CN109142748A - Human prostate specific antigen detection method and its kit - Google Patents
Human prostate specific antigen detection method and its kit Download PDFInfo
- Publication number
- CN109142748A CN109142748A CN201810894178.3A CN201810894178A CN109142748A CN 109142748 A CN109142748 A CN 109142748A CN 201810894178 A CN201810894178 A CN 201810894178A CN 109142748 A CN109142748 A CN 109142748A
- Authority
- CN
- China
- Prior art keywords
- specific antigen
- electrode
- solution
- human prostate
- gold nano
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
- G01N33/6893—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 diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The present invention discloses a kind of human prostate specific antigen detection method and its kit.The present invention is using human prostate specific antigen as test object, high quantum production rate gold nano cluster electrogenerated chemiluminescence technology and immuno analytical method are organically combined, high quantum production rate gold nano cluster electrogenerated chemiluminescence probe is prepared using reduction method, using nano material of manganese dioxide as electrogenerated chemiluminescence quencher, the redox reaction of the ascorbic acid and manganese dioxide that are generated using enzyme linked immunoassay restores electrochemiluminescence signal, for a kind of high-performance electrogenerated chemiluminescence human prostate specific antigen detection method based on high quantum production rate gold nano cluster probe.It is 5 × 10 to the human prostate specific antigen detection range of linearity‑4 The pg/mL of pg/mL ~ 50, detection are limited to 0.11 fg/mL.Have the characteristics that quick, accurate, high sensitivity, selectivity and stability are good, amount of samples is few, there is preferable potential applicability in clinical practice.
Description
Technical field
The present invention relates to a kind of electrogenerated chemiluminescence human prostatic specifics based on high quantum production rate gold nano cluster probe
Property antigen detection method and its detection kit, belong to analytical chemistry and field of nanometer technology.
Background technique
The clinical analysis of tumor markers is most important for the early diagnosis of cancer and proteomics research, while
Deepen the understanding to the relevant disease organism process of cancer.Human prostate specific antigen (prostate specific
Antigen, PSA) it is a kind of serine protease generated by prostate epithelial cell secretion, there is clinical meaning most of
It can all be increased in prostate cancer, it is meaningful to the diagnosis for not having Symptomatic prostate cancer in early days.Studies have shown that in prostate
In cancer patient, most PSA are bonding state, and Free PSA/t-PSA ratio increases lower than normal person or benign prostate
Raw patient.Therefore, Free PSA is detected, screening and diagnosis of prostate cancer can be improved by calculating Free PSA and t-PSA ratio
Specificity.But forefront is avoided there is the barrier between a kind of blood-epithelium around normal prostate duct system
The PSA that glandular epithelium generates is directly entered among blood, and the PSA almost all in serum is produced by prostate epithelial cell
Raw, thus PSA content is considerably less in blood, this causes difficulty to the early diagnosis of prostate cancer to a certain extent.At present
Many kinds have been developed in immunoassay method for detecting human prostate specific antigen content in serum, such as enzyme linked immunological point
Analysis method, radio immunoassay, fluoroimmunoassay, chemiluminescence immunoassay and mass spectral analysis etc..These methods
Sensitivity is extremely limited, therefore, needs to develop a kind of method detection human prostate highly sensitive, specificity is good, easy to operate
Specific antigen.Electrogenerated chemiluminescence immunoassay is a kind of novel immune detection method quickly grown in recent years, oneself is facing
Bed analysis and the fields such as medicine, immune are widely applied, which has that easy to operate, quick, selectivity is good, the range of linearity is wide,
And many excellent performance characteristics such as easy to control, and electrogenerated chemiluminescence technology is not necessarily to excitation light source, background signal bottom, not only
It is possible to prevente effectively from photobleaching and background interference problem in conventional fluorescent analysis, and it is more clever compared to conventional method of analysis
It is quick.
Novel light-emitting body is found, the electrogenerated chemiluminescence system for developing function admirable is the important of Electrochemiluminescprocess process
Research direction.Quantum dot or nanocluster, because having good optically and electrically characteristic, research and development, photosensitive biography in luminescent material
Building of sensor etc. causes more and more concerns.In numerous illuminators, gold nano cluster is due to its excellent light
Stability, hypotoxicity and good water solubility etc., in biomarker, medical imaging, biosensor, catalysis and photoelectronics etc.
Field is widely used.2011, Zhu person of outstanding talent et al. reported cathode electricity of the gold nano cluster in potassium peroxydisulfate system for the first time
Chemiluminescence phenomenon is caused, in the same year, Chen Guonan et al. also reports the electroluminescent chemistry of anode of the gold nano cluster in triethylamine system
Luminescence phenomenon.The concern of numerous researchers is attracted subsequently, based on the electrogenerated chemiluminescence system of gold nano cluster.But by
Limitation in its electrogenerated chemiluminescence intensity is small, quantum yield is low, luminous mechanism is not known the problems such as, gold nano cluster is electroluminescent
The research in chemiluminescence field is also considerably less.In view of the above-mentioned problems, high quantum has been made by nanoscale regulation in this seminar
The gold nano cluster probe new method of yield, and detection and the small molecule biological thiol etc. for being applied to cell release dopamine
Highly sensitive, the quick detection of biomolecule.For the urgent need of clinical tumor diagnosing and treating, design and prepare based on novel
The high quantum production rate electrochemiluminescimmunosensor immunosensor of functionalization gold nano cluster and the detection for being used for tumor markers
With more important Academic innovations and clinical value.
The present invention provides a kind of, and the electrogenerated chemiluminescence human prostate based on high quantum production rate gold nano cluster probe is special
Specific Antigen detection method, and the detection kit based on this method preparation.
Summary of the invention
It is an object of the present invention to provide a kind of, and the electroluminescent chemistry based on high quantum production rate gold nano cluster probe is sent out
Light human prostate specific antigen detection method.
It is another object of the present invention to provide a kind of electroluminescent chemistry based on high quantum production rate gold nano cluster probe
Shine human prostate specific antigen detection kit.
To achieve the goals above, the invention adopts the following technical scheme: a kind of be based on high quantum production rate gold nano cluster
The electrogenerated chemiluminescence human prostate specific antigen detection method of probe, it is characterised in that: first in electrode face finish gold
Nanocluster prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe by reduction method, and further in electrode table
Nano material of manganese dioxide is modified in face, as electrochemiluminescence signal quencher；In the fixed people forefront of blank ELISA Plate
Gland specific antigen-antibody is added human prostate specific antigen, adds the human prostate specific antigen of biotin labeling
Antibody forms sandwich immunoassay compound；Streptavidin-alkaline phosphatase is added, adds 2- phosphoric acid-L-AA trisodium
Salt generates ascorbic acid by alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt；Reaction solution is taken out, it will
Electrode immerses in reaction solution, and redox reaction occurs for the ascorbic acid in the manganese dioxide and reaction solution of electrode surface；Acquisition
The electrochemiluminescence signal of modified electrode realizes human prostate specific antigen according to the change of electrochemiluminescence signal
Detection.
The reduction method prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe and uses following electrochemistry also
Former method or chemical reduction method preparation:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode be to electrode, Ag/AgCl is reference electrode, by above-mentioned electrode insertion 0.1 mol/L, pH 7.4 phosphate delay
It rushes in solution, applies negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrification
Learn the gold nano cluster probe that shines；
(2) gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the gold nano cluster probe modification electrode of chemical reduction method preparation.
The electrode face finish nano material of manganese dioxide is prepared using following electrochemical deposition methods or drop-coating:
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in the KMnO of 1:1 V/V4-H2SO4In solution, electricity
Manganese dioxide is deposited, sedimentation potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, and cleaning is dried with nitrogen；
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to 1.25
ML, 0.1 mol/L, pH 6.0 buffer solution in, the deionized water volume that keeps solution last is added into ultrasound 30 after 5 mL
Min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters again, obtain dioxy
Change manganese nanometer sheet solution；Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
The method in the fixed human prostate specific antigen antibody of blank ELISA Plate is self-assembly method: taking blank enzyme mark
Plate, in blank ELISA Plate sample well be added 1 ~ 5 mg/mL, pH 8.5 50 ~ 100 μ L of dopamine solution, in 4 DEG C react 1 ~
5 h are gently rinsed with water after reaction, are patted dry；It is molten that 0.1 ~ 0.5 mg/mL human prostate specific antigen antibody is added dropwise again
Liquid, 4 DEG C of 8 ~ 12 h of incubation, rinsing；The bovine serum albumin solution of 50 ~ 100 μ L 1wt% ~ 5wt% is added, is reacted in 4 DEG C
0.5 ~ 2 h, washing, pats dry.
The addition human prostate specific antigen adds the human prostate specific antigen antibody of biotin labeling,
The method for forming sandwich immunoassay compound are as follows: in the example reaction hole for being fixed with human prostate specific antigen abzyme target
Human prostate specific antigen sample is added, covers sealing plate film, gently oscillation mixes, and 37 DEG C of incubation min of 30 min ~ 60 are washed
It washs, pats dry；The 50 μ L of human prostate specific antigen antibody of 0.1 ~ 10 μ g/mL biotin labeling is added, 37 DEG C incubate 30
The min of min ~ 60, washing, pats dry.
Addition Streptavidin-the alkaline phosphatase adds 2- phosphoric acid-L-AA trisodium salt method are as follows:
Every hole is separately added into 0.1 ~ 1 U/mL Streptavidin-alkalinity of 50 μ L in the ELISA Plate hole for forming sandwich immunoassay compound
Phosphatase and 5 ~ 10 mmol/L 2- phosphoric acid-L-AA trisodium-salt solution, gently oscillation mixes, and is protected from light, 37 DEG C of temperature
Educate the min of 30 min ~ 60.
The electrochemiluminescence signal method of the acquisition modified electrode is as follows: it is tested using three-electrode system, with
Modified electrode is working electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, and buffer solution is phosphate buffer
Or Tris-HCl buffer solution, electrolyte used are KCl or KNO3；The insertion of above-mentioned electrode is contained into over cure acid ion coreaction
In the buffer solution of agent, apply certain scanning voltage, working electrode surface generates electrogenerated chemiluminescence radiation, photomultiplier tube
High pressure is set as 600 ~ 800 V, detects electrogenerated chemiluminescence radiation signal.
The detection that human prostate specific antigen is realized according to the change of electrochemiluminescence signal are as follows: electrode surface
The electrogenerated chemiluminescence intensity value of electrochemiluminescence signal collected and the logarithm of human prostate specific antigen concentration
5 × 10-4In a linear relationship in the range of the pg/mL of pg/mL ~ 50, detection is limited to 0.11 fg/mL.
Electrogenerated chemiluminescence human prostate-specific of the present invention based on high quantum production rate gold nano cluster probe is anti-
The detection kit of former detection method, which is characterized in that including gold nano cluster solution, nano material of manganese dioxide, people forefront
Gland specific antigen-antibody fixes the human prostatic specific of ELISA Plate, human prostate specific antigen standard items, biotin labeling
Property antigen-antibody, Streptavidin-alkaline phosphatase, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence
Test electrolyte solution.
The fixed ELISA Plate of the human prostate specific antigen antibody are as follows: blank ELISA Plate is taken, in blank ELISA Plate sample
50 ~ 100 μ L of dopamine solution that 1 ~ 5 mg/mL pH 8.5 is added in sample wells is used after reaction in 4 DEG C of 1 ~ 5 h of reaction
Water gently rinses, and pats dry；It is added dropwise 0.1 ~ 0.5 mg/mL human prostate specific antigen antibody-solutions again, 4 DEG C of 8 ~ 12 h of incubation,
Rinsing；The bovine serum albumin solution of 50 ~ 100 μ L 1wt% ~ 5wt% is added, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry；
The cleaning solution is phosphate buffer or Tris-HCl buffer solution；The electrogenerated chemiluminescence tests electrolyte solution
Buffer solution containing potassium peroxydisulfate.
Specifically, the present invention uses following specific technical solution: it is electroluminescent to prepare high quantum production rate gold nano cluster first
Chemiluminescence probe using manganese dioxide as electrogenerated chemiluminescence quencher, and then utilizes enzyme linked immunoassay, anti-in sandwich immunoassay
On the basis of answering, generated based on Streptavidin-alkaline phosphatase enzyme solutions and 2- phosphoric acid-L-AA trisodium reactant salt anti-
Thus the oxygen chemical original reacting recovery electrochemiluminescence signal of bad hematic acid and manganese dioxide develops a kind of based on high quantum production rate
The electrogenerated chemiluminescence immune analysis method of gold nano cluster probe, the height for tumor markers human prostate specific antigen
Effect detection.The present invention the following steps are included:
(1) prepared by high quantum production rate gold nano cluster probe
High quantum production rate gold nano cluster probe preparation method is as follows:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode are to electrode, and Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into buffer solution, apply negative potential voltage
(within the scope of -1.4 V of V ~ -2) carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrochemical luminescence gold nano cluster probe.
(2) it is anti-that gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution
5 ~ 30 min(preferably 0.1 mol/L sodium borohydride solution is answered to react 5 min), obtain the gold nano cluster of chemical reduction method preparation
Probe modification electrode.
The gold nano cluster material is functional modification gold nano cluster, such as: N- acetylation-L-cysteine-gold
Nanocluster, bovine serum albumin(BSA)-gold nano cluster etc..
(2) electrochemiluminescence signal acquisition method
By polishing electrode, then it is sequentially placed into HNO3It is cleaned by ultrasonic in solution, dehydrated alcohol and deionized water, N2Drying.Using three
Electrode system is tested, and using modified glassy carbon electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode,
Above-mentioned electrode is inserted into the buffer solution containing coreagent.Using step pulse method, initial potential is 0 V, burst length
For 10 s, termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as the V of 600 V ~ 800, detects work
Make the electrochemiluminescence signal of electrode surface generation.
The electrode is glass-carbon electrode, screen printing electrode or ITO electrode etc..Above-mentioned coreagent be over cure acid group from
Son, concentration range are 0.01 ~ 1 mol/L, preferably 0.1 mol/L.The buffer solution is phosphate buffer or Tris-HCl
Buffer solution, added electrolyte is KCl or KNO in buffer solution3, concentration is 0.01 ~ 1 mol/L, preferably 0. 1 mol/
L。
(3) nano material of manganese dioxide method of modifying
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in KMnO4-H2SO4In (1:1 V/V) solution, electrification
Deposition manganese dioxide is learned, sedimentation potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, preferably -0.2 V, 300
S is rinsed well with distilled water later, is dried with nitrogen spare.
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to
In the buffer solution of 1.25 mL, 0.1 mol/L pH 6.0, the volume that addition deionized water keeps solution last is at ultrasonic after 5 mL
30 min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters, obtain again
Manganese dioxide nano-plates solution.Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
(4) sandwich immunoassay reaction system constructs
Specific step is as follows for the building of sandwich immunoassay reaction system: (1) blank ELISA Plate being set blank well, (blank control wells are not loaded
Product, remaining each step operation are identical), standard sample wells, sample to be tested hole.Blank ELISA Plate is taken, in blank ELISA Plate sample well
The dopamine solution (the Tris-HCl buffer solution of pH 8.5 dissolves) of 50 μ L, 1 ~ 5 mg/mL is added, reacts 1 ~ 5 in 4 DEG C
H is gently rinsed with water after reaction, is patted dry.0.01 ~ 0.5 mg/mL human prostate specific antigen antibody-solutions are added dropwise again,
4 DEG C of 8 ~ 12 h of incubation, rinsing.The bovine serum albumin solution of 50 ~ 200 μ L 1% ~ 5% is added, in 4 DEG C of 0.5 ~ 2 h of reaction,
Washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, such as after the reaction was completed
This is repeated 4 times, and pats dry).Then 50 μ L of human prostate specific antigen standard solution is added in standard sample wells, it is anti-in sample
50 μ L samples are added in Ying Kongzhong, cover sealing plate film, and gently oscillation mixes, 37 DEG C of 30 ~ 60 min of incubation, and washing pats dry, then plus
Enter the human prostate specific antigen antibody 50 μ L of 0.1 ~ 10 μ g/mL biotin labeling, 37 DEG C of 30 ~ 60 min of incubation are washed
It washs, pats dry.0.1 ~ 1 U/mL Streptavidin -50 μ L of alkaline phosphatase enzyme solutions is added in every hole, and gently oscillation mixes, 37 DEG C of temperature
30 ~ 60 min are educated, is washed, is patted dry with the 10 Tris-HCl buffers of mM pH=7.3.
(5) measurement of human prostate specific antigen
Human prostate specific antigen detecting step is as follows: toward the ELISA Plate of the sandwich immunoassay reaction system of above-mentioned building
Middle addition 5 ~ 10 mmol/L 2- phosphoric acid -50 μ L of L-AA trisodium-salt solution, adds 10 Tris- of mM pH=8.0
50 μ L of HCl buffer solution, gently oscillation mixes, and 37 DEG C are protected from light 25 ~ 30 min.By above-mentioned manganese dioxide/gold nano group
Cluster modified electrode, which immerses, impregnates 4 ~ 10 min in above-mentioned reaction solution, rinsed well after taking-up with distilled water, N2Drying.Collecting work
The electrochemiluminescence signal that electrode surface generates maps to human prostate specific antigen concentration with electrochemiluminescence signal
Draw standard curve.
(6) kit
A kind of electrogenerated chemiluminescence human prostate specific antigen detection reagent based on high quantum production rate gold nano cluster probe
Box, including gold nano cluster solution, nano material of manganese dioxide, the fixed ELISA Plate of human prostate specific antigen antibody,
Human prostate specific antigen standard items, the human prostate specific antigen antibody of biotin labeling, Streptavidin-alkalinity phosphorus
Sour enzyme, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence test electrolyte solution.
Compared with prior art, the invention has the benefit that
The present invention is using high quantum production rate gold nano cluster probe as electrogenerated chemiluminescence material, with nano-manganese dioxide for electroluminescentization
Luminescence quenchers are learned, ascorbic acid is generated using enzyme linked immunoassay and restores its electrochemiluminescence signal realization human prostate spy
The detection of Specific Antigen.The present invention is high to the detection sensitivity of human prostate specific antigen, easy to operate, can effectively avoid inspection
The interference of actual sample complexity composition during survey, thus specificity is good, accuracy is high.Also, the present invention is at low cost, production letter
List, stability is good, sensitivity is good, and the range of linearity is wide by (5 × 10-4The pg/mL of pg/mL ~ 50), detection limits low (0.11 fg/mL),
With good market value.
Detailed description of the invention
Fig. 1 is electrogenerated chemiluminescence-time plot of gold nano cluster probe modification glass-carbon electrode.
Fig. 2 is manganese dioxide/gold nano cluster probe modification glass-carbon electrode electrogenerated chemiluminescence-time plot.
Fig. 3 is electrogenerated chemiluminescence-time plot (human prostate spy of present invention detection human prostate specific antigen
Specific Antigen concentration is 50 pg/mL).
Linear relationship of the Fig. 4 between electrogenerated chemiluminescence Strength Changes and human prostate specific antigen log concentration value
Figure.
Specific embodiment
The present invention is further elaborated in the following with reference to the drawings and specific embodiments, and the present invention is not limited thereto.
Human prostate specific antigen antibody used in the present invention (write from memory picogram Biotechnology Co., Ltd in Wuhan), people forefront
Gland specific antigen (write from memory picogram Biotechnology Co., Ltd in Wuhan), the human prostate specific antigen antibody of biotin labeling
(the PSA antibody of biotin labeling, write from memory picogram Biotechnology Co., Ltd in Wuhan), (Wuhan is rich for Streptavidin-alkaline phosphatase
Shi De bioengineering Co., Ltd), 2- phosphoric acid-L-AA trisodium salt (Sigma-Aldrich company) is prior art production
Product.
Embodiment 1
The 20 mg/mL chlorauric acid solution of NaOH and 0.4 mL of 0.6 mL, 0.5 moL/L is taken to be added to 4 mL, 0.08 mol/L
N-acetyl-L-cysteine solution in, mixing is placed in 37 DEG C of thermostatic water baths and is incubated for 3 hours.To after reaction, use
The bag filter that molecular weight is 3000 obtains N-acetyl-L-cysteine protection after purification to 24 h of reaction solution dialysis purification
Gold nano cluster solution is kept in dark place in 4 DEG C of refrigerators.
Embodiment 2
By the glass-carbon electrode of 3 mm of diameter 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
The gold nano cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and room temperature is dry
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, obtain gold nano cluster probe
Modified electrode.Above-mentioned electrode is inserted into the 0.1 mol/L pH 7.4 containing 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl
In phosphate buffer solution.Using step pulse method, initial potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V,
Burst length is 1 s.Photomultiplier tube high pressure is set as 750 V, the electrogenerated chemiluminescence letter that detection working electrode surface generates
Number, obtain electrochemical luminescence signals (see figure 1).
Embodiment 3
By the glass-carbon electrode of 3 mm of diameter 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
The gold nano cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and room temperature is dry
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, obtain gold nano cluster probe
Modified glassy carbon electrode.Using three-electrode system, using gold nano cluster probe modification glass-carbon electrode as working electrode, platinum electrode is
To electrode, Ag/AgCl is reference electrode, using chronoamperometry, in KMnO4-H2SO4In (1:1 V/V) solution, electro-deposition two
Manganese oxide, sedimentation potential are -0.2 V, and the time is 300 s, and obtained electrode is that manganese dioxide/gold nano cluster modifies glass carbon
Electrode is rinsed well with distilled water later, N2Gas gently dries up spare.The insertion of above-mentioned electrode is contained into 0.1 mol/L persulfuric acid
In 0.1 mol/L pH, 7.4 phosphate buffer solution of potassium and 0.1 mol/L KCl.Using step pulse method, initial potential
For 0 V, the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as 750 V,
The electrochemiluminescence signal that working electrode surface generates is detected, electrochemical luminescence signals (see figure 2) is obtained.
Embodiment 4
Take blank ELISA Plate, be added in blank ELISA Plate sample well 50 μ L, 5 mg/mL dopamine solution (pH's 8.5
The dissolution of Tris-HCl buffer solution), in 4 DEG C of 2 h of reaction, is gently rinsed, patted dry with water after reaction.100 μ g/ are added dropwise again
ML human prostate specific antigen antibody-solutions, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, to remove electrode
The antibody of surface non-specific adsorption.It is subsequently added into 1% 50 μ L of bovine serum albumin solution, in 4 DEG C of 1 h of reaction, reaction
Washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so weight after the completion
It is 4 times multiple, pat dry), obtain the fixed ELISA Plate of human prostate specific antigen antibody.
Embodiment 5
It is added 50 pg/mL's in the standard sample wells of the fixed ELISA Plate of human prostate specific antigen antibody prepared by embodiment 4
50 μ L of human prostate specific antigen standard solution, covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, washing,
It pats dry, adds human prostate specific antigen antibody 50 μ L, 37 DEG C of 30 min of incubation of 0.5 μ g/mL biotin labeling,
Washing, pats dry.Every hole is added 0.2 U/mL Streptavidin -50 μ L(37 DEG C of alkaline phosphatase enzyme solutions and preheats 30 min), gently
Light oscillation mixes, 37 DEG C of 45 min of incubation, and washing pats dry.The 2- acid phosphorus-L- of 8 mmol/L is added into above-mentioned ELISA Plate again
50 μ L(37 DEG C of ascorbic acid trisodium-salt solution preheats 30 min), 50 μ L of the Tri-Hcl of pH=8.0 is added, is gently vibrated
It mixes, 37 DEG C are protected from light 25 min.Liquid in each hole is taken out, is added in the EP pipe of 2 mL, the electricity that will be handled well
Pole, which is put into each corresponding concentration EP pipe, impregnates 10 min, is rinsed well after taking-up with distilled water, N2Drying.Above-mentioned electrode is inserted
Enter in 0.1 mol/L pH, 7.4 phosphate buffer solution containing 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl.It adopts
With step pulse method, initial potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photoelectricity times
Increase pipe high pressure and is set as 750 V, the electrochemiluminescence signal that detection working electrode surface generates, obtained electrochemical luminescence letter
Number than plus the solution of human prostate specific antigen significantly increases (Fig. 3).
Embodiment 6
Take blank ELISA Plate, by blank ELISA Plate set blank well (sample is not added in blank control wells, remaining each step operation is identical),
Standard sample wells, sample to be tested hole.Be added in each hole of blank ELISA Plate 50 μ L, 5 mg/mL dopamine solution (pH's 8.5
The dissolution of Tris-HCl buffer solution), in 4 DEG C of 2 h of reaction, is gently rinsed, patted dry with water after reaction.200 μ g/ are added dropwise again
ML human prostate specific antigen antibody-solutions, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, to remove electrode
The antibody of surface non-specific adsorption.It is subsequently added into 1% 50 μ L of bovine serum albumin solution, in 4 DEG C of 1 h of reaction, reaction
Washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so weight after the completion
It is 4 times multiple, pat dry).Then the 50 μ L of human prostate specific antigen standard solution of various concentration, lid are added in standard sample wells
Upper sealing plate film, gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, adds the people of 0.5 μ g/mL biotin labeling
Prostate-specific antigen antibody 50 μ L, 37 DEG C of 30 min of incubation, washing, pat dry.It is affine that 0.2 U/mL strepto- is added in every hole
50 μ L(37 DEG C of element-alkaline phosphatase enzyme solutions preheats 30 min), gently oscillation mixes, 37 DEG C of 45 min of incubation, washs, and claps
It is dry.2- acid phosphorus -50 μ L(37 DEG C of L-AA trisodium-salt solution preheating 30 of 8 mmol/L is added into above-mentioned ELISA Plate again
Min), 50 μ L of the Tri-Hcl of pH=8.0 is added, gently oscillation mixes, and 37 DEG C are protected from light 25 min.It will be in each hole
Liquid takes out, and is added in the EP pipe of 2 mL, the electrode handled well is put into each corresponding concentration EP pipe and impregnates 10 min.
It is rinsed well after taking-up with distilled water, N2Drying.The insertion of above-mentioned electrode is contained into 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L
In 0.1 mol/L pH, 7.4 phosphate buffer solution of KCl.Using step pulse method, initial potential is 0 V, burst length
For 10 s, termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as 750 V, detects working electrode table
The electrochemiluminescence signal that face generates is 5 × 10 in human prostate specific antigen concentration-4The model of the pg/mL of pg/mL ~ 50
The logarithm and electrogenerated chemiluminescence intensity value for enclosing interior human prostate specific antigen concentration are in good linear relationship, detection limit
Fig. 4 is seen for 0.11 fg/mL().
Embodiment 7
Take the KMnO of 500 μ L, 10 mmol/L4Solution is added to 2-(the N- morpholines of 1.25 mL, 0.1 mol/L pH 6.0
Generation) in ethanesulfonic acid (MES) buffer solution, volume that deionized water keeps solution last is added into 30 min of ultrasound after 5 mL, end
12000 r.p.m are centrifuged 10 min afterwards, are washed with water 3 times, are scattered in 2.5 mL deionized waters again, obtain manganese dioxide nano
Piece solution.
Embodiment 8
Kit application method: (1) the gold nano cluster drop for the N-acetyl-L-cysteine protection for taking 5 μ L embodiments 1 to prepare
It is added in the glassy carbon electrode surface handled well, drying at room temperature.And the electrode is further immersed in 0.1 mol/L sodium borohydride solution
Middle reaction 5 minutes, obtains gold nano cluster probe modification glass-carbon electrode.The MnO for taking 6 μ L embodiments 7 to prepare2Nanometer sheet solution
It is added dropwise in above-mentioned gold nano cluster probe modification glassy carbon electrode surface, drying at room temperature, obtains manganese dioxide/gold nano cluster modification
Glass-carbon electrode.(2) the fixed ELISA Plate of human prostate specific antigen antibody prepared by Example 4, it is (empty to be set to blank well
Sample is not added in white control wells, remaining each step operation is identical), standard sample wells, sample to be tested hole.In each Kong Zhongjia of blank ELISA Plate
The dopamine solution (the Tris-HCl buffer solution of pH 8.5 dissolves) for entering 50 μ L, 5 mg/mL, in 4 DEG C of 2 h of reaction, reaction
After gently rinsed with water, pat dry.200 μ g/mL human prostate specific antigen antibody-solutions are added dropwise again, 4 DEG C are incubated for 12
h.It is gently rinsed after incubation with water, to remove the antibody of electrode surface non-specific adsorption.It is subsequently added into 50 μ L 1%
BSA solution, in 4 DEG C of 1 h of reaction, washing (carefully takes sealing plate film off, discards liquid, dry, every hole is filled it up with after the reaction was completed
Cleaning solution discards after standing 30 seconds, is so repeated 4 times, pats dry).Then the people forefront of various concentration is added in standard sample wells
50 μ L of gland specific antigen standard solution, covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry,
Human prostate specific antigen antibody 50 μ L, 37 DEG C of 30 min of incubation of 0.5 μ g/mL biotin labeling are added, are washed,
It pats dry.The 50 μ L(37 DEG C of Streptavidin-alkaline phosphatase enzyme solutions that 0.2 U/mL is added in every hole preheats 30 min), gently shake
Mixing, 37 DEG C of 45 min of incubation are swung, washing pats dry.The 2- acid phosphorus-L- that 8 mmol/L are added into above-mentioned ELISA Plate again is anti-bad
50 μ L(37 DEG C of hematic acid trisodium-salt solution preheats 30 min), 50 μ L of the Tri-Hcl of pH=8.0 is added, gently oscillation mixes,
37 DEG C are protected from light 25 min.(3) liquid in each hole is taken out, is added in the EP pipe of 2 mL, the electrode that will be handled well
It is put into each corresponding concentration EP pipe and impregnates 10 min.It is rinsed well after taking-up with distilled water, N2Drying.Above-mentioned electrode is inserted into
In 0.1 mol/L pH, 7.4 phosphate buffer solution containing 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl.Using
Step pulse method, initial potential are 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier transit
Pipe high pressure is set as 750 V, the electrochemiluminescence signal that detection working electrode surface generates, in human prostate specific antigen
Concentration is 5 × 10-4 The logarithm of human prostate specific antigen concentration and electroluminescent chemistry are sent out in the range of the pg/mL of pg/mL ~ 50
Intensity variation value is in good linear relationship, and detection is limited to 0.11 fg/mL.
The foregoing is merely exemplary embodiments of the invention, are not intended to limit the invention, all in essence of the invention
Made any modification within mind and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of electrogenerated chemiluminescence human prostate specific antigen detection side based on high quantum production rate gold nano cluster probe
Method, it is characterised in that: first in electrode face finish gold nano cluster, high quantum production rate gold nano cluster is prepared by reduction method
Electrogenerated chemiluminescence probe, and further in electrode face finish nano material of manganese dioxide, as electrogenerated chemiluminescence
Signal quencher；In the fixed human prostate specific antigen antibody of blank ELISA Plate, it is added human prostate specific antigen, then plus
Enter the human prostate specific antigen antibody of biotin labeling, forms sandwich immunoassay compound；Streptavidin-alkalinity phosphorus is added
Sour enzyme adds 2- phosphoric acid-L-AA trisodium salt, passes through alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA
Trisodium salt generates ascorbic acid；Reaction solution is taken out, electrode is immersed in reaction solution, in the manganese dioxide and reaction solution of electrode surface
Ascorbic acid occur redox reaction；The electrochemiluminescence signal for acquiring modified electrode, believes according to electrogenerated chemiluminescence
Number change realize human prostate specific antigen detection.
2. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the reduction method prepares the spy of high quantum production rate gold nano cluster electrogenerated chemiluminescence
Needle is prepared using following electrochemical reducings or chemical reduction method:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode be to electrode, Ag/AgCl is reference electrode, by above-mentioned electrode insertion 0.1 mol/L, pH 7.4 phosphate delay
It rushes in solution, applies negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrification
Learn the gold nano cluster probe that shines；
(2) gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the gold nano cluster probe modification electrode of chemical reduction method preparation.
3. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the electrode face finish nano material of manganese dioxide uses following electrochemistry
Sedimentation or drop-coating preparation:
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in the KMnO of 1:1 V/V4-H2SO4In solution, electricity
Manganese dioxide is deposited, sedimentation potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, and cleaning is dried with nitrogen；
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to 1.25
ML, 0.1 mol/L, pH 6.0 buffer solution in, the deionized water volume that keeps solution last is added into ultrasound 30 after 5 mL
Min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters again, obtain dioxy
Change manganese nanometer sheet solution；Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
4. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the method in the fixed human prostate specific antigen antibody of blank ELISA Plate
For self-assembly method: taking blank ELISA Plate, the dopamine solution of 1 ~ 5 mg/mL, pH 8.5 is added in blank ELISA Plate sample well
50 ~ 100 μ L are gently rinsed with water after reaction, are patted dry in 4 DEG C of 1 ~ 5 h of reaction；Before 0.1 ~ 0.5 mg/mL people is added dropwise again
Column gland specific antigen-antibody solution, 4 DEG C of 8 ~ 12 h of incubation, rinsing；The ox blood that 50 ~ 100 μ L 1wt% ~ 5wt% are added is pure
Protein solution, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry.
5. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the addition human prostate specific antigen, adds the people of biotin labeling
Prostate-specific antigen antibody, the method for forming sandwich immunoassay compound are as follows: anti-being fixed with human prostate specific antigen
Human prostate specific antigen sample is added in the example reaction hole of body ELISA Plate, covers sealing plate film, gently oscillation mixes, and 37
DEG C incubate the min of 30 min ~ 60, washing, pat dry；The human prostate specific antigen of 0.1 ~ 10 μ g/mL biotin labeling is added
Antibody 50 μ L, 37 DEG C of incubation min of 30 min ~ 60, washing, pat dry.
6. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the addition Streptavidin-alkaline phosphatase, it is anti-to add 2- phosphoric acid-L-
The method of bad hematic acid trisodium salt are as follows: every hole is separately added into the 0.1 ~ 1 of 50 μ L in the ELISA Plate hole for forming sandwich immunoassay compound
U/mL Streptavidin-alkaline phosphatase and 5 ~ 10 mmol/L 2- phosphoric acid-L-AA trisodium-salt solution, gently oscillation is mixed
It is even, it is protected from light, 37 DEG C of incubation 30 min ~ 60 min.
7. a kind of electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe according to claim 1
Gland specific antigen detection method, it is characterized in that the electrochemiluminescence signal method of the acquisition modified electrode is as follows: using
Three-electrode system is tested, and using modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, is delayed
Rushing solution is phosphate buffer or Tris-HCl buffer solution, and electrolyte used is KCl or KNO3；The insertion of above-mentioned electrode is contained
In the buffer solution for having over cure acid ion coreagent, apply certain scanning voltage, working electrode surface generates electroluminescentization
Luminous radiation is learned, photomultiplier tube high pressure is set as 600 ~ 800 V, detects electrogenerated chemiluminescence radiation signal.
8. any a kind of electrogenerated chemiluminescence based on high quantum production rate gold nano cluster probe according to claim 1 ~ 7
Human prostate specific antigen detection method, it is characterized in that described realize human prostate according to the change of electrochemiluminescence signal
The detection of specific antigen are as follows: before the electrogenerated chemiluminescence intensity value of electrode surface electrochemiluminescence signal collected and people
The logarithm of column gland specific antigen concentration is 5 × 10-4In a linear relationship in the range of the pg/mL of pg/mL ~ 50, detection is limited to
0.11 fg/mL。
9. a kind of any electrogenerated chemiluminescence people forefront based on high quantum production rate gold nano cluster probe claim 1-8
The detection kit of gland specific antigen detection method, which is characterized in that including gold nano cluster solution, manganese dioxide nano material
Before material, human prostate specific antigen antibody fix the people of ELISA Plate, human prostate specific antigen standard items, biotin labeling
It is column gland specific antigen-antibody, Streptavidin-alkaline phosphatase, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electroluminescent
Chemiluminescent assay electrolyte solution.
10. the electrogenerated chemiluminescence human prostate according to claim 9 based on high quantum production rate gold nano cluster probe
The detection kit of specific antigen detection method, it is characterized in that the fixed ELISA Plate of the human prostate specific antigen antibody
Are as follows: blank ELISA Plate is taken, 50 ~ 100 μ of dopamine solution of 1 ~ 5 mg/mL pH 8.5 is added in blank ELISA Plate sample well
L is gently rinsed with water after reaction, is patted dry in 4 DEG C of 1 ~ 5 h of reaction；0.1 ~ 0.5 mg/mL human prostatic specific is added dropwise again
Property antigen-antibody solution, 4 DEG C of 8 ~ 12 h of incubation, rinsing；The bovine serum albumin(BSA) that 50 ~ 100 μ L 1wt% ~ 5wt% are added is molten
Liquid, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry；The cleaning solution is phosphate buffer or Tris-HCl buffer solution；Institute
The electrogenerated chemiluminescence test electrolyte solution stated is the buffer solution containing potassium peroxydisulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810894178.3A CN109142748A (en) | 2018-08-08 | 2018-08-08 | Human prostate specific antigen detection method and its kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810894178.3A CN109142748A (en) | 2018-08-08 | 2018-08-08 | Human prostate specific antigen detection method and its kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109142748A true CN109142748A (en) | 2019-01-04 |
Family
ID=64791965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810894178.3A Pending CN109142748A (en) | 2018-08-08 | 2018-08-08 | Human prostate specific antigen detection method and its kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109142748A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110596065A (en) * | 2019-09-25 | 2019-12-20 | 福建医科大学 | Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material |
| CN111208183A (en) * | 2020-01-13 | 2020-05-29 | 成都医学院 | ECL sandwich immunosensing-based antigen detection kit |
| CN111518170A (en) * | 2020-05-09 | 2020-08-11 | 新乡医学院 | FRET-based PSA fluorescent probe, preparation method and application thereof |
| CN113322305A (en) * | 2021-06-04 | 2021-08-31 | 青岛科技大学 | Based on gold nanocluster/MnO2Preparation and application of electrochemical luminescence sensor of nanoflower |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120309018A1 (en) * | 2008-12-05 | 2012-12-06 | Myriad Genetics, Incorporated | Cancer detection markers |
| CN104931698A (en) * | 2015-05-17 | 2015-09-23 | 济南大学 | Preparation method and application of NP-NiGd@Au-based gastric cancer marker gold nano-cluster electrogenerated chemiluminescence sensor |
| CN105675697A (en) * | 2016-01-19 | 2016-06-15 | 济南大学 | Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens |
| CN106248597A (en) * | 2016-08-29 | 2016-12-21 | 南开大学 | A kind of visible detection method based on noble metal nano particles and application |
| CN106483110A (en) * | 2016-09-21 | 2017-03-08 | 安徽师范大学 | A kind of biological sensor, its preparation method and purposes |
| CN106706607A (en) * | 2017-02-07 | 2017-05-24 | 福建医科大学 | High-quantum-yield electrochemiluminescence gold nano-cluster probe and preparation method of high-quantum-yield electrochemiluminescence gold nano-cluster probe |
| CN106872546A (en) * | 2017-02-07 | 2017-06-20 | 福建医科大学 | Electrochemical reducing prepares high quantum production rate electrochemical luminescence gold nano cluster probe |
| CN107589099A (en) * | 2017-09-03 | 2018-01-16 | 福建医科大学 | 6 purinethol detection methods and its kit based on gold nano cluster |
-
2018
- 2018-08-08 CN CN201810894178.3A patent/CN109142748A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120309018A1 (en) * | 2008-12-05 | 2012-12-06 | Myriad Genetics, Incorporated | Cancer detection markers |
| CN104931698A (en) * | 2015-05-17 | 2015-09-23 | 济南大学 | Preparation method and application of NP-NiGd@Au-based gastric cancer marker gold nano-cluster electrogenerated chemiluminescence sensor |
| CN105675697A (en) * | 2016-01-19 | 2016-06-15 | 济南大学 | Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens |
| CN106248597A (en) * | 2016-08-29 | 2016-12-21 | 南开大学 | A kind of visible detection method based on noble metal nano particles and application |
| CN106483110A (en) * | 2016-09-21 | 2017-03-08 | 安徽师范大学 | A kind of biological sensor, its preparation method and purposes |
| CN106706607A (en) * | 2017-02-07 | 2017-05-24 | 福建医科大学 | High-quantum-yield electrochemiluminescence gold nano-cluster probe and preparation method of high-quantum-yield electrochemiluminescence gold nano-cluster probe |
| CN106872546A (en) * | 2017-02-07 | 2017-06-20 | 福建医科大学 | Electrochemical reducing prepares high quantum production rate electrochemical luminescence gold nano cluster probe |
| CN107589099A (en) * | 2017-09-03 | 2018-01-16 | 福建医科大学 | 6 purinethol detection methods and its kit based on gold nano cluster |
Non-Patent Citations (1)
| Title |
|---|
| 彭花萍 等: "二氧化锰/金纳米团簇电致化学发光复合体系用于谷胱甘肽的检测", 《第十三届全国电分析化学学术会议 会议论文摘要集》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110596065A (en) * | 2019-09-25 | 2019-12-20 | 福建医科大学 | Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material |
| CN110596065B (en) * | 2019-09-25 | 2021-10-15 | 福建医科大学 | Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material |
| CN111208183A (en) * | 2020-01-13 | 2020-05-29 | 成都医学院 | ECL sandwich immunosensing-based antigen detection kit |
| CN111518170A (en) * | 2020-05-09 | 2020-08-11 | 新乡医学院 | FRET-based PSA fluorescent probe, preparation method and application thereof |
| CN113322305A (en) * | 2021-06-04 | 2021-08-31 | 青岛科技大学 | Based on gold nanocluster/MnO2Preparation and application of electrochemical luminescence sensor of nanoflower |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108287187B (en) | Electrochemical luminescence sensor | |
| CN109142748A (en) | Human prostate specific antigen detection method and its kit | |
| CN106706607B (en) | High quantum production rate electrogenerated chemiluminescence gold nano cluster probe and preparation method thereof | |
| Dai et al. | Electrochemical sensor for immunoassay of carcinoembryonic antigen based on thionine monolayer modified gold electrode | |
| CN107121462B (en) | A kind of preparation method vulcanizing the dual titania-doped insulin optical electro-chemistry sensor of decrease cadmium sulfide/carbon of Cu/SiO 2 | |
| CN106501240B (en) | Electrochemiluminescsensor sensor and its preparation method and application with dual signal source | |
| CN106596969A (en) | Production method, product, detection method and application of electrochemiluminescence immunosensor | |
| CN108802139A (en) | A kind of electrogenerated chemiluminescence method of detection glutathione | |
| CN109142331A (en) | A kind of electrogenerated chemiluminescence method and its kit for carcinomebryonic antigen detection | |
| CN104655855A (en) | Preparation method and application of tumor marker electrochemiluminescence immunoassay sensor based on multifunctional carbon nitride material | |
| CN109613244B (en) | Preparation method and application of Ag @ Pt-CuS labeled immunosensor | |
| CN108827948A (en) | Acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe | |
| CN105842460B (en) | A kind of preparation method of the electrochemiluminescimmunosensor immunosensor based on silver-colored hydridization bismuth sulfide | |
| CN110554027A (en) | preparation method and application of immunosensor for promoting gold nanocluster electroluminescent response based on iron oxide array coreaction | |
| CN105606681B (en) | A kind of preparation method and application of the biosensor based on golden copper-multi-walled carbon nanotube-manganese dioxide structure | |
| CN110441295A (en) | One kind is based on ferritin encapsulation Ir (ppy)3Biosensor preparation method | |
| CN109164090A (en) | The electrochemiluminescdetection detection method and its kit of tumor necrosis factor α | |
| CN107044978B (en) | Glutathione electrogenerated chemiluminescence measuring method based on gold nano cluster probe | |
| CN106053823B (en) | A kind of spectral type electrogenerated chemiluminescence immunologic detection method based on CdZnSe ternary quantum dots | |
| CN111504909B (en) | Photoelectrochemical biosensor for label-free detection of alpha fetoprotein, preparation method and application thereof | |
| CN103884707A (en) | Luminol and trisruthenium-based potential-resolved electrochemiluminescence detection method and application thereof | |
| CN113252747A (en) | Preparation method of self-powered sensor | |
| CN102435736A (en) | Method for measuring antigen of ovarian cancer embryo by electrochemical luminescence (ECL) immunosensor | |
| CN104865240A (en) | Disposable nanometer electrogenerated chemiluminescence two-component immune sensor and preparation method thereof | |
| CN108872209A (en) | Alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190104 |
|
| WD01 | Invention patent application deemed withdrawn after publication |