CN109470757B - Electrochemical detection method for prostate specific antigen detection - Google Patents
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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Abstract
The electrochemical immunosensor electrical signal marker for detecting the prostate specific antigen is protease, a catalytic substrate of the protease is polypeptide, and the sequence characteristics of the polypeptide are as follows: the third amino acid in the direction of the carbon terminus of the cleavage site is histidine H. The detection method for detecting the prostate antigen adopts the electric signal marker of the electrochemical immunosensor for detecting the prostate specific antigen, and comprises the following steps of A: preparing a secondary antibody/carbon nano tube/protease complex; b: preparing a working electrode; c: the working electrode was soaked in PBS solution containing copper ions and a polypeptide substrate for 2 hours, and then electrochemical testing was performed using linear sweep voltammetry. The electric signal marker has obvious electric signal, can meet the requirement of detecting the concentration of prostate specific antigen in human serum, does not use biphenyl substances in the catalytic substrate, and is harmless to workers and the environment.
Description
Technical Field
The invention relates to a prostate specific antigen detection method, in particular to an electrochemical immunosensor signal marker for prostate specific antigen detection and a detection method, belonging to the technical field of biochemistry.
Background
The prostate specific antigen is a biomarker of prostate diseases, and the concentration of the prostate specific antigen in serum is closely related to the occurrence of prostate cancer. For example, the concentration of prostate specific antigen in normal human serum is below 4 ng/mL, but when it is above 10 ng/mL, the prostate patient is most likely to have prostate cancer. Therefore, the detection of prostate specific antigen is of great significance in the early diagnosis of prostate diseases. At present, the method for clinically detecting the prostate specific antigen is mainly an enzyme-linked immunosorbent assay, but the method needs to use a special instrument, is expensive, has complex detection procedures and higher detection cost, and needs to use biphenyl compounds as a detection substrate, and the biphenyl compounds have a certain carcinogenic effect, so that the development of a novel method for detecting the prostate specific antigen has very important significance.
The electrochemical immunosensor has the advantages of convenience in use, high measurement precision, simplicity in maintenance, low cost and the like, is one of effective methods for detecting protein, and has wide application prospects in bioanalysis. In this detection system, a target protein can be captured by an antibody 1 (primary antibody) on the surface of an electrode, and then the captured protein is recognized and outputted with a signal by an enzyme-labeled antibody 2 (secondary antibody). The enzyme is a common electric signal marker for signal output of an electrochemical immunosensor, and mainly comprises catalase, glucose oxidase and alkaline phosphomonoesterase at present. However, these natural enzymes have some potential disadvantages, such as poor stability, high cost, and difficult preparation. In addition, the metal catalytic centers of catalase and glucose oxidase are buried in the middle of protein, so that electron transfer is not easy to directly occur on the surface of an electrode, and a special enzyme catalytic substrate (such as hydrogen peroxide, glucose and the like) is required to be adopted; the catalytic product of the alkaline phosphomonoesterase has poor oxidation resistance, is unstable in air, has weak electric signal, and is easy to form polymer to passivate an electrode. These factors severely limit the practical application of electrochemical immunosensors.
Disclosure of Invention
The invention aims to overcome the problems in the existing prostate specific antigen detection and provides an electrochemical immunosensor electric signal marker for prostate specific antigen detection and a detection method.
An electrochemical immunosensor electrical signal marker for prostate specific antigen detection, wherein the electrical signal marker is protease, a catalytic substrate of the protease is polypeptide, and the polypeptide can be cut by the protease; the sequence characteristics of the polypeptide are: the third amino acid of the cleavage site in the direction of the carbon terminus is histidine H; further, the method comprises the following steps of; the protease and a secondary antibody corresponding to the prostate specific antigen are compounded on the carbon nano tube to form a secondary antibody/carbon nano tube/protease compound.
The detection method for detecting the prostate specific antigen adopts the electric signal marker of the electrochemical immunosensor for detecting the prostate specific antigen, and comprises the following steps:
a: the preparation of the secondary antibody/carbon nano tube/protease complex comprises the following substeps:
a1: pretreating the carbon nano tube;
boiling a multi-walled carbon nanotube for 12 hours by using 2M nitric acid, cooling, then centrifugally washing for three times, heating the centrifuged multi-walled carbon nanotube in a nitric acid/sulfuric acid mixed solution for 12 hours at the heating temperature of 60-100 ℃, centrifugally washing for multiple times until the pH value is close to 7, and finally, drying the centrifugate in vacuum to obtain a shorter carbon nanotube;
a2: preparing a secondary antibody/carbon nano tube/protease complex;
activating carboxyl on the surface of the carbon nano tube obtained in the step A1 by using EDC/NHS, centrifugally separating to remove redundant EDC and NHS, adding a secondary antibody, carrying out oscillation reaction for 1 hour, adding protease, continuing oscillation for 12 hours, centrifugally washing the obtained precipitate for three times by using secondary water to obtain a secondary antibody/carbon nano tube/protease compound, and storing at low temperature for later use;
b: preparation of working electrode
B1: modifying carboxylated graphene on the surface of a glassy carbon electrode: dropwise adding the carboxylated graphene solution to the surface of a glassy carbon electrode, and naturally airing;
b2: preparing a primary anti-modification electrode, namely activating the electrode obtained in the step B1 by using EDC/NHS, washing the surface of the electrode by using secondary water, adding primary anti-drop on the surface of the electrode, and washing the electrode by using the secondary water after 2 hours;
b3: capturing the prostate specific antigen, namely soaking the electrode obtained in the step B2 in a PBS (phosphate buffer solution) containing the prostate specific antigen, washing the surface of the electrode with distilled water, and airing;
b4: capturing a secondary antibody/carbon nanotube/protease complex, namely soaking the electrode obtained in the step B3 in a PBS (phosphate buffer solution) containing the secondary antibody/carbon nanotube/protease complex, washing the surface of the electrode with distilled water, and airing;
c: and (3) soaking the electrode obtained in the step B4 in a PBS (phosphate buffer solution) containing copper ions and a polypeptide substrate for 2 hours, and then performing electrochemical test by adopting linear sweep voltammetry.
Further, the secondary antibody/carbon nanotube/protease complex obtained in the step a3 is stored at a low temperature for later use, wherein the storage temperature is 4 ℃.
Further, the glassy carbon electrode in step B1 was 3 mm in diameter.
Further, in the electrochemical test in the step C, a three-electrode system is adopted, and the electrode prepared in the step B4 is used as a working electrode.
The invention has the positive and beneficial technical effects that: compared with the current commercialized enzyme-linked immunosorbent assay, the method of the invention omits a large-scale expensive detection instrument. The electrochemical sensor has small instrument volume and small sensing electrode array, so that the present invention has convenient detection, greatly lowered entity threshold for developing prostate specific antigen detection, wide detection range, obvious catalytic product signal, high stability, sensitivity, detection limit, etc. and no biphenyl matter in the catalytic substrate.
Drawings
FIG. 1 is a schematic diagram of the detection of an electrochemical immunosensor using protease as a signal marker.
FIG. 2 is a mass spectrum of a product produced by hydrolysis of a substrate polypeptide catalyzed by a secondary antibody/carbon nanotube/trypsin complex in the absence of complex copper ions.
FIG. 3 is a mass spectrum of the product of the hydrolysis of substrate polypeptide catalyzed by the secondary antibody/carbon nanotube/trypsin complex in the presence of copper ion complex.
FIG. 4 is a linear scanning voltammogram of a primary anti-modified electrode after being subjected to modification steps of prostate specific antigen and secondary antibody/carbon nanotube/protease complex with different concentrations and then being soaked in a substrate solution for 2 hours. The concentration of the prostate specific antigen is 0, 0.01, 0.1, 0.5, 1, 2, 5, 10 and 20 ng/mL in sequence.
FIG. 5 is a graph of peak current of the sensor plotted linearly with prostate specific antigen concentration.
FIG. 6 is a linear scanning voltammogram of different proteins measured by the sensor.
FIG. 7 is a linear scanning voltammogram of a sensor for measuring prostate specific antigen in serum.
Detailed Description
In order to more fully explain the implementation of the present invention, examples of the implementation of the present invention are provided. These examples are merely illustrative of the process and do not limit the scope of the present invention, and the present invention is described by the following examples, but not limited to the following examples, and any modified embodiments are included in the technical scope of the present invention.
The electric signal marker is protease, the protease and a second antibody corresponding to prostate specific antigen are modified on a carbon nano tube to form a second antibody/carbon nano tube/protease complex, the substrate of the protease is polypeptide, and the sequence characteristics of the polypeptide are as follows: the third amino acid in the direction of the carbon terminus of the cleavage site is histidine H. The various abbreviations in the present invention represent the following substances: EDC: (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride); NHS: n-hydroxysuccinimide; MES: (2- (N-morpholine) ethanesulfonic acid monohydrate); PBS: phosphoric acid buffer solution; PSA, prostate specific antigen.
Example 1: preparation of secondary antibody/carbon nano tube/protease complex
A1: pretreating the carbon nano tube;
20 mg of the multi-walled carbon nanotube was boiled in 50 mL of 2M nitric acid for 12 hours, cooled, and then centrifuged and washed three times at 14000 rpm for 5 minutes each time. Heating the centrifuged multi-walled carbon nanotube in 50 mL of nitric acid/sulfuric acid (volume ratio is 1: 3) mixed solution for 12 hours at the heating temperature of 60-120 ℃, centrifuging and washing for multiple times until the pH value is close to 7, and finally drying the centrifugate in vacuum to obtain a shorter carbon nanotube;
a2: preparing a secondary antibody/carbon nano tube/protease complex;
adding the carbon nanotube (1.5 mg) obtained from A1 into 1 mL of MES buffer solution, performing ultrasonic treatment for 30 minutes, then adding 1 mL of MES (pH 7.4, 10 mM) buffer solution containing 0.4M EDC and 0.1M NHS, performing centrifugal separation to remove redundant EDC and NHS, adding 150 muL of 0.1 mug/mL secondary antibody into the centrifugal precipitate, performing oscillation reaction for 1 hour, then adding 150 muL of 1 mg/mL trypsin, continuing oscillation for 12 hours, performing centrifugal washing on the obtained precipitate for three times by secondary water to obtain a secondary antibody/carbon nanotube/protease compound, dispersing by using 1 mL of PBS buffer solution, and storing at low temperature for later use;
example 2: activity assay of Secondary antibody/carbon nanotube/protease Complex
After mixing 5. mu.L of the secondary antibody/carbon nanotube/protease complex obtained in step A2 with 100. mu.L of PBS buffer solution (pH 7.4, 0.2M) containing 2 mM of the polypeptide GARGGH and reacting at room temperature for 1 hour, the reaction solution was diluted to 500. mu.L with PBS containing or not containing 0.5mM of copper ions, and centrifuged at 14000 rpm, and the supernatant was subjected to mass spectrometry in which the mass spectrometry pattern was negative ion mode. As can be seen from fig. 2, the synthesized secondary antibody/carbon nanotube/protease complex can catalyze the hydrolysis of the polypeptide GARGGH, and the main peak at 268.1035 Da is the mass spectrum peak of the hydrolysate GGH (fig. 2), which indicates that trypsin modified to the carbon nanotube can catalyze the hydrolysis of the polypeptide GARGGH; when copper ions were added, a new mass spectrum peak (FIG. 3) appeared at 330.0253 Da, corresponding to GGH-Cu (II) complex, indicating that the hydrolysate GGH formed a complex with Cu (II).
Example 3: response of the sensor to prostate specific antigen
B: preparation of working electrode
B1: modifying carboxylated graphene on the surface of a glassy carbon electrode, namely dripping 5 mu L of 0.5 mg/mL carboxylated graphene solution on the surface of the glassy carbon electrode with the diameter of 3 mm, and naturally airing;
b2: preparing a primary anti-modification electrode, namely soaking the electrode obtained from B1 in PBS buffer solution containing 0.4M EDC and 0.1M NHS for 15 minutes, washing the surface of the electrode with secondary water, and drying the electrode with nitrogen; then dripping 10 mu L of primary antibody on the surface of the electrode, washing the electrode with secondary water after reacting for 2 hours, soaking the electrode in PBS (phosphate buffer solution) containing 0.1% BSA (w/v) for 30 minutes, taking out the electrode, washing the surface of the electrode with the secondary water, and drying the electrode by nitrogen;
b3: detecting the capture of the target object, namely dripping 10 mu L of PBS solution containing prostate specific antigen with certain concentration on the surface of the electrode obtained by B2, washing the electrode with secondary water after reacting for 30 minutes, and drying the electrode with nitrogen;
b4: capturing the secondary antibody/carbon nanotube/protease complex, namely dripping 10 mu L of the secondary antibody/carbon nanotube/protease complex on the surface of the electrode obtained from B3, washing the surface of the electrode with distilled water after reacting for 30 minutes, and drying the electrode with nitrogen;
c: electrochemical testing; soaking the electrode obtained from B4 in 30 μ L of PBS solution containing 0.5mM copper ions and 0.5mM polypeptide GARGGH for 2 hours, and performing electrochemical test by linear sweep voltammetry;
the electrochemical detection adopts a three-electrode system, the electrode obtained from B4 is a working electrode, a saturated Ag/AgCl electrode is a reference electrode, and a Pt electrode is an auxiliary electrode. The test results are shown in fig. 4. The curves are the results of cyclic voltammetry tests of the working electrode through steps B1-B3, and the oxidation peaks in the graphs are generated by electrocatalytic oxidation of water by the enzyme-catalytically generated GGH-Cu (II) complex. As can be seen from the graph, the catalytic peak current increases with the increase in the concentration of prostate-specific antigen, indicating that the amount of GGH-Cu (II) complex ultimately produced depends on the concentration of prostate-specific antigen, and FIG. 5 is a relationship between the oxidation current and the concentration of prostate-specific antigen. As can be seen from FIG. 5, the current intensity increased linearly with the increase of the concentration of prostate specific antigen (0.01-2 ng/mL), indicating that the method can be used for the quantitative detection of prostate specific antigen. The detection limit is 0.01 ng/mL. The above results indicate that the secondary antibody/carbon nanotube/protease complex can be used as an electrical signal marker of an electrochemical sensor.
Examples of responses to different proteins:
the prostate specific antigen in step B3 was changed to the other protein to be tested, and the conditions in the other steps were not changed, and the results of the experiment are shown in fig. 6. As can be seen in fig. 6, no catalytic peak was generated by the other proteins. Thus, the method can selectively detect prostate specific antigen. Curves 1 to 5 in fig. 6 correspond to: IgG (immunoglobulin G), AFP (alpha-fetoprotein), thrombobin (Thrombin), HSA (human serum albumin), PSA (prostate specific antigen). The concentration of prostate specific antigen was 5 ng/mL and the concentration of other proteins was 100 ng/mL.
Example 4: detection of prostate specific antigen in serum
The prostate specific antigen in step B3 was replaced with a human serum sample, and the serum was diluted 10-fold with a standard containing different amounts of prostate specific antigen during the test, and the conditions in the other steps were not changed, and the results of the experiment are shown in fig. 7. As can be seen from FIG. 7, when the concentration of the added prostate specific antigen standard sample is 0 ng/mL, the serum sample generates a small catalytic peak, and the surface serum sample contains prostate specific antigen, and the catalytic peak signal is obviously enhanced with the increase of the concentration of the added prostate specific antigen standard sample. The method can be used for detecting prostate specific antigen in serum. The concentration of prostate specific antigen in the diluted serum sample was calculated to be 0.11 ng/mL according to the standard curve of FIG. 5.
Similar results were obtained when papain was used for trypsin and the corresponding polypeptide substrate was GARGGH, or when dipeptidyl peptidase-IV was used for trypsin and GPGGH was used for the corresponding polypeptide substrate. The technical scheme of the invention can be realized by adopting other proteases and corresponding polypeptides, wherein the polypeptides can be cut by the proteases, and the third amino acid of the cutting site towards the carbon terminal direction is histidine H. Trypsin is preferred for this application in view of its low cost and ready availability, which makes it easier to clinically establish large-scale use.
After the embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended that all simple modifications, equivalent changes and modifications made to the above embodiments based on the technical spirit of the present invention shall fall within the technical scope of the present invention, and the present invention shall not be limited to the embodiments illustrated in the description.
Sequence listing
<110> Anyang college of teachers and schools
<120> electrochemical immunosensor electric signal marker for prostate specific antigen detection and detection method
<160>2
<170>SIPOSequenceListing 1.0
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<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Ala Arg Gly Gly His
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<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Pro Gly Gly His
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Claims (4)
1. The detection method for the prostate specific antigen adopts an electrochemical immunosensor electric signal marker for prostate specific antigen detection, the electric signal marker is protease, a catalytic substrate of the protease is polypeptide, the polypeptide can be cut by the protease, and the sequence characteristics of the polypeptide are as follows: the third amino acid from the cleavage site in the direction of the carbon terminus is histidine H, characterized by comprising the following steps:
a: the preparation of the secondary antibody/carbon nano tube/protease complex comprises the following substeps:
a1: pretreating the carbon nano tube;
boiling a multi-walled carbon nanotube for 12 hours by using 2M nitric acid, cooling, then centrifugally washing for three times, heating the centrifuged multi-walled carbon nanotube in a nitric acid/sulfuric acid mixed solution for 12 hours at the heating temperature of 60-100 ℃, centrifugally washing for multiple times until the pH value is close to 7, and finally, drying the centrifugate in vacuum to obtain a shorter carbon nanotube;
a2: preparing a secondary antibody/carbon nano tube/protease complex;
activating carboxyl on the surface of the carbon nano tube obtained in the step A1 by using EDC/NHS, centrifugally separating to remove redundant EDC and NHS, adding a secondary antibody, carrying out oscillation reaction for 1 hour, adding protease, continuing oscillation for 12 hours, centrifugally washing the obtained precipitate for three times by using secondary water to obtain a secondary antibody/carbon nano tube/protease compound, and storing at low temperature for later use;
b: preparation of working electrode
B1: modifying carboxylated graphene on the surface of a glassy carbon electrode: dropwise adding the carboxylated graphene solution to the surface of a glassy carbon electrode, and naturally airing;
b2: preparing a primary anti-modification electrode, namely activating the electrode obtained in the step B1 by using EDC/NHS, washing the surface of the electrode by using secondary water, adding primary anti-drop on the surface of the electrode, and washing the electrode by using the secondary water after 2 hours;
b3: capturing the prostate specific antigen, namely soaking the electrode obtained in the step B2 in a PBS (phosphate buffer solution) containing the prostate specific antigen, washing the surface of the electrode with distilled water, and airing;
b4: capturing a secondary antibody/carbon nanotube/protease complex, namely soaking the electrode obtained in the step B3 in a PBS (phosphate buffer solution) containing the secondary antibody/carbon nanotube/protease complex, washing the surface of the electrode with distilled water, and airing;
c: and (3) soaking the electrode obtained in the step B4 in a PBS (phosphate buffer solution) containing copper ions and a polypeptide substrate for 2 hours, and then performing electrochemical test by adopting linear sweep voltammetry.
2. The method for detecting a prostate-specific antigen according to claim 1, wherein: and B, storing the secondary antibody/carbon nano tube/protease complex obtained in the step A3 at a low temperature for later use, wherein the storage temperature is 4 ℃.
3. The method for detecting a prostate-specific antigen according to claim 1, wherein: the glassy carbon electrode in step B1 was 3 mm in diameter.
4. The method for detecting a prostate-specific antigen according to claim 1, wherein: in the step C, a three-electrode system is adopted in the electrochemical test, and the electrode prepared in the step B4 is used as a working electrode.
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