4-VPA, other reactants and ions. And finally, freeze drying for 12h to obtain the carbon dots with double-bonded modified surfaces for later use.
(3) Adding 320 mu L of 1.25mg/mL AFP, 0.5mmol NIPAAm and 4-VPBA into 15mL carbonate buffer solution with pH of 8.5, dissolving by ultrasonic, introducing nitrogen to remove oxygen, and performing magnetic stirring prepolymerization at 25 ℃ for 40 min; dispersing 20mg of carbon dot powder subjected to surface double-bonding modification in 15mL of carbonate buffer solution with the pH value of 8.5; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol EDGMA and 13.25mg APS, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction for 4h at 60 ℃. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally, eluting the template protein by using 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the FMIP and FNIP fluorescence are almost the same; and (4) freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
The FNIP preparation process is the same as FMIP preparation process except that alpha-fetoprotein is not added during the synthesis process.
Example 4:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 20mL of phosphate buffer solution with the pH value of 5, sequentially adding 200 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to perform covalent condensation reaction, and the reaction was magnetically stirred for 4 hours. After the reaction, the reaction solution was dialyzed in water for 48 hours to remove unreacted 4-VPA, other reactants and ions. And finally, freeze drying for 12h to obtain the carbon dots with double-bonded modified surfaces for later use.
(3) Adding 320 mu L of 1.25mg/mL AFP, 0.5mmol NIPAAm and APBA into 15mL dimethyl sulfoxide solution, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 40 min; dispersing 20mg of carbon dot powder subjected to surface double-bonding modification into 15mL of dimethyl sulfoxide solution; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol of MBA and 13.25mg of APS, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction at 60 ℃ for 4 h. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally, eluting the template protein by using 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the FMIP and FNIP fluorescence are almost the same; and freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
The FNIP preparation process is the same as FMIP preparation process except that alpha-fetoprotein is not added during the synthesis process.
Example 5:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 20mL of phosphate buffer solution with the pH value of 5, sequentially adding 200 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to perform covalent condensation reaction, and the reaction was magnetically stirred for 4 hours. After the reaction was completed, the reaction solution was dialyzed against water for 48 hours to remove unreacted 4-VPA, other reactants and ions. And finally, freeze drying for 12h to obtain the carbon dots with double-bonded modified surfaces for later use.
(3) Adding 320 mu L of 1.25mg/mL AFP, 0.5mmol OEG and 0.5mmol 4-VPBA into 15mL pure water solution, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 40 min; dispersing 20mg of carbon dot powder with surface double-bonding modification in 15mL of pure water; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol of MBA and 20mg of APS, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction for 4h at 60 ℃. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally eluting the template proteins with 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the FMIP and FNIP fluorescence are nearly identical; and freeze-drying the obtained product to obtain the fluorescent nano-molecular imprinting bionic sensor.
The FNIP preparation process is the same as FMIP preparation process except that alpha-fetoprotein is not added during the synthesis process.
Example 6:
(1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use.
(2) Ultrasonically dispersing 20mg of carbon dot powder into 15mL of phosphate buffer solution with the pH value of 5, sequentially adding 200 mu L of 1M EDC and NHS, and magnetically stirring for reacting for 20 min; mu.L of 0.25M 4-VPA was added to the above solution to carry out covalent condensation reaction, and the reaction was carried out for 4 hours with magnetic stirring. After the reaction is finished, dialyzing the reaction solution for 48 hours to remove unreacted 4-VPA, other reactants and ions. And finally, freeze drying for 12h to obtain the carbon dots with double-bonded modified surfaces for later use.
(3) Adding 320 mu L of 1.25mg/mL AFP, 0.5mmol NIPAAm and 0.5mmol 4-VPBA into 15mL pure water, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 50 min; dispersing 20mg of carbon dot powder with surface double-bonding modification in 15mL of pure water; then mixing the pre-assembled solution with the carbon dot solution with double-bond modified surface, adding 0.5mmol of MBA and 13.25mg of APS, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction at 60 ℃ for 4 h. Centrifuging at 10,000rpm/min for 5min, collecting precipitate, and washing with pure water for 5 times; finally, eluting the template protein by using 10% (v/v) HAc-10% (w/v, mg/mL) SDS until the fluorescence of the imprinted biomimetic sensor and the fluorescence of the non-imprinted biomimetic sensor are almost the same; and (4) freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
The preparation process of the non-imprinted sensor is the same as the preparation process of the imprinted sensor except that alpha-fetoprotein is not added in the synthesis process.
Test examples
Test example 1: synthesis process optimization
In the process of preparing the protein MIP, factors such as the volumes of the functional monomer, the cross-linking agent and the pore-foaming agent, the dosage of carbon points and the like have great influence on the adsorption performance of the sensor. Therefore, the influence factors of the sensor are examined in the experiment, and the results are shown in table 1.
Table 1 fluorescence nanometer molecular imprinting bionic sensor preparation method process optimization table
Test example 2: transmission electron microscopy characterization
Fig. 2 is a transmission electron micrograph of an imprinted sensor prepared in example 6. The particle sizes of the FMIP and the FNIP are about 30-40nm, the size is uniform, the dispersity is good, the shape is nearly circular, and the appearance is clear and visible.
Test example 3: evaluation of adsorption property of fluorescence nano bionic sensor
(1) In order to examine the ability of FMIP, FNIP to identify AFP, the product prepared in example 6 was examined for adsorption properties. 24 parts of FMIPs and FNIPs, 5mg, were each precisely weighed and dispersed in 2mL of a pH 8.5 alpha-fetoprotein phosphate buffer solution at concentrations of 0, 10, 20, 40, 60, 80, 100, and 120ng/mL, and shaken at 28 ℃ for 1 hour to measure the fluorescence intensity of FMIP and FNIP at the optimal emission wavelength. The relationship between fluorescence intensity and concentration was fitted using the Stern-Volmer equation (equation 1), and the measurements were performed in triplicate and adsorption curves were plotted, see FIG. 3.
F0/F＝1+KSV[Q](1)
Wherein F0And F represents fluorescence intensity before and after AFP is adsorbed by FMIP and FNIP, respectively, Q represents AFP concentration, and K representsSVIs the fluorescence quenching constant.
As shown in FIG. 3, it can be seen that the fluorescence intensity quenching increases with the increase of the AFP concentration in FMIP and FNIP, and when the AFP concentration is 100ng/mL, the fluorescence intensity does not decrease any more and the adsorption reaches saturation. At the same time, under the same AFP concentration, the fluorescence quenching of FMIP is larger than that of FNIP, mainly because the marking site on the surface of FMIP has specific interaction with AFP, and the adsorption of FNIP to AFP is mainly the physical adsorption of marking materials. As can be seen from B in FIG. 3 and D in FIG. 3, the fluorescence intensity of FMIP and FNIP is linear with AFP concentration, ranging from 10 to 100ng/mL, with a detection limit of 0.474 ng/mL. The Imprinting Factor (IF) (formula 2) is calculated to be 3.67, which shows that FMIP has good recognition capability on the target protein AFP.
IF＝KFMIP/KFNIP(2)
Wherein KFMIP、KFNIPFluorescence quenching constants of FMIP and FNIP, respectively
(2) To examine the recognition rate of FMIP for AFP, adsorption kinetics studies were performed on the product prepared in example 6. Precisely weighing 24 parts of FMIPs and FNIPs of about 5mg respectively, dispersing in 2mL of alpha-fetoprotein phosphate buffer solution of 60ng/mL, shaking at 28 deg.C, measuring fluorescence intensity of FMIPs at optimal emission wavelength at 0, 5, 10, 20, 30, 40, 50 and 60min respectively, and taking F as reference0Plot F on ordinate and time on abscissa.
The results are shown in FIG. 4, where it is seen that in 60ng/mL AFP phosphate buffer solution, when the adsorption time reaches 30min, a large amount of AFP interacts with the recognition site on FMIP, resulting in a significant decrease in its fluorescence intensity. When the adsorption reached 50min, the fluorescence intensity tended to be substantially stable, indicating that FMIP reached equilibrium for AFP adsorption. The FMIP rapidly reaches the adsorption balance mainly because the nano imprinting sensor prepared by the surface imprinting method has a thin and uniform imprinting layer, a large specific surface area and a high mass transfer rate. The FNIP fluorescence intensity change is not obvious as FMIP, mainly because FMIP has AFP specific recognition sites, and the adsorption of AFP by FNIP is physical adsorption.
Test example 3: selectivity test
According to the preparation method of example 6, about 18 parts of FMIP and FNIP were precisely weighed and dispersed in 2mL of 60ng/mL of a solution of similar proteins of different structures including the target protein alpha-fetoprotein, glycoprotein horseradish peroxidase (HRP), Transferrin (TF) and non-glycoprotein cytochrome C (Cyt C), lysosome (Lys) and Bovine Serum Albumin (BSA), and after shaking at 28 ℃ for 50min, the fluorescence intensity of FMIP at the optimal emission wavelength was measured. With F0Plot F on ordinate and protein species on abscissa. As shown in FIG. 5, FMIP has a strong fluorescence response to AFP, but no obvious fluorescence response to other glycoproteins and non-glycoproteins, and the ratio of the change of the fluorescence intensity of FMIP to AFP to the change of the fluorescence intensity of TF, HRP, BSA and OB is 4.128, 4.880, 2.359 and 1.673, respectively, which shows that FMIP has a good specific recognition ability to AFP, mainly because FMIP has a site for AFP specific binding. While FNIP does not quench AFP and other structural analogs significantly, primarily due to the adsorptive nature of FNIPIn nonspecific adsorption. Therefore, the synthesized FMIP has a good imprinting effect on the AFP and can be used for selective recognition of the AFP.
Claims (1)
1. The application of the fluorescent nano molecular imprinting bionic sensor in preparing a liver cancer diagnosis kit is characterized in that the fluorescent nano molecular imprinting bionic sensor is prepared by the following method: (1) 1.051g of citric acid and 335 mu L of ethylenediamine are added into 20mL of ultrapure water and stirred to be dissolved; transferring the obtained solution into a 50mL polytetrafluoroethylene autoclave, and reacting for 5h at 200 ℃; naturally cooling to room temperature, dialyzing with pure water for 48h, and freeze-drying for 12h to obtain carbon dots for later use; (2) ultrasonically dispersing 20mg of carbon dot powder into 15mL of phosphate buffer solution with pH =5, sequentially adding 200 μ L of 1M EDC and NHS, and reacting for 20min by magnetic stirring; adding 300 mu L of 0.25M 4-vinylaniline into the solution for covalent condensation reaction, and reacting for 4 hours under magnetic stirring; after the reaction is finished, dialyzing the reaction solution for 48 hours; finally, freeze drying for 12h to obtain carbon dots with double-bonded modified surfaces for later use; (3) adding 320 mu L of 1.25mg/mL AFP, 0.5mmol NIPAAm and 0.5mmol 4-VPBA into 15mL pure water, ultrasonically dissolving, introducing nitrogen to remove oxygen, and magnetically stirring at 25 ℃ for prepolymerization for 50 min; dispersing 20mg of carbon dot powder with surface double-bonding modification in 15mL of pure water; then mixing the pre-polymerized solution with a carbon dot solution with double-bond modification on the surface, adding 0.5mmol of N, N-methylene-bisacrylamide and 13.25mg of potassium persulfate, introducing nitrogen to remove oxygen for 15min, magnetically stirring, and carrying out polymerization reaction for 4h at 60 ℃; centrifuging at 10000 rpm for 5min, collecting precipitate, and washing with pure water for 5 times; finally, eluting the template protein by using 10% (v/v) HAc-10% (w/v) SDS until the fluorescence of the imprinted biomimetic sensor and the fluorescence of the non-imprinted biomimetic sensor are almost the same; and freeze-drying the obtained product to obtain the fluorescent nano molecular imprinting bionic sensor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710592816.1A CN107607498B (en) | 2017-07-19 | 2017-07-19 | Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710592816.1A CN107607498B (en) | 2017-07-19 | 2017-07-19 | Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107607498A CN107607498A (en) | 2018-01-19 |
| CN107607498B true CN107607498B (en) | 2020-06-02 |
Family
ID=61059773
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710592816.1A Expired - Fee Related CN107607498B (en) | 2017-07-19 | 2017-07-19 | Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107607498B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856402B (en) * | 2019-03-11 | 2021-08-10 | 中国科学院兰州化学物理研究所 | Preparation of molecularly imprinted sensor for AFP online detection in human serum |
| CN112022902A (en) * | 2019-05-18 | 2020-12-04 | 云南伦扬科技有限公司 | Preparation method and application of carbon-point modified fluconazole eucalyptus oil microemulsion gel |
| CN111099575B (en) * | 2020-01-09 | 2021-05-28 | 郑州大学 | Lysosome targeted polarity and viscosity dual-response fluorescent carbon dot, preparation method and application |
| CN111321205A (en) * | 2020-03-11 | 2020-06-23 | 昆明理工大学 | MiRNA detection method |
| CN111329846A (en) * | 2020-04-24 | 2020-06-26 | 云南伦扬科技有限公司 | Vaginal sterilization adhesive film and preparation method thereof |
| CN111603570B (en) * | 2020-07-06 | 2021-04-30 | 南京工业大学 | Carbon-point-modified hollow copolymer nano particle, preparation method and application thereof, drug delivery system and application thereof |
| CN115029132B (en) * | 2022-05-27 | 2023-05-02 | 重庆师范大学 | Preparation method of novel dopamine functional fluorescent carbon dots, product and application thereof |
| CN115849351B (en) * | 2022-12-29 | 2024-04-30 | 华侨大学 | Modified graphene quantum dot, preparation method, graphene quantum dot/photo-curing resin composite material and polaroid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103570871A (en) * | 2013-10-21 | 2014-02-12 | 南京医科大学 | Mesoporous molecular sieve SBA-15 composite nano surface imprinted polymer and preparation method thereof |
| CN103923647A (en) * | 2014-04-21 | 2014-07-16 | 中国石油大学(北京) | Nitrogen-doped high-luminescent carbon quantum dot and preparation method thereof |
| CN104062275A (en) * | 2014-06-25 | 2014-09-24 | 东华大学 | MWCNTs-QDs-based nano fluorescence bionic sensor and preparation method thereof |
-
2017
- 2017-07-19 CN CN201710592816.1A patent/CN107607498B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103570871A (en) * | 2013-10-21 | 2014-02-12 | 南京医科大学 | Mesoporous molecular sieve SBA-15 composite nano surface imprinted polymer and preparation method thereof |
| CN103923647A (en) * | 2014-04-21 | 2014-07-16 | 中国石油大学(北京) | Nitrogen-doped high-luminescent carbon quantum dot and preparation method thereof |
| CN104062275A (en) * | 2014-06-25 | 2014-09-24 | 东华大学 | MWCNTs-QDs-based nano fluorescence bionic sensor and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| A fluorescent molecularly-imprinted polymer gate with temperature and pH as inputs for detection of alpha-fetoprotein;Paramita Karfa et al.;《Biosensors and Bioelectronics》;20151202;第78卷;454-456、462 * |
| ENZYME-ANALOGUE BUILT POLYMERS, 17*. INVESTIGATIONS ON THE RACEMIC RESOLUTION OF AMINO ACIDS;GUNTER WULFF et al.;《Reactive Polymers》;19840331;第2卷(第3期);168 * |
| Immobilization of Carbon Dots in Molecularly Imprinted Microgels for Optical Sensing of Glucose at Physiological pH;Hui Wang et al.;《ACS Appl. Mater. Interfaces》;20150706;第7卷(第29期);15737 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107607498A (en) | 2018-01-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107607498B (en) | Fluorescent nano molecular imprinting bionic sensor and preparation method and application thereof | |
| Tarannum et al. | Molecularly imprinted polymers as receptors for assays of antibiotics | |
| Wang et al. | Surface-imprinted fluorescence microspheres as ultrasensitive sensor for rapid and effective detection of tetracycline in real biological samples | |
| Liao et al. | An ultrasensitive ELISA method for the detection of procalcitonin based on magnetic beads and enzyme-antibody labeled gold nanoparticles | |
| Büyüktiryaki et al. | Phosphoserine imprinted nanosensor for detection of Cancer Antigen 125 | |
| WO2010137532A1 (en) | Detection method and quantification method of detection target | |
| Wang et al. | Epitope imprinted polymer nanoparticles containing fluorescent quantum dots for specific recognition of human serum albumin | |
| CN104316679B (en) | The application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay | |
| WO2009158657A2 (en) | Molecularly imprinted polymers for detecting microorganisms | |
| Garcia-Cruz et al. | Molecularly imprinted nanoparticles-based assay (MINA)–detection of leukotrienes and insulin | |
| Battaglia et al. | Detection of canine and equine procalcitonin for sepsis diagnosis in veterinary clinic by the development of novel MIP-based SPR biosensors | |
| Saeki et al. | Signalling molecular recognition nanocavities with multiple functional groups prepared by molecular imprinting and sequential post-imprinting modifications for prostate cancer biomarker glycoprotein detection | |
| CN111089968A (en) | Multi-index time-resolved fluorescence immunoassay detection method of single detection line | |
| CN113189064B (en) | Glycopeptide-based fluorescent molecularly imprinted polymer, preparation method and application in screening and detecting glycoprotein | |
| Liu et al. | Photografted poly (methyl methacrylate)-based high performance protein microarray for hepatitis B virus biomarker detection in human serum | |
| CN108801989B (en) | Aptamer-molecularly imprinted fluorescent sensor for detecting cytochrome c and preparation method thereof | |
| CN107132206B (en) | Rapid detection method for virus activity | |
| CN110437490B (en) | Thermo-sensitive molecularly imprinted quantum dot for detecting cytochrome c and preparation method and application thereof | |
| CN115753716A (en) | Fluorescence biosensor for detecting Golgi protein 73 | |
| CN108760694B (en) | Detection method of cytochrome c based on aptamer-molecularly imprinted fluorescent sensor | |
| CN111426667A (en) | Fluorescence method for β -lactoglobulin detection based on quantum dot-aptamer-graphene oxide | |
| Zhu et al. | Double imprinting-based electrochemical detection of mimetic exosomes | |
| CN109517170A (en) | Bovine serum albumin(BSA) magnetism bionic immune analysis kit and its application | |
| RU2684325C1 (en) | Method of producing conjugate based on magnetic metall-carbon nanoparticles suitable for diagnostic and analytical target using nmr-relaxometry as a detection method | |
| CN114486822A (en) | Method for detecting antigen-antibody interaction by FRET technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200602 Termination date: 20210719 |