CN102226810A

CN102226810A - Electrochemical immunoassay method based on Dopamine embedded liposome

Info

Publication number: CN102226810A
Application number: CN2011100758383A
Authority: CN
Inventors: 仲召阳; 王东; 卿毅; 戴楠; 李梦侠; 关伟; 金丰; 单锦露
Original assignee: Third Affiliated Hospital of TMMU
Current assignee: Third Military Medical University TMMU; Third Affiliated Hospital of TMMU
Priority date: 2011-03-28
Filing date: 2011-03-28
Publication date: 2011-10-26

Abstract

The invention discloses an electrochemical immunoassay method based on Dopamine embedded small single-layer liposome. The method comprises the following steps: ProGRP antibodies are subjected to a specific immunization reaction with ProGRPs in a sample to be detected; a conjugate of the dopamine embedded small single-layer liposome and the ProGRP antibodies, which is added and is demulsified by a surfactant, releases electroactive molecules Dopamine; an MWNT (multi-walled carbon nanotube) modified GCE (glassy carbon electrode) is treated as a working electrode, and the concentration of the ProGRP in the sample to be detected is detected through the signal intensity of a linear scanning current-voltage peak current of Dopamine. The electrochemical immunoassay method provided by the invention has a linear response range of 50 to 1000 pg/ml and a low detection limit of 16 pg/ml, so the method has the advantages of good specificity and high sensitivity, and has an important meaning to the SCLC (small-cell lung cancer) diagnosis.

Description

Electro-chemistry immunity detection method based on liposome embedded dopamine

Technical field

The present invention relates to the electro-chemistry immunity field, particularly a kind of electro-chemistry immunity detection method of the small unilamellar vesicle based on the embedding dopamine.

Background technology

Lung cancer is one of modal malignant tumour of China, and it is the first that M ﹠ M occupies the whole world, and be the trend that increases year by year.Small-cell carcinoma of the lung (small-cell lung cancer, SCLC) be the highest lung cancer of grade malignancy, case accounts for 15～20% of all lung cancer cases, tumour cell has that differentiation degree is low, the doubling time is short, di is high, be prone to special biological behaviours such as DISTANT METASTASES IN in early days, extremely sensitive to radiation and chemotherapy, the most of patients PD is fast, and is dead in a short time, therefore, carry out early diagnosis, formulate the rational therapy scheme and assess curative effect particularly important to small-cell carcinoma of the lung.The main method that is used for the lung cancer auxiliary diagnosis at present has rabat, CT and phlegm cytology checking etc., and the detection of blood serum tumor markers has bigger practical value owing to characteristics such as the acceptance of taking a sample simply, be easy at aspects such as tumor screening, diagnosis, evaluation curative effects.

Gastrin releasing peptide (GRP) is to isolate a kind of secretory gastrointestinal hormone of Gastrin that has by McDonald etc. in 1978 from the gastric tissue of pig, is effective sercretogogue or regulates peptide, intestines, the brain peptide be made up of 27 amino acid.The researcher finds that gastrin releasing peptide (GRP) can be present in the neuroendocrine tissue of the nerve fibre of normal brain, stomach and fetus lung.Also secrete gastrin releasing peptide (GRP) in many small-cell carcinoma of the lung (SCLC) cell line and the tumor tissues, and be rich in gastrin releasing peptide (GRP) acceptor, therefore, gastrin releasing peptide (GRP) can be used as the mark of small-cell carcinoma of the lung.But, detect so be difficult in the serum because gastrin releasing peptide (GRP) can be degraded fast by expeptidase.Gastrin precursor release peptide (ProGRP) is the front body structure of gastrin releasing peptide (GRP), because Stability Analysis of Structures, therefore can be in blood plasma stably express, and the level of gastrin precursor release peptide can reflect the level and gastrin releasing peptide (GRP) the expression of gene level of gastrin releasing peptide (GRP) indirectly, so gastrin precursor release peptide (ProGRP) receives publicity day by day as the clinical value of new mark at aspects such as small-cell carcinoma of the lung (SCLC) diagnosis and progression of disease monitorings.Japan scholar mountain pass studies show that the susceptibility of this mark is 76%, and specificity is 97%, positive findings predicted value 94%, and negative findings predicted value 87%, the level and the therapeutic response of gastrin precursor release peptide (ProGRP) have correlativity preferably.Comprehensive report result both domestic and external, gastrin precursor release peptide (ProGRP) has the characteristics of susceptibility height, high specificity as the new label of small-cell carcinoma of the lung (SCLC), and can reflect accurately that small-cell carcinoma of the lung (SCLC) state of an illness reaches the reaction to chemotherapy.Therefore, to the diagnosis of small-cell carcinoma of the lung (SCLC) be had than high sensitive, the gastrin precursor release peptide of specificity and accuracy rate can be clinical identification small-cell carcinoma of the lung (SCLC) reference frame is provided as the blood serum tumor markers of prompting small-cell carcinoma of the lung (SCLC).Gastrin precursor release peptide not only can be used for the early diagnosis of small-cell carcinoma of the lung (SCLC) as blood serum tumor markers, also helps to judge result of treatment and the early stage tumor recurrence of finding.At present, adopt enzyme-linked immunoassay method to measure gastrin precursor release peptide (ProGRP) both at home and abroad and be in the clinical practice stage, but euzymelinked immunosorbent assay (ELISA) sensitivity is not high, is difficult for detecting trace gastrin precursor release peptide (ProGRP), the accuracy that influence detects small-cell carcinoma of the lung (SCLC).

Electrochemiluminescence (ECL) is by voltage or the current signal that applies certain waveform on electrode, carry out between the product of electrolytic reaction or with system in the coexistence component reaction produce chemiluminescent phenomenon.Electrochemiluminescence (ECL) is that with the difference of chemiluminescence (CL) electrochemiluminescence (ECL) is that electricity starts luminescence-producing reaction, and chemiluminescence (CL) is to start luminescence-producing reaction by compound.Therefore, electrochemiluminescence (ECL) reaction is accurately control easily, has dirigibility and stability.Electrochemical immunoanalytical method (Electrochemical Immunoassay, ECIA) be a kind of immunoassay new method that immunological technique is combined with Electrochemical Detection, specific reaction based on antigen and antibody, with some not only easily mensuration but also material mark with high susceptibility on specific antigen or antibody molecule, enhancing enlarge-effect by these labels shows the character and the content of antigen in the reactive system or antibody, has the high sensitivity of luminesceence analysis and the high degree of specificity of antigen-antibody reaction concurrently.The label of electrochemical immunoanalytical mainly contains two classes: biology enzyme and electroactive substance.Electroactive substance as label is to have the metallic ion of electrochemical redox character and electroactive organic functions group.

Liposome, be when being scattered in water by amphipathic molecule such as phosphatide and sphingolipid, the hydrophobic tail of molecule is tended to flock together and is avoided water, and hydrophilic head is exposed to the vesicle with bilayer structure that water forms, can become the carrier of various functional moleculars by finishing and inner embedding, and the release of these functional moleculars is controlled.Therefore, provide a kind of electro-chemistry immunity detection method based on liposome embedded electric active molecule highly sensitive, that specificity is good to be used for detecting gastrin precursor release peptide (ProGRP), significant to early diagnosis, state of illness monitoring, judging prognosis and formulation proper treatment scheme thereof and the assessment curative effect of small-cell carcinoma of the lung (SCLC).

Summary of the invention

In view of this, the invention provides a kind of electro-chemistry immunity detection method of the small unilamellar vesicle based on the embedding dopamine.This detection method adopts double antibody sandwich method, make gastrin precursor release peptide (ProGRP) generation specific immune response in immobilized gastrin precursor release peptide (ProGRP) antibody and the testing sample, use the small unilamellar vesicle of embedding dopamine and the common couplings of gastrin precursor release peptide (ProGRP) antibody to detect again, use surfactant TritionX-100 breakdown of emulsion then, discharge the electric active molecule dopamine, utilize multi-walled carbon nano-tubes (MWNT) modified glassy carbon as working electrode, detect the concentration of gastrin precursor release peptide in the testing sample by the linear sweep volt-ampere peak current signal intensity that detects dopamine.

In order to realize the foregoing invention purpose, the invention provides following technical scheme:

The invention provides a kind of electro-chemistry immunity detection method, comprise the steps:

Step 1: the small unilamellar vesicle of embedding dopamine and protein antibodies to be checked be the preparation of couplings altogether: the small unilamellar vesicle that dropwise adds the embedding dopamine in glutaraldehyde solution, dialyse behind the incubation, add protein antibodies solution to be checked, the reaction back adds the confining liquid incubation, and the small unilamellar vesicle and the protein antibodies to be checked that make the embedding dopamine are total to couplings;

Step 2: contain the standard items incubation that variable concentrations is treated Reichl's test to immobilized the adding in the microwell plate of protein antibodies to be checked, add the confining liquid incubation, wash with PBS, after the small unilamellar vesicle of the described embedding dopamine of adding and protein antibodies to be checked are total to the couplings incubation in microwell plate, wash with PBS solution, add surfactant breakdown of emulsion at room temperature, make detection solution; From microwell plate, get described detection solution, transfer in the electro-chemical test pond, adopt linear sweep voltammetry, with the multi-walled carbon nano-tubes modified glassy carbon is working electrode, and the platinum electrode conduct is to electrode, and saturated calomel electrode is as contrast electrode, carry out the electrochemiluminescence test, with the concentration for the treatment of Reichl's test in the standard items is horizontal ordinate, is ordinate with corresponding galvanochemistry peak to peak current, the production standard curve;

Step 3: add sample incubation to be checked in the microwell plate of protein antibodies to be checked to immobilized, carry out the electrochemiluminescence test, record the galvanochemistry peak to peak current intensity of testing sample, read the corresponding concentration for the treatment of Reichl's test from typical curve as step 2.

The preparation method of the small unilamellar vesicle of embedding dopamine of the present invention, comprise the steps: potpourri and chloroform-methanol mixed solution mixing with dipalmitoyl phosphatidylcholine and cephalin, sonicated, behind the decompression rotary evaporation, add the phosphate buffer solution that contains dopamine, swelling makes the multilamellar liposome vesica of embedding dopamine, with the multilamellar liposome vesica sonicated of described embedding dopamine, makes the small unilamellar vesicle of embedding dopamine.

Liposome: be that the hydrophobic tail of molecule is tended to flock together, and avoids water when being scattered in water by amphipathic molecule such as phosphatide and sphingolipid, and hydrophilic head is exposed to water, the vesicle with bilayer structure of formation, diameter 25～1000nm does not wait.

Phosphatide is the lipid that a class contains phosphoric acid, mainly contains two big class phosphatide in the body, and the phosphatide that is made of glycerine is called glycerophosphatide (phosphoglyceride); By the phosphatide that sphingosine constitutes, be called sphingomyelins (sphingolipid).Have the hydrophilic head (hydrophilic head) of substituted radical (containing ammonia alkali or the alcohols) formation that links to each other by phosphoric acid and the hydrophobic tail (hydrophobictail) that constitutes by fatty acid chain.The hydrophilic head of phosphatide is positioned at the film surface in biological membrane, and hydrophobic tail is positioned at the film inboard.Phosphoglyceride (phosphoglycerides) main chain is a glycerol-3-phosphate, and two other hydroxyl in the glycerol molecule is all by the esterification of fatty acid institute, and phosphate group can be formed various phosphoglycerides after the different micromolecular compound esterification of various structures again.What in-vivo content was more is phosphatid ylcholine (lecithin), phosphatidyl-ethanolamine (cephalin), phosphatidylserine, phosphatidyl glycerol, diphosphatidylglycerol (heart phosphide) and phosphinositides etc., and each phosphatide can have some kinds because of the fatty acid of forming is different.

The phosphatide that is used to prepare liposome has natural phospholipid, as soybean lecithin, egg yolk lecithin etc.; Synthetic phospholipid is as two palmityl phosphatidyl cholines, distearoyl phosphatidylcholine etc.Lecithin, chemical name is a phosphatid ylcholine, is the one group of filemot oil material that is present among animal vegetable tissue and the yolk, its constituent comprises phosphoric acid, choline, fatty acid, glycerine, glycolipid, triglyceride and phosphatide.DPPC, dipalmitoyl-sn-glycero-3-phosphocholine, dipalmitoyl phosphatidylcholine, a kind of chemical reagent can be used for the synthetic fat plastid.Cephalin, the general designation of phosphatidyl-ethanolamine and phosphatidylserine, a kind of phosphatide by glycerine, fatty acid, phosphoric acid and monoethanolamine are formed extensively distributes in vivo, is enriched in brain and spinal cord especially.

Liposome reaches according to its size and the number of plies (number of bilayer in the liposome) generally is divided three classes: multilayer capsule (MLV), big individual layer capsule (LUV) and little individual layer capsule (SUV), its principal ingredient is a phosphatide.Multilayer capsule (MLV), particle diameter is between 1～5 μ m; Big individual layer capsule (LUV) is the large unilamellar vesicles capsule, and particle diameter is at 0.1～l μ m; Little individual layer capsule (SUV), about 0.02～0.08 μ m of particle diameter.As preferably, the present invention selects small unilamellar vesicle for use, is convenient to breakdown of emulsion in follow-up test.Liposome has amphipathic property and very strong adaptability and harmless natural component, and it wraps up wide range, and lipophilic substance, amphiprotic substance and water-soluble substances composition can be wrapped, and therefore can be used as the carrier of medicine or electroactive material.

At present, the correlative study about liposome has related to drug delivery, film engineering science, fields such as biology sensor.Usually, the size of liposome is compared other several nano particles from diameter tens nanometers to tens micron, and its preparation is simple, can effectively restrain non-specific absorption and good biocompatibility.The most important thing is that liposome can become the carrier of various functional moleculars by finishing and inner embedding, and the release of these functional moleculars is controlled.Utilize liposome as the multifunctional carrier in the immunoassays, with different semiochemicalses, be wrapped in the liposome interior aqueous phase as electroactive material, optical active substance, biology enzyme albumen etc., functional group can be modified by various physics or chemical method in its surface, makes the liposome of functionalization be widely used in the structure of immunosensor and gene sensor.

The preparation method of liposome has multiple, and film dispersion method is a kind of preparation method of classics, and it can form multilamelar liposome, obtains small unilamellar vesicle (small unilamellar vesicle) after sonicated.This method is easy and simple to handle, the liposome structure typical case, and therefore, the present invention adopts film dispersion method, prepares liposome with two palmityl phosphatidyl cholines and cephalin.

Dopamine (Dopamine), chemical name are 4-(2-ethylamino-) benzene-1, and the 2-glycol is called for short DA, and chemical formula is C ₈H ₁₁NO ₂, molal weight is 153.178g/mol.Dopamine is a kind of crucial neurotransmitter in hypothalamus and the pituitary gland, is the precursor of neuraminidase (NA), has good electrochemical activity, helps cell to transmit pulse.Therefore, dopamine (Dopamine) is applied to have the characteristics of safety non-toxic in the electro-chemistry immunity detection technique as electric active molecule.The present invention selects for use dopamine as electric active molecule, is embedded in the small unilamellar vesicle, and the electro-chemistry immunity detection method based on the small unilamellar vesicle of embedding dopamine is provided, and detects the concentration of gastrin precursor release peptide in the testing sample.

In order to remove nonspecific binding site, the present invention adopts the confining liquid bovine serum albumin(BSA).

In order to guarantee abundant combination, it is 20～40 ℃ that the present invention selects the temperature of incubation for use.

In order to guarantee abundant combination, it is 40～120min that the present invention selects the time of incubation for use.

As preferably, described surfactant is TritonX-100.

Preferably, the preparation method of the small unilamellar vesicle of embedding dopamine of the present invention comprises the steps:

Step 1: be 3: 1 potpourri 1mg of dipalmitoyl phosphatidylcholine (DPPC) and cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 with mol ratio, sonicated makes W/O emulsion;

Step 2: the decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, after adding 2mL contains the phosphate buffer solution of 0.1g/mL dopamine, swelling 40～80min in 40～60 ℃ water-bath, violent mixing makes the multilamellar liposome vesica of embedding dopamine;

Step 3: with the multilamellar liposome vesica sonicated of described embedding dopamine, the centrifugal 10～20min of 8000rpm makes the small unilamellar vesicle of embedding dopamine.

As preferably, the unilamellar liposome of the described embedding dopamine that makes is scattered in the PBS solution, in 4 ℃ of preservations.

The present invention also provides the small unilamellar vesicle of the embedding dopamine that described preparation method makes.

As preferably, the preparation method of described multi-walled carbon nano-tubes modified glassy carbon is that glass-carbon electrode is through Al ₂O ₃Stick with paste polishing back supersound washing, make and handle the back glass-carbon electrode, handle multi-walled carbon nano-tubes with red fuming nitric acid (RFNA)-concentrated sulphuric acid mixed solution, washing final vacuum drying, be scattered in the DMF solution, sonicated makes multi-walled carbon nano-tubes solution, described multi-walled carbon nano-tubes drips of solution is added in glass-carbon electrode surface, described processing back, makes described multi-walled carbon nano-tubes modified glassy carbon electrode.

Preferably, the preparation method of described multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode comprises the steps:

Step 1: (Ф=4mm) is successively through the Al of 1.0,0.3,0.05 μ m for glass-carbon electrode ₂O ₃It is clean with distilled water flushing to stick with paste the polishing back, and supersound washing in distilled water, acetone, distilled water respectively again places under the room temperature and dries, and makes to handle the back glass-carbon electrode;

Step 2: the red fuming nitric acid (RFNA)-concentrated sulphuric acid mixed solution that with volume ratio is 1: 1 is handled multi-walled carbon nano-tubes (MWNT) 5h in 80 ℃, and washing final vacuum drying makes and handles back multi-walled carbon nano-tubes (MWNT);

Step 3: get the described processing back multi-walled carbon nano-tubes of 5.0mg (MWNT) and be scattered in the DMF solution of 5mL 4～6%, ultrasonic 30min makes multi-walled carbon nano-tubes (MWNT) solution;

Step 4: get the described MWNT drips of solution of 6～10 μ L and be added in glass-carbon electrode surface, described processing back, under room temperature, place and dry, make described multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode, in 4 ℃ refrigerator, preserve standby.

Dimethyl formamide (DMF) molecular formula is C ₃H ₇NO, molecular weight is 73.10, is a kind of transparency liquid, energy and water and most of organic solvent dissolve each other, and are the common solvent of chemical reaction.

Carbon nano-tube is the C that continues ₆₀The another allotrope of the carbon of Fa Xianing is a kind of One-dimensional Quantum material with special construction (radial dimension is a nanometer scale, and axial dimension is a micron dimension, and all seal basically at the pipe two ends) afterwards.Carbon nano-tube has typical stratiform hollow structure feature, has certain included angle between the synusia of formation carbon nano-tube.The pipe shaft of carbon nano-tube is the director circle tubular construction, is made up of hexagon carbocyclic ring microstructure unit, and terminal cap moiety perhaps is called the many wall constructions of polygon taper by containing the polygonized structure that pentagonal carbocyclic ring is formed.The distance that is maintained fixed between layer and the layer is about 0.34nm, and diameter is generally 2～20nm.Its radial dimension is less, the external diameter of pipe generally in several nanometers to tens nanometers, the internal diameter of pipe is littler, what have has only about 1nm; And its length is generally at micron order, and length and diameter are bigger than very, can reach 10 ³～10 ⁶Therefore, carbon nano-tube is considered to a kind of typical monodimension nanometer material.The unique texture of carbon nano-tube has determined it to have many special physics and chemical property.The C=C covalent bond of forming carbon nano-tube is the most stable chemical bond of nature, and institute is so that carbon nano-tube has the very mechanical property of excellence.Carbon nano-tube had both had the intrinsic person's character of carbon materials, had the conduction and the thermal conductivity of metal material again, the heat-resisting and corrosion resistance of stupalith, the stitchability of textile fibres, and the lightweight of macromolecular material, workability.Carbon nano-tube can be divided into according to the classification of the number of plies of graphene film: Single Walled Carbon Nanotube (Single-walled nanotubes, SWNT) and multi-walled carbon nano-tubes (Multi-walled nanotubes, MWNT).Therefore, in the electro-chemistry immunity detection method of the small unilamellar vesicle based on described embedding dopamine provided by the invention, select for use multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode as working electrode.

As preferably, described linear sweep voltammetry sweep limit is-0.2～0.8V, sweeps speed and is 50mV/s.

As preferably, the Reichl's test for the treatment of of the present invention is a tumor-marker albumen.

Tumor markers (Tumor Marker): produce by tumor tissues self, can reflect that tumour exists and a class biochemical substances of growth, they or be not present in normal adult tissue and be detected in embryonic tissue, or the content in tumor tissues substantially exceeds the content in normal structure, their existence or quantitative change can be pointed out the character of tumour, so as to understanding tissue generation, cell differentiation, the cell function of tumour, judge and the treatment guidance with the diagnosis, classification, the prognosis that help tumour.Mainly contain embryonal antigen, sugar antigen, natural autoantigen, cytokeratin, tumour relevant enzyme, hormone and some oncogene etc.Tumor-marker albumen is to be produced by tumor tissues self, can reflect that tumour exists and the protein matter of growth.

Electrochemical immunoanalytical method (Electrochemical Immunoassay, ECIA) be a kind of immunoassay new method that immunological technique is combined with Electrochemical Detection, specific reaction based on antigen and antibody, with some not only easily mensuration but also material mark with high susceptibility on specific antigen or antibody molecule, enhancing enlarge-effect by these labels shows the character and the content of antigen in the reactive system or antibody, has the high sensitivity of luminesceence analysis and the high degree of specificity of antigen-antibody reaction concurrently.Therefore, electrochemical detection method provided by the invention can be used for detecting tumor-marker albumen by the specific reaction of antigen-antibody.

Preferably, described tumor-marker albumen comprises gastrin precursor release peptide, neuronspecific enolase, CYFRA21-1, AFP, APT, CEA, CA242, CA125, CA199, CA153, CA724, CA50, f-PSA, t-PSA, Free β-hCG, SCCA, β 2-MG.

Tumor-marker albumen produces different according to tumor tissues, comprise differentiation antigen; Embryonal antigen (AFP, CEA); Isodynamic enzyme (NSE); Hormone (HCG); Tissure specific antigen (PSA, freePSA); Mucin, glycoprotein, glycolipid (CA125); Often can check mark: AFP, CEA, CA199, CA724, CA125, CA153, NSE, Cyfra21-1, PSA, free PSA on the electrochemiluminescence instrument.

AFP, APT: main related neoplasms: hepatocellular carcinoma and germinocarcinoma; Other related neoplasms: ECC, teratoma of ovary, cancer of the stomach, cancer of bile ducts, cancer of pancreas etc.

CEA: main related neoplasms: the tumor markers of wide spectrum; Other related neoplasms: be common in lung cancer, colorectal cancer, cancer of pancreas, cancer of the stomach, breast cancer, medullary carcinoma of thyroid gland etc.

CA242: main related neoplasms: cancer of pancreas, stomach, colon cancer; Other related neoplasms: liver cancer, the cancer of the esophagus, lung cancer.

CA125: main related neoplasms: oophoroma; Other related neoplasms: lung cancer, cancer of pancreas, breast cancer, liver cancer, gastrointestinal cancer, the cancer of the uterus.

CA199: main related neoplasms: cancer of pancreas, cholangiocarcinoma, colorectal cancer; Other related neoplasms: liver cancer, carcinoma of gallbladder, cholangiocarcinoma etc.

CA153: main related neoplasms: the first-selected mark of breast cancer; Other related neoplasms: lung cancer, oophoroma, adenocarcinoma of lung, colorectal cancer etc. all can increase.

CA724: main related neoplasms: one of best tumor markers of cancer of the stomach; Other related neoplasms: other gastrointestinal cancers, breast cancer, lung cancer, oophoroma etc. are also had different recall rates.

CA50: main related neoplasms: the mark of pancreas and knot, the carcinoma of the rectum; Other related neoplasms: cancer of the stomach, carcinoma of gallbladder, liver cancer, lung cancer, breast cancer.

NSE, gastrin precursor release peptide: main related neoplasms: small-cell carcinoma of the lung; Other related neoplasms: adenocarcinoma of lung, maxicell lung cancer.

CYFRA21-1: main related neoplasms: lung squamous cancer, cervical carcinoma, the cancer of the esophagus; Other related neoplasms: carcinoma of urinary bladder, nasopharyngeal carcinoma, oophoroma, gastrointestinal cancer.

F-PSA: main related neoplasms: prostate cancer; Other related neoplasms: some gynecological tumor and breast cancer.

T-PSA: main related neoplasms: prostate cancer; Other related neoplasms: some gynecological tumor, polycystic ovary syndrome, breast cancer.

Free β-hCG: main related neoplasms: gynecological tumor and non-smart originality carcinoma of testis; Other related neoplasms: breast cancer, smart originality carcinoma of testis, lung cancer, liver cancer etc.

SCCA: main related neoplasms: SCC; Other related neoplasms: lung squamous cancer, incidence squama cancer, the cancer of the esophagus and external genital squamous cell carcinoma etc.

β 2-MG: main related neoplasms: the complementary mark of malignant tumour, chronic lymphocytic leukemia, lymphocyte sarcoma, Huppert's disease etc. are particularly evident.Other related neoplasms: also as seen raise in lung cancer, breast cancer, gastrointestinal cancer and the cervix cancer.

Preferably, the described Reichl's test for the treatment of is a gastrin precursor release peptide.

As preferably, a kind of electro-chemistry immunity detection method provided by the invention comprises the steps:

Step 1: the small unilamellar vesicle of embedding dopamine and gastrin precursor release peptide (ProGRP) antibody is the preparation of couplings altogether: the small unilamellar vesicle that dropwise adds described embedding dopamine in the glutaraldehyde solution of 0.5mL 2.0～3.0%, stirring reaction 40～80min at room temperature, under 4 ℃, place the phosphate buffer 12h that dialyses, remove excessive glutaraldehyde molecule, make the small unilamellar vesicle of the embedding dopamine of aldehyde radicalization; Get gastrin precursor release peptide (ProGRP) antibody-solutions of 200～300 μ L, slowly join in the small unilamellar vesicle of embedding dopamine of the described aldehyde radicalization of 200～300 μ L, fully descend lasting stirring reaction 1～2h at 25 ℃ behind the mixing, the bovine serum albumin solution that adds 40～80 μ L 0.25%, the unreacted aldehyde radical in small unilamellar vesicle surface with the embedding dopamine that seals described aldehyde radicalization, and at 25 ℃ of following incubation reaction 1～2h, the small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that make the embedding dopamine are total to couplings, and is standby in 4 ℃ of preservations;

Step 2: add sample to be checked in the microwell plate of ProGRP antibody to immobilized,, add 80～120 μ L 0.25%BSA solution again, to seal non-specific unusual binding site in 37 ℃ of reaction 1h; Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA; The small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that add 80～120 μ L embedding dopamines in microwell plate are total to couplings, react 1h down at 37 ℃, wash 3 times with PBS solution, to remove the liposome of non-specific absorption, the TritonX-100 solution that adds 80～120 μ L 1.2mmol/L is more at room temperature cultivated 20min, make the small unilamellar vesicle of described embedding dopamine and the liposome breakdown of emulsion in the common couplings of gastrin precursor release peptide (ProGRP) antibody, make detection solution; Get the described detection solution of 80～120 μ L from immobilized the microwell plate of gastrin precursor release peptide (ProGRP) antibody, transfer in the electro-chemical test pond, adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with multi-walled carbon nano-tubes (MWNT) modified glassy carbon for preparing is working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, carry out the electrochemiluminescence test, with the concentration for the treatment of Reichl's test in the standard items is horizontal ordinate, with corresponding galvanochemistry peak to peak current is ordinate, the production standard curve;

The invention provides a kind of electro-chemistry immunity detection method of the small unilamellar vesicle based on the embedding dopamine.Specific immune response takes place by gastrin precursor release peptide (ProGRP) in gastrin precursor release peptide (ProGRP) antibody and the testing sample in this detection method, add the small unilamellar vesicle of embedding dopamine and the common couplings of gastrin precursor release peptide (ProGRP) antibody, through surfactant TritonX-100 breakdown of emulsion, discharge the electric active molecule dopamine, utilize multi-walled carbon nano-tubes (MWNT) modified glassy carbon as working electrode, detect the concentration of gastrin precursor release peptide in the testing sample by the linear sweep volt-ampere peak current signal intensity that detects dopamine.Electro-chemistry immunity detection method linear response range provided by the invention is 50～1000pg/mL, is limited to 16pg/mL under detecting, and specificity is good, and is highly sensitive, significant to the diagnosis of small-cell carcinoma of the lung (SCLC).

Description of drawings

Fig. 1 shows among the embodiment 5 gastrin precursor release peptide (ProGRP) typical curve that the electro-chemistry immunity detection method based on the small unilamellar vesicle of described embedding dopamine makes, horizontal ordinate is the concentration of gastrin precursor release peptide (ProGRP) standard items, and unit is pg/mL; Ordinate is for detecting the peak current average that obtains dopamine (DA), and unit is μ A.

Fig. 2 shows among the embodiment 6 gastrin precursor release peptide (ProGRP) typical curve that the electro-chemistry immunity detection method based on the small unilamellar vesicle of described embedding dopamine makes, horizontal ordinate is the concentration of gastrin precursor release peptide (ProGRP) standard items, and unit is pg/mL; Ordinate is for detecting the peak current average that obtains dopamine (DA), and unit is μ A.

Fig. 3 shows among the embodiment 7 gastrin precursor release peptide (ProGRP) typical curve that the electro-chemistry immunity detection method based on the small unilamellar vesicle of described embedding dopamine makes, horizontal ordinate is the concentration of gastrin precursor release peptide (ProGRP) standard items, and unit is pg/mL; Ordinate is for detecting the peak current average that obtains dopamine (DA), and unit is μ A.

Fig. 4 shows among the embodiment 8 gastrin precursor release peptide (ProGRP) typical curve that the electro-chemistry immunity detection method based on the small unilamellar vesicle of described embedding dopamine makes, horizontal ordinate is the concentration of gastrin precursor release peptide (ProGRP) standard items, and unit is pg/mL; Ordinate is for detecting the peak current average that obtains dopamine (DA), and unit is μ A.

Embodiment

The invention discloses a kind of electro-chemistry immunity detection method of the small unilamellar vesicle based on the embedding dopamine, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.

Below in conjunction with embodiment, further set forth the present invention:

Dipalmitoyl phosphatidylcholine among the present invention (DPPC), cephalin (PE), dopamine (DA), gastrin precursor release peptide (ProGRP), gastrin precursor release peptide (ProGRP) antibody, N, dinethylformamide (DMF) is all available from U.S. Sigma company; Multi-walled carbon nano-tubes (MWNT) is available from Chengdu Organical Chemical Co., Ltd., Chinese Academy of Sciences; The human serum sample is all from the 3rd tumor center of affiliated hospital of Third Military Medical University, 156 of test set (patients serum), and wherein the man is 78,78 of woman; 100 of control groups (normal human serum), wherein the man is 50,50 of woman.

The small unilamellar vesicle of embodiment 1 preparation embedding dopamine

Be 3: 1 potpourri 1mg of dipalmitoyl phosphatidylcholine (DPPC) and cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 with mol ratio, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, after adding 2mL contains the phosphate buffer solution of 0.1g/mL dopamine, and swelling 80min in 40 ℃ water-bath, violent mixing makes the multilamellar liposome vesica of embedding dopamine; With the multilamellar liposome vesica sonicated of described embedding dopamine, the centrifugal 15min of 8000rpm makes the small unilamellar vesicle of embedding dopamine.The small unilamellar vesicle of the described embedding dopamine that makes is scattered in the PBS solution, in 4 ℃ of preservations.

The small unilamellar vesicle of embodiment 2 preparation embedding dopamines

Be 3: 1 potpourri 1mg of dipalmitoyl phosphatidylcholine (DPPC) and cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 with mol ratio, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, after adding 2mL contains the phosphate buffer solution of 0.1g/mL dopamine, and swelling 60min in 50 ℃ water-bath, violent mixing makes the multilamellar liposome vesica of embedding dopamine; With the multilamellar liposome vesica sonicated of described embedding dopamine, the centrifugal 10min of 8000rpm makes the small unilamellar vesicle of embedding dopamine.The small unilamellar vesicle of the described embedding dopamine that makes is scattered in the PBS solution, in 4 ℃ of preservations.

The small unilamellar vesicle of embodiment 3 preparation embedding dopamines

Be 3: 1 potpourri 1mg of dipalmitoyl phosphatidylcholine (DPPC) and cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 with mol ratio, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, after adding 2mL contains the phosphate buffer solution of 0.1g/mL dopamine, and swelling 40min in 60 ℃ water-bath, violent mixing makes the multilamellar liposome vesica of embedding dopamine; With the multilamellar liposome vesica sonicated of described embedding dopamine, the centrifugal 20min of 8000rpm makes the small unilamellar vesicle of embedding dopamine.The small unilamellar vesicle of the described embedding dopamine that makes is scattered in the PBS solution, in 4 ℃ of preservations.

The Electrochemical Detection of the small unilamellar vesicle of embodiment 4 embedding dopamines

The small unilamellar vesicle of the embedding dopamine of preparation among the embodiment 1 to 3 is carried out electron-microscope scanning, and the small unilamellar vesicle clear-cut of described embedding dopamine, particle diameter are 0.02～0.08 μ m.

Control group 1: prepare the not small unilamellar vesicle of embedding dopamine: the dipalmitoyl phosphatidylcholine (DPPC) and the potpourri 1mg of cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 that with the ratio of amount of substance are 3: 1, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, behind the adding 2mL phosphate buffer solution, and swelling 80min in 40 ℃ water-bath, violent mixing makes the multilamellar liposome vesica; With described multilamellar liposome vesica sonicated, the centrifugal 15min of 8000rpm makes the not small unilamellar vesicle of embedding dopamine.The described small unilamellar vesicle that makes is scattered in the PBS solution, in 4 ℃ of preservations.

Control group 2: prepare the not small unilamellar vesicle of embedding dopamine: the dipalmitoyl phosphatidylcholine (DPPC) and the potpourri 1mg of cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 that with the ratio of amount of substance are 3: 1, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, behind the adding 2mL phosphate buffer solution, and swelling 60min in 50 ℃ water-bath, violent mixing makes the multilamellar liposome vesica; With described multilamellar liposome vesica sonicated, the centrifugal 10min of 8000rpm makes the not small unilamellar vesicle of embedding dopamine.The described small unilamellar vesicle that makes is scattered in the PBS solution, in 4 ℃ of preservations.

Control group 3: prepare the not small unilamellar vesicle of embedding dopamine: the dipalmitoyl phosphatidylcholine (DPPC) and the potpourri 1mg of cephalin (PE) and the chloroform-methanol mixed solution mixing that the 10mL volume ratio is 6: 1 that with the ratio of amount of substance are 3: 1, sonicated makes W/O emulsion; The decompression rotary evaporation is removed the organic solvent in the described W/O emulsion, behind the adding 2mL phosphate buffer solution, and swelling 40min in 60 ℃ water-bath, violent mixing makes the multilamellar liposome vesica; With described multilamellar liposome vesica sonicated, the centrifugal 20min of 8000rpm makes the not small unilamellar vesicle of embedding dopamine.The described small unilamellar vesicle that makes is scattered in the PBS solution, in 4 ℃ of preservations.

Small unilamellar vesicle to the not embedding dopamine of the small unilamellar vesicle of the embedding dopamine of embodiment 1 to 3 preparation and control group 1 to 3 preparation carries out Electrochemical Detection:

In the small unilamellar vesicle of the not embedding dopamine that the small unilamellar vesicle of the embedding dopamine of preparation and control group 1 to 3 prepare in embodiment 1 to 3, the TritonX-100 solution that adds 80～120 μ L 1.2mmol/L is at room temperature cultivated 20min, make the small unilamellar vesicle of the embedding dopamine of preparation among the embodiment 1 to 3 and the small unilamellar vesicle breakdown of emulsion of the not embedding dopamine that control group 1 to 3 prepares, get corresponding breakdown of emulsion solution 80～120 μ L and carry out Electrochemical Detection.Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with the glass-carbon electrode is working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, carry out the electrochemiluminescence test, detect the linear sweep volt-ampere peak current signal intensity of dopamine.The results are shown in Table 1.

Table 1 Electrochemical Detection result

Group	Embodiment 1	Control group 1	Embodiment 2	Control group 2	Embodiment 3	Control group 3
							Current signal (μ A)	1.2	0.07	1.4	0.03	1.4	0.07
Dopamine	Have	-	Have	-	Have	-

Comprehensive The above results as can be known, the small unilamellar vesicle of the embedding dopamine of embodiment 1 to 3 preparation, clear in the Electronic Speculum bottom profiled, particle diameter is 0.02～0.08 μ m.The employing linear sweep voltammetry is carried out Electrochemical Detection to the small unilamellar vesicle of the embedding dopamine of embodiment 1 to 3 preparation, small unilamellar vesicle is behind the TritonX-100 breakdown of emulsion, all there is current signal to produce, the small unilamellar vesicle of proof embodiment 1 to 3 preparation all is embedded with dopamine, and the current signal of Electrochemical Detection has stability and repeatability.

Embodiment 5 detects gastrin precursor release peptide (ProGRP) based on the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine

The small unilamellar vesicle of embedding dopamine and gastrin precursor release peptide (ProGRP) antibody is the preparation of couplings altogether:

The small unilamellar vesicle that in the glutaraldehyde solution of 0.5mL 2.0%, dropwise adds the embedding dopamine of embodiment 1 preparation, stirring reaction 40min at room temperature, under 4 ℃, place the phosphate buffer 12h that dialyses, remove excessive glutaraldehyde molecule, make the small unilamellar vesicle of the embedding dopamine of aldehyde radicalization; Get gastrin precursor release peptide (ProGRP) antibody-solutions of 200 μ L, slowly join in the small unilamellar vesicle of embedding dopamine of the described aldehyde radicalization of 200 μ L, fully descend lasting stirring reaction 1h at 25 ℃ behind the mixing, the bovine serum albumin solution that adds 40 μ L 0.25%, the unreacted aldehyde radical in small unilamellar vesicle surface with the embedding dopamine that seals described aldehyde radicalization, and at 25 ℃ of following incubation reaction 1h, the small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that make the embedding dopamine are total to couplings, and is standby in 4 ℃ of preservations.

Detect the making of gastrin precursor release peptide (ProGRP) typical curve based on the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine:

To immobilized 50,100,250 of the 80 μ L that add in the microwell plate of gastrin precursor release peptide antibody, 500, gastrin precursor release peptide (ProGRP) standard solution of 1000pg/mL is in 37 ℃ of reaction 1h, add 80 μ L 0.25%BSA solution again, to seal non-specific unusual binding site; Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA.

The small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that add 80 μ L embedding dopamines in microwell plate are total to couplings, react 1h down at 37 ℃, wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 80 μ L 1.2mmol/L is at room temperature cultivated 20min, makes the small unilamellar vesicle of described embedding dopamine and the liposome breakdown of emulsion in the common couplings of gastrin precursor release peptide (ProGRP) antibody, makes solution to be measured; Get the described solution to be measured of 80 μ L from immobilized the microwell plate of gastrin precursor release peptide (ProGRP) antibody, transfer in the electro-chemical test pond.

Preparation multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode: (Ф=4mm) is successively through the Al of 1.0,0.3,0.05 μ m for glass-carbon electrode ₂O ₃It is clean with distilled water flushing to stick with paste the polishing back, and supersound washing in distilled water, acetone, distilled water respectively again places under the room temperature and dries, and makes to handle the back glass-carbon electrode; Red fuming nitric acid (RFNA)-the concentrated sulphuric acid mixed solution that with volume ratio is 1: 1 is handled multi-walled carbon nano-tubes (MWNT) 5h in 80 ℃, and washing final vacuum drying makes and handles back multi-walled carbon nano-tubes (MWNT); Get the described processing back multi-walled carbon nano-tubes of 5.0mg (MWNT) and be scattered in the DMF solution of 5mL 4%, ultrasonic 30min makes multi-walled carbon nano-tubes (MWNT) solution; Get the described MWNT drips of solution of 6 μ L and be added in glass-carbon electrode surface, described processing back, under room temperature, place and dry, make described multi-walled carbon nano-tubes (MWNT) modified glassy carbon, in 4 ℃ refrigerator, preserve standby.

Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with the MWNT modified glassy carbon for preparing is working electrode, the platinum electrode conduct is to electrode, and saturated calomel electrode (SCE) is carried out the electrochemiluminescence test as contrast electrode, detect the linear sweep volt-ampere peak current signal intensity of dopamine, the production standard curve, as shown in Figure 1, the equation of typical curve is: I=0.0013C _ProGRP+ 1.0729.

Based on gastrin precursor release peptide (ProGRP) among the electro-chemistry immunity detection method test sample human serum sample of the small unilamellar vesicle of embedding dopamine: the human serum sample is all from the 3rd tumor center of affiliated hospital of Third Military Medical University, 156 of test set (patient), wherein the man is 78,78 of woman; 100 of control groups (normal human serum), wherein the man is 50,50 of woman.

To the immobilized blood serum sample solution that adds 100 μ L in the microwell plate of gastrin precursor release peptide antibody respectively,, add 100 μ L 0.25%BSA solution again to seal non-specific unusual binding site in 37 ℃ of reaction 1h.Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA.

In microwell plate, add the small unilamellar vesicle of 80 μ L embedding dopamines and the common coupling solution of gastrin precursor release peptide antibody, react 1h down at 37 ℃.Wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 80 μ L 1.2mmol/L is at room temperature cultivated 20min makes the liposome breakdown of emulsion, makes solution to be measured.Then, respectively from getting the described solution to be measured of 80 μ L the microwell plate of ProGRP antibody and transfer in the electro-chemical test pond with immobilized.Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with three-electrode system, the MWNT modified glassy carbon electrode that promptly prepares is a working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, by detecting the peak current intensity (parallel detection three times) of DA.Calculate the mean value of the peak current intensity of the dopamine that obtains, and compare, obtain the concentration of corresponding gastrin precursor release peptide (ProGRP) with typical curve.

Embodiment 6 detects gastrin precursor release peptide (ProGRP) based on the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine

The small unilamellar vesicle that in the glutaraldehyde solution of 0.5mL 3.0%, dropwise adds the embedding dopamine of embodiment 1 preparation, stirring reaction 50min at room temperature, under 4 ℃, place the phosphate buffer 12h that dialyses, remove excessive glutaraldehyde molecule, make the small unilamellar vesicle of the embedding dopamine of aldehyde radicalization; Get gastrin precursor release peptide (ProGRP) antibody-solutions of 250 μ L, slowly join in the small unilamellar vesicle of embedding dopamine of the described aldehyde radicalization of 250 μ L, fully descend lasting stirring reaction 2h at 25 ℃ behind the mixing, the bovine serum albumin solution that adds 50 μ L 0.25%, the unreacted aldehyde radical in small unilamellar vesicle surface with the embedding dopamine that seals described aldehyde radicalization, and at 25 ℃ of following incubation reaction 2h, the small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that make the embedding dopamine are total to couplings, and is standby in 4 ℃ of preservations.

To immobilized 50,100,250 of the 100 μ L that add in the microwell plate of gastrin precursor release peptide antibody, 500, gastrin precursor release peptide (ProGRP) standard solution of 1000pg/mL is in 37 ℃ of reaction 1h, add 100 μ L 0.25%BSA solution again, to seal non-specific unusual binding site; Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA.

The small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that add 100 μ L embedding dopamines in microwell plate are total to couplings, react 1h down at 37 ℃, wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 100 μ L 1.2mmol/L is at room temperature cultivated 20min, makes the small unilamellar vesicle of described embedding dopamine and the liposome breakdown of emulsion in the common couplings of gastrin precursor release peptide (ProGRP) antibody, makes solution to be measured; Get the described solution to be measured of 100 μ L from immobilized the microwell plate of gastrin precursor release peptide (ProGRP) antibody, transfer in the electro-chemical test pond.

Preparation multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode: (Ф=4mm) is successively through the Al of 1.0,0.3,0.05 μ m for glass-carbon electrode ₂O ₃It is clean with distilled water flushing to stick with paste the polishing back, and supersound washing in distilled water, acetone, distilled water respectively again places under the room temperature and dries, and makes to handle the back glass-carbon electrode; Red fuming nitric acid (RFNA)-the concentrated sulphuric acid mixed solution that with volume ratio is 1: 1 is handled multi-walled carbon nano-tubes (MWNT) 5h in 80 ℃, and washing final vacuum drying makes and handles back multi-walled carbon nano-tubes (MWNT); Get the described processing back multi-walled carbon nano-tubes of 5.0mg (MWNT) and be scattered in the DMF solution of 5mL 5%, ultrasonic 30min makes multi-walled carbon nano-tubes (MWNT) solution; Get the described MWNT drips of solution of 8 μ L and be added in glass-carbon electrode surface, described processing back, under room temperature, place and dry, make described multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode, in 4 ℃ refrigerator, preserve standby.

Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with the MWNT modified glassy carbon electrode for preparing is working electrode, the platinum electrode conduct is to electrode, and saturated calomel electrode (SCE) is carried out the electrochemiluminescence test as contrast electrode, detect the linear sweep volt-ampere peak current signal intensity of dopamine, the production standard curve, as shown in Figure 2, the equation of typical curve is: I=0.001C _ProGRP+ 1.099.

Based on gastrin precursor release peptide (ProGRP) in the electro-chemistry immunity detection method human body blood serum sample of the small unilamellar vesicle of embedding dopamine: the human serum sample is all from the 3rd tumor center of affiliated hospital of Third Military Medical University, 156 of test set (patient), wherein the man is 78,78 of woman; 100 of control groups (normal human serum), wherein the man is 50,50 of woman.

In microwell plate, add the small unilamellar vesicle of 100 μ L embedding dopamines and the common coupling solution of gastrin precursor release peptide antibody, react 1h down at 37 ℃.Wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 100 μ L 1.2mmol/L is at room temperature cultivated 20min makes the liposome breakdown of emulsion, makes solution to be measured.Then, respectively from getting the described solution to be measured of 100 μ L the microwell plate of gastrin precursor release peptide antibody and transfer in the electro-chemical test pond with immobilized.Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with three-electrode system, the MWNT modified glassy carbon electrode that promptly prepares is a working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, by detecting the peak current intensity (parallel detection three times) of dopamine.Calculate the mean value of the peak current intensity of dopamine, and compare, obtain the concentration of corresponding gastrin precursor release peptide (ProGRP) with typical curve.

Embodiment 7 detects gastrin precursor release peptide (ProGRP) based on the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine

The small unilamellar vesicle that in the glutaraldehyde solution of 0.5mL 2.5%, dropwise adds the embedding dopamine of embodiment 2 preparations, stirring reaction 60min at room temperature, under 4 ℃, place the phosphate buffer 12h that dialyses, remove excessive glutaraldehyde molecule, make the small unilamellar vesicle of the embedding dopamine of aldehyde radicalization; Get gastrin precursor release peptide (ProGRP) antibody-solutions of 300 μ L, slowly join in the small unilamellar vesicle of embedding dopamine of the described aldehyde radicalization of 300 μ L, fully descend lasting stirring reaction 1.5h at 25 ℃ behind the mixing, the bovine serum albumin solution that adds 70 μ L 0.25%, the unreacted aldehyde radical in small unilamellar vesicle surface with the embedding dopamine that seals described aldehyde radicalization, and at 25 ℃ of following incubation reaction 1.5h, the small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that make the embedding dopamine are total to couplings, and is standby in 4 ℃ of preservations.

To immobilized 50,100,250 of the 120 μ L that add in the microwell plate of gastrin precursor release peptide antibody, 500, gastrin precursor release peptide (ProGRP) standard solution of 1000pg/mL is in 37 ℃ of reaction 1h, add 120 μ L 0.25%BSA solution again, to seal non-specific unusual binding site; Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA.

The small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that add 120 μ L embedding dopamines in microwell plate are total to couplings, react 1h down at 37 ℃, wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 120 μ L 1.2mmol/L is at room temperature cultivated 20min, makes the small unilamellar vesicle of described embedding dopamine and the liposome breakdown of emulsion in the common couplings of gastrin precursor release peptide (ProGRP) antibody, makes solution to be measured; Get the described solution to be measured of 120 μ L from immobilized the microwell plate of gastrin precursor release peptide (ProGRP) antibody, transfer in the electro-chemical test pond.

Preparation multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode: (Ф=4mm) is successively through the Al of 1.0,0.3,0.05 μ m for glass-carbon electrode ₂O ₃It is clean with distilled water flushing to stick with paste the polishing back, and supersound washing in distilled water, acetone, distilled water respectively again places under the room temperature and dries, and makes to handle the back glass-carbon electrode; Red fuming nitric acid (RFNA)-the concentrated sulphuric acid mixed solution that with volume ratio is 1: 1 is handled multi-walled carbon nano-tubes (MWNT) 5h in 80 ℃, and washing final vacuum drying makes and handles back multi-walled carbon nano-tubes (MWNT); Get the described processing back multi-walled carbon nano-tubes of 5.0mg (MWNT) and be scattered in the DMF solution of 5mL 6%, ultrasonic 30min makes multi-walled carbon nano-tubes (MWNT) solution; Get the described MWNT drips of solution of 9 μ L and be added in glass-carbon electrode surface, described processing back, under room temperature, place and dry, make described multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode, in 4 ℃ refrigerator, preserve standby.

Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with the MWNT modified glassy carbon electrode for preparing is working electrode, the platinum electrode conduct is to electrode, and saturated calomel electrode (SCE) is carried out the electrochemiluminescence test as contrast electrode, detect the linear sweep volt-ampere peak current signal intensity of dopamine, the production standard curve, as shown in Figure 3, the equation of typical curve is: I=0.001C _ProGRP+ 1.104.

Based on gastrin precursor release peptide (ProGRP) in the electro-chemistry immunity detection method human body blood serum sample of the small unilamellar vesicle of embedding dopamine: the human serum sample is all from the 3rd tumor center of affiliated hospital of Third Military Medical University, 156 of test set (patient), wherein the man is 78,78 of woman; 100 of control groups (normal human serum), wherein the man is 50,50 of woman

In microwell plate, add the small unilamellar vesicle of 120 μ L embedding dopamines and the common coupling solution of gastrin precursor release peptide antibody, react 1h down at 37 ℃.Wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 120 μ L 1.2mmol/L is at room temperature cultivated 20min makes the liposome breakdown of emulsion, makes solution to be measured.Then, respectively from getting the described solution to be measured of 120 μ L the microwell plate of gastrin precursor release peptide antibody and transfer in the electro-chemical test pond with immobilized.Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with three-electrode system, the MWNT modified glassy carbon electrode that promptly prepares is a working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, by detecting the peak current intensity (parallel detection three times) of DA.Calculate the mean value of the peak current intensity of dopamine, and compare, obtain the concentration of corresponding gastrin precursor release peptide (ProGRP) with typical curve.

Embodiment 8 detects gastrin precursor release peptide (ProGRP) based on the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine

The small unilamellar vesicle that in the glutaraldehyde solution of 0.5mL 2.0%, dropwise adds the embedding dopamine of embodiment 3 preparations, stirring reaction 80min at room temperature, under 4 ℃, place the phosphate buffer 12h that dialyses, remove excessive glutaraldehyde molecule, make the small unilamellar vesicle of the embedding dopamine of aldehyde radicalization; Get gastrin precursor release peptide (ProGRP) antibody-solutions of 275 μ L, slowly join in the small unilamellar vesicle of embedding dopamine of the described aldehyde radicalization of 275 μ L, fully descend lasting stirring reaction 1.75h at 25 ℃ behind the mixing, the bovine serum albumin solution that adds 80 μ L 0.25%, the unreacted aldehyde radical in small unilamellar vesicle surface with the embedding dopamine that seals described aldehyde radicalization, and at 25 ℃ of following incubation reaction 1.75h, the small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that make the embedding dopamine are total to couplings, and is standby in 4 ℃ of preservations.

To immobilized 50,100,250 of the 110 μ L that add in the microwell plate of gastrin precursor release peptide antibody, 500, gastrin precursor release peptide (ProGRP) standard solution of 1000pg/mL is in 37 ℃ of reaction 1h, add 110 μ L 0.25%BSA solution again, to seal non-specific unusual binding site; Reaction is finished the back and is washed with PBS and remove unconjugated sample protein and BSA.

The small unilamellar vesicle and gastrin precursor release peptide (ProGRP) antibody that add 110 μ L embedding dopamines in microwell plate are total to couplings, react 1h down at 37 ℃, wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 110 μ L 1.2mmol/L is at room temperature cultivated 20min, makes the small unilamellar vesicle of described embedding dopamine and the liposome breakdown of emulsion in the common couplings of gastrin precursor release peptide (ProGRP) antibody, makes solution to be measured; Get the described solution to be measured of 110 μ L from immobilized the microwell plate of gastrin precursor release peptide (ProGRP) antibody, transfer in the electro-chemical test pond.

Preparation multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode: (Ф=4mm) is successively through the Al of 1.0,0.3,0.05 μ m for glass-carbon electrode ₂O ₃It is clean with distilled water flushing to stick with paste the polishing back, and supersound washing in distilled water, acetone, distilled water respectively again places under the room temperature and dries, and makes to handle the back glass-carbon electrode; Red fuming nitric acid (RFNA)-the concentrated sulphuric acid mixed solution that with volume ratio is 1: 1 is handled multi-walled carbon nano-tubes (MWNT) 5h in 80 ℃, and washing final vacuum drying makes and handles back multi-walled carbon nano-tubes (MWNT); Get the described processing back multi-walled carbon nano-tubes of 5.0mg (MWNT) and be scattered in the DMF solution of 5mL6%, ultrasonic 30min makes multi-walled carbon nano-tubes (MWNT) solution; Get the described MWNT drips of solution of 10 μ L and be added in glass-carbon electrode surface, described processing back, under room temperature, place and dry, make described multi-walled carbon nano-tubes (MWNT) modified glassy carbon electrode, in 4 ℃ refrigerator, preserve standby.

Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with the MWNT modified glassy carbon electrode for preparing is working electrode, the platinum electrode conduct is to electrode, and saturated calomel electrode (SCE) is carried out the electrochemiluminescence test as contrast electrode, detect the linear sweep volt-ampere peak current signal intensity of dopamine, the production standard curve, as shown in Figure 4, the equation of typical curve is: I=0.001C _ProGRP+ 1.064.

In microwell plate, add the liposome of 110 μ L embedding dopamines and the common coupling solution of gastrin precursor release peptide antibody, react 1h down at 37 ℃.Wash 3 times with PBS solution, to remove the liposome of non-specific absorption.

The TritonX-100 solution that adds 110 μ L 1.2mmol/L is at room temperature cultivated 20min makes the liposome breakdown of emulsion, makes solution to be measured.Then, respectively from getting the described solution to be measured of 110 μ L the microwell plate of ProGRP antibody and transfer in the electro-chemical test pond with immobilized.Adopt linear sweep voltammetry (sweep limit-0.2～0.8V, sweep speed and be 50mV/s), with three-electrode system, the MWNT modified glassy carbon electrode that promptly prepares is a working electrode, the platinum electrode conduct is to electrode, saturated calomel electrode (SCE) is as contrast electrode, by detecting the peak current intensity (parallel detection three times) of dopamine.Calculate the mean value of the peak current intensity of dopamine, and compare, obtain the concentration of corresponding gastrin precursor release peptide (ProGRP) with typical curve.

Embodiment 9 is based on the comparison of the electro-chemistry immunity detection method and the ELISA detection method of the small unilamellar vesicle of embedding dopamine

The human serum sample is all from the 3rd tumor center of affiliated hospital of Third Military Medical University, 156 of test set (patients serum), and wherein the man is 78,78 of woman; 100 of control groups (normal human serum), wherein the man is 50,50 of woman.

ELISA detects gastrin precursor release peptide (ProGRP): the ELISA kit is available from the sharp paddy bio tech ltd in Shanghai.

With among the embodiment 5 to 8 based on the testing result of the electro-chemistry immunity detection method of the small unilamellar vesicle of embedding dopamine relatively, blood serum sample is the test set patients serum, totally 156 samples, wherein the male patient is 78,78 of female patients are equally divided into 13 groups, 6 of every group of serum samples, each 3 of masculinity femininity serum samples the results are shown in Table 2.

Two kinds of detection methods of table 2 detect gastrin precursor release peptide (ProGRP) in patient's serum

The blood serum sample numbering	ELISA(pg/mL)	Electro-chemistry immunity (pg/mL)
			1	114.8	89.2
2	156.1	205.7
			3	179.8	251.3
4	247.5	206.1
			5	291.7	326.9
6	468.4	427.2
			7	499.1	549.8
8	527.3	510.5
			9	684.6	635.3
10	700.8	684.1
			11	753.9	801.7
12	824.5	910.2
			13	890.3	855.7

Electro-chemistry immunity detection method and ELISA detection method comparative test result show that the correlativity of two kinds of detection methods is good, and linear equation is y=0.987x+14.85, and related coefficient is R=0.9839, and P＜0.01, two kind of a method is remarkable positive correlation.

The electro-chemistry immunity detection method: linear response range is 50-1000pg/mL, detects lower limit: 16pg/mL; The ELISA detection method: sensing range is 100pg/mL-1000pg/mL, lowest detectable limit: 50pg/mL.The electro-chemistry immunity detection method of the small unilamellar vesicle based on the embedding dopamine provided by the invention is compared with the ELISA detection method, and highly sensitive, specificity and accuracy are good, and be significant to the diagnosis of small-cell carcinoma of the lung.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

1. an electro-chemistry immunity detection method is characterized in that, comprises the steps:

Step 1: the small unilamellar vesicle of preparation embedding dopamine and protein antibodies to be checked be couplings altogether;

Step 2: contain the abundant combination of standard items that variable concentrations is treated Reichl's test to immobilized the adding in the microwell plate of described protein antibodies to be checked, remove nonspecific binding site, the small unilamellar vesicle that adds described embedding dopamine in microwell plate is total to couplings with protein antibodies to be checked and fully combines, add surfactant breakdown of emulsion at room temperature, make detection solution; From microwell plate, get described detection solution, transfer in the electro-chemical test pond, adopt linear sweep voltammetry, carry out the electrochemiluminescence test, with concentration and the corresponding galvanochemistry peak to peak current drawing standard curve for the treatment of Reichl's test in the standard items;

2. electro-chemistry immunity detection method as claimed in claim 1 is characterized in that, is working electrode with the multi-walled carbon nano-tubes modified glassy carbon.

3. electro-chemistry immunity detection method as claimed in claim 2 is characterized in that, the preparation method of described multi-walled carbon nano-tubes modified glassy carbon is that glass-carbon electrode is through Al ₂O ₃Stick with paste polishing back supersound washing, make and handle the back glass-carbon electrode, handle multi-walled carbon nano-tubes with red fuming nitric acid (RFNA)-concentrated sulphuric acid mixed solution, be scattered in the DMF solution, sonicated, make multi-walled carbon nano-tubes solution, described multi-walled carbon nano-tubes drips of solution is added in glass-carbon electrode surface, described processing back, make described multi-walled carbon nano-tubes modified glassy carbon electrode.

4. electro-chemistry immunity detection method as claimed in claim 1 is characterized in that, described surfactant is TritonX-100.

5. electro-chemistry immunity detection method as claimed in claim 1 is characterized in that, described linear sweep voltammetry sweep limit is-0.2～0.8V, sweeps speed and is 50mV/s.

6. as each described electro-chemistry immunity detection method of claim 1 to 5, it is characterized in that the described Reichl's test for the treatment of is a tumor-marker albumen.

7. electro-chemistry immunity detection method as claimed in claim 6, it is characterized in that described tumor-marker albumen comprises gastrin precursor release peptide, neuronspecific enolase, CYFRA21-1, AFP, APT, CEA, CA242, CA125, CA199, CA153, CA724, CA50, f-PSA, t-PSA, Free β-hCG, SCCA, β 2-MG.

8. electro-chemistry immunity detection method as claimed in claim 7 is characterized in that, the described Reichl's test for the treatment of is a gastrin precursor release peptide.