CN110563882B - Preparation method and application of nano artificial antibody targeting cardiac troponin I - Google Patents
Preparation method and application of nano artificial antibody targeting cardiac troponin I Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a nanometer artificial antibody of a targeted cardiac troponin I. By combining a molecular imprinting technology and taking the whole protein or polypeptide fragment of the troponin I as a template, the specificity and the selectivity of the nano artificial antibody to the troponin I are further improved. Affinity constant K of screened nano artificial antibody to troponin IDThe value reaches 6.26 multiplied by 10‑11M, is equivalent to antibody and has good selectivity. The obtained nano artificial antibody can be combined with magnetic nano particles, an integral column or a microfluidic chip to realize the high selective enrichment of low-abundance cardiac troponin I in a serum sample, and can realize the high-sensitivity detection of the cardiac troponin I by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit, test paper strips and the like.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a nano artificial antibody of a targeted cardiac troponin I.
Background
Acute myocardial infarction is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery. Clinically, severe and persistent poststernal pain, rest and incomplete relief of nitrate medicines are caused, and the increased activity of serum myocardial enzyme and progressive electrocardiogram change are accompanied, so that arrhythmia, shock or heart failure can occur, and the life can be threatened. China has a remarkable rising trend in recent years, newly issues at least 50 ten thousand every year, and finds out at least 200 ten thousand. The rapid diagnostic reagent of cardiac troponin I (cTnI) can be used for rapid auxiliary diagnosis in sudden myocardial infarction and is increasingly applied.
The level of peripheral blood cTnI is normally low: (0-0.3 mu g/L), after the acute myocardial infarction occurs, the molecular weight of the compound is small, the compound is quickly released into blood, and the concentration of the compound is quickly increased. However, due to the complex components of serum samples, the detection of low abundance cTnI in real samples still lacks selectivity and specificity.
The natural antibody is widely applied to selective recognition and detection of troponin, however, the natural antibody is mainly obtained by animal immunization, has the defects of high cost, low preparation efficiency, long screening period, difficult changeability and preservation, immunogenicity and the like, and the monoclonal antibody also has the problem of large batch performance difference, and has great limitation in practical application.
Disclosure of Invention
The present invention aims to overcome the defects of the prior art and provide a preparation method and a use of a nano-artificial antibody targeting cardiac troponin I, wherein the protein-flexible polymer interaction is researched by simulating the protein-protein interaction, and the monomer sequence structure and the spatial distribution of the flexible polymer are adjusted to create the artificial antibody based on the nano-particles with the protein-like high specificity and the reversible binding property.
The technical scheme is as follows:
a method for preparing a nano artificial antibody targeting cardiac troponin I comprises the following steps: n-isopropyl acrylamide, N-tert-butyl acrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N' -methylene bisacrylamide as a cross-linking agent, cardiac troponin I holoprotein or a polypeptide fragment of the cardiac troponin I as a template molecule, and the non-biological nano artificial antibody is obtained by eluting the template molecule after polymerization under the action of an initiator.
The polypeptide fragment of the cardiac troponin I is an N-terminal polypeptide fragment or a C-terminal polypeptide fragment.
Preferably, the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinylimidazole, N- (3-dimethylaminopropyl) methacrylamide, acrylamide, (3-acrylamidopropyl) trimethylammonium chloride, N- (2-aminoethyl) acrylamide and N- (3-aminopropyl) methacrylate hydrochloride.
Preferably, N-isopropylacrylamide is used in an amount of 5 to 60 wt.%, N-tert-butylacrylamide is used in an amount of 5 to 50 wt.%, charged functional monomer is used in an amount of 0.5 to 20 wt.%, and N, N' -methylenebisacrylamide is used in an amount of 0.5 to 10 wt.%.
Preferably, the polymerization method is any one of inverse emulsion polymerization, precipitation polymerization, or radical polymerization.
Preferably, the initiator is ammonium persulfate or azobisisobutyronitrile.
Preferably, the polymerization reaction temperature under the nitrogen atmosphere is 25-70 ℃, and the polymerization reaction time is 3-36 h.
Preferably, the template molecule elution solution is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or the elution of the template molecule is realized by changing the temperature and pH value.
Preferably, the particle size of the nanoparticle artificial antibody is 10-3000 nm.
The nanoparticle artificial antibody of the cardiac troponin I prepared by the method can be used for realizing high-selectivity enrichment of low-abundance cardiac troponin I in a serum sample by combining magnetic nanoparticles, an integral column or a microfluidic chip, and realizing high-sensitivity detection of the cardiac troponin by combining a high-sensitivity Raman spectrum, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit, a test strip, an electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection methods.
The invention uses biomembrane interference technique (BLI) to determine the affinity between the nanoparticle artificial antibody and the cardiac troponin I, the biosensor fixes the cardiac troponin I, and then the affinity is determinedThe detection nano particles are placed in a detection pool, and when the interaction of the artificial antibody and the troponin I occurs, the thickness of a biological layer is increased; the binding dissociation time is used as an abscissa, the increment of the signal intensity caused by the drift of the interference curve is used as an ordinate, a standard curve is drawn, and the binding affinity K between the nanoparticle artificial antibody and the troponin I is fittedDAnd association and dissociation rates konAnd kdis。
The technical effects obtained by the invention are as follows:
(1) the cardiac troponin I nanoparticle artificial antibody obtained by the method has higher affinity and good selectivity, and can be applied to enrichment and detection of cardiac troponin I in serum instead of biological antibodies; the artificial antibody is a polymer prepared by a chemical method, has high stability, long service life and strong capability of resisting severe environment, and overcomes the defects of long preparation period, easy inactivation, high cost, immunogenicity and the like of the traditional biological antibody.
(2) The invention combines nanotechnology and biomembrane interference technology to establish a high-flux screening system for the artificial antibody of the cardiac troponin I. The method can obtain affinity K between the nanoparticles and cardiac troponin I within 20 minDAnd association and dissociation rates konAnd kdis. Greatly shortens the screening time and is also suitable for the high-throughput screening of the artificial antibodies of other antigens.
(3) The artificial antibody prepared by the method can be repeatedly used, and the cost is greatly reduced; and the synthesis process and the regeneration process are simple, and the method is suitable for detection and application of various cardiac troponins I.
(4) The artificial antibody prepared by the method has wide application, can realize high-selectivity enrichment of low-abundance cardiac troponin I in a serum sample by combining magnetic nanoparticles, an integral column or a microfluidic chip, and realizes high-sensitivity detection of the cardiac troponin by combining methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, an ELISA (enzyme-linked immunosorbent assay) kit, a chemiluminescence kit, a test strip, an electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT (computed tomography) detection, nuclear magnetic detection and the like.
Drawings
FIG. 1 is the scanning electron microscope image of nanometer artificial antibody with different particle sizes.
Fig. 2 is a real-time binding dissociation curve and its fit line between nano-artificial antibodies of different concentrations and troponin I.
FIG. 3 shows that 1/K between the imprinted polymer nanoparticle artificial antibody and the non-imprinted polymer nanoparticle and different proteinsDThe values are compared to a graph.
Fig. 4 is a scanning electron microscope image of a nano-artificial antibody monolith.
FIG. 5 is a graph comparing the fluorescence intensity of pure water and a quantum dot solution under irradiation of an ultraviolet lamp.
Fig. 6 is a transmission electron microscope image of a quantum dot.
Fig. 7 is a scanning electron microscope image of magnetic nanoparticles of a nano-artificial antibody.
Detailed Description
In order to make those skilled in the art better understand the technical scheme of the present invention, the following describes in detail the preparation method and use of a nano-artificial antibody targeting cardiac troponin I provided by the present invention with reference to the examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
The first embodiment is as follows: preparation of cardiac troponin I artificial antibody based on nanoparticle three-dimensional structure modified reconstructed recognition region
1. Design and synthesis of nanoparticles
N-isopropylacrylamide (58-X mol%), charged monomer (3-acrylamidopropyl) trimethylammonium chloride (X mol%), N-tert-butylacrylamide (35 mol%), crosslinker N, N' -methylenebisacrylamide (7 mol%) and sodium dodecylsulfonate (10mg) were dissolved in water to give a total monomer concentration of 130 mM. After the initiator was added, polymerization was carried out at 65 ℃ for 3 hours under a nitrogen atmosphere with a magnetic stirrer. The polymerized solution was purified by dialysis with an excess of pure water, and the polymer nanoparticles were obtained after freeze-drying.
2. Preliminary screening of Artificial antibodies
Select to makeTroponin I is used as a target object, molecularly imprinted polymer nanoparticles are used as bionic antibodies, the affinity between nanoparticle artificial antibodies and cardiac troponin I is determined by utilizing a biomembrane interference technology (BLI), and the biosensor is used for fixing cardiac troponin I in interaction molecules to form a biomembrane layer. The nanoparticles to be tested are then placed in a detection cell and the thickness of the biolayer increases when the interaction of the artificial antibody and troponin I takes place. The binding dissociation time is used as an abscissa, the increment of the signal intensity caused by the drift of the interference curve is used as an ordinate, a standard curve is drawn, and the binding affinity K between the nanoparticles and the troponin I is fittedDAnd association and dissociation rates konAnd kdis。
3. Method for improving artificial antibody K by molecular imprinting technologyDValue and selectivity thereof
In the preliminary screening, the protein has high K with troponinDAfter the nanoparticles are in value, in order to further increase their KDValue and selectivity, the experiment adopts a molecular imprinting method to improve the specificity and the selectivity of the artificial antibody. The synthesis of imprinted polymers (MIPs) is as follows: n-isopropylacrylamide (28 mol%), N- (3-aminopropyl) methacrylic acid (30 mol%), N-tert-butylacrylamide (35 mol%), N, N' -methylenebisacrylamide (7 mol%), sodium dodecylsulfate (10mg) and 20mg of the troponin polypeptide fragment were dissolved in water to give a total monomer concentration of 130 mM. After the initiator was added, polymerization was carried out at 45 ℃ for 12 hours under a nitrogen atmosphere with a magnetic stirrer. Then 0.04mol of NaCl is added and the stirring is continued for 30 minutes at room temperature to elute the template polypeptide, finally, the polymerized solution is purified by dialysis with excessive pure water (water is changed three times per day), and after freeze drying, the molecularly imprinted polymer nano-particles are obtained, and the scanning electron microscope picture of the nano artificial antibody with different particle diameters is shown in figure 1.
Determination of affinity constant K of molecular imprinting artificial antibody (MIP) and non-imprinting polymer (NIP) on adsorption of troponin I by biomembrane interference technology (BLI)DValue and affinity constant K for the adsorption of hetero-proteins (human serum albumin, myoglobin, fibrinogen and trypsin)DThe results are shown in fig. 2, fig. 3 and table 1, indicating that the nano-artificial antibody has high affinity and selectivity for troponin I, comparable to the natural antibody.
Table 1 shows the binding affinity constants of the imprinted polymer nanoparticle artificial antibody and the non-imprinted polymer nanoparticle for the target troponin I and the impurity interfering proteins myoglobin, human serum albumin, fibrinogen and trypsin, respectively.
TABLE 1
Example two: troponin I nano artificial antibody as ligand is combined with monolithic column stationary phase
First, it is necessary to bond a silane coupling agent to the inner wall of the capillary column. After the inner wall of the capillary column is washed clean by acetone and pure water in sequence, sodium hydroxide solution with the concentration of 1mol/L is pumped through the capillary column to fully and uniformly fill the capillary column, and then the capillary column is placed in a drying oven at 120 ℃. And then taking out the capillary column after the reaction is finished, and washing the sodium hydroxide in the capillary column by using pure water until the pH value of the pumped solution of the capillary column is neutral. The capillary column was then further rinsed with acetone and blown dry with nitrogen, and the capillary column was placed into an oven at 120 ℃ for thorough drying without sealing. 0.1g of silane coupling agent 3-methacryloxypropyltrimethoxysilane was dissolved in 0.9g of anhydrous toluene in a brown vial, and 1-2 mg of a polymerization inhibitor 2, 2-biphenyl-1-picrylhydrazino was added. After pumping it through a dry capillary column, the end of the capillary column was sealed and placed in an oven at 120 ℃ for reaction. Finally the capillary column was flushed with acetone and dried with nitrogen.
Then synthesizing a nanoparticle artificial antibody monolithic column, uniformly mixing 240mg of monomer glycidyl methacrylate, 160mg of coupling agent ethylene glycol dimethacrylate, 100mg of troponin I nanoparticle artificial antibody, pore-forming agents lauryl alcohol and cyclohexanol, and adding 4mg of initiator Azoisobutyronitrile (AIBN). The mixed solution was sonicated for 1 to 2 minutes and then purged with nitrogen, whereupon the mixed solution was pumped into a capillary column, sealed at both ends, and placed in a water bath at 60 ℃ for 24 hours. After completion of the reaction, the monolith was washed with methanol to remove the porogen and characterized by scanning electron microscopy, see fig. 4.
Example three: selective enrichment of low-abundance troponin in serum sample by using nano artificial antibody monolithic column
Pumping a serum sample containing troponin I with the concentration of 0.001-10 mu g/L into a nano artificial antibody monolithic column through a micro-injection pump at the flow rate of 0.1-0.5 mu L/min for selective enrichment, and eluting impurity protein non-specifically adsorbed on the monolithic column by using a 100mM phosphate buffer solution (pH 7) after the enrichment. And eluting the enriched troponin I, and calculating the enrichment times and the recovery rate. Finally, the obtained enrichment multiple is 500, and the recovery rate is 98%.
Example four: preparation of double-emission fluorescent quantum dot test strip based on nano artificial antibody
(1) Synthesis of blue fluorescent carbon quantum dots
Weighing 4.2g of citric acid solid in a 50mL centrifuge tube, adding 40mL of water to completely dissolve the solid, adding 1.34mL of ethylenediamine solution (the molar ratio of citric acid to ethylenediamine is 1:1), stirring to uniformly disperse the solution, transferring the solution to a polytetrafluoroethylene reaction kettle, and placing the reaction kettle in an oven at 200 ℃ for reaction for 5 hours; after the reaction is finished, cooling to room temperature, dialyzing for 1 day by using a dialysis membrane with the molecular weight cutoff of 1000Da, and collecting for later use after the dialysis is finished. The synthesized fluorescent carbon quantum dots appeared blue (fig. 5), and their transmission electron microscopy is shown in fig. 6.
(2) Synthesis of red fluorescent carbon quantum dots
Adding 0.1g of m-phenylenediamine and 1mL of ammonia water into 10mL of ethanol solution, ultrasonically stirring for 10min to dissolve the m-phenylenediamine, transferring the obtained solution into a polytetrafluoroethylene reaction kettle, placing the polytetrafluoroethylene reaction kettle into a drying oven, reacting for 6h at 180 ℃, naturally cooling to room temperature after the reaction is finished, and diluting the obtained carbon quantum dots to 909.61mg/L by using ethanol to obtain the red fluorescent quantum dots.
(3) Preparation of nano artificial antibody dual-emission fluorescent quantum dot test strip
Dissolving 4.2g of citric acid solid with 40mL of water, adding 1.34mL of ethylenediamine solution, mixing uniformly, reacting for 5h in a polytetrafluoroethylene reaction kettle at 200 ℃, cooling to room temperature after the reaction is finished, and dialyzing for 1 day by using a dialysis bag of 1000Da to obtain the carbon quantum dots. Adding 500 mu L TEOS into a mixed solution of 8.5mL of ethanol solution and 1.5mL of water, magnetically stirring for 10min, adding 3mL of synthesized blue fluorescent quantum dots (carbon quantum dots) into a reaction system, stirring for 30min, finally adding 500 mu L of ammonia water, stirring for 4h, centrifuging for 10min at 12000r/min after the reaction is finished, washing precipitates for multiple times by using ethanol and water to obtain nanoparticles with the carbon quantum dots encapsulated in silicon dioxide, wherein the silicon dioxide nanoparticles show blue fluorescence. The red quantum dots and the troponin I template are molecularly imprinted on the surface of the nano artificial antibody to form red fluorescent molecularly imprinted nanoparticles (MIP), the MIP which shows red fluorescence is wrapped on the surface of the silica nanoparticles which show blue fluorescence, then the template molecules are eluted, and due to the superposition of fluorescence, the nano artificial antibody dual-emission fluorescence quantum dots show purple fluorescence.
And then the protein to be detected is combined with the outermost cavity after being contacted with the nano artificial antibody dual-emission fluorescent quantum dot, and simultaneously, the fluorescence quenching of the outermost red fluorescent quantum dot is caused, so that the nano artificial antibody dual-emission fluorescent quantum dot is changed from purple fluorescence to blue fluorescence. The cut filter paper strip is soaked in the prepared nano artificial antibody dual-emission fluorescent quantum dot solution, then a sample to be detected is dripped on the test paper strip, and the ultraviolet lamp is used for irradiating the sample to be detected, so that the visual detection of the sample to be detected can be realized.
Example five:
an appropriate amount of (N- (3-aminopropyl) methacrylate hydrochloride) (APM) and an appropriate amount of N, N' -methylenebisacrylamide (Bis) were weighed and prepared into mixed solutions having concentrations of 19.8mg/mL and 20.0 mg/mL. 41.42mg of N-isopropylacrylamide and 10mg of sodium dodecyl sulfate are weighed into a 50mL centrifuge tube. 33.06mg of N-t-butylacrylamide was weighed out and dissolved in 1mL of ethanol, and the whole was put into a centrifuge tube. The centrifuge tube solution was made up to approximately 50mL with deionized water. And (3) adding 100 mu L of APM and Bis mixed solution into a centrifuge tube, and shaking up. The solution in 50mL centrifuge tube was added to a three-necked flask and sonicated for 30min, and its SEM image is shown in FIG. 7. 20mg of N-terminal polypeptide fragment of cardiac troponin I was added, and after adding an initiator ammonium persulfate, polymerization was carried out at 45 ℃ for 12 hours with a magnetic stirrer under nitrogen atmosphere. And then adding 0.04mol of NaCl, continuously stirring for 30 minutes at room temperature to elute the template polypeptide, finally purifying the polymerized solution by dialysis with excessive pure water (changing water three times a day), and freeze-drying to obtain the nano artificial antibody of the targeted cardiac troponin I.
And adding a proper amount of magnetic balls into the reaction solution to obtain the magnetic nano artificial antibody.
The invention discloses a preparation method and application of a nanometer artificial antibody of a targeted cardiac troponin I, wherein a plurality of functional monomers and a cross-linking agent are polymerized into a gel nanometer particle artificial antibody by different components, and the affinity and the selectivity of the artificial antibody to the cardiac troponin I are regulated and controlled by changing the proportion of the functional monomers. By combining a molecular imprinting technology and taking the whole protein or polypeptide fragment of the troponin I as a template, the specificity and the selectivity of the nano artificial antibody to the troponin I are further improved. Affinity constant K of screened nano artificial antibody to troponin IDThe value reaches 6.26 multiplied by 10-11M, is equivalent to antibody and has good selectivity. The invention provides a controllable preparation method of artificial antibody nano particles, and the artificial antibody gel nano particles with uniform particle size and controllable size and shape are obtained. The obtained nano artificial antibody can realize high-selectivity enrichment of low-abundance cardiac troponin I in a serum sample by combining with magnetic nano particles, an integral column or a microfluidic chip, and can realize high-sensitivity detection of the cardiac troponin I by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit, a test strip, an electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT detection, nuclear magnetic detection and the like.
The present invention is not limited to the above-described examples, and various changes can be made without departing from the spirit and scope of the present invention within the knowledge of those skilled in the art.
Claims (8)
1. A preparation method of a nano artificial antibody targeting cardiac troponin I is characterized in that N-isopropylacrylamide, N-tert-butylacrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N' -methylenebisacrylamide as a cross-linking agent, a cardiac troponin I holoprotein or a polypeptide fragment of the cardiac troponin I as a template molecule are polymerized and then eluted to obtain the nano artificial antibody targeting cardiac troponin I; the dosage of N-isopropyl acrylamide is 5-60 wt%, the dosage of N-tert-butyl acrylamide is 5-50 wt%, the dosage of charged functional monomer is 0.5-20 wt%, and the dosage of N, N' -methylene bisacrylamide is 0.5-10 wt%;
the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinyl imidazole, N- (3-dimethylaminopropyl) methacrylamide, (3-acrylamidopropyl) trimethyl ammonium chloride, N- (2-aminoethyl) acrylamide and N- (3-aminopropyl) methacrylate hydrochloride.
2. The method for preparing the nano artificial antibody targeting cardiac troponin I according to claim 1, wherein the polymerization process is inverse emulsion polymerization or precipitation polymerization.
3. The method for preparing the nano artificial antibody targeting the cardiac troponin I according to claim 1, wherein the initiator is ammonium persulfate or azobisisobutyronitrile.
4. The method for preparing the nano artificial antibody targeting the cardiac troponin I according to claim 2, wherein the polymerization temperature is 25 to 70 ℃ and the polymerization time is 3 to 36 hours under the nitrogen atmosphere.
5. The method for preparing the nano artificial antibody targeting the cardiac troponin I according to claim 1, wherein the elution solution of the template molecule is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or the elution of the template molecule is achieved by changing the temperature and pH value.
6. The method for preparing the nano artificial antibody targeting cardiac troponin I according to claim 1, wherein the nano artificial antibody has a particle size of 10 to 3000 nm.
7. The nano-artificial antibody targeting the cardiac troponin I, which is prepared according to any one of claims 1 to 6, is characterized in that the nano-artificial antibody targeting the cardiac troponin I is used for realizing high-selectivity enrichment of low-abundance cardiac troponin I in a serum sample by combining with magnetic nanoparticles, an integral column or a microfluidic chip, and realizing high-sensitivity detection of the cardiac troponin by combining with high-sensitivity Raman spectroscopy, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit, a test strip, an electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection methods.
8. A preparation method of a double-emission fluorescent quantum dot test strip of a nano artificial antibody targeting cardiac troponin I prepared by the method of any one of claims 1 to 6 is characterized by comprising the following specific steps:
(1) synthesis of blue fluorescent carbon quantum dots
Weighing 4.2g of citric acid solid in a 50mL centrifuge tube, adding 40mL of water to completely dissolve the solid, adding 1.34mL of ethylenediamine solution, wherein the molar ratio of the citric acid to the ethylenediamine is 1:1, stirring to uniformly disperse the citric acid and the ethylenediamine, transferring the liquid to a polytetrafluoroethylene reaction kettle, and placing the reaction kettle in an oven at 200 ℃ for reaction for 5 hours; cooling to room temperature after the reaction is finished, dialyzing for 1 day by using a dialysis membrane with the molecular weight cutoff of 1000Da, and collecting for later use after the dialysis is finished;
(2) synthesis of red fluorescent carbon quantum dots
Adding 0.1g of m-phenylenediamine and 1mL of ammonia water into 10mL of ethanol solution, ultrasonically stirring for 10min to dissolve the m-phenylenediamine, transferring the obtained solution into a polytetrafluoroethylene reaction kettle, placing the polytetrafluoroethylene reaction kettle into a drying oven, reacting for 6h at 180 ℃, naturally cooling to room temperature after the reaction is finished, and diluting the obtained carbon quantum dots to 909.61mg/L by using ethanol to obtain red fluorescent carbon quantum dots;
(3) preparation of nano artificial antibody dual-emission fluorescent quantum dot test strip
Dissolving 4.2g of citric acid solid with 40mL of water, adding 1.34mL of ethylenediamine solution, uniformly mixing, reacting for 5 hours in a polytetrafluoroethylene reaction kettle at 200 ℃, cooling to room temperature after the reaction is finished, and dialyzing for 1 day by using a dialysis bag of 1000Da to obtain carbon quantum dots; adding 500 mu L TEOS into a mixed solution of 8.5mL of ethanol solution and 1.5mL of water, magnetically stirring for 10min, adding 3mL of synthesized blue fluorescent carbon quantum dots into a reaction system, stirring for 30min, finally adding 500 mu L of ammonia water, stirring for 4h, centrifuging for 10min at 12000r/min after the reaction is finished, washing precipitates for multiple times by using ethanol and water to obtain nano particles encapsulating the carbon quantum dots in silicon dioxide, wherein the silicon dioxide nano particles show blue fluorescence; molecularly imprinting the red fluorescent carbon quantum dots and the troponin I template on the surface of the nano artificial antibody to form a molecularly imprinted nano MIP (molecular imprinted polymer) showing red fluorescence, wrapping the MIP showing red fluorescence on the surface of the silica nano particles showing blue fluorescence, eluting the template molecules, and enabling the nano artificial antibody dual-emission fluorescent quantum dots to show purple fluorescence due to the superposition of the fluorescence;
then, when the protein to be detected is contacted with the nano artificial antibody dual-emission fluorescent quantum dots, the protein to be detected can be combined with the cavity on the outermost layer, and simultaneously, the fluorescence quenching of the red fluorescent quantum dots on the outermost layer is caused, so that the purple fluorescence of the nano artificial antibody dual-emission fluorescent quantum dots is changed into blue fluorescence; the cut filter paper strip is soaked in the prepared nano artificial antibody dual-emission fluorescent quantum dot solution, then a sample to be detected is dripped on the test paper strip, and the ultraviolet lamp is used for irradiating the sample to be detected, so that the visual detection of the sample to be detected can be realized.
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| 心肌肌钙蛋白I的分子印迹电化学生物传感器;李成学 等;《生物加工过程》;20120531;第10卷(第3期);61-66 * |
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