CN114184789B - Prostate specific antigen detection probe and kit for detecting prostate specific antigen - Google Patents
Prostate specific antigen detection probe and kit for detecting prostate specific antigen Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
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Abstract
The invention provides a prostate specific antigen detection probe and a prostate specific antigen detection kit, and belongs to the technical field of biological sensing. In the invention, the surface of the Pd-containing organic complex L-Pd has amino groups, can be chemically combined with carboxyl groups of the prostate specific antigen secondary antibody through NH-CO bonds, and has good stability; meanwhile, the Pd-containing organic complex L-Pd has good water solubility, and can greatly improve the biocompatibility of the prostate specific antigen detection probe. In the invention, the L-Pd formed after self-assembly of the organic ligand L and the (ethandiene) palladium dinitrate has a supermolecular nano cage structure, and has excellent catalytic oxidation effect on TMB, not only because the metal Pd in the organic ligand L has the catalytic effect of noble metal, but also the nano cage structure has a certain enrichment effect on TMB, thereby further achieving the purpose of signal amplification.
Description
Technical Field
The invention relates to the technical field of biological sensing, in particular to a prostate specific antigen detection probe and a prostate specific antigen detection kit.
Background
In common malignant tumors of male urinary system and reproductive organs, the occurrence rate of prostate cancer is as high as 95%, and the sixth disease rate of male malignant tumors is also listed, and the disease is greatly affected by genetic factors. Prostate cancer not only causes damage to the quality of life of the patient, but also causes great threat to physical health and life safety. By detecting the biomarker with specificity and sensitivity to the prostate cancer, the aim of early diagnosis and monitoring of the prostate cancer can be achieved, so that the morbidity and the severity of the prostate cancer are reduced, the quality of life is effectively ensured, and the survival rate is improved.
Therefore, the early diagnosis and monitoring of the prostate cancer can be achieved by judging the nature and the state of the prostate cancer according to the content and the change condition of the prostate specific antigen. In medicine, diagnosis of diseases by quantitative detection of prostate-specific antigen in serum has become a current clinical focus and hotspot. Prostate specific antigen is detected by a chemobiological immunosensor using specific binding of the antibody antigen. The method has the advantages of high sensitivity, low cost, simple and convenient operation and high detection speed, and is an important method for detecting the biomarker.
For traditional enzyme-linked immunosorbent assays, detection antibodies are usually labeled with a native enzyme. However, 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 nanoenzymes have poor water solubility, resulting in reduced biocompatibility, which increases difficulty in ELISA detection of prostate specific antigen.
Disclosure of Invention
In view of the above, the present invention aims to provide a prostate specific antigen detection probe and a kit for detecting a prostate specific antigen. The prostate specific antigen detection probe provided by the invention has good water solubility.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a prostate specific antigen detection probe, which comprises a prostate specific antigen secondary antibody and a Pd-containing organic complex L-Pd chemically combined with the prostate specific antigen secondary antibody;
the Pd-containing organic complex L-Pd is obtained by self-assembling an organic ligand L and (ethandiene) palladium dinitrate, wherein the organic ligand L has a structure shown in a formula I:
preferably, the preparation method of the Pd-containing organic complex L-Pd comprises the following steps:
(1) Mixing 4, 7-dibromo-2, 1, 3-benzothiadiazole, bis- (4-pyridine) -amine, a catalyst and an organic solvent for a coordination reaction to obtain an organic ligand L;
(2) Mixing organic ligand L, (ethanamine diene) palladium dinitrate and water, and performing self-assembly to obtain Pd-containing organic complex L-Pd.
Preferably, the mass ratio of the organic ligand L to the (ethandiene) palladium (II) dinitrate is 1:1.5-2.
Preferably, the temperature of the coordination reaction is 160-180 ℃ and the time is 4-6 days;
the self-assembly temperature is 45-55 ℃ and the self-assembly time is 10-20 h.
Preferably, the mass ratio of the secondary prostate specific antigen antibody to the Pd-containing organic complex L-Pd is 1:5-10.
The invention provides a preparation method of the prostate specific antigen detection probe, which comprises the following steps:
mixing the prostate specific antigen secondary antibody with a carboxyl activating agent for carboxyl activation to obtain a carboxyl-activated prostate specific antigen secondary antibody;
and mixing the activated carboxyl-containing prostate specific antigen secondary antibody with Pd-containing organic complex L-Pd, and carrying out chemical combination to obtain the prostate specific antigen detection probe.
The invention provides application of a prostate specific antigen detection probe in preparation of a kit for detecting prostate specific antigens.
The invention provides a kit for detecting a prostate specific antigen, which comprises the prostate specific antigen detection probe, a prostate specific antigen primary antibody, a nonspecific protein and a chromogenic substrate.
Preferably, the chromogenic substrate is 3,3', 5' -tetramethylbenzidine.
Preferably, the buffer solution also comprises phosphate buffer solution with pH value of 7.0-7.4 and phosphate buffer solution with pH value of 4-4.5.
The invention provides a prostate specific antigen detection probe, which comprises a prostate specific antigen secondary antibody and Pd-containing organic complex L-Pd chemically combined with the prostate specific antigen secondary antibody, wherein the Pd-containing organic complex L-Pd is obtained by self-assembling an organic ligand L and (ethandiene) palladium dinitrate, and the organic ligand L has a structure shown in a formula I. In the invention, the surface of the Pd-containing organic complex L-Pd has amino groups, can be chemically combined with carboxyl groups of the prostate specific antigen secondary antibody through NH-CO bonds, and has good stability; meanwhile, the Pd-containing organic complex L-Pd has good water solubility, and can greatly improve the biocompatibility of the prostate specific antigen detection probe. In the invention, the L-Pd formed after self-assembly of the organic ligand L and the (ethandiene) palladium dinitrate has a supermolecular nano cage structure, has excellent catalytic oxidation effect on TMB, not only has the catalytic effect of noble metal on metal Pd, but also has a certain enrichment effect on TMB due to the nano cage structure, thereby further achieving the purpose of signal amplification.
In addition, the L-Pd has the effect of similar oxidase only under the catalysis of ultraviolet light, and not only can stop the whole catalytic reaction simply and quickly by ending the ultraviolet light, but also can simplify the operation steps, and has better controllability and wider application range.
The invention provides a kit for detecting a prostate specific antigen, which comprises a prostate specific antigen detection probe, a prostate specific antigen primary antibody, a nonspecific protein and a chromogenic substrate. In the invention, the prostate specific antigen primary antibody, the prostate specific antigen to be detected and the prostate specific antigen detection probe can form an antibody-antigen-antibody sandwich type biosensor, wherein the prostate specific antigen detection probe can specifically recognize the prostate specific antigen, has good enzyme activity, can catalyze chromogenic substrates to develop colors, has good linear relation in the concentration range of the prostate specific antigen within 0.0001-1000ng/mL along with the increase of the concentration of the prostate specific antigen, has the advantages of high sensitivity, low cost, quick detection and low detection limit, and has a good linear relation in the lowest detection limit within the concentration range of the prostate specific antigen within 0.0001-1000 ng/mL.
Drawings
FIG. 1 is a flow chart of the preparation of L ligand and L-Pd and a structural simulation;
FIG. 2 is a transmission electron microscope image of L-Pd obtained in example 3;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the L ligand obtained in example 3;
FIG. 4 is a graph showing the absorbance contrast of the PSA of example 3 when it is irradiated with ultraviolet light;
FIG. 5 is a graph showing the effect of pH of phosphate buffer solution on the absorbance of PSA in example 3;
FIG. 6 is a graph showing the effect of temperature on the ability of L-Pd to catalyze the oxidation of TMB in example 3;
FIG. 7 is a graph showing the absorbance versus concentration of PSA antigen for example 3;
FIG. 8 is a linear relationship between absorbance and the logarithmic value of PSA antigen concentration in example 3;
FIG. 9 is a graph showing the effect of interferents on prostate specific antigen detection results;
FIG. 10 shows the results of prostate specific antigen detection of prostate epithelial cells expressing PSA;
FIG. 11 shows peak absorbance change of the chemical-biological immunosensor after long-term placement.
Detailed Description
The invention provides a prostate specific antigen detection probe, which comprises a prostate specific antigen secondary antibody and a Pd-containing organic complex L-Pd chemically combined with the prostate specific antigen secondary antibody;
the Pd-containing organic complex L-Pd is obtained by self-assembling an organic ligand L and (ethandiene) palladium dinitrate, wherein the organic ligand L has a structure shown in a formula I:
in the present invention, the preparation method of the Pd-containing organic complex L-Pd preferably comprises the following steps:
(1) Mixing 4, 7-dibromo-2, 1, 3-benzothiadiazole, bis- (4-pyridine) -amine, a catalyst and an organic solvent for a coordination reaction to obtain an organic ligand L;
(2) Mixing organic ligand L, (ethanamine diene) palladium dinitrate and water, and performing self-assembly to obtain Pd-containing organic complex L-Pd.
The invention mixes 4, 7-dibromo-2, 1, 3-benzothiadiazole, bis- (4-pyridine) -amine, catalyst and organic solvent to carry out coordination reaction, thus obtaining the organic ligand L. In the present invention, the catalyst is preferably anhydrous copper sulfate and anhydrous potassium carbonate, and the molar ratio of the anhydrous copper sulfate to the anhydrous potassium carbonate is preferably 10:8-9.
In the present invention, a compounding accelerator is preferably added to the compounding reaction, and the compounding accelerator is preferably 18-crown-6.
In the present invention, the organic solvent is preferably diphenyl ether.
In the invention, the molar ratio of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the bis- (4-pyridine) -amine is preferably 1:2.5-3.5, more preferably 1:3; the molar ratio of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the coordination accelerator is preferably 1:0.01-0.02; the molar ratio of the 4, 7-dibromo-2, 1, 3-benzothiadiazole to the anhydrous copper sulfate is preferably 1:4-5.
In the present invention, the mixing means is preferably stirring mixing, and the temperature of the stirring mixing is preferably 50 ℃ and the time is preferably 30min.
In the present invention, the temperature of the compounding reaction is preferably 160 to 180 ℃, more preferably 170 ℃; the time is preferably 4 to 6 days, more preferably 5 days.
After the complexing reaction, the invention preferably carries out post-treatment on the obtained complexing reaction liquid, and the post-treatment preferably comprises the following steps:
removing the organic solvent of the coordination reaction liquid, and eluting and purifying the obtained residues to obtain an eluent;
and sequentially performing rotary evaporation and drying on the obtained eluent to obtain the organic ligand L.
In the present invention, the mode of removing the organic solvent of the reaction mixture is preferably pressure distillation.
In the present invention, the eluting purification comprises:
the neutral alumina is used as a stationary phase and dichloromethane is used as a mobile phase for first elution, and then the mixed solution of THF and dichloromethane is used as a mobile phase for second elution. In the present invention, the volume ratio of THF to dichloromethane at the second elution is preferably 1:1.
In the present invention, the particle size of the stationary phase is preferably 100 to 200 mesh. In the present invention, the purpose of the first elution is to remove by-products, after which the resulting residue is visible as blue-violet fluorescence under uv light.
In the present invention, the eluent obtained after the second elution has yellow fluorescence under ultraviolet conditions.
In the present invention, the temperature of the rotary evaporation is preferably 60 ℃; in the present invention, the drying means is preferably vacuum drying.
In the present invention, the organic ligand L is insoluble in water.
After the organic ligand L is obtained, the organic ligand L, (ethandiene) palladium (II) dinitrate and water are mixed, and self-assembly is carried out, so that the Pd-containing organic complex L-Pd is obtained.
In the present invention, the (ethanamine diene) palladium (II) dinitrate has the structure shown in formula II:
in the present invention, the mass ratio of the organic ligand L to the (ethandiene) palladium (II) dinitrate is preferably 1:1.5 to 2, more preferably 1:1.65.
In the present invention, the self-assembly temperature is preferably 45 to 55 ℃, more preferably 50 ℃; the time is preferably 10 to 20 hours, more preferably 12 to 15 hours.
The organic ligand L, (ethanamine diene) palladium (II) dinitrate self-assembles to form L-Pd with supermolecule nano cage structure.
After the self-assembly, the present invention preferably performs a post-treatment of the resulting self-assembled product, the post-treatment preferably comprising:
and washing and drying the self-assembled product to obtain the Pd-containing organic complex L-Pd solid.
In the present invention, the washing detergent is preferably acetone, and the number of times of washing is preferably 3.
In the present invention, the drying means is preferably vacuum drying, and the drying temperature is preferably 50 ℃.
In the present invention, the mass ratio of the secondary prostate specific antigen to the Pd-containing organic complex L-Pd is preferably 1:5 to 10, more preferably 1:6 to 8.
The invention provides a preparation method of the prostate specific antigen detection probe, which comprises the following steps:
mixing the prostate specific antigen secondary antibody with a carboxyl activating agent for carboxyl activation to obtain a carboxyl-activated prostate specific antigen secondary antibody;
and mixing the activated carboxyl-containing prostate specific antigen secondary antibody with Pd-containing organic complex L-Pd, and carrying out chemical combination to obtain the prostate specific antigen detection probe.
The invention mixes the prostate specific antigen secondary antibody with a carboxyl activating agent to activate carboxyl, and obtains the prostate specific antigen secondary antibody with activated carboxyl. In the present invention, the carboxyl activator is preferably EDC and NHS, and the mass ratio of EDC to NHS is preferably 1:1.
In the present invention, the temperature of the carboxyl group activation is preferably 0 to 4 ℃, more preferably 4 ℃, and the time is preferably 1 to 2 hours.
After the activated carboxyl-containing prostate specific antigen secondary antibody is obtained, the activated carboxyl-containing prostate specific antigen secondary antibody is mixed with Pd-containing organic complex L-Pd for chemical combination, so that the prostate specific antigen detection probe is obtained. In the present invention, the temperature of the chemical bonding is preferably 0 to 4 ℃, and the time is preferably 18 to 30 hours, more preferably 24 hours.
After the chemical binding, the present invention preferably dialyzes and lyophilizes the resulting chemically bound product. In the present invention, the dialysis is preferably performed in ultrapure water, the molecular weight cut-off of the dialysis is preferably 1000, and the dialysis time is preferably 3 days.
The invention provides an application of the prostate specific antigen detection probe or the prostate specific antigen detection probe prepared by the preparation method in preparation of a kit for detecting the prostate specific antigen.
The invention provides a kit for detecting a prostate specific antigen, which comprises the prostate specific antigen detection probe, a prostate specific antigen primary antibody, a nonspecific protein and a chromogenic substrate.
In the present invention, the chromogenic substrate is preferably 3,3', 5' -Tetramethylbenzidine (TMB).
In the present invention, the nonspecific protein is preferably bovine serum albumin.
In the present invention, the kit for detecting a prostate-specific antigen further preferably comprises a phosphate buffer having a pH of 7.0 to 7.4 and a phosphate buffer having a pH of 4 to 4.5.
In the present invention, the method for detecting a prostate-specific antigen preferably comprises the steps of:
performing first incubation on the first antigen of the prostate specific antigen 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 a prostate specific antigen 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, performing a chromogenic reaction under the ultraviolet light condition, and testing the absorbance peak value of the chromogenic solution at 550-800 nm;
obtaining the concentration of the prostate specific antigen in the sample to be tested 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 prostate-specific antigen and the absorbance peak.
The invention carries out first incubation on the first antigen of the prostate specific antigen in an incubator to obtain a first incubation product. In the present invention, the incubator is preferably a 96-well elisa plate. The 96-well elisa plate is preferably washed before the first incubation, and the washing detergent is preferably water and phosphate buffer with ph=7.0 to 7.4.
The present invention has no particular requirement for the primary prostate specific antigen, and the primary prostate specific antigen known to those skilled in the art can be used.
In the present invention, the concentration of the primary prostate-specific antigen is preferably 2. Mu.g/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 bovine serum albumin is preferably 1wt%. The invention uses the nonspecific protein to block the unbound primary antibody.
In the present invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 45 to 60 minutes.
In the present invention, the unbound material is preferably removed by washing with a phosphate buffer having ph=7.0 to 7.4.
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 25 to 37 ℃, more preferably 28 to 30 ℃, and the time is preferably 1.5 to 2 hours.
In the present invention, the unbound material is preferably removed by washing with a phosphate buffer having ph=7.0 to 7.4.
After the third incubation product is obtained, a prostate specific antigen detection probe is added into the third incubation product, and fourth incubation is carried out to remove unbound materials, so that a fourth incubation product is obtained. In the present invention, the concentration of the prostate-specific antigen detection probe is preferably 1. Mu.g/mL. In the present invention, the temperature of the fourth incubation is preferably 25 to 37 ℃, more preferably 28 to 30 ℃, and the time is preferably 1h.
In the present invention, the unbound material is preferably removed by washing with a phosphate buffer having ph=7.0 to 7.4.
After the fourth incubation product is obtained, a chromogenic substrate is added into the fourth incubation product, a chromogenic reaction is carried out under the condition of ultraviolet light, and the absorbance peak value of the chromogenic solution at 550-800nm is tested. In the present invention, the chromogenic substrate is preferably 3,3', 5' -Tetramethylbenzidine (TMB). In the present invention, the concentration of the chromogenic substrate is preferably 75mM.
In the present invention, the wavelength of the ultraviolet light is preferably 325nm. In the present invention, the color reaction temperature is preferably room temperature, and the time is preferably 10 to 15 minutes.
The absorbance of the color-developing solution is preferably measured by using an ultraviolet spectrophotometer.
After the absorbance peak value is obtained, the concentration of the prostate specific antigen 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 prostate-specific antigen and the absorbance peak.
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 prostate specific antigen in a gradient concentration comprising 10 -4 、10 -3 、10 -2 、0.1、1、10、100、1000ng/mL。
The method comprises the steps of taking a standard substance solution of the prostate specific antigen 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 the prostate specific antigen with gradient concentration, drawing by taking the logarithm of the prostate specific antigen as an abscissa and taking the absorbance value as an ordinate, and obtaining a linear relation curve of the logarithm of the prostate specific antigen concentration and the absorbance value.
Specifically, the relevant data of the standard curve are shown in table 1.
TABLE 1 Standard Curve correlation data
In the present invention, the lower limit of detection of the prostate-specific antigen is 30fg mL -1 (S/N=3), the detection range is 0.0001-1000ng mL -1 。
The following examples are provided to illustrate a prostate specific antigen detection probe and a kit for detecting a prostate specific antigen according to the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of prostate specific antigen detection probes
(1) Synthesis of L ligand: 4, 7-dibromo-2, 1, 3-benzothiadiazole (0.3 g,1.02 mmol) and bis- (4-pyridin) -amine (1 g,6 mmol) were mixed in a 100mL round bottom flask, 18-crown-6 (0.01 g,0.04 mmol), anhydrous copper sulfate (0.16 g,10 mmol) and anhydrous potassium carbonate (1.12 g,8.16 mmol) were added, and finally 40mL diphenyl ether was added as solvent. Degassing with nitrogen, sealing, stirring at 50deg.C for 30min, heating to 170deg.C, stirring under nitrogen for 4 days, distilling under reduced pressure to remove diphenyl ether, and drying. Eluting with neutral alumina as stationary phase and dichloromethane as mobile phase until the side product is removed (blue-violet fluorescence is visible under ultraviolet irradiation), eluting with mixed solution of THF and dichloromethane as mobile phase, and obtaining yellow fluorescent substance as target product ligand L.
(2) Synthesis of L-Pd: the resulting eluate containing L was collected, subjected to rotary evaporation in a water bath at 60℃to remove THF and methylene chloride, and dried in vacuo to give a water-insoluble ligand. Then, 20mg of ligand L and 33mg of (ethandiene) palladium (II) dinitrate were dissolved in a 10mL round bottom flask, and 4mL of ultra-pure water was added thereto and stirred at 55℃for 12 hours. And (3) washing the obtained product with acetone for three times after drying, and performing vacuum drying at 50 ℃ after completion to obtain the water-soluble L-Pd with the oxidase-like effect under ultraviolet irradiation, and preserving for later use.
(3)Ab 2 Synthesis of @ L-Pd: 0.4mg Ab was taken 2 The carboxyl groups of the secondary antibody were activated by dissolving in 4mL of ultrapure water, adding 5mM EDC and NHS, and stirring at 4℃for 1 hour. After activation was complete, 4mg of L-Pd was added and stirring was continued at 4℃for 24h. Taking a dialysis bag with the molecular weight of 1000, placing the mixed solution in the step (1) into the dialysis bag after activation, stirring and dialyzing in a beaker filled with 100mL of ultrapure water, dialyzing for 3d at 4 ℃, and removing excess L-Pd, wherein the ultrapure water in the beaker is replaced every 8 hours. After the dialysis was completed, the resulting solution was re-fixed to 4mL and frozen for use, diluted ten-fold when used.
Method for detecting (II) prostate specific antigen
(1) Preparing a standard 96-well ELISA plate, and cleaning by sequentially using pure water and phosphate buffer solution with pH value of 7.0-7.4;
(2) 100. Mu.L of Ab was used at a concentration of 2. Mu.g/mL 1 Adding the solution into the enzyme-labeled wells treated in the step (1), and standing overnight at 4 ℃ for incubation;
(3) Slowly cleaning the enzyme-labeled hole in the step (2) for three times by using phosphate buffer solution with pH=7.0-7.4, adding 1% BSA solution for blocking, and slowly cleaning the phosphate buffer solution for three times again after 1 h;
(4) Sequentially adding 100 mu L of PSA solution with the concentration of 0.0001-1000ng/mL into the enzyme-labeled wells treated by the step (3), incubating for 2 hours at 37 ℃, and slowly washing for three times by using phosphate buffer with the pH value of 7.0-7.4;
(5) 100. Mu.L of Ab was taken at a concentration of 1. Mu.g/mL 2 After adding @ L-Pd solution to the treated microplate wells of (4) and incubating at 37℃for 1 hour, the wells were washed three times slowly with phosphate buffer solution having pH=7.0-7.4, 100. Mu.L of 75mM TMB solution was added, 200. Mu.L of phosphate buffer solution having pH=4.0-4.5 was added, and the wells were left to stand at room temperature for 10 minutes under irradiation of ultraviolet light having a wavelength of 325nm.
The application method of the water-soluble chemical biological immunosensor for detecting the prostate specific antigen comprises the following specific steps:
(1) Placing the mixed solution in the ELISA plate in a micro cuvette after ultraviolet irradiation, placing the micro cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 550-800nm, and recording the absorbance peak when the peak appears in the 660nm range;
(2) Recording absorbance peaks corresponding to the prostate specific antigen at different concentrations;
(3) The concentration of the prostate specific antigen in the sample to be tested is obtained by using a standard curve method, the detection range is 0.0001-1000ng/mL, and the LOD reaches 30fg/mL (S/N=3).
Example 2
Preparation of prostate specific antigen detection probes
(1) Synthesis of L ligand: 4, 7-dibromo-2, 1, 3-benzothiadiazole (1 g,3 mmol) and bis- (4-pyridin) -amine (1 g,6 mmol) were mixed in a 100mL round bottom flask, 18-crown-6 (0.01 g,0.04 mmol), anhydrous copper sulfate (0.16 g,10 mmol) and anhydrous potassium carbonate (1.12 g,8.16 mmol) were added, and finally 40mL diphenyl ether was added as solvent. Degassing with nitrogen, sealing, stirring at 50deg.C for 30min, heating to 170deg.C, stirring under nitrogen for 4 days, distilling under reduced pressure to remove diphenyl ether, and drying. Eluting with neutral alumina as stationary phase and dichloromethane as mobile phase until the side product is removed (blue-violet fluorescence is visible under ultraviolet irradiation), eluting with mixed solution of THF and dichloromethane as mobile phase, and obtaining yellow fluorescent substance as target product ligand L.
(2) Synthesis of L-Pd: the resulting eluate containing L was collected, subjected to rotary evaporation in a water bath at 60℃to remove THF and methylene chloride, and dried in vacuo to give a water-insoluble ligand. Then, 20mg of ligand L and 33mg of (ethandiene) palladium (II) dinitrate were dissolved in a 10mL round bottom flask, and 4mL of ultra-pure water was added thereto and stirred at 55℃for 12 hours. And (3) washing the obtained product with acetone for three times after drying, and performing vacuum drying at 50 ℃ after completion to obtain the water-soluble L-Pd with the oxidase-like effect under ultraviolet irradiation, and preserving for later use.
(3)Ab 2 Synthesis of @ L-Pd: 0.4mg Ab was taken 2 Dissolving in 4mL of ultrapure water, adding 5mM EDC and NHS, stirring at 4deg.C for 1 hr, and activating carboxyl group of the secondary antibody. After activation was complete, 4mg of L-Pd was added and stirring was continued at 4℃for 24h. Taking a dialysis bag with the molecular weight of 1000, placing the mixed solution in the step (1) into the dialysis bag after activation, stirring and dialyzing in a beaker filled with 100mL of ultrapure water, dialyzing for 3d at 4 ℃, and removing excess L-Pd, wherein the ultrapure water in the beaker is replaced every 8 hours. After the dialysis was completed, the resulting solution was re-fixed to 4mL and frozen for use, diluted ten-fold when used.
Method for detecting (II) prostate specific antigen
(1) Preparing a standard 96-well ELISA plate, and cleaning by sequentially using pure water and phosphate buffer solution with pH value of 7.0-7.4;
(2) 100. Mu.L of Ab1 solution with the concentration of 2. Mu.g/mL is added into the enzyme-labeled well treated by the method (1), and the mixture is incubated at the temperature of 4 ℃ overnight;
(3) Slowly washing the enzyme-labeled hole in the step (2) for three times by using phosphate buffer with pH=7.0-7.4, adding 1% BSA solution for blocking, and slowly washing the phosphate buffer for three times again after 1 h;
(4) Sequentially adding 100 mu L of PSA solution with the concentration of 0.0001-1000ng/mL into the enzyme-labeled wells treated by the step (3), incubating for 2 hours at 37 ℃, and slowly washing three times by using phosphate buffer with the pH of 7.0-7.4;
(5) 100. Mu.L of Ab2@L-Pd solution with the concentration of 1. Mu.g/mL is added into the treated enzyme-labeled well (4), and after incubation is carried out for 1h at 37 ℃, the enzyme-labeled well is slowly washed three times by using phosphate buffer with the pH of 7.0-7.4, 100. Mu.L of TMB solution with the pH of 75mM is added, 200. Mu.L of phosphate buffer with the pH of 4.0-4.5 is added, and the enzyme-labeled well is placed for 10min at room temperature under the irradiation of ultraviolet lamp with the wavelength of 325nm.
The application method of the water-soluble chemical biological immunosensor for detecting the prostate specific antigen comprises the following specific steps:
(1) Placing the mixed solution in the ELISA plate in a micro cuvette after ultraviolet irradiation, placing the micro cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 550-800nm, and recording absorbance peaks when peaks appear in the 660nm range;
(2) Recording absorbance peaks corresponding to the prostate specific antigen at different concentrations;
(3) The concentration of the prostate specific antigen in the sample to be tested is obtained by using a standard curve method, the detection range is 0.0001-1000ng/mL, and the LOD reaches 30fg/mL (S/N=3).
Example 3
Preparation of prostate specific antigen detection probes
(1) Synthesis of L ligand: 4, 7-dibromo-2, 1, 3-benzothiadiazole (0.6 g,2.04 mmol) and bis- (4-pyridin) -amine (1 g,6 mmol) were mixed in a 100mL round bottom flask, 18-crown-6 (0.01 g,0.04 mmol), anhydrous copper sulfate (0.16 g,10 mmol) and anhydrous potassium carbonate (1.12 g,8.16 mmol) were added, and finally 40mL diphenyl ether was added as solvent. Degassing with nitrogen, sealing, stirring at 50deg.C for 30min, heating to 170deg.C, stirring under nitrogen for 4 days, distilling under reduced pressure to remove diphenyl ether, and drying. Eluting with neutral alumina as stationary phase and dichloromethane as mobile phase until the side product is removed (blue-violet fluorescence is visible under ultraviolet irradiation), eluting with mixed solution of THF and dichloromethane as mobile phase, and obtaining yellow fluorescent substance as target product ligand L.
(2) Synthesis of L-Pd: the resulting eluate containing L was collected, subjected to rotary evaporation in a water bath at 60℃to remove THF and methylene chloride, and dried in vacuo to give a water-insoluble ligand. Then, 20mg of ligand L and 33mg of (ethandiene) palladium (II) dinitrate were dissolved in a 10mL round bottom flask, and 4mL of ultra-pure water was added thereto and stirred at 55℃for 12 hours. And (3) washing the obtained product with acetone for three times after drying, and performing vacuum drying at 50 ℃ after completion to obtain the water-soluble L-Pd with the oxidase-like effect under ultraviolet irradiation, and preserving for later use.
(3)Ab 2 Synthesis of @ L-Pd: 0.4mg Ab was taken 2 The carboxyl groups of the secondary antibody were activated by dissolving in 4mL of ultrapure water, adding 5mM EDC and NHS, and stirring at 4℃for 1 hour. After activation was complete, 4mg of L-Pd was added and stirring was continued at 4℃for 24h. Taking a dialysis bag with the molecular weight of 1000, placing the mixed solution in the step (1) into the dialysis bag after activation, stirring and dialyzing in a beaker filled with 100mL of ultrapure water, dialyzing for 3d at 4 ℃, and removing excess L-Pd, wherein the ultrapure water in the beaker is replaced every 8 hours. After dialysis, the obtained solution is re-fixed to 4mL for freezing, and diluted when in useTen times as many as possible.
Method for detecting (II) prostate specific antigen
(1) Preparing a standard 96-well ELISA plate, and cleaning by sequentially using pure water and phosphate buffer solution with pH value of 7.0-7.4;
(2) 100. Mu.L of Ab was used at a concentration of 2. Mu.g/mL 1 Adding the solution into the enzyme-labeled wells treated in the step (1), and standing overnight at 4 ℃ for incubation;
(3) Slowly cleaning the enzyme-labeled hole in the step (2) for three times by using phosphate buffer solution with pH=7.0-7.4, adding 1% BSA solution for blocking, and slowly cleaning the phosphate buffer solution for three times again after 1 h;
(4) Sequentially adding 100 mu L of PSA solution with the concentration of 0.0001-1000ng/mL into the enzyme-labeled wells treated by the step (3), incubating for 2 hours at 37 ℃, and slowly washing for three times by using phosphate buffer with the pH value of 7.0-7.4;
(5) 100. Mu.L of Ab was taken at a concentration of 1. Mu.g/mL 2 Adding @ L-Pd solution into the treated enzyme-labeled well (4), incubating for 1h at 37 ℃, slowly washing three times by using phosphate buffer solution with pH=7.0-7.4, adding 100 mu L of 75mM TMB solution, adding 200 mu L of phosphate buffer solution with pH=4.0-4.5, and standing for 10min at room temperature under irradiation of ultraviolet lamp with 325nm wavelength.
The application method of the water-soluble chemical biological immunosensor for detecting the prostate specific antigen comprises the following specific steps:
(1) Placing the mixed solution in the ELISA plate in a micro cuvette after ultraviolet irradiation, placing the micro cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 550-800nm, and recording the absorbance peak when the peak appears in the 660nm range;
(2) Recording absorbance peaks corresponding to the prostate specific antigen at different concentrations;
(3) The concentration of the prostate specific antigen in the sample to be tested is obtained by using a standard curve method, the detection range is 0.0001-1000ng/mL, and the LOD reaches 30fg/mL (S/N=3).
FIG. 1 is a schematic diagram and a structural simulation of the preparation of L ligand and L-Pd, wherein (A) is a schematic diagram and (B) is a schematic diagram and a structural simulation of the preparation of L ligand and L-Pd (B). Wherein the structure of the L-Pd is simulated as an octahedral supermolecule nano cage.
FIG. 2 is a transmission electron microscope image of L-Pd in the step (2), wherein the L-Pd supermolecule nanocages are small in size, about 1-2nm in size and distributed in a dot shape.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the L ligand in the step (1) of example 3, wherein 8.5ppm is a peak of ortho-nitrogen hydrogen on the L-square benzene ring, 7.5ppm is a peak of meta-nitrogen hydrogen on the L-square benzene ring, and 7.0ppm is hydrogen on the central benzene ring of the L ligand. Since diphenyl ether is a high boiling point solvent and is difficult to remove, small amounts of impurity peaks such as solvent peaks appear at 7.0-7.5 ppm. This nuclear magnetic resonance hydrogen spectrum illustrates the successful synthesis of ligand L.
FIG. 4 is a graph showing the comparison of absorbance at concentrations of 0.01, 0.1, 1ng/mL, respectively, of UV light, showing that the ability to catalyze the oxidation of TMB in L-Pd is induced by UV light, which greatly enhances the oxidase-like ability of L-Pd, which is very weak in the absence of UV light.
Fig. 5 shows the absorbance change of the PSA solution at 10ng/mL after changing the pH of the phosphate buffer solution used, and it can be seen that ph=4.0 to 4.5 is the optimum pH range for catalytic oxidation of TMB by L-Pd.
FIG. 6 is a graph showing the effect of temperature on the ability of L-Pd to catalyze the oxidation of TMB. As the temperature increases, the ability of the L-Pd to catalyze the oxidation of TMB gradually decreases, so that the room temperature of 20-25 ℃ is the optimal temperature range from the cost point of view.
FIG. 7 shows that the antigen concentrations were taken at 10 -4 、10 -3 、10 -2 Working curves of absorbance as a function of PSA antigen concentration after 0.1, 1, 10, 100, 1000 ng/mL. It can be seen that Ab increases with increasing PSA concentration 2 The content of @ L-Pd is increased, the catalytic oxidation capability of TMB is improved, the blue color is gradually deepened, and the absorbance of the system is gradually increased.
FIG. 8 is a linear relationship between absorbance and the logarithmic value of PSA antigen concentration. It can be seen that the absorbance is well-linearly related to the logarithmic value of the antigen concentration (ng/mL), where R 2 Since 0.9983, the PSA content can be accurately quantified based on the change in absorbance.
Example 4
The Prostate Specific Antigen (PSA) of example 3 was replaced with a common interferent in serum, and the detection method of the prostate specific antigen was the same as that of example 3, except that the method of example 3 was used. Among the common interferents are Bovine Serum Albumin (BSA), OVA (ovalbumin), LZ (lysozyme), AA (ascorbic acid), SUC (sucrose) and GLC (glucose), and PSA solutions and mixed solutions of all the above interferents with PSA. The concentration of the interferents was 100ng/mL.
FIG. 9 is a graph showing the effect of interferents on prostate specific antigen detection results. The serum interferent has no great influence on absorbance, and meanwhile, the content detection of the PSA is not influenced by the serum interferent, so that the chemical biosensor has the advantages of good selectivity, strong anti-interference capability and potential of application in practical detection.
Example 5
The procedure of example 3 was repeated except that the prostate-specific antigen (PSA) of example 3 was replaced with prostate epithelial cells expressing PSA, and the method of detecting a prostate-specific antigen was the same as that of example 3. Wherein, prostate epithelial cells were taken separately: normal prostate epithelial cells (RWPE-1), PSA-low expressing prostate epithelial cells (DU 145 and PC 3), and PSA-high expressing prostate epithelial cells (LNCaP and 22RV 1).
FIG. 10 shows the results of prostate specific antigen detection of prostate epithelial cells expressing PSA. The calculated PSA content in the five cells meets the expectations, and the PSA content in RWPE-1, DU145 and PC3 is far lower than the PSA content in LNCaP and 22RV1, so that the chemical biosensor can be well applied to detection of actual samples.
Example 6
The Prostate Specific Antigen (PSA) of example 3 was replaced with a PSA solution at a concentration of 1ug/mL, and the prepared sensor was left for 3 weeks to measure the peak change in absorbance at days 1, 2, 3, 4, 5, 6, 7, 14, 21, respectively, and the prepared chemobiological immunosensor was used in the same manner as in example 3, except that example 3 was used.
As shown in FIG. 11, the sensor has good stability, and the measured result is less affected by time, wherein the absorbance on day 21 is about 84.82% on day 1, which indicates that the sensor has good detection capability after long-term placement, and is simple and quick to use.
Example 7
The procedure of example 3 was repeated except that the Prostate Specific Antigen (PSA) of example 3 was replaced with an artificial serum sample to which 0.1, 1 and 10ng/mL of PSA solution were added, and the prepared chemical-biological immunosensor was used in the same manner as in example 3.
Example 7 is the detection of PSA concentration in an artificial serum sample using a labeled recovery method to assess the feasibility and accuracy of the sensor to detect an actual sample for analysis, the results are shown in table 1.
TABLE 1 detection results by the labeled recovery method
It can be seen from table 1 that the recovery rate of the sensor is 99.8% -100.2% and the RSD value is 2.4% -8.8%, which indicates that the sensor can be applied to the actual detection of PSA content and has great potential in the diagnosis and analysis of prostate cancer.
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 (5)
1. A kit for detecting a prostate specific antigen comprises a prostate specific antigen detection probe, a prostate specific antigen primary antibody, a non-specific protein and a chromogenic substrate;
the prostate specific antigen detection probe comprises a prostate specific antigen secondary antibody and a Pd-containing organic complex L-Pd which is chemically combined with the prostate specific antigen secondary antibody;
the Pd-containing organic complex L-Pd is obtained by self-assembling an organic ligand L and (ethandiene) palladium dinitrate, wherein the organic ligand L has a structure shown in a formula I:
the mass ratio of the prostate specific antigen secondary antibody to the Pd-containing organic complex L-Pd is 1:5-10;
the temperature of the chemical combination is 0-4 ℃ and the time is 18-30 hours;
the preparation method of the prostate specific antigen detection probe comprises the following steps:
mixing the prostate specific antigen secondary antibody with a carboxyl activating agent for carboxyl activation to obtain a carboxyl-activated prostate specific antigen secondary antibody;
mixing the activated carboxyl-containing prostate specific antigen secondary antibody with Pd-containing organic complex L-Pd, and carrying out chemical combination to obtain a prostate specific antigen detection probe;
the chromogenic substrate is 3,3', 5' -tetramethylbenzidine.
2. The kit for detecting prostate specific antigen according to claim 1, wherein the preparation method of the Pd-containing organic complex L-Pd comprises the steps of:
(1) Mixing 4, 7-dibromo-2, 1, 3-benzothiadiazole, bis- (4-pyridine) -amine, a catalyst and an organic solvent for a coordination reaction to obtain an organic ligand L;
(2) Mixing organic ligand L, (ethanamine diene) palladium dinitrate and water, and performing self-assembly to obtain Pd-containing organic complex L-Pd.
3. The kit for detecting the prostate specific antigen according to claim 2, wherein the mass ratio of the organic ligand L to the (ethandiene) palladium dinitrate is 1:1.5-2.
4. The kit for detecting prostate specific antigen according to claim 2, wherein the temperature of the coordination reaction is 160-180 ℃ for 4-6 days;
the self-assembly temperature is 45-55 ℃ and the self-assembly time is 10-20 h.
5. The kit for detecting prostate specific antigen according to claim 1, further comprising a phosphate buffer solution with a pH value of 7.0 to 7.4 and a phosphate buffer solution with a pH value of 4 to 4.5.
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