CN114354525B - Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin for non-diagnostic purpose - Google Patents
Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin for non-diagnostic purpose Download PDFInfo
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Abstract
The invention provides an alpha-bungarotoxin detection probe and a method for detecting alpha-bungarotoxin in a non-diagnostic purpose, belonging to the technical field of biological sensing. The invention takes the hydrogen bond organic framework material formed by 1,3,6, 8-tetra (4-carboxybenzene) pyrene as the enzyme material for catalyzing the color development of the color development agent, has rich carboxyl on the surface, can be chemically combined with the nucleic acid aptamer of the alpha-bungarotoxin modified with amino through CO-NH bond, and improves the stability of the probe. The invention provides a method for detecting alpha-bungarotoxin without diagnosis purpose, which utilizes an alpha-bungarotoxin detection probe to specifically identify the alpha-bungarotoxin and catalyze chromogenic substrates to develop color, and the absorbance detected by ultraviolet analysis is enhanced along with the increase of the concentration of the alpha-bungarotoxin and is between 0.0001 and 316ng mL ‑1 Has good linear relation in the concentration range of the alpha-bungarotoxin.
Description
Technical Field
The invention relates to the technical field of biological sensing, in particular to an alpha-bungarotoxin detection probe and a method for detecting alpha-bungarotoxin by non-diagnostic purposes.
Background
The bungarous is the fourth biggest venomous snake on land and is also the most toxic snake in China, and more than ten people can die due to 1mg toxin of one adult bungarous. The toxin contained in the bungarose venom mainly comprises proteins and polypeptides, and the main components are neurotoxin, including alpha-bungarotoxin (alpha-BGT), beta-bungarotoxin (beta-BGT), kappa-bungarotoxin (kappa-BGT), gamma-bungarotoxin (gamma-BGT), phospholipase A and other enzymes. When bitten by bungarous, the user does not feel pain and sleeps, and when the user is slightly poisoned, the user can paralyze the body part, if toxin acts on the neuromuscular junction, the nerve conduction route can be blocked, so that the striated muscles cannot shrink normally, respiratory paralysis is caused, and the acting time is about 40 minutes to 2 hours, or up to 24 hours. The mortality rate of bungaromas bites is extremely high before the application of antivenin.
In the past decades, the detection of snake venom has evolved greatly, creating a number of methods for detecting snake venom. Such as radioimmunoassay, immunoelectrophoresis, fluoroimmunoassay, enzyme-linked immunosorbent assay, etc. Among them, enzyme-linked immunosorbent assay (ELISA) has the advantages of high specificity, rapidness and convenience, and has been widely used. The enzyme-linked immunosorbent assay requires the use of toxin secondary antibody-natural enzyme as a probe conjugate to recognize the toxin and color the color-developing agent, but the existing natural enzymes such as horseradish peroxidase, laccase and phosphoalkaline lipase have the defects of poor stability and weak catalytic activity when used for ELISA detection.
The existing nano enzyme such as ferroferric oxide, single-atom iron nitrogen carbon and the like has the outstanding advantages of good stability, low cost, strong catalytic activity, strong affinity to a substrate and the like. Based on the excellent performance of the nano-enzyme, the nano-enzyme is used as a powerful substitute for natural enzyme in the traditional enzyme-linked immunosorbent assay. However, the existing nano-enzyme is easy to agglomerate in water, so that the application of the nano-enzyme in a biosensor is limited.
Disclosure of Invention
In view of the above, the present invention aims to provide an alpha-bungarotoxin detection probe, a method for detecting alpha-bungarotoxin for non-diagnostic purposes. The alpha-bungarotoxin detection probe provided by the invention has good dispersibility.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an alpha-bungarotoxin detection probe, which comprises an alpha-bungarotoxin nucleic acid aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin nucleic acid aptamer;
the alpha-bungarotoxin aptamer is modified with amino;
the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxybenzene) pyrene.
Preferably, the mass ratio of the alpha-bungarotoxin aptamer to the hydrogen bond organic framework material is 1:50-1000.
The invention provides a preparation method of the alpha-bungarotoxin detection probe, which comprises the following steps:
providing a hydrogen bond organic framework material with an organic ligand of 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and activating carboxyl to obtain a hydrogen bond organic framework material with activated carboxyl;
and mixing the hydrogen bond organic framework material with activated carboxyl and the nucleic acid aptamer of the alpha-bungarotoxin modified with amino, and carrying out chemical combination to obtain the alpha-bungarotoxin detection probe.
Preferably, the mass ratio of the hydrogen bond organic framework material of the activated carboxyl to the nucleic acid aptamer of the alpha-bungarotoxin modified with the amino is 300-500:1.
The invention provides a kit for detecting alpha-bungarotoxin, which comprises the alpha-bungarotoxin detection probe, an alpha-bungarotoxin primary antibody, non-specific proteins and a chromogenic substrate.
Preferably, the chromogenic substrate is 3,3', 5' -tetramethylbenzidine.
The invention provides a method for detecting alpha-bungarotoxin for non-diagnostic purposes, which comprises the following steps:
first incubating the alpha-bungarotoxin primary antibody to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unbound material to obtain a second incubation product;
adding a sample to be detected into the second incubation product, performing third incubation, and removing unbound materials to obtain a third incubation product;
adding the alpha-bungarotoxin detection probe into the third incubation product, performing fourth incubation, and removing unbound materials to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product to perform a chromogenic reaction, and testing the absorbance peak value of the chromogenic solution at 500-800 nm;
obtaining the concentration of the alpha-bungarotoxin in the sample to be detected according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the concentration of the alpha-bungarotoxin and the absorbance peak value.
Preferably, the color reaction is performed in an acetate buffer.
Preferably, the color reaction is performed under normal light conditions or 365nm laser irradiation.
Preferably, the time of the color reaction is 10 to 60 minutes.
The invention provides an alpha-bungarotoxin detection probe, which comprises an alpha-bungarotoxin nucleic acid aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin nucleic acid aptamer; the alpha-bungarotoxin aptamer is modified with amino; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxybenzene) pyrene. The hydrogen bond organic framework material formed by 1,3,6, 8-tetra (4-carboxybenzene) pyrene has rich carboxyl on the surface, can be chemically combined with the nucleic acid aptamer of the alpha-bungarotoxin modified with amino through CO-NH bond, and improves the stability of the probe. Meanwhile, the hydrogen bond organic framework material is easy to disperse in water, has good dispersion stability, avoids the defect of agglomeration of the existing metal-based nano enzyme in water, and has the advantages of high biocompatibility and low toxicity; compared with the alpha-bungarotoxin secondary antibody, the nucleic acid aptamer of the alpha-bungarotoxin modified with amino has the advantages of high stability and low production cost.
The invention provides a kit for detecting alpha-bungarotoxin, which comprises an alpha-bungarotoxin detection probe, an alpha-bungarotoxin primary antibody, non-specific proteins and a chromogenic substrate. In the invention, the alpha-bungarotoxin first antibody, the alpha-bungarotoxin to be detected and the alpha-bungarotoxin detection probe can form an antibody-antigen-antibody sandwich type biosensor, wherein the alpha-bungarotoxin detection probe can specifically identify the alpha-bungarotoxin and has good enzyme activity, can catalyze a chromogenic substrate to develop color, and can realize detection of the alpha-bungarotoxin.
The invention provides a method for detecting alpha-silver ring for non-diagnostic purposeThe invention uses the alpha-bungarotoxin detection probe to specifically identify the alpha-bungarotoxin and catalyze the chromogenic substrate to develop color, the absorbance detected by ultraviolet analysis is enhanced along with the increase of the concentration of the alpha-bungarotoxin and is between 0.0001 and 316ng mL -1 Has good linear relation in the concentration range of the alpha-bungarotoxin, and the lowest detection lower limit is 0.033fg/mL. The detection method provided by the invention has high stability and strong anti-interference capability, and visual alpha-bungarotoxin detection can be realized only by a one-step color development method.
Drawings
FIG. 1 is an X-ray diffraction pattern of HOF obtained in example 1;
FIG. 2 is the H obtained in example 1 4 Infrared spectrograms of TBAPy and HOF;
FIG. 3 is a transmission electron microscope image of the HOF obtained in example 1;
FIG. 4 is a graph showing the results of the anti-interference test of the colorimetric immunosensor obtained in example 2;
FIG. 5 is a graph showing the absorption spectrum of ultraviolet and visible light of the α -BGT of example 3 at different concentrations;
FIG. 6 shows the standard curves obtained in example 3 and example 4;
FIG. 7 is a graph showing the absorption spectrum of ultraviolet and visible light of the α -BGT of example 4 at different concentrations.
Detailed Description
The invention provides an alpha-bungarotoxin detection probe, which comprises an alpha-bungarotoxin nucleic acid aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin nucleic acid aptamer; the alpha-bungarotoxin aptamer is modified with amino; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxybenzene) pyrene. In the present invention, the alpha-bungarotoxin detection probe is abbreviated as HOF@NH 2 -α-BGT-apt。
In the invention, the mass ratio of the alpha-bungarotoxin aptamer to the hydrogen bond organic framework material is preferably 1:50-1000, more preferably 1:200-800, and even more preferably 1:400-600.
In the present invention, the amino group-modified nucleic acid aptamer of alpha-bungarotoxinThe source of (c) is preferably commercially available. As a specific example of the present invention, the amino-modified aptamer to α -bungarotoxin was purchased from Beijing engine biotechnology Co. In the invention, the amino group-modified nucleic acid aptamer of the alpha-bungarotoxin has the following sequence number: 5-GCGAGGTGTTCGAGAGTTAGGGGCGACATGACCAAACGTT-3, modified at the 5-terminus with NH 2 。
The source of the hydrogen bond organic framework material is not particularly required, and the hydrogen bond organic framework material of which the organic ligand is 1,3,6, 8-tetra (4-carboxybenzene) pyrene conventionally and commercially available in the field can be used or prepared by self. When the hydrogen bond organic framework material is prepared by itself, the preparation method preferably includes:
under an alkaline environment, mixing 4-methoxycarbonyl phenylboronic acid, 1,3,6, 8-tetrabromopyrene and a catalyst, and performing a coupling reaction to obtain 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene;
in an alkaline environment, carrying out hydrolysis reaction on the 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene, and acidizing to obtain 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
and (3) performing self-assembly reaction on the 1,3,6, 8-tetra (4-carboxybenzene) pyrene to obtain a hydrogen bond organic framework material (HOF).
In an alkaline environment, 4-methoxycarbonyl phenylboronic acid, 1,3,6, 8-tetrabromopyrene and a catalyst are mixed for coupling reaction, so that 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene is obtained. In the present invention, the molar ratio of the 4-methoxycarbonylphenylboronic acid to the 1,3,6, 8-tetrabromopyrene is preferably 1 to 2:1, more preferably 1.5 to 1.75:1. In the present invention, the coupling reaction is preferably carried out in an organic solvent, preferably dioxane.
The present invention preferably uses potassium carbonate to provide the alkaline environment. In the present invention, the catalyst is preferably tetrakis (triphenylphosphine) palladium, and the mass ratio of 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene to the catalyst is preferably 50:1.
In the present invention, the temperature of the coupling reaction is 25 to 100 ℃, more preferably 50 to 75 ℃; the time is preferably 24 to 96 hours, more preferably 48 to 72 hours.
After the coupling reaction, the present invention preferably performs a post-treatment on the resulting coupling reaction solution. In the present invention, the post-treatment preferably includes the steps of:
mixing the coupling reaction liquid with a quenching agent, and performing a quenching reaction to obtain a quenching reaction liquid;
and extracting an organic phase, drying and removing an organic solvent from the quenching reaction liquid in sequence to obtain the pure 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene.
In the invention, the quenching agent is preferably a mixed solution of ice water and concentrated hydrochloric acid, and the volume ratio of the ice water to the concentrated hydrochloric acid is preferably 3:1.
In the present invention, the extractant used for the extraction is preferably chloroform. In the present invention, the drying agent used for drying is preferably magnesium sulfate. The invention preferably removes the organic solvent by means of vacuum drying.
In the present invention, the reaction process of the coupling reaction is shown in formula 1.
After the 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene is obtained, the 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene is subjected to hydrolysis reaction in an alkaline environment, and the 1,3,6, 8-tetra (4-carboxybenzene) pyrene is obtained after acidification. The alkaline environment is preferably provided by KOH in the present invention. In the present invention, the hydrolysis reaction is preferably carried out in a mixed solution of tetrahydrofuran, dioxane and water, and the volume ratio of tetrahydrofuran, dioxane and water in the mixed solution is preferably 5:2:2.
In the present invention, the temperature of the hydrolysis reaction is preferably 25 to 100 ℃, more preferably 50 to 75 ℃; the time is preferably 2 to 36 hours, more preferably 12 to 24 hours.
After the hydrolysis reaction, the organic solvent in the hydrolysis reaction liquid is preferably removed, and the mode of removing the organic solvent is preferably vacuum drying.
In the present invention, the acidification comprises:
the resulting hydrolysis reaction product was dissolved in water and the pH was adjusted to 2 using concentrated HCl.
After acidification, the obtained acidified product is preferably washed and dried in sequence to obtain the pure 1,3,6, 8-tetra (4-carboxybenzene) pyrene product.
In the invention, the preparation process of the 1,3,6, 8-tetra (4-carboxybenzene) pyrene is shown as a formula 2.
After the 1,3,6, 8-tetra (4-carboxylbenzene) pyrene is obtained, the 1,3,6, 8-tetra (4-carboxylbenzene) pyrene is subjected to self-assembly reaction, and a hydrogen bond organic framework material is obtained. In the present invention, the self-assembly reaction is preferably performed in a mixed solution of N, N-dimethylformamide and methanol. In the invention, the methanol can activate the pore canal of 1,3,6, 8-tetra (4-carboxybenzene) pyrene.
In the present invention, the temperature of the self-assembly reaction is preferably room temperature, and the time is preferably 12 hours.
After the self-assembly reaction, the invention preferably uses ethanol and acetone to wash and purify the obtained product.
The invention provides a preparation method of the alpha-bungarotoxin detection probe, which comprises the following steps:
providing a hydrogen bond organic framework material with an organic ligand of 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and activating carboxyl to obtain a hydrogen bond organic framework material with activated carboxyl;
and mixing the hydrogen bond organic framework material with activated carboxyl and the nucleic acid aptamer of the alpha-bungarotoxin modified with amino, and carrying out chemical combination to obtain the alpha-bungarotoxin detection probe.
In the invention, the sources of the hydrogen bond organic framework material of which the organic ligand is 1,3,6, 8-tetra (4-carboxybenzene) pyrene are the same as the above, and the detailed description is omitted.
The hydrogen bond organic framework material is mixed with a carboxyl activating agent for carboxyl activation, so that the hydrogen bond organic framework material for activating carboxyl is obtained. In the invention, the carboxyl activating agent is preferably a mixed solution of NHS and EDC, and the mass ratio of NHS to EDC in the mixed solution is preferably 1:1.
In the present invention, the temperature of the carboxyl group activation is preferably room temperature, and the time is preferably 30 to 150min, more preferably 120min.
After the carboxyl is activated, the invention mixes the hydrogen bond organic framework material of the activated carboxyl and the nucleic acid aptamer of the alpha-bungarotoxin modified with amino, and carries out chemical combination to obtain the alpha-bungarotoxin detection probe. In the invention, the mass ratio of the hydrogen bond organic framework material for activating carboxyl to the nucleic acid aptamer of the alpha-bungarotoxin modified with amino is preferably 300-500:1, and more preferably 500:1, 450:1, 400:1, 350:1 or 300:1.
In the present invention, the mixing means is preferably stirring mixing. In the present invention, the temperature of the chemical bonding is preferably room temperature, and the time is preferably 6 to 24 hours, more preferably 12 hours. In the chemical bonding process, the carboxyl of the hydrogen bond organic framework material of the activated carboxyl reacts with the nucleic acid aptamer of the alpha-bungarotoxin modified with the amino to generate a CO-NH bond.
After the chemical combination, the invention preferably carries out centrifugation and washing on the obtained product to obtain the alpha-bungarotoxin detection probe.
In the present invention, the rate of centrifugation is preferably 8000rpm and the time is preferably 15min. In the present invention, the washing detergent is preferably a phosphate buffer solution having ph=7.2, and the number of times of washing is preferably 3.
The invention provides a kit for detecting alpha-bungarotoxin, which comprises the alpha-bungarotoxin detection probe, an alpha-bungarotoxin primary antibody, non-specific proteins and a chromogenic substrate.
The present invention is not particularly limited to the above-mentioned primary antibody against α -bungarotoxin, and may be carried out using a conventional primary antibody against α -bungarotoxin commercially available in the art. In the present invention, the concentration of the α -bungarotoxin primary antibody is preferably 1 μg/mL.
In the present invention, the nonspecific protein is preferably bovine serum albumin.
In the present invention, the chromogenic substrate is preferably 3,3', 5' -tetramethylbenzidine. In the present invention, the concentration of 3,3', 5' -tetramethylbenzidine is preferably 1 to 25mM, more preferably 10mM.
In the present invention, the kit for detecting α -bungarotoxin preferably further comprises a buffer solution and a washing solution. In the present invention, the buffer solution is preferably an acetic acid buffer solution, and the pH value of the acetic acid buffer solution is preferably 4. In the present invention, the washing solution is preferably a phosphate buffer solution containing tween-20, the content of tween-20 is preferably 0.05wt%, and the pH value of the phosphate buffer solution is preferably 7.2.
The invention provides a method for detecting alpha-bungarotoxin for non-diagnostic purposes, which comprises the following steps:
first incubating the alpha-bungarotoxin primary antibody in an incubator to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unbound material to obtain a second incubation product;
adding a sample to be detected into the second incubation product, performing third incubation, and removing unbound materials to obtain a third incubation product;
adding an alpha-bungarotoxin detection probe into the third incubation product, performing fourth incubation, and removing unbound materials to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product to perform a chromogenic reaction, and testing the absorbance peak value of the chromogenic solution at 500-800 nm;
obtaining the concentration of the alpha-bungarotoxin in the sample to be detected according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the concentration of the alpha-bungarotoxin and the absorbance peak value.
The invention carries out first incubation on the alpha-bungarotoxin primary antibody in an incubator to obtain a first incubation product. In the present invention, the incubator is preferably a 96-well elisa plate. In the present invention, the concentration of the α -bungarotoxin primary antibody is preferably 1 μg/mL.
In the present invention, the temperature of the first incubation is preferably 4℃and the time is preferably 8 to 12 hours.
After the first incubation product is obtained, non-specific proteins are added into the first incubation product, second incubation is carried out, and unbound materials are removed, so that a second incubation product is obtained. In the present invention, the nonspecific protein is preferably bovine serum albumin. In the present invention, the concentration of the nonspecific protein is preferably 1wt%. In the present invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 15 to 120min, more preferably 60min.
In the present invention, the manner of removing unbound material is preferably washing with a phosphate buffer containing tween-20. The present invention uses the non-specific protein to block unbound primary antibodies.
After the second incubation product is obtained, adding a sample to be detected into the second incubation product, performing third incubation, and removing unbound materials to obtain a third incubation product. In the present invention, the sample to be tested is preferably a serum sample. In the present invention, the temperature of the third incubation is preferably room temperature, and the time is preferably 1h.
In the present invention, the manner of removing unbound material is preferably washing with a phosphate buffer containing tween-20.
After the third incubation product is obtained, adding an alpha-bungarotoxin detection probe into the third incubation product, performing fourth incubation, and removing unbound materials to obtain a fourth incubation product. In the present invention, the temperature of the fourth incubation is preferably room temperature and the time is preferably 1h. In the present invention, the manner of removing unbound material is preferably washing with a phosphate buffer containing tween-20.
After the fourth incubation product is obtained, a chromogenic substrate is added into the fourth incubation product to carry out a chromogenic reaction, and the absorbance peak value of the chromogenic liquid at 500-800 nm is tested. In the present invention, the chromogenic substrate is preferably 3,3', 5' -tetramethylbenzidine. In the present invention, the color reaction is preferably performed in an acetic acid buffer solution. In the present invention, the color reaction is preferably carried out under normal light conditions or 365nm laser irradiation. In the present invention, the time of the color reaction is preferably 10 to 60 minutes, more preferably 30 minutes. The invention uses an ultraviolet spectrophotometer to test absorbance.
After the absorbance peak value is obtained, the concentration of the alpha-bungarotoxin in the sample to be detected is obtained according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the concentration of the alpha-bungarotoxin and the absorbance peak value.
As a specific embodiment of the present invention, the method for drawing the standard curve preferably includes the following steps:
standard solutions of alpha-bungarotoxin are provided in gradient concentrations including 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 316ng/mL.
The method comprises the steps of taking a standard substance solution of alpha-bungarotoxin with gradient concentration as a sample to be tested, testing according to the detection method of the invention, obtaining an absorbance peak value corresponding to the standard substance solution of alpha-bungarotoxin with gradient concentration, drawing by taking the logarithm of the alpha-bungarotoxin as an abscissa and taking the absorbance value as an ordinate, and obtaining a linear relation curve of the logarithm of the beta-bungarotoxin concentration and the absorbance value.
Specifically, when the color reaction is performed under normal light conditions, the relevant data of the standard curve are shown in table 1.
TABLE 1 Standard Curve for color development under Normal light conditions
The relevant data for the standard curve are shown in Table 2 when the color reaction is carried out under 365nm laser irradiation.
TABLE 2 Standard Curve for color development under 365nm laser irradiation
In the invention, the lower detection limit of the alpha-bungarotoxin is 0.033fg mL -1 (S/N=3), the detection range is 0.0001-316ng mL -1 。
The following examples are provided to illustrate a probe for detecting alpha-bungarotoxin and a method for detecting alpha-bungarotoxin for non-diagnostic purposes, but are not to be construed as limiting the scope of the invention.
Example 1
Preparation of alpha-bungarotoxin detection probes
(1) First, 5g of 4-methoxycarbonylphenylboronic acid, 2.85g of 1,3,6, 8-tetrabromopyrene, 0.1g of tetrakis (triphenylphosphine) palladium and 6g of potassium carbonate were dissolved in 100mL of dioxane, under N 2 Stirring at 85deg.C under protection for 72 hr. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v=3:1), extracting with chloroform to collect an organic phase, finally drying with magnesium sulfate and removing the organic solvent by vacuum drying to obtain a product of 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene;
(2) 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene was first dissolved in 100mL of a mixed solution of tetrahydrofuran/dioxane/water (v/v=5:2:2), then 1g KOH was added, and the mixture was stirred at 85℃under reflux for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL H was added 2 O was stirred at room temperature for 2h. The pH was adjusted to 2 with concentrated HCl. The yellow solid obtained is collected by filtration, washed with water for a plurality of times and dried in vacuum to obtain 1,3,6, 8-tetra (4-carboxybenzene) pyrene (H) 4 TBAPy)；
(3) 150mg of 1,3,6, 8-tetra (4-carboxybenzene) pyrene is dissolved in 22.5mL of N, N-dimethylformamide solution, 90mL of methanol is added, stirring is carried out for 10min, after standing for 12h at room temperature, ethanol and acetone are used for purifying for several times to obtain hydrogen bond organic framework material (HOF);
(4) Dispersing 1mg of HOF prepared in the step (2) in 1mL of phosphoric acid buffer solution, and adding 100. Mu.L of NHS/E with concentration of 0.1MDC (NHS: edc=1:1) was mixed and carboxyl groups were activated for 2h at room temperature. Then 100. Mu.L of 2. Mu.M aminated alpha-bungarotoxin aptamer (NH) 2 - α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffer solution at ph=7.2 to give hof@nh 2 -a-BGT-apt biological probe.
The X-ray diffraction pattern of the obtained HOF is shown in FIG. 1. From the X-ray diffraction results, a strong diffraction peak was seen at about 2θ=4.7, which is consistent with literature reports, demonstrating successful synthesis of HOFs. Since HOF is an organic framework material formed by self-assembling organic ligands through hydrogen bond acting force and pi-pi stacking acting force, the stability of HOF in water is critical to the application of HOF, and HOF is dissolved in H 2 O and acetic acid buffer (ph=4) were placed for 7 days and XRD analysis was performed thereon, and it was seen that it was in H 2 The O and acetic acid buffer solution are stable in the water, and a foundation is laid for further application.
1,3,6, 8-tetra (4-carboxylbenzenepyrene) (H 4 TBAPy) and hydrogen bond organic framework material (HOF) are shown in figure 2. From the infrared spectrum of HOF, it can be derived at 1605cm -1 The successful synthesis of hydrogen bonded organic framework materials is further explained due to the framework vibration of the pyrene ring in the HOF framework.
The transmission electron microscope images of the obtained HOFs are shown in FIG. 3, and (A), (B), (C) and (D) in FIG. 3 are transmission electron microscope images at different magnifications. From fig. 3, it can be seen that HOF is a rod-like structure and is uniformly distributed with a smooth surface.
Method for detecting alpha-bungarotoxin for non-diagnostic purposes
(1) Adding 100 mu L of alpha-BGT primary antibody with the concentration of 1 mu g/mL into a 96-well ELISA plate, and incubating overnight at 4 ℃;
(2) Slowly washing the ELISA plate in step (1) three times by using a phosphate buffer solution containing Tween-20, and adding a 1% bovine serum albumin solution to block unbound antibodies; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; the phosphate buffer solution containing tween-20, wherein the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) Respectively dripping the alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plate treated in the step (2), incubating for 1h at room temperature, and cleaning the ELISA plate holes with a phosphate buffer containing Tween-20 for three times after incubation is completed;
(4) 100. Mu.L of HOF@NH 2 Dropwise adding the biological probe of the alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate three times by using phosphate buffer solution containing Tween-20 after incubation is completed;
(5) 150 mu L of 3,3', 5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution are added into an ELISA plate, and the reaction is carried out for 30min at room temperature under 365nm laser irradiation, so that the colorimetric immunosensor for detecting the alpha-BGT with different concentrations is obtained.
(6) Adding 150 mu L of 3,3', 5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution into an ELISA plate, and reacting for 30min at room temperature to obtain colorimetric immune biosensors for detecting alpha-BGT with different concentrations;
the concentration of 3,3', 5' -tetramethylbenzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0.
Placing the color development solution in the 96-hole ELISA plate in a micro cuvette, and placing the micro cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500 nm;
recording absorbance peaks corresponding to alpha-BGT under different concentrations;
the concentration of alpha-BGT in the sample to be detected is obtained by using a working curve method, and the result shows that the linear range is 0.0001-316ng/mL, and the lower detection Limit (LOD) is as low as 0.033fg mL -1 (S/N＝3)。
Example 2
Preparation of alpha-bungarotoxin detection probes
(1) First, 5g of 4-methoxycarbonylphenylboronic acid, 2.85g of 1,3,6, 8-tetrabromopyrene, 0.1g of tetrakis (triphenylphosphine) palladium and 6g of potassium carbonate were dissolved in 100mL of dioxane, under N 2 Stirring at 85deg.C under protection for 72 hr. The reaction product was then poured into a solution of ice water and concentrated hydrochloric acid (v/v=3:1), the organic phase was collected by extraction with chloroform, finally dried over magnesium sulfate and the organic solvent was removed by vacuum drying, finally obtaining the product 1,3,6, 8-tetrad(4- (methoxycarbonyl) phenyl) pyrene;
(2) 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene was first dissolved in 100mL of a mixed solution of tetrahydrofuran/dioxane/water (v/v=5:2:2), then 1g KOH was added, and the mixture was stirred at 85℃under reflux for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL H was added 2 O was stirred at room temperature for 2h. The pH was adjusted to 2 with concentrated HCl. The yellow solid obtained is filtered and collected, washed by water for a plurality of times, and dried in vacuum to obtain 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
(3) 150mg of 1,3,6, 8-tetra (4-carboxybenzene) pyrene is dissolved in 22.5mL of N, N-dimethylformamide solution, 90mL of methanol is added, stirring is carried out for 10min, after standing for 12h at room temperature, ethanol and acetone are used for purifying for several times to obtain hydrogen bond organic framework material (HOF);
(4) 1mg of the HOF prepared in the step (2) was dispersed in 1mL of a phosphate buffer solution, and 100. Mu.L of a mixed solution of NHS/EDC (NHS: EDC=1:1) at a concentration of 0.1M was added thereto to activate carboxyl groups at room temperature for 2 hours. Then 100. Mu.L of 2. Mu.M aminated alpha-bungarotoxin aptamer (NH) 2 - α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffer solution at ph=7.2 to give hof@nh 2 -a-BGT-apt biological probe.
Method for detecting alpha-bungarotoxin for non-diagnostic purposes
(1) Adding 100 mu L of alpha-BGT primary antibody with the concentration of 1 mu g/mL into a 96-well ELISA plate, and incubating overnight at 4 ℃;
(2) Slowly washing the ELISA plate in step (1) three times by using a phosphate buffer solution containing Tween-20, and adding a 1% bovine serum albumin solution to block unbound antibodies; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; the phosphate buffer solution containing tween-20, wherein the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) Mixing 90 mu L of alpha-BGT solution with the concentration of 1ng/mL with 10 mu L of Agkistrodon acutus (D.acutus), trimeresurus albolabris (TSV-PA), louis rotundus (T.mucross quats), agkistrodon halys (Agkistrodon), agkistrodon halys (KC), naja, agkistrodon halys (BGFT) and beta-bungarous (beta-BGT) toxin uniformly, respectively dripping the mixture into the enzyme-labeled plate treated in the step (2), incubating the mixture for 1h at room temperature, and washing the enzyme-labeled plate holes with phosphate buffer solution containing Tween-20 for three times after incubation is completed;
(4) 100. Mu.L of HOF@NH 2 Dropwise adding the biological probe of the alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate three times by using phosphate buffer solution containing Tween-20 after incubation is completed;
(5) 150 mu L of 3,3', 5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution are added into an ELISA plate, and the reaction is carried out for 30min at room temperature under 365nm laser irradiation, wherein the concentration of the 3,3', 5' -tetramethyl benzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0, so that the result of the anti-interference experiment of the alpha-BGT colorimetric immunosensor is obtained. The results obtained are shown in FIG. 4.
From fig. 4, it can be seen that the common venomous snake toxins have negligible interference to the colorimetric immune biosensor constructed by us, which indicates that the colorimetric immune biosensor constructed by us for detecting alpha-BGT has high stability and selectivity and has clinical application value.
Example 3
Preparation of alpha-bungarotoxin detection probes
(1) First, 5g of 4-methoxycarbonylphenylboronic acid, 2.85g of 1,3,6, 8-tetrabromopyrene, 0.1g of tetrakis (triphenylphosphine) palladium and 6g of potassium carbonate were dissolved in 100mL of dioxane, under N 2 Stirring at 85deg.C under protection for 72 hr. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v=3:1), extracting with chloroform to collect an organic phase, finally drying with magnesium sulfate and removing the organic solvent by vacuum drying to obtain a product of 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene;
(2) 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene was first dissolved in 100mL of a mixed solution of tetrahydrofuran/dioxane/water (v/v=5:2:2), then 1g KOH was added, and the mixture was stirred at 85℃under reflux for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL H was added 2 O was stirred at room temperature for 2h. The pH was adjusted to 2 with concentrated HCl. Collecting yellow solid by filtration, washing with water for several times, and vacuum dryingTo 1,3,6, 8-tetrakis (4-carboxylbenzenepyrene);
(3) 150mg of 1,3,6, 8-tetra (4-carboxybenzene) pyrene is dissolved in 22.5mL of N, N-dimethylformamide solution, 90mL of methanol is added, stirring is carried out for 10min, after standing for 12h at room temperature, ethanol and acetone are used for purifying for several times to obtain hydrogen bond organic framework material (HOF);
(4) 1mg of the HOF prepared in the step (2) was dispersed in 1mL of a phosphate buffer solution, and 100. Mu.L of a mixed solution of NHS/EDC (NHS: EDC=1:1) at a concentration of 0.1M was added thereto to activate carboxyl groups at room temperature for 2 hours. Then 100. Mu.L of 2. Mu.M aminated alpha-bungarotoxin aptamer (NH) 2 - α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffer solution at ph=7.2 to give hof@nh 2 -a-BGT-apt biological probe.
Method for detecting alpha-bungarotoxin for non-diagnostic purposes
(1) Adding 100 mu L of alpha-BGT primary antibody with the concentration of 1 mu g/mL into a 96-well ELISA plate, and incubating overnight at 4 ℃;
(2) Slowly washing the ELISA plate in step (1) three times by using a phosphate buffer solution containing Tween-20, and adding a 1% bovine serum albumin solution to block unbound antibodies; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; the phosphate buffer solution containing tween-20, wherein the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) Respectively dripping the alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plate treated in the step (2), incubating for 1h at room temperature, and washing the ELISA plate holes three times by using a phosphate buffer containing Tween-20 after incubation is completed;
(4) 100. Mu.L of HOF@NH 2 Dropwise adding the biological probe of the alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate three times by using phosphate buffer solution containing Tween-20 after incubation is completed;
(5) 150. Mu.L of 3,3', 5' -tetramethylbenzidine, which has a concentration of 10mM, and 150. Mu.L of an acetic acid buffer solution having a pH of 4.0, were added to the ELISA plate. Under 365nm laser irradiation, reacting for 30min at room temperature to obtain the colorimetric immune biosensor for detecting the alpha-BGT with different concentrations.
The ultraviolet-visible absorption spectrum of different concentrations of alpha-BGT is shown in FIG. 5. As can be seen from fig. 5, the constructed ELISA biosensor detects different concentrations of alpha-BGT using an ultraviolet-visible spectrophotometer at 652nm, with the absorbance value gradually increasing as the concentration of alpha-BGT toxin increases. The resulting standard curve is shown in fig. 6 with UV conditions, y=0.0868x+0.6262. It can be seen that the absorbance has a good linear relationship with the logarithm of the alpha-BGT concentration, the correlation coefficient (R 2 ) 0.9982 and LOD of 0.033 fg.mL -1 (S/n=3), showing good linearity and lower LOD values.
Example 4
Preparation of alpha-bungarotoxin detection probes
(1) 5g of 4-methoxycarbonylphenylboronic acid, 2.85g of 1,3,6, 8-tetrabromopyrene, 0.1g of tetrakis (triphenylphosphine) palladium and 6g of potassium carbonate are initially dissolved in 100mL of dioxane and stirred at 85℃for 72h under N2. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v=3:1), extracting with chloroform to collect an organic phase, finally drying with magnesium sulfate and removing the organic solvent by vacuum drying to obtain a product of 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene;
(2) 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene was first dissolved in 100mL of a mixed solution of tetrahydrofuran/dioxane/water (v/v=5:2:2), then 1g KOH was added, and the mixture was stirred at 85℃under reflux for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL H was added 2 O was stirred at room temperature for 2h. The pH was adjusted to 2 with concentrated HCl. The yellow solid obtained is filtered and collected, washed by water for a plurality of times, and dried in vacuum to obtain 1,3,6, 8-tetra (4-carboxybenzene) pyrene; the phosphate buffer solution containing tween-20, wherein the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) 150mg of 1,3,6, 8-tetra (4-carboxybenzene) pyrene is dissolved in 22.5mL of N, N-dimethylformamide solution, 90mL of methanol is added, stirring is carried out for 10min, after standing for 12h at room temperature, ethanol and acetone are used for purifying for several times to obtain hydrogen bond organic framework material (HOF);
(4) 1mg of the HOF prepared in the step (2) was dispersed in 1mL of a phosphate buffer solution, and 100. Mu.L of a mixed solution of NHS/EDC (NHS: EDC=1:1) at a concentration of 0.1M was added thereto to activate carboxyl groups at room temperature for 2 hours. Then 100. Mu.L of 2. Mu.M aminated alpha-bungarotoxin aptamer (NH) 2 - α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffer solution at ph=7.2 to give hof@nh 2 -a-BGT-apt biological probe.
Method for detecting alpha-bungarotoxin for non-diagnostic purposes
(1) Adding 100 mu L of alpha-BGT primary antibody with the concentration of 1 mu g/mL into a 96-well ELISA plate, and incubating overnight at 4 ℃;
(2) Slowly washing the ELISA plate in step (1) three times by using a phosphate buffer solution containing Tween-20, and adding a 1% bovine serum albumin solution to block unbound antibodies; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) Respectively dripping the alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plate treated in the step (2), incubating for 1h at room temperature, and washing the ELISA plate holes three times by using a phosphate buffer containing Tween-20 after incubation is completed;
(4) 100. Mu.L of HOF@NH 2 Dropwise adding the biological probe of the alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate three times by using phosphate buffer solution containing Tween-20 after incubation is completed;
(5) 150. Mu.L of 3,3', 5' -tetramethylbenzidine, which has a concentration of 10mM, and 150. Mu.L of an acetic acid buffer solution having a pH of 4.0, were added to the ELISA plate. And reacting for 30min at room temperature to obtain the colorimetric immune biosensor for detecting the alpha-BGT with different concentrations.
And (3) taking a developing solution in the 96-hole ELISA plate in a micro cuvette, placing the micro cuvette in an ultraviolet spectrophotometer for testing, and recording absorbance peaks corresponding to alpha-BGT under different concentrations, wherein the scanning range is 800-500 nm. The results are shown in FIG. 7. As can be seen from FIG. 7, the absorbance value gradually increased with increasing concentration of alpha-BGT toxinAdding. The resulting standard curve is shown in fig. 6 for the standard curve under the without UV conditions, y=0.0675x+0.5127. It can be seen that the absorbance has a good linear relationship with the logarithm of the alpha-BGT concentration, the correlation coefficient (R 2 ) 0.9982 and LOD of 0.033 fg.mL -1 (S/n=3), showing good linearity and lower LOD values.
Example 5 labeled recovery experiment
In order to verify the feasibility and practicality of the constructed immune biosensor, a labeling recovery experiment is also performed. A blank sample of human serum was taken, to which were added 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL of the α -bungarotoxin standard of known concentration, respectively, and the results were measured using our constructed immunosensor and calculated for recovery, as shown in Table 1.
TABLE 1 alpha-bungarose labeled recovery experiment results in human serum samples
As can be seen from table 1: the recovery rate of the immune biosensor for detecting the alpha-bungarotoxin is 99.34.0% -105.00%, and the standard deviation is 2.50% -3.78%, so that the analysis accuracy and reliability of the immune biosensor for detecting the alpha-bungarotoxin in a human serum sample are acceptable, and the immune biosensor has potential application value in clinical diagnosis.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. An alpha-bungarotoxin detection probe comprising an alpha-bungarotoxin nucleic acid aptamer and a hydrogen bond organic framework material chemically bound to the alpha-bungarotoxin nucleic acid aptamer;
the alpha-bungarotoxin aptamer is modified with amino;
the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
the preparation method of the alpha-bungarotoxin detection probe comprises the following steps:
providing a hydrogen bond organic framework material with an organic ligand of 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and activating carboxyl to obtain a hydrogen bond organic framework material with activated carboxyl;
mixing the hydrogen bond organic framework material of the activated carboxyl and the nucleic acid aptamer of the alpha-bungarotoxin modified with the amino, and carrying out chemical combination to obtain an alpha-bungarotoxin detection probe;
the mass ratio of the hydrogen bond organic framework material for activating carboxyl to the nucleic acid aptamer of the alpha-bungarotoxin modified with amino is 300-500:1.
2. The alpha-bungarotoxin detection probe of claim 1, wherein the mass ratio of the alpha-bungarotoxin aptamer to the hydrogen bond organic framework material is 1:50-1000.
3. A method for preparing the α -bungarotoxin detection probe of claim 1 or 2, comprising the steps of:
providing a hydrogen bond organic framework material with an organic ligand of 1,3,6, 8-tetra (4-carboxybenzene) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and activating carboxyl to obtain a hydrogen bond organic framework material with activated carboxyl;
mixing the hydrogen bond organic framework material of the activated carboxyl and the nucleic acid aptamer of the alpha-bungarotoxin modified with the amino, and carrying out chemical combination to obtain an alpha-bungarotoxin detection probe;
the mass ratio of the hydrogen bond organic framework material for activating carboxyl to the nucleic acid aptamer of the alpha-bungarotoxin modified with amino is 300-500:1.
4. A kit for detecting α -bungarotoxin, comprising the α -bungarotoxin detection probe of claim 1 or 2 or the α -bungarotoxin detection probe prepared by the method of claim 3, an α -bungarotoxin primary antibody, a non-specific protein, and a chromogenic substrate.
5. The kit for detecting α -bungarotoxin according to claim 4, wherein the chromogenic substrate is 3,3', 5' -tetramethylbenzidine.
6. A method for detecting α -bungarotoxin for non-diagnostic purposes comprising the steps of:
first incubating the alpha-bungarotoxin primary antibody to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unbound material to obtain a second incubation product;
adding a sample to be detected into the second incubation product, performing third incubation, and removing unbound materials to obtain a third incubation product;
adding the alpha-bungarotoxin detection probe according to claim 1 or 2 or the alpha-bungarotoxin detection probe prepared by the preparation method according to claim 3 into the third incubation product, performing fourth incubation, and removing unbound materials to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product to perform a chromogenic reaction, and testing the absorbance peak value of the chromogenic solution at 500-800 nm;
obtaining the concentration of the alpha-bungarotoxin in the sample to be detected according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the concentration of the alpha-bungarotoxin and the absorbance peak value.
7. The method according to claim 6, wherein the color reaction is performed in an acetate buffer.
8. The method according to claim 6, wherein the color reaction is performed under normal light conditions or 365nm laser irradiation.
9. The method according to claim 7 or 8, wherein the time for the color reaction is 10 to 60 minutes.
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