CN113687063B - Glycoprotein dynamic light scattering immunization method based on phenylboronic acid crosslinking agent - Google Patents
Glycoprotein dynamic light scattering immunization method based on phenylboronic acid crosslinking agent Download PDFInfo
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Abstract
The invention relates to a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent, wherein a magnetic carrier marked by the phenylboronic acid cross-linking agent and an immune recognition element is used as a dynamic light scattering signal enhancement probe, average hydration kinetic particle size change of a solution before and after a target glycoprotein is used as a bridge to form a multi-layer sandwich structure is used as a dynamic light scattering signal output, and the content of the glycoprotein in a sample to be detected is measured and reacted by utilizing the hydration kinetic diameter change. The glycoprotein dynamic light scattering immunization method based on the phenylboronic acid crosslinking agent is simple in operation steps, high-sensitivity detection of glycoprotein in a complex sample can be realized in a short time, the prepared immunomagnetic beads can separate and enrich target protein from a complex sample matrix, interference of the sample matrix on subsequent detection is effectively eliminated, the problem of difficulty in preparation of a paired antibody can be solved, simple, convenient, rapid and timely detection of glycoprotein is realized, and the glycoprotein dynamic light scattering immunization method is worthy of further popularization and use.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent.
Background
In recent years, boric acid is widely applied to the construction of functional materials as an important ligand, and is widely applied to a sensor for detecting biomolecules of cis-diol, the pH switch property of boric acid affinity enables phenylboronic acid to become an important molecule for identifying and enriching substances containing the cis-diol, and the unique property of combination of the boric acid and the cis-diol enables the phenylboronic acid as an antibody substitute for biomimetic immunological analysis, so that the detection of the cis-diol substances (glycoprotein) can be realized on the basis of saving one antibody, and the requirement on antibody pairing is greatly reduced.
The dynamic light scattering technology, also known as photon correlation spectrum or quasi-elastic light scattering, is a technology widely applied to particle size and particle size distribution research, homogeneous immunosensors based on dynamic light scattering have drawn more and more attention in the detection of various chemical and biological targets due to their ultra-high sensitivity and specificity, and magnetic nano materials have become a class of nano materials which are currently researched and applied more due to their unique physical and chemical properties, and have excellent performances such as good dispersibility and enrichment capacity, good biocompatibility, strong controllability and the like, so that the dynamic light scattering technology is widely applied to immunological analysis, separation and purification of proteins and the like.
The immunoassay is one of the most widely applied glycoprotein analysis methods, at present, enzyme-linked immunosorbent assay (ELISA) has the dominant position in the detection aspect of pathogenic bacteria and protein biomarkers with the unique advantages of high flux, good robustness and the like, however, a pair of completely matched antibodies with high affinity and specificity to a target is a necessary condition for establishing a high-sensitivity and specific immunoassay method, but obtaining the highly matched antibodies has great difficulty, and in addition, the traditional sandwich ELISA uses an enzyme-catalyzed chromogenic substrate to generate a colored product as a signal output, and the sensitivity is usually between ng/mL and g/mL; meanwhile, the natural enzyme has poor storage stability and environmental tolerance, so that the development of a novel detection method which is sensitive, free of enzyme and free of antibody matching is of great significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent, which has the advantages of saving one antibody, solving the problem of difficulty in preparing a paired antibody, realizing high sensitivity and timely detection of glycoprotein, and solving the problems of complex detection operation steps, low antigen-antibody utilization and reaction efficiency, low sensitivity, unstable signals, false positive signals and the like of the traditional enzyme-linked immunosorbent assay of the main detection method of glycoprotein.
In order to achieve the purpose, the invention provides the following technical scheme: a glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent, wherein a magnetic carrier marked by the phenylboronic acid cross-linking agent and an immune recognition element is used as a dynamic light scattering signal enhancement probe, average hydration kinetic particle size change of a solution before and after a target glycoprotein is used as a bridge to form a multi-layer sandwich structure is used as a dynamic light scattering signal output, and the content of the glycoprotein in a sample to be detected in a reaction is determined by utilizing the hydration kinetic diameter change, and the method comprises the following steps:
(1) Magnetic probe: marking the surface of the carboxylated magnetic carrier by the glycoprotein specificity immune recognition element by adopting an EDC one-step method to obtain a magnetic probe marked by the specificity immune recognition element;
(2) Boric acid crosslinking agent: covalently coupling phenylboronic acid through a carrier to form a boric acid cross-linking agent complex for glycoprotein recognition;
(3) Detection of glycoprotein: adding a magnetic probe marked by a specific immune recognition element and a phenylboronic acid cross-linking agent into a target solution to be detected, reacting at 37 ℃ for 15-30min, determining the average hydration kinetic diameter of the solution on a Malvern nanometer particle size analyzer, and determining the content of glycoprotein in a sample to be detected by using the change of the hydration kinetic diameter.
Further, the method is directed to a monoclonal antibody or an aptamer, etc., which are specific for the desired glycoprotein.
Further, the magnetic carrier is Fe304 nano-microsphere, jin Ci particle, silicon-coated magnet, magnetic bead and the like.
Further, the boric acid crosslinking agent is a compound formed by connecting protein, nano particles and polymer serving as a carrier with phenylboronic acid, and the like.
Further, in the step (1), an EDC one-step method is used to label the glycoprotein-specific immune recognition element on the surface of the magnetic carrier to form a magnetic immune probe, and the specific preparation method of the carboxylated magnetic carrier labeled with the specific immune recognition element comprises the following steps: adding a magnetic carrier into a buffer solution with the pH value of 6.0-8.0PB (0.01 mol/L), adding a specific immune recognition element, stirring at room temperature for reaction, repeatedly adding 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) for 3 times, and after the reaction is finished and magnetic attraction is carried out for 5-20min, discarding the supernatant; adding acid hydrolyzed casein with mass volume fraction of 2%, adding EDC, stirring at room temperature for 1h, magnetically attracting for 5-20min, and removing supernatant; then adding 6-amino-3-pyridine phenylboronic acid with the mass volume fraction of 2%, stirring for 1h at room temperature, magnetically attracting for 5-20min, then discarding the supernatant, washing PB precipitate for 3 times, redissolving the precipitate PB7.4, and storing at 4 ℃.
Further, the step (2) of forming the boric acid cross-linking agent compound by coupling biphenyl boric acid as a carrier specifically comprises the following operations: the carboxyl phenylboronic acid or the amino phenylboronic acid is fixed on a carrier such as protein, polymer, nano particle and the like by an EDC method to form the phenylboronic acid cross-linking agent.
Further, diluting the glycoprotein to a proper concentration by PB7.5 (0.01 mol/L), adding 200 mu L of the glycoprotein into a glass tube, adding a certain amount of magnetic nanoprobe, and reacting for 5-20min at 37 ℃; and magnetically attracting for 5-20min, then removing the supernatant, ultrasonically washing for 2-3 times, then adding 800L of a phenylboronic acid cross-linking agent, ultrasonically reacting for 5-20min, measuring the change of the average hydration kinetic particle size, and further determining the content of the glycoprotein through the change of signals.
Further, the marking amount of the specific immune recognition element marked magnetic carrier is determined by the maximum variation of the average hydrated particle size of the solution.
Further, the dosage of the magnetic nanoprobe is determined by the maximum variation of the average hydrated particle size of the solution.
Further, the dosage of the phenylboronic acid crosslinking agent is determined by the maximum variation of the average hydrated particle size of the solution.
Compared with the prior art, the technical scheme of the application has the following beneficial effects:
1. according to the glycoprotein dynamic light scattering immunization method based on the phenylboronic acid crosslinking agent, the unique property of combination of boric acid and cis-diol is utilized, the identification unit phenylboronic acid crosslinking agent is designed for identification of glycoprotein cis-diol, and the unique property of combination of boric acid and cis-diol enables identification molecules phenylboronic acid to be used as an antibody substitute for biomimetic immunological analysis, so that multivalent combination and high-sensitivity detection of cis-diol substances (glycoprotein) can be realized on the basis of saving one antibody, and the requirement on antibody pairing is greatly reduced.
2. According to the glycoprotein dynamic light scattering immunization method based on the phenylboronic acid crosslinking agent, an immune probe formed by labeling a magnetic carrier by using a target protein specific immune recognition element is used as a dynamic light scattering signal enhancement probe, the phenylboronic acid crosslinking agent and the immune probe output a dynamic light scattering signal by using the average hydration kinetic particle size change of a solution before and after a multilayer sandwich structure is formed by using target glycoprotein as a bridge, the content of glycoprotein in a sample to be detected is measured and reacted by using the hydration kinetic diameter change, the operation steps are simple, the high-sensitivity detection of glycoprotein in a complex sample can be realized in a short time, the prepared immunomagnetic beads can separate and enrich target protein from the complex sample matrix, and the interference of the sample matrix on subsequent detection is effectively eliminated.
Drawings
FIG. 1 is a schematic diagram of the process of the present invention;
FIG. 2 is a standard curve of a dynamic light scattering homogeneous immunoassay of alpha-fetoprotein based on Fe304 magnetic microspheres;
FIG. 3 is a standard curve of dynamic light scattering homogeneous immunoassay of amino-terminal pro-brain natriuretic peptide based on Fe304 magnetic microspheres;
FIG. 4 is a standard curve of a dynamic light scattering homogeneous immunoassay for chorionic gonadotropin based on Fe304 magnetic microspheres.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-4, a dynamic light scattering immunization method for glycoprotein based on a phenylboronic acid cross-linking agent, wherein the phenylboronic acid cross-linking agent and a magnetic carrier marked by an immune recognition element are used as dynamic light scattering signal enhancement probes, average hydration kinetic particle size changes of a solution before and after a target glycoprotein is used as a bridge to form a "multilayer sandwich" structure are output as dynamic light scattering signals, and the content of glycoprotein in a sample to be detected is measured and reacted by using the hydration kinetic diameter changes, includes the following steps:
(1) Magnetic probe: marking the surface of the carboxylated magnetic carrier by the glycoprotein specificity immune recognition element by adopting an EDC one-step method to obtain a magnetic probe marked by the specificity immune recognition element;
(2) Boric acid crosslinking agent: covalently coupling phenylboronic acid through a carrier to form a boric acid cross-linking agent complex for identification of the glycoprotein;
(3) Detection of glycoprotein: adding a magnetic probe marked by a specific immune recognition element and a phenylboronic acid cross-linking agent into a target solution to be detected, reacting at 37 ℃ for 15-30min, determining the average hydration kinetic diameter of the solution on a Malvern nanometer particle size analyzer, and determining the content of glycoprotein in a sample to be detected by using the change of the hydration kinetic diameter.
The method aims at a specific immune recognition element monoclonal antibody or an aptamer and the like of target glycoprotein; the magnetic carrier is Fe304 nanometer microsphere, jin Ci particle, silicon coated magnet, magnetic bead, etc.; the boric acid cross-linking agent is a compound formed by connecting protein, nano particles and polymer serving as a carrier with phenylboronic acid, and the like; diluting glycoprotein with PB7.5 (0.01 mol/L) to appropriate concentration, adding 200 μ L into glass tube, adding a certain amount of magnetic nanoprobe, and reacting at 37 deg.C for 5-20min; and magnetically attracting for 5-20min, then removing the supernatant, ultrasonically washing for 2-3 times, then adding 800L of a phenylboronic acid cross-linking agent, ultrasonically reacting for 5-20min, measuring the change of the average hydration kinetic particle size, and further determining the content of the glycoprotein through the change of signals.
In the step (1), an EDC one-step method is adopted to mark the glycoprotein specificity immune recognition element on the surface of the magnetic carrier to form a magnetic immune probe, and the specific preparation method of the carboxylated magnetic carrier marked by the specificity immune recognition element comprises the following steps: adding a magnetic carrier into a buffer solution with the pH value of 6.0-8.OPB (0.01 mol/L), adding a specific immune recognition element, stirring at room temperature for reaction, repeatedly adding 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) for 3 times, magnetically attracting for 5-20min after the reaction is completed, and then removing a supernatant; adding acid hydrolyzed casein with mass volume fraction of 2%, adding EDC, stirring at room temperature for 1h, magnetically attracting for 5-20min, and removing supernatant; then adding 6-amino-3-pyridine phenylboronic acid with the mass volume fraction of 2%, stirring for 1h at room temperature, magnetically attracting for 5-20min, then discarding the supernatant, washing PB precipitate for 3 times, redissolving the precipitate PB7.4, and storing at 4 ℃.
In the preferred technical scheme: the magnetic probe marked by the immune recognition element in the step (1) adopts different marking amounts for obtaining high sensitivity, and finally the marking amount of the immune recognition element is determined by obtaining a maximum particle size increment signal through incubation with a target substance and a phenylboronic acid cross-linking agent; the final dosage of the magnetic carrier marked by the immune recognition element is determined by setting different reaction input quantities, and determining the average hydration kinetic diameter of the solution after the reaction is finished, so as to obtain the maximum particle size increase signal and determine the optimal use concentration of the magnetic nanoprobe.
Forming a boric acid cross-linking agent compound by coupling biphenyl boric acid serving as a carrier in the step (2), and specifically comprising the following operations: the carboxyl phenylboronic acid or the amino phenylboronic acid is fixed on a carrier such as protein, polymer, nano particle and the like by an EDC method to form the phenylboronic acid cross-linking agent.
And (3) determining the dosage of the phenylboronic acid cross-linking agent in the step (3) by incubating the target substance and the magnetic nano-probe to obtain a maximum particle size increase signal after determining the labeling quantity of the magnetic probe labeled by the immune recognition element and the magnetic nano-probe.
Each buffer solution and the complex solution are filtered through a 0.22 mu m filter membrane before use.
It is to be noted that the method is suitable for proteins with glycosylation, and is particularly suitable for trace detection of target analytes.
In the method, a signal output substrate is a magnetic carrier, 1) when the content of glycoprotein in a solution is zero or extremely low, an immune recognition element on the magnetic carrier cannot or cannot be combined with a target antigen, and a phenylboronic acid cross-linking agent cannot be combined with glycoprotein, so that a three-in-one multilayer sandwich structure of a magnetic nanoprobe-antigen-phenylboronic acid cross-linking agent cannot be formed, and the average hydration kinetic diameter of the solution is slightly increased or not increased compared with a blank value (the magnetic carrier marked with the immune recognition element); 2) When the content of glycoprotein in the solution is increased, the immune recognition element on the magnetic carrier is combined with a target antigen, and then the phenylboronic acid cross-linking agent can be combined with the glycoprotein, so that the proportion of a three-in-one multilayer sandwich structure formed by the magnetic nanoprobe-antigen-phenylboronic acid cross-linking agent is gradually increased, and the average hydration kinetic diameter of the solution is increased more and more than a blank value (the magnetic carrier marked with the immune recognition element); 3) Along with the change of the content of the glycoprotein, the increase of the average hydration kinetic diameter of the solution is changed linearly, and finally, the quantitative and sensitive detection of the glycoprotein is realized.
Preparation method of phosphate buffer solution (PBS, 0.05M, pH7.4): 40g of NaCl, 13.5g of Na2HP04, 1.0g of KH2P04 and 1.0g of KCl were dissolved in 1L of ultrapure water.
Example 1 application of magnetic nanospheres to detection of alpha-fetoprotein content
Preparation of 1-carboxylated Fe304 magnetic microspheres
1) Preparation of oleic acid Fe304 magnetic beads
and a, synthesizing Fe304 magnetic beads, namely adding 300mL of ultrapure water into a 500mL three-neck flask, introducing N2 to remove oxygen in water, preheating for 20min at 50 ℃, adding 3.2g of FeCl2. H20 and 5.2g of FeCl3, magnetically stirring, uniformly mixing, adding 25mL of ammonia water, quickly changing yellow solution into black, reacting for 30min at a constant temperature of 50 ℃, separating the synthesized magnetic beads through magnetic attraction, washing for 3-5 times by using the ultrapure water until the pH value of the solution is neutral after the magnetic attraction, and re-dissolving in 300mL of the ultrapure water.
b, oleic acid modification of Fe304 magnetic beads, adding the magnetic beads obtained in the previous step into a three-neck flask, introducing N2, stirring and dropwise adding 2.4mL of oleic acid, adsorbing the oil-acidified magnetic beads on the wall of a stirrer or a flask due to hydrophobic property, reacting at 70 ℃ for 3 hours, gradually clarifying black solution, stopping reaction, pouring out the solution in the beaker, adding 300mL of ethanol to elute the adsorbed oleic acid magnetic beads, magnetically adsorbing for 3 minutes to remove an ethanol solvent, and repeating the step for 4 times until the ethanol liquid level has no floating oleic acid layer.
2) Synthesis of carboxylated Fe304 magnetic microspheres
Placing 5mg polymaleic anhydride-1-octadecene amphiphilic chain and 10mg oily Fe304 magnetic beads with the particle size of about 10nm into a sample injection bottle, adding 120L chloroform, performing vortex mixing, adding 250L 10mg/mL sodium dodecyl sulfate aqueous solution, performing ultrasonic emulsification on the mixture, and setting ultrasonic parameters as follows: the power is 8%, the time is 2min, the ultrasonic treatment is carried out for 5s at an interval of 10s, after the ultrasonic emulsification is finished, the mixture is placed in a 60 ℃ oven for 4h to volatilize chloroform, centrifugation is carried out for 15min at 13500rpm, finally, the synthesized phenylboronic acid Fe304 magnetic microsphere carrier is suspended in 1mL of ultrapure water for later use, the estimated centrifugation loss rate is 20%, and therefore the concentration of the obtained phenylboronic acid Fe304 magnetic microsphere carrier is 12mg/mL.
2 preparation of Fe304 microsphere probe for detecting antibody mark
Adding 10 μ L of the synthesized carboxylated Fe304 nano-microspheres into 200 μ L of pH8.0PB, adding 3 μ g of detection antibody corresponding to alpha-fetoprotein, and stirring at room temperature for reaction for 30min; and (2) continuously stirring for reaction, adding 0.2 mu g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at room temperature for 30min, supplementing twice, magnetically attracting for 5min, then removing the supernatant, then adding acid hydrolyzed casein with the mass volume fraction of 2%, adding 0.2 mu g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at room temperature for 1h, magnetically attracting for 5min, then removing the supernatant, then adding 2% by mass of 6-amino-3-pyridylbenzoic acid, stirring at room temperature for 1h, magnetically attracting for 5min, then removing the supernatant, washing PB precipitates for 3 times, and re-dissolving the precipitated PB7.4 at 4 ℃ for storage.
Preparation of 3-phenylboronic acid cross-linking agent
The 8arm PEG 3-APBA complex was synthesized as a phenylboronic acid crosslinking agent by covalent coupling of 8arm PEG-succinimidyl glutarate and 3-aminophenyl boron hydrochloride (3-APBA), and specifically, 166.08mg of 3-APBA was added to a 4ml pb7.4 (0.01M) solution, and the pH of the solution was adjusted to alkaline, then, 800mg of 8arm PEG-succinimidyl glutarate was dissolved in 16mL pb7.4 (0.01M), and then, it was added to the above dissolved 3-APBA solution, and the reaction was stirred at room temperature for 4 hours, and after completion of the reaction, the prepared 8arm PEG @3-APBA was dialyzed in 0.01M PBS for 3 days, and finally, the obtained 8arm PEG @3-APBA was quantified and stored at 4 ℃.
4 detecting the content of alpha-fetoprotein
When the novel dynamic light scattering immunodetection method is used for detecting the content of alpha fetoprotein, the method is implemented by the following steps: the detection method of the invention is used for detecting and analyzing results.
1) Diluting alpha fetoprotein antigen positive samples which are quantified by chemiluminescence in a hospital with PBS7.4 respectively, and determining the concentration according to the required actual detection limit;
2) And placing the diluted alpha-fetoprotein antigen into a refrigerator at 4 ℃ for later use.
3) The content of glycoprotein is detected by the detection method.
4) And (6) analyzing the result.
The solutions were tested for corresponding mean hydration kinetic diameters on a malvern nano-particle size analyzer using 15 different concentrations of standards 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, 0.98, 0.49, 0.245, 0.12, 0.06, and 0pg/mL prepared as described above.
And drawing a standard curve by taking the particle size of the solution as a vertical coordinate and the alpha-fetoprotein concentration (pg/mL) as a horizontal coordinate, solving a linear equation, substituting the particle size increase value of the sample into the standard curve when detecting the actual sample, reading the concentration of the corresponding sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the alpha-fetoprotein in the sample.
Example 2 glycoprotein-alpha fetoprotein as test substance
6 mu L of alpha-fetoprotein detection antibody-labeled Fe304 microsphere probe reacts with 200 mu L of alpha-fetoprotein samples with different concentrations for 5min, the mixture is magnetically absorbed for 5min, supernatant is discarded, PB7.5 (0.01 mol/L) ultrasonic washing is carried out for 2-3 times, 800 mu L of 8arm PEG @3-APBA with 0.2mg/mL is added for redissolution and ultrasonic treatment, after incubation for 5min at 25 ℃, the change of the average hydration kinetic diameter of the solution is measured by a Malvern nanometer particle size analyzer at 25 ℃, the average value is calculated and then substituted into a standard curve to obtain the concentration of the alpha-fetoprotein in a sample to be detected, and the specific experimental result is as follows: the linear standard curve is y =19.68ln (x) +246, r =0.9875, see fig. 2, and the minimum detection limit of the method is defined as the mean hydrated particle size at 20 first standards (the mean hydrated particle size of the solution at 0 standard time) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the required antigen concentration, calculated from the standard curve as the minimum detection line of 0.33pg/mL.
Example 3 application of gold magnetic microspheres to detection of alpha-fetoprotein content
1 preparation of gold magnetic Probe labeled with detection antibody
Adding 10 μ L Jin Ci nanoparticles (10 mg/mL) into 200 μ L pH8.0PB, adding 4 μ g detection antibody corresponding to alpha-fetoprotein, and stirring at room temperature for reaction for 30min; and (2) continuously stirring for reaction, adding 0.2 mu g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at room temperature for 30min, supplementing twice, magnetically attracting for 5min after the reaction is finished, discarding the supernatant, then adding acid hydrolyzed casein with the mass volume fraction of 2%, adding 0.2 mu g of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at room temperature for 1h, magnetically attracting for 5min, discarding the supernatant, then adding 6-amino-3-pyridylbenzoic acid with the mass volume fraction of 2%, stirring at room temperature for 1h, magnetically attracting for 5min, discarding the supernatant, washing PB precipitates for 3 times, and redissolving the precipitates PB7.4 at 4 ℃ for storage.
2 detecting the content of alpha-fetoprotein
When the novel dynamic light scattering immunodetection method is used for detecting the content of alpha fetoprotein, the novel dynamic light scattering immunodetection method is implemented through the following steps: the detection method of the invention is used for detecting and analyzing results.
1) Respectively diluting alpha fetoprotein antigen positive samples which are quantified by chemiluminescence in a hospital by PBS7.4, and determining the concentration according to the required actual detection limit;
2) And placing the diluted alpha-fetoprotein antigen into a refrigerator at 4 ℃ for later use.
3) The content of glycoprotein is detected by the detection method.
4) And (6) analyzing the result.
The solutions were tested for corresponding mean hydration kinetic diameters on a malvern nano-particle size analyzer using 15 different concentrations of standards 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, 0.98, 0.49, 0.245, 0.12, 0.06, and 0pg/mL prepared as described above.
And drawing a standard curve by taking the particle size of the solution as a vertical coordinate and the alpha-fetoprotein concentration (pg/mL) as a horizontal coordinate, solving a linear equation, substituting the particle size increase value of the sample into the standard curve when detecting the actual sample, reading the concentration of the corresponding sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the alpha-fetoprotein in the sample.
Example 4 glycoprotein-human chorionic gonadotropin as test substance
6 mu L of Fe304 microsphere probe marked by human chorionic gonadotropin detection antibody reacts with 200 mu L of human chorionic gonadotropin with different concentrations for 5min, the magnetic attraction is carried out for 5min, supernatant is discarded, PB7.5 (0.01 mol/L) ultrasonic washing is carried out for 2-3 times, then 800 mu L0.2mg/mL of 8arm PEG @3-APBA is added for redissolution and ultrasonic treatment, after incubation for 5min at 25 ℃, the average hydration kinetic diameter change of the solution is measured by a Malvern nanometer particle size analyzer at 25 ℃, the average value is calculated and then substituted into a standard curve to obtain the concentration of the human chorionic gonadotropin a sample to be detected, and the specific experimental result is as follows: the linear standard curve is y =11.774ln (x) +252.1, r =0.9845, see fig. 2, and the minimum detection limit of the method is defined as the average hydrated particle size at 20 first standards (0 standard time solution average hydrated particle size) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), and the required antigen concentration is calculated by the standard curve as 0.15mlU/mL for the minimum detection line.
Example 5 glycoprotein-amino terminal pro-brain natriuretic peptide as test substance
6 mu L of amino-terminal pro-brain natriuretic peptide detection antibody-labeled Fe304 microsphere probe reacts with 200 mu L of amino-terminal pro-brain natriuretic peptide with different concentrations for 5min, the mixture is magnetically absorbed for 5min, supernatant is discarded, PB7.5 (0.01 mol/L) ultrasound is used for washing for 2-3 times, 800 mu L of 8arm PEG @3-APBA with 0.2mg/mL is added for redissolution and ultrasound, after incubation for 5min at 25 ℃, the average hydration kinetic diameter change of the solution is measured by a Malvern nanometer particle size analyzer at 25 ℃, the average value is calculated and then substituted into a standard curve to obtain the concentration of the amino-terminal pro-brain natriuretic peptide in a sample to be detected, and the specific experimental results are as follows: the linear standard curve is y =15.608ln (x) +284.91, r =0.9881, see fig. 2, and the minimum detection limit of the method is defined as the average hydrated particle size at 20 first standards (0 standard time solution average hydrated particle size) plus 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), and the required antigen concentration, calculated from the standard curve, is 14fg/mL for the minimum detection line.
Example 6 Synthesis of BSA @ CPBA as a Benzenboronic acid crosslinker
Coupling of Bovine Serum Albumin (BSA) and Carboxyphenylboronic acid (CPBA) was synthesized by the EDC method by first dissolving 31.43mg of CPBA in 1.0mL of N, N-Dimethylformamide (DMF), adding 108.92mg of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 130.8mg of N-hydroxysuccinimide (NHS) and stirring at room temperature for 6h, then dissolving 250mg of BSA in 10mL of PB7.5 (0.01M) and then adding the above activated CPBA solution, stirring overnight at room temperature to adjust the pH to 7.5, and finally dialyzing the prepared BSA @ CPBA in 0.01M PBS for 3 days and storing at 4 ℃ for use.
Example 7 Synthesis of CuNCs @ MBA as a Benzoboronic acid crosslinker
CuNCs was synthesized according to the previously reported method by first dissolving 32mg of mercaptophenylboronic acid (MBA) in a mixed solution containing 7.0mL of DMF and 1.0mL of deionized water, sonicating for 40min, filtering the solution with a 0.22 μm filter, then dissolving 5.0mg of CuS04 in 0.5mL of ultrapure water, adding to the dissolved MBA solution, stirring at room temperature for 3 hours, and storing the synthesized CuNCs @ PBA at 4 ℃ for use.
Example 8 Synthesis of Si02@3-APBA as a Benzoboronic acid crosslinker
The Si02@3-APBA complex was synthesized by covalent coupling of Si02 and 3-aminobenzeneborate (3-APBA), 200. Mu.L of 10mg/mL SiO2 was dissolved in 2.0mL of PB 6.0 (0.01M), followed by addition of 150L of 50mg/mL of 3-APBA and adjustment of pH to 6.0, stirring at room temperature for 30min, followed by addition of 25g of EDC to the above solution and repetition of 3 times, after completion of the reaction, centrifugation at 15000rpm for 15min, followed by washing twice with PB7.4 (0.01M), and finally the precipitate was resuspended in 500. Mu.L of PB pH7.4 (0.01M) and stored at 4 ℃ for use.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A glycoprotein dynamic light scattering immunization method based on a phenylboronic acid cross-linking agent is characterized in that a magnetic carrier marked by the phenylboronic acid cross-linking agent and an immune recognition element is used as a dynamic light scattering signal enhancement probe, average hydration kinetic particle size change of a solution before and after a target glycoprotein is used as a bridge to form a multi-layer sandwich structure is used as a dynamic light scattering signal to be output, the method for determining the content of glycoprotein in a sample to be detected by using hydration kinetic diameter change comprises the following steps:
(1) Magnetic probe: marking the surface of the carboxylated magnetic carrier by the glycoprotein specificity immune recognition element by adopting an EDC one-step method to obtain a magnetic probe marked by the specificity immune recognition element;
(2) Boric acid crosslinking agent: covalently coupling phenylboronic acid through a carrier to form a boric acid cross-linking agent complex for glycoprotein recognition; the boric acid crosslinking agent is a compound formed by connecting protein, nano particles or polymer serving as a carrier with phenylboronic acid;
(3) Detection of glycoprotein: adding a magnetic probe marked by a specific immune recognition element and a phenylboronic acid cross-linking agent into a target solution to be detected, reacting at 37 ℃ for 15-30min, determining the average hydration kinetic diameter of the solution on a Malvern nanometer particle size analyzer, and determining the content of glycoprotein in a sample to be detected by using the change of the hydration kinetic diameter;
in the step (1), an EDC one-step method is adopted to mark a glycoprotein specificity immune recognition element on the surface of a magnetic carrier to form a magnetic immune probe, and the specific preparation method of the carboxylated magnetic carrier marked by the specificity immune recognition element comprises the following steps: adding a magnetic carrier into a buffer solution of pH6.0-8.0PB, adding a specific immune recognition element, stirring at room temperature for reaction, repeatedly adding 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for 3 times, magnetically attracting for 5-20min after the reaction is completed, and then removing a supernatant; then adding acid hydrolyzed casein with the mass volume fraction of 2%, adding EDC, stirring for 1h at room temperature, magnetically attracting for 5-20min, and then discarding the supernatant; then adding 2% mass volume fraction 6-amino-3-pyridine phenylboronic acid, stirring at room temperature for 1h, magnetically attracting for 5-20min, removing supernatant, washing PB precipitate for 3 times, redissolving the precipitate PB7.4, and storing at 4 ℃; the concentration of the PB buffer solution is 0.01mol/L;
the step (2) of forming the boric acid cross-linking agent compound by coupling biphenyl boric acid as a carrier specifically comprises the following operations: fixing carboxyl phenylboronic acid or aminophenylboronic acid on protein, polymer or nano-particle by an EDC method to form a phenylboronic acid cross-linking agent;
diluting the glycoprotein to a proper concentration by using PB7.5, adding 200 mu L of the glycoprotein into a glass tube, adding a certain amount of magnetic nano probe, and reacting for 5-20min at 37 ℃; magnetically attracting for 5-20min, then discarding the supernatant, ultrasonically washing for 2-3 times, then adding 800L of phenylboronic acid cross-linking agent, ultrasonically reacting for 5-20min, measuring the average hydration kinetic particle size change, and further determining the content of glycoprotein through the change of signals; the concentration of the PB7.5 is 0.01mol/L.
2. The dynamic light scattering immunization method of glycoproteins based on phenylboronic acid cross-linking agent according to claim 1, wherein: the method is directed to a monoclonal antibody or aptamer specific for the glycoprotein of interest.
3. The dynamic light scattering immunization method of glycoproteins based on phenylboronic acid cross-linking agent according to claim 1, wherein: the magnetThe sexual carrier is Fe 3 O 4 Nano-microspheres, jin Ci particles, silicon-coated magnets or magnetic beads.
4. The dynamic light scattering immunization method of glycoproteins based on phenylboronic acid cross-linking agent according to claim 3, wherein: and determining the labeling quantity of the specific immune recognition element labeled magnetic carrier through the maximum variation of the average hydrated particle size of the solution.
5. The dynamic light scattering immunization method of glycoproteins based on phenylboronic acid cross-linking agent according to claim 3, wherein: and determining the dosage of the magnetic nanoprobe according to the maximum variation of the average hydrated particle size of the solution.
6. The dynamic light scattering immunization method of glycoproteins based on phenylboronic acid cross-linking agent according to claim 3, wherein: the dosage of the phenylboronic acid crosslinking agent is determined by the maximum variation of the average hydrated particle size of the solution.
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