CN114354525A - Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin in non-diagnosis purpose - Google Patents
Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin in non-diagnosis purpose Download PDFInfo
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Abstract
The invention provides an alpha-bungarotoxin detection probe and a method for detecting alpha-bungarotoxin for non-diagnosis purposes, belonging to the technical field of biosensing. According to the invention, a hydrogen bond organic framework material formed by 1,3,6, 8-tetra (4-carboxyphenyl) pyrene is used as an enzyme material for catalyzing the color development of the color developing agent, the surface of the enzyme material is provided with abundant carboxyl, and the enzyme material can be chemically combined with an aptamer of alpha-bungarotoxin modified with amino through a CO-NH bond, so that the stability of the probe is improved. The invention provides a method for detecting alpha-bungarotoxin with a non-diagnostic purpose, which specifically recognizes the alpha-bungarotoxin by utilizing an alpha-bungarotoxin detection probe and catalyzes a 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 the concentration is 0.0001-316ng mL‑1Has a good linear relationship in the concentration range of the alpha-bungarotoxin.
Description
Technical Field
The invention relates to the technical field of biosensing, in particular to an alpha-bungarotoxin detection probe and a method for detecting alpha-bungarotoxin for non-diagnosis purposes.
Background
The bungarus multicinctus is the fourth poisonous snake on land and is also the snake with the highest toxicity in China, and 1mg of toxin of an adult bungarus multicinctus can cause more than ten people to die. The main components of the toxins contained in the bungarus fasciatus venom are protein and polypeptide, and the toxins mainly comprise neurotoxin, including enzymes such as alpha-bungarus fasciatus toxin (alpha-BGT), beta-bungarus fasciatus toxin (beta-BGT), kappa-bungarus fasciatus toxin (kappa-BGT), gamma-bungarus fasciatus toxin (gamma-BGT), phospholipase A and the like. The snake bite can not feel pain but sleepy, the body part is paralyzed when the snake bite is slightly poisoned, if the toxin acts on the joint position of the nerve muscle, the nerve conduction route can be blocked, so that the striated muscle can not normally contract, the breathing paralysis is caused, and the acting time is about 40 minutes to 2 hours or as long as 24 hours. Before the application of the anti-snake venom serum, the death rate of the bite of the bungarus multicinctus is extremely high.
The detection of snake venom has been greatly developed over the past decades and many methods for detecting snake venom have been established. Such as radioimmunoassay, immunoelectrophoresis, fluorescence immunoassay, enzyme-linked immunosorbent assay, etc. Among them, enzyme-linked immunosorbent assay (ELISA) has the advantages of high specificity, rapidness, simplicity and convenience, and has been widely used. The enzyme-linked immunosorbent assay needs to use a toxin secondary antibody-natural enzyme as a probe conjugate to identify toxin and enable a color developing agent to develop color, but the prior natural enzymes such as horseradish peroxidase, laccase and alkali-phosphate lipase have the defects of poor stability and weak catalytic activity when used for ELISA detection.
The existing nano enzyme such as ferroferric oxide, monatomic iron nitrogen carbon and the like has the outstanding advantages of good stability, low cost, strong catalytic activity, strong affinity to substrates 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 α -bungarotoxin detection probe and a method for detecting α -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 aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin 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-carboxyphenyl) 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-carboxyphenyl) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and performing carboxyl activation to obtain a hydrogen bond organic framework material for activating carboxyl;
and mixing the hydrogen bond organic framework material for activating carboxyl and the aptamer of 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 for activating carboxyl to the aptamer of alpha-bungarotoxin modified with amino is 300-500: 1.
The invention provides a kit for detecting alpha-bungarotoxin, which comprises the alpha-bungarotoxin detection probe, an alpha-bungarotoxin first antibody, a non-specific protein and a chromogenic substrate.
Preferably, the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine.
The invention provides a method for detecting alpha-bungarotoxin for non-diagnostic purposes, which comprises the following steps:
performing first incubation on the alpha-bungarotoxin first antibody to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unconjugated substances to obtain a second incubation product;
adding a sample to be tested into the second incubation product, performing third incubation, and removing unconjugated substances to obtain a third incubation product;
adding the alpha-bungarotoxin detection probe into the third incubation product, performing fourth incubation, and removing unconjugated substances to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product, carrying out 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 relation curve of logarithm of alpha-bungarotoxin concentration and absorbance peak value.
Preferably, the color reaction is performed in an acetate buffer.
Preferably, the color reaction is performed under normal light conditions or under 365nm laser irradiation.
Preferably, the time of the color development reaction is 10-60 min.
The invention provides an alpha-bungarotoxin detection probe, which comprises an alpha-bungarotoxin aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin 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-carboxyphenyl) pyrene. The hydrogen bond organic framework material formed by 1,3,6, 8-tetra (4-carboxyphenyl) pyrene has rich carboxyl on the surface, and can be chemically combined with the aptamer of alpha-bungarotoxin modified with amino through a CO-NH bond, so that the stability of the probe is improved. Meanwhile, the hydrogen bond organic framework material is easy to disperse in water and has good dispersion stability, the defect of agglomeration of the existing metal-based nano enzyme in water is avoided, and the hydrogen bond organic framework material has the advantages of high biocompatibility and low toxicity; compared with an alpha-bungarotoxin secondary antibody, the aptamer of the alpha-bungarotoxin modified with the 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 first antibody, a non-specific protein 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 biosensor, wherein the alpha-bungarotoxin detection probe can specifically recognize the alpha-bungarotoxin, has good enzyme activity, can catalyze a chromogenic substrate to develop color, and realizes the detection of the alpha-bungarotoxin.
The invention provides a method for detecting alpha-bungarotoxin with a non-diagnostic purpose, which specifically recognizes the alpha-bungarotoxin by utilizing an alpha-bungarotoxin detection probe and catalyzes a 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 the concentration is 0.0001-316ng mL-1Has good linear relation in the concentration range of the alpha-bungarotoxin, and the lowest detection lower limit is 0.033 fg/mL. The detection method provided by the invention has high stability and strong anti-interference capability, and can realize visual alpha-bungarotoxin detection 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 shows H obtained in example 14Infrared spectrograms of TBAPy and HOF;
FIG. 3 is a transmission electron micrograph of the HOF obtained in example 1;
FIG. 4 shows the anti-interference test results of the colorimetric immunobiosensor obtained in example 2;
FIG. 5 is a graph of the UV-VIS absorption spectra of α -BGT of example 3 at different concentrations;
FIG. 6 is a graph showing the standard curves obtained in examples 3 and 4;
FIG. 7 is a graph of the UV-VIS absorption spectra of α -BGT of example 4 at different concentrations.
Detailed Description
The invention provides an alpha-bungarotoxin detection probe, which comprises alpha-A bungarotoxin aptamer and a hydrogen bonding organic framework material chemically bound to the alpha-bungarotoxin 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-carboxyphenyl) pyrene. In the present invention, the alpha-bungarotoxin detection probe is abbreviated as HOF @ NH2-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 source of the aptamer of α -bungarotoxin modified with an amino group is preferably commercially available. As a specific example of the present invention, the aptamer of alpha-bungarotoxin modified with an amino group is purchased from Biotech, Inc. of Kyoto engine, Beijing. In the invention, the sequence number of the aptamer of alpha-bungarotoxin modified with amino is as follows: 5-GCGAGGTGTTCGAGAGTTAGGGGCGACATGACCAAACGTT-3, modified with NH at the 5-terminal2。
The invention has no special requirement on the source of the hydrogen bond organic framework material, and the hydrogen bond organic framework material with 1,3,6, 8-tetra (4-carboxyphenyl) pyrene as the conventional organic ligand in the field can be used or prepared by self. When the hydrogen bonding organic framework material is prepared by itself, the preparation method preferably comprises:
under an alkaline environment, mixing 4-methoxycarbonylphenylboronic acid, 1,3,6, 8-tetrabromopyrene and a catalyst, and carrying out 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 acidifying to obtain 1,3,6, 8-tetra (4-carboxyphenyl) pyrene;
and carrying out self-assembly reaction on the 1,3,6, 8-tetra (4-carboxyphenyl) pyrene to obtain the hydrogen bond organic framework material (HOF).
In an alkaline environment, 4-methoxycarbonylphenylboronic acid, 1,3,6, 8-tetrabromopyrene and a catalyst are mixed for a coupling reaction to obtain the 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene. In the invention, the molar ratio of the 4-methoxycarbonylphenylboronic acid to the 1,3,6, 8-tetrabromopyrene is preferably 1-2: 1, and more preferably 1.5-1.75: 1. In the present invention, the coupling reaction is preferably carried out in an organic solvent, which is preferably dioxane.
The invention preferably uses potassium carbonate to provide the alkaline environment. In the invention, the catalyst is preferably tetrakis (triphenylphosphine) palladium, and the mass ratio of the 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene to the catalyst is preferably 50: 1.
In the invention, the temperature of the coupling reaction is 25-100 ℃, and more preferably 50-75 ℃; the time is preferably 24 to 96 hours, and more preferably 48 to 72 hours.
After the coupling reaction, the present invention preferably performs a post-treatment on the obtained coupling reaction solution. In the present invention, the post-treatment preferably comprises the steps of:
mixing the coupling reaction solution with a quenching agent, and carrying out quenching reaction to obtain a quenching reaction solution;
and sequentially extracting an organic phase, drying and removing the organic solvent from the quenching reaction liquid to obtain a pure product of the 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene.
In the present invention, the quenching agent is preferably a mixture 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 organic solvent is preferably removed by vacuum drying.
In the invention, the reaction process of the coupling reaction is shown as 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-carboxyphenyl) 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 performed 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 invention, the temperature of the hydrolysis reaction is preferably 25-100 ℃, and more preferably 50-75 ℃; the time is preferably 2 to 36 hours, and more preferably 12 to 24 hours.
After the hydrolysis reaction, the organic solvent in the hydrolysis reaction solution is preferably removed in the present invention, and the organic solvent is preferably removed by vacuum drying.
In the present invention, the acidification comprises:
the resulting hydrolysis product was dissolved in water and the pH adjusted to 2 using concentrated HCl.
After the acidification, the invention preferably washes and dries the obtained acidification product in sequence to obtain the pure product of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene.
In the invention, the preparation process of the 1,3,6, 8-tetra (4-carboxyphenyl) pyrene is shown as a formula 2.
After the 1,3,6, 8-tetra (4-carboxyphenyl) pyrene is obtained, the 1,3,6, 8-tetra (4-carboxyphenyl) pyrene is subjected to self-assembly reaction to obtain the hydrogen bond organic framework material. In the present invention, the self-assembly reaction is preferably carried out in a mixed solution of N, N-dimethylformamide and methanol. In the present invention, the methanol can activate the pore channels of 1,3,6, 8-tetrakis (4-carboxyphenyl) 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 product is preferably washed and purified by using ethanol and acetone.
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-carboxyphenyl) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and performing carboxyl activation to obtain a hydrogen bond organic framework material for activating carboxyl;
and mixing the hydrogen bond organic framework material for activating carboxyl and the aptamer of alpha-bungarotoxin modified with amino, and carrying out chemical combination to obtain the alpha-bungarotoxin detection probe.
In the present invention, the source of the hydrogen bonding organic framework material of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene as the organic ligand is the same as that described above, and the description thereof is omitted.
The invention mixes the hydrogen bond organic framework material with a carboxyl activating agent to carry out carboxyl activation, thus obtaining the hydrogen bond organic framework material for activating carboxyl. In the present invention, the carboxyl activating agent is preferably a mixture of NHS and EDC, and the mass ratio of NHS to EDC in the mixture is preferably 1: 1.
In the invention, the temperature for activating the carboxyl is preferably room temperature, and the time is preferably 30-150 min, and more preferably 120 min.
After the carboxyl is activated, the invention mixes the hydrogen bond organic framework material of the activated carboxyl and the aptamer of the alpha-bungarotoxin modified with the amino for 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 aptamer of 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 is preferably performed by stirring. In the invention, the temperature of the chemical bonding is preferably room temperature, and the time is preferably 6-24 h, and more preferably 12 h. In the chemical combination process, the carboxyl of the hydrogen bond organic framework material for activating the carboxyl reacts with the aptamer of alpha-bungarotoxin modified with amino to generate a CO-NH bond.
After the chemical combination, the invention preferably centrifuges and washes the obtained product to obtain the alpha-bungarotoxin detection probe.
In the present invention, the rate of the centrifugation is preferably 8000rpm, and the time is preferably 15 min. In the present invention, the washing detergent is preferably a phosphoric acid buffer solution having a pH of 7.2, and the number of washing is preferably 3.
The invention provides a kit for detecting alpha-bungarotoxin, which comprises the alpha-bungarotoxin detection probe, a first alpha-bungarotoxin antibody, a non-specific protein and a chromogenic substrate.
The first antibody to alpha-bungarotoxin is not particularly required in the present invention, and a commercially available first antibody to alpha-bungarotoxin is used as is conventional in the art. In the present invention, the concentration of the first antibody to alpha-bungarotoxin is preferably 1. mu.g/mL.
In the present invention, the nonspecific protein is preferably bovine serum albumin.
In the present invention, the chromogenic substrate is preferably 3,3',5,5' -tetramethylbenzidine. In the present invention, the concentration of the 3,3',5,5' -tetramethylbenzidine is preferably 1 to 25mM, and more preferably 10 mM.
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 of the acetic acid buffer solution is preferably 4. In the present invention, the washing solution is preferably a phosphate buffer containing tween-20, the content of tween-20 is preferably 0.05 wt%, and the pH value of the phosphate buffer is preferably 7.2.
The invention provides a method for detecting alpha-bungarotoxin for non-diagnostic purposes, which comprises the following steps:
performing first incubation on the alpha-bungarotoxin first antibody in an incubator to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unconjugated substances to obtain a second incubation product;
adding a sample to be tested into the second incubation product, performing third incubation, and removing unconjugated substances to obtain a third incubation product;
adding an alpha-bungarotoxin detection probe into the third incubation product, performing fourth incubation, and removing unconjugated substances to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product, carrying out 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 relation curve of logarithm of alpha-bungarotoxin concentration and absorbance peak value.
According to the invention, the first antibody of alpha-bungarotoxin is subjected to first incubation in an incubator to obtain a first incubation product. In the present invention, the incubator is preferably a 96-well microplate. In the present invention, the concentration of the first antibody to alpha-bungarotoxin is preferably 1. mu.g/mL.
In the invention, the temperature of the first incubation is preferably 4 ℃, and the time is preferably 8-12 h.
After the first incubation product is obtained, the invention adds non-specific protein into the first incubation product, carries out second incubation, and removes unconjugated substance to obtain a second incubation product. In the present invention, the nonspecific protein is preferably bovine serum albumin. In the present invention, the concentration of the nonspecific protein is preferably 1 wt%. In the invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 15-120 min, and more preferably 60 min.
In the present invention, the mode of removing the unconjugated substance 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, the sample to be tested is added into the second incubation product, the third incubation is carried out, and the unconjugated substance is removed, so that a third incubation product is obtained. 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 1 h.
In the present invention, the mode of removing the unconjugated substance is preferably washing with a phosphate buffer containing tween-20.
After the third incubation product is obtained, the alpha-bungarotoxin detection probe is added into the third incubation product, fourth incubation is carried out, and unconjugated substances are removed 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 1 h. In the present invention, the mode of removing the unconjugated substance is preferably washing with a phosphate buffer containing tween-20.
After the fourth incubation product is obtained, adding a chromogenic substrate into the fourth incubation product to perform chromogenic reaction, and testing the absorbance peak value of chromogenic solution at 500-800 nm. In the present invention, the chromogenic substrate is preferably 3,3',5,5' -tetramethylbenzidine. In the present invention, the color development reaction is preferably carried out in an acetic acid buffer solution. In the present invention, the color development reaction is preferably carried out under normal light conditions or under 365nm laser irradiation. In the present invention, the time of the color reaction is preferably 10 to 60min, and more preferably 30 min. The invention uses an ultraviolet spectrophotometer to test the 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 relation curve of logarithm of alpha-bungarotoxin concentration and absorbance peak value.
As a specific embodiment of the present invention, the method for drawing the standard curve preferably includes the following steps:
providing a standard solution of α -bungarotoxin at a gradient concentration comprising 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 316 ng/mL.
The alpha-bungarotoxin standard solution with gradient concentration is used as a sample to be tested, the test is carried out according to the detection method of the invention, the absorbance peak value corresponding to the alpha-bungarotoxin standard solution with gradient concentration is obtained, the logarithm of the alpha-bungarotoxin is used as the abscissa, the absorbance value is used as the ordinate, and the linear relation curve of the logarithm of the beta-bungarotoxin concentration and the absorbance value is obtained.
Specifically, when the color reaction is performed under normal illumination conditions, the data associated with the standard curve are shown in table 1.
TABLE 1 Standard Curve of color development under Normal light conditions
When the color reaction is performed under 365nm laser irradiation, the relevant data of the standard curve are shown in Table 2.
TABLE 2365 nm Standard Curve of color development under laser irradiation
In the invention, the lower limit of the detection of the alpha-bungarotoxin is 0.033fg mL-1(S/N is 3) and the detection range is 0.0001-316ng mL-1。
The following examples are provided to illustrate in detail an α -bungarotoxin detection probe and a method for detecting α -bungarotoxin for non-diagnostic purposes, but they should not be construed as limiting the scope of the present invention.
Example 1
Firstly, preparing alpha-bungarotoxin detection probe
(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 N2Stirring for 72h at 85 ℃ under protection. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v is 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 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. about.5: 2:2), followed by addition of 1g of KOH,the mixture was stirred at 85 ℃ under reflux for 12h and dried under vacuum to remove the organic solvent. Then 100mL of H was added2O was stirred at room temperature for 2 h. The pH was adjusted to 2 with concentrated HCl. The resulting yellow solid was collected by filtration, washed several times with water, and dried under vacuum to give 1,3,6, 8-tetrakis (4-carboxyphenyl) pyrene (H)4TBAPy)；
(3) Dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in 22.5mL of N, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(4) 1mg of HOF prepared in step (2) was dispersed in 1mL of phosphoric acid 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 to activate the carboxyl group at room temperature for 2 hours. Then 100. mu.L of aminated alpha-bungarotoxin aptamer (NH) at a concentration of 2. mu.M was added2- α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffered solution pH 7.2 to give HOF @ NH2-a biological probe for α -BGT-apt.
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 observed around 2 θ 4.7, which is consistent with the literature report, indicating the successful synthesis of HOF. Because HOF is an organic framework material formed by self-assembling organic ligands through hydrogen bond acting force and pi-pi accumulation acting force, the stability of HOF in water is crucial to the application of HOF, and HOF is dissolved in H2O and acetic acid buffer (pH 4) were left for 7 days and then XRD analysis was performed, which revealed that it was in H2The O and the acetic acid buffer solution are stable, and a foundation is laid for further application of the O and the acetic acid buffer solution.
1,3,6, 8-tetra (4-carboxyphenyl) pyrene (H)4TBAPy) and hydrogen bonding organic framework material (HOF) as shown in fig. 2. From the infrared spectrum of HOF, 1605cm-1Further illustrating the successful synthesis of hydrogen bonded organic framework materials due to the skeletal vibrations of the pyrene ring in the HOF framework.
The transmission electron micrograph of the obtained HOF is shown in FIG. 3, and (A), (B), (C), and (D) in FIG. 3 are transmission electron micrographs at different magnifications. It can be seen from fig. 3 that HOF is a rod-like structure, and is uniformly distributed, and its surface is smooth.
(II) 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 enzyme label plate, and incubating overnight at 4 ℃;
(2) slowly washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; in the phosphate buffer solution containing tween-20, the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) respectively dripping alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plates treated in the step (2), incubating for 1h at room temperature, and washing the ELISA plate holes for three times by using phosphate buffer solution containing Tween-20 after incubation is finished;
(4) 100 μ L of HOF @ NH2Dripping a biological probe of alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) adding 150 mu L of 3,3',5,5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting for 30min at room temperature under 365nm laser irradiation to obtain the colorimetric immune biosensor for detecting alpha-BGT with different concentrations.
(6) Adding 150 mu L of 3,3',5,5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting at room temperature for 30min to obtain the colorimetric immune biosensor for detecting alpha-BGT with different concentrations;
the concentration of the 3,3',5,5' -tetramethylbenzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0.
Placing a chromogenic solution in a 96-hole enzyme label plate in a micro cuvette, and placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500 nm;
recording absorbance peak values corresponding to the alpha-BGT under different concentrations;
by working curve method to obtainThe results of the concentration of alpha-BGT in the sample to be detected show that the linear range is 0.0001-316ng/mL, and the lower limit of detection (LOD) is as low as 0.033fg mL-1(S/N＝3)。
Example 2
Firstly, preparing alpha-bungarotoxin detection probe
(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 N2Stirring for 72h at 85 ℃ under protection. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v is 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 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 of KOH was added, and the mixture was stirred under reflux at 85 ℃ for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL of H was added2O was stirred at room temperature for 2 h. The pH was adjusted to 2 with concentrated HCl. Filtering and collecting the obtained yellow solid, washing with water for several times, and drying in vacuum to obtain 1,3,6, 8-tetra (4-carboxyphenyl) pyrene;
(3) dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in 22.5mL of N, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(4) 1mg of HOF prepared in step (2) was dispersed in 1mL of phosphoric acid 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 to activate the carboxyl group at room temperature for 2 hours. Then 100. mu.L of aminated alpha-bungarotoxin aptamer (NH) at a concentration of 2. mu.M was added2- α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffered solution pH 7.2 to give HOF @ NH2-a biological probe for α -BGT-apt.
(II) 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 enzyme label plate, and incubating overnight at 4 ℃;
(2) slowly washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; in the phosphate buffer solution containing tween-20, the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) uniformly mixing 90 mu L of alpha-BGT solution with the concentration of 1ng/mL with 10 mu L of Agkistrodon acutus (D.acutus), phyllostachys chinensis (TSV-PA), magnetite head (T.macrostemus), Agkistrodon halys (Agkistrodon), King Cobra (KC), cobra (Naja), bungarus multicinctus (BGFT) and beta-bungarus multicinctus (beta-BGT) toxins with the concentration of 10 mu g/mL, dripping the mixture into the enzyme label plate treated in the step (2), incubating the mixture at room temperature for 1h, and washing the plate holes of the enzyme label plate with phosphate buffer solution containing Tween-20 for three times after the incubation is finished;
(4) 100 μ L of HOF @ NH2Dripping a biological probe of alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) adding 150 mu L of 3,3',5,5' -tetramethylbenzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting for 30min at room temperature under 365nm laser irradiation, wherein the concentration of the 3,3',5,5' -tetramethylbenzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0, so as to obtain the anti-interference experimental result of the alpha-BGT colorimetric immune biosensor. The results are shown in FIG. 4.
As can be seen from FIG. 4, the interference of the common snake venom toxin to the constructed colorimetric immune biosensor is basically negligible, which shows that the constructed colorimetric immune biosensor for detecting alpha-BGT has high stability and selectivity and has clinical application value.
Example 3
Firstly, preparing alpha-bungarotoxin detection probe
(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 N2Stirring for 72h at 85 ℃ under protection. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v is 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 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 of KOH was added, and the mixture was stirred under reflux at 85 ℃ for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL of H was added2O was stirred at room temperature for 2 h. The pH was adjusted to 2 with concentrated HCl. Filtering and collecting the obtained yellow solid, washing with water for several times, and drying in vacuum to obtain 1,3,6, 8-tetra (4-carboxyphenyl) pyrene;
(3) dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in 22.5mL of N, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(4) 1mg of HOF prepared in step (2) was dispersed in 1mL of phosphoric acid 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 to activate the carboxyl group at room temperature for 2 hours. Then 100. mu.L of aminated alpha-bungarotoxin aptamer (NH) at a concentration of 2. mu.M was added2- α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffered solution pH 7.2 to give HOF @ NH2-a biological probe for α -BGT-apt.
(II) 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 enzyme label plate, and incubating overnight at 4 ℃;
(2) slowly washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20; in the phosphate buffer solution containing tween-20, the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) respectively dripping alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plates treated in the step (2), incubating for 1h at room temperature, and washing the ELISA plate holes for three times by using phosphate buffer solution containing Tween-20 after the incubation is finished;
(4) 100 μ L of HOF @ NH2Dripping a biological probe of alpha-BGT-apt into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) mu.L of 3,3',5,5' -tetramethylbenzidine and 150. mu.L of acetic acid buffer solution are added to the microplate, the concentration of the 3,3',5,5' -tetramethylbenzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0. And (3) reacting for 30min at room temperature under 365nm laser irradiation to obtain the colorimetric immune biosensor for detecting the alpha-BGT with different concentrations.
The ultraviolet and visible light absorption spectrums of the alpha-BGT with different concentrations are shown in FIG. 5. As can be seen from FIG. 5, the constructed ELISA biosensor uses an ultraviolet-visible spectrophotometer to detect different concentrations of alpha-BGT under the condition of 652nm, and the absorbance value gradually increases with the increase of the concentration of the alpha-BGT toxin. The resulting standard curve is shown in fig. 6 with the standard curve under UV conditions, y being 0.0868x + 0.6262. As can be seen, the absorbance has a good linear relationship with the logarithm of the concentration of alpha-BGT, and the correlation coefficient (R)2) 0.9982, LOD 0.033 fg. mL-1(S/N ═ 3), showing good linearity and lower LOD values.
Example 4
Firstly, preparing alpha-bungarotoxin detection probe
(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 were initially dissolved in 100mL of dioxane and stirred at 85 ℃ for 72h under the protection of N2. Then pouring the reaction product into a solution of ice water and concentrated hydrochloric acid (v/v is 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 1,3,6, 8-tetra (4- (methoxycarbonyl) phenyl) pyrene;
(2) first, 1,3,6, 8-tetrakis (4- (methoxycarbonyl) phenyl) pyrene was dissolved in 100mL of tetrahydrofuran/dioxane/water (v)To the mixed solution of/v ═ 5:2:2), then 1g KOH was added, and the mixture was stirred under reflux at 85 ℃ for 12 hours, and the organic solvent was removed by vacuum drying. Then 100mL of H was added2O was stirred at room temperature for 2 h. The pH was adjusted to 2 with concentrated HCl. Filtering and collecting the obtained yellow solid, washing with water for several times, and drying in vacuum to obtain 1,3,6, 8-tetra (4-carboxyphenyl) pyrene; in the phosphate buffer solution containing tween-20, the content of tween-20 is 0.05 percent, and the pH value of the phosphate buffer solution is 7.2;
(3) dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in 22.5mL of N, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(4) 1mg of HOF prepared in step (2) was dispersed in 1mL of phosphoric acid 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 to activate the carboxyl group at room temperature for 2 hours. Then 100. mu.L of aminated alpha-bungarotoxin aptamer (NH) at a concentration of 2. mu.M was added2- α -BGT-apt), stirred at room temperature for 12h, then centrifuged at 8000rpm for 15min and washed three times with phosphate buffered solution pH 7.2 to give HOF @ NH2-a biological probe for α -BGT-apt.
(II) 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 enzyme label plate, and incubating overnight at 4 ℃;
(2) slowly washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) respectively dripping alpha-BGT solution with the concentration of 0.0001-316ng/mL into the ELISA plates treated in the step (2), incubating for 1h at room temperature, and washing the ELISA plate holes for three times by using phosphate buffer solution containing Tween-20 after the incubation is finished;
(4) 100 μ L of HOF @ NH2Dripping a biological probe of-alpha-BGT-apt into the enzyme label plate treated in the step (3), incubating for 1h at room temperature, and after the incubation is finished, adding a probe containing vomitWashing the enzyme label plate for three times by using a-20-DEG C phosphate buffer solution;
(5) mu.L of 3,3',5,5' -tetramethylbenzidine and 150. mu.L of acetic acid buffer solution are added to the microplate, the concentration of the 3,3',5,5' -tetramethylbenzidine is 10mM, and the pH of the acetic acid buffer solution is 4.0. Reacting at room temperature for 30min to obtain the colorimetric immune biosensor for detecting the alpha-BGT with different concentrations.
And (3) placing the chromogenic solution in the 96-hole enzyme label plate in a micro cuvette, placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500nm, and recording the absorbance peak value corresponding to the alpha-BGT under different concentrations. The results are shown in FIG. 7. As can be seen from fig. 7, the absorbance values gradually increased with increasing concentration of α -BGT toxin. The resulting standard curve is shown in figure 6 for without UV, with y being 0.0675x + 0.5127. As can be seen, the absorbance has a good linear relationship with the logarithm of the concentration of alpha-BGT, and the correlation coefficient (R)2) 0.9982, LOD 0.033 fg. mL-1(S/N ═ 3), showing good linearity and lower LOD values.
Example 5 spiking recovery experiment
In order to verify the feasibility and the practicability of the constructed immune biosensor, a labeling recovery experiment is also carried out. A blank sample of human serum was taken, and alpha-bungarotoxin standards of known concentrations, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, and 100ng/mL, were added thereto, and detection was performed using the immunobiosensor constructed by us and the recovery rate was calculated, and the results are shown in Table 1.
TABLE 1 results of standard recovery experiments for alpha-bungarus multicinctus in human serum samples
As can be seen from table 1: the recovery rate of the immune biosensor for detecting alpha-bungarus multicinctus toxin provided by the invention is 99.34.0% -105.00%, and the standard deviation is 2.50% -3.78%, which shows that the analysis accuracy and reliability of the immune biosensor for detecting alpha-bungarus multicinctus in human serum samples are acceptable, and the immune biosensor has potential application value in clinical diagnosis.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An alpha-bungarotoxin detection probe, comprising an alpha-bungarotoxin aptamer and a hydrogen bond organic framework material chemically combined with the alpha-bungarotoxin 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-carboxyphenyl) pyrene.
2. The alpha-bungarotoxin detection probe as claimed in 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 an α -bungarotoxin assay probe according to 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-carboxyphenyl) pyrene;
mixing the hydrogen bond organic framework material with a carboxyl activating agent, and performing carboxyl activation to obtain a hydrogen bond organic framework material for activating carboxyl;
and mixing the hydrogen bond organic framework material for activating carboxyl and the aptamer of alpha-bungarotoxin modified with amino, and carrying out chemical combination to obtain the alpha-bungarotoxin detection probe.
4. The preparation method according to claim 3, wherein the mass ratio of the hydrogen bond organic framework material for activating carboxyl groups to the aptamer modified with amino-alpha-bungarotoxin is 300-500: 1.
5. A kit for detecting alpha-bungarotoxin, comprising an alpha-bungarotoxin detection probe according to claim 1 or 2 or an alpha-bungarotoxin detection probe prepared by the preparation method according to claim 3 or 4, an alpha-bungarotoxin first antibody, a non-specific protein, and a chromogenic substrate.
6. The kit for detecting α -bungarotoxin according to claim 5, wherein said chromogenic substrate is 3,3',5,5' -tetramethylbenzidine.
7. A method for detecting α -bungarotoxin for non-diagnostic purposes, comprising the steps of:
performing first incubation on the alpha-bungarotoxin first antibody to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unconjugated substances to obtain a second incubation product;
adding a sample to be tested into the second incubation product, performing third incubation, and removing unconjugated substances to obtain a third incubation product;
adding the alpha-bungarotoxin detecting probe of claim 1 or 2 or the alpha-bungarotoxin detecting probe prepared by the preparation method of claim 3 or 4 into the third incubation product, performing a fourth incubation, and removing unconjugated substances to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product, carrying out 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 relation curve of logarithm of alpha-bungarotoxin concentration and absorbance peak value.
8. The detection method according to claim 7, wherein the color reaction is carried out in an acetic acid buffer.
9. The detection method according to claim 7, wherein the color reaction is performed under normal light conditions or under 365nm laser irradiation.
10. The detection method according to claim 8 or 9, wherein the time for the color development reaction is 10 to 60 min.
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