CN105784658B - A method for rapid detection of tumor-associated small peptide MUC1 - Google Patents
A method for rapid detection of tumor-associated small peptide MUC1 Download PDFInfo
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- CN105784658B CN105784658B CN201610184950.3A CN201610184950A CN105784658B CN 105784658 B CN105784658 B CN 105784658B CN 201610184950 A CN201610184950 A CN 201610184950A CN 105784658 B CN105784658 B CN 105784658B
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 23
- 102100034256 Mucin-1 Human genes 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title abstract description 18
- 206010028980 Neoplasm Diseases 0.000 title abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 27
- 108091023037 Aptamer Proteins 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 108010008707 Mucin-1 Proteins 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 229910021389 graphene Inorganic materials 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 230000006978 adaptation Effects 0.000 abstract description 3
- 238000009396 hybridization Methods 0.000 abstract description 2
- 238000011896 sensitive detection Methods 0.000 abstract 1
- 201000011510 cancer Diseases 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 5
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000439 tumor marker Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000000184 acid digestion Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
This patent discloses the methods of quickly detection tumour correlation small peptide MUC1 a kind of, are related to fluorescence probe detection technique field.The characteristics of using aptamers and target small peptide specific recognition, specific adaptation body sequence is designed, to achieve the purpose that object specific recognition；By combining the hybridization reaction of two kinds of hairpin structure probes to complete the strategy of signal amplification, to realize the Sensitive Detection of target small peptide.This method includes four steps such as probe initial denaturation-hybrid reaction-background quenching-fluorescence detection, easy to operate quick, time saving and energy saving, provides a kind of new method for the detection of tumour correlation small peptide MUC1.
Description
Technical field
The present invention relates to fluorescence probe detection technique fields, and more specifically one kind is with aptamers specific recognition and two
The fluorescence detection small peptide method that the mode of kind hair fastener probe hybridization reaction amplification constructs.
Background technique
Gradually high trend was presented in national cancer morbidity and the death rate in recent years, therefore found quick, quantitative, clever
The method of quick detection tumor marker is extremely important in early detection and treatment monitoring.Cancer-associated tumor marker at present
Mainly there are enzyme, hormone, antigen, small peptide and microRNA etc..Small peptide is as the product after the hydrolysis of human body internal protein in amino acid
Digestion, absorb etc. play an important role.After human body cell generates canceration, cancer cell metabolism and normal cell
Very big difference is had, cancer cell can also generate small peptide substance, thus by the contents level of detection small peptide, people being capable of energy
Discovery cancerous tumor cell in time, to achieve the purpose that disease early warning.
Small peptide MUC1, is called CA15-3, participate in signal transmission and regulation immune cell growth in terms of have
Important function.MUC1 has expression in most people adenocarcinoid, and the serum cancer mark quality testing being still commercialized
Antigen in survey method.The MUC1 of tumor tissues can generate abnormality in cancer cell surfaces, that is, glycosylate relative to normal cell not
Completely.Since MUC1 specificity is higher than general antigen, sensibility is higher than carcinomebryonic antigen, thus by as cancer in terms of cancer detection
Disease marker is applied.
In order to solve the problems, such as that small peptide also can be carried out detection at low concentrations, small peptide identification and signal amplification strategy become outstanding
It is important.However, the method for detecting small peptide at present, such as enzyme linked immunosorbent assay (ELISA), the surface plasma resonance without label, liquid
Phase chromatography-mass spectroscopy and the method for electrochemical analysis although sensitivity and specificity with higher, but require a series of
Fixation, separation and cleaning step, it is time-consuming, laborious.Therefore developing one kind can specific recognition and low concentration
The method of small peptide is of great significance.It is anti-that the characteristics of adaptation physical efficiency is specifically bound with target molecule and hair fastener probe hybridize chain type
The advantage of amplified signal is answered just to can solve the above problem, and easy to operate, time saving and energy saving.
Summary of the invention
The technical problem to be solved by the present invention is to construct a kind of specific recognition small peptide MUC1 and be able to achieve follow-up signal
The small peptide detection method of amplification.
A kind of method of quick detection tumour correlation small peptide MUC1, it is characterized in that the following steps are included:
(1) probe initial denaturation
Firstly, the hair fastener probe 1 and hair fastener probe 2 and aptamers, English name of Fluoresceincarboxylic acid FAM label are
Aptamer is incubated for 0.5 h respectively at 95 oC；These three solution are placed into 1 h at room temperature；
(2) hybrid reaction
The overall reaction mixeding liquid volume that following steps carry out is 200 μ L, and Mucin1 and the concentration of various concentration are
The aptamers that the hair fastener probe 2 and concentration that the hair fastener probe 1 of 10-50 nM, concentration are 10-50 nM are 5-10 nM are mixed in slow
It rushes in solution, mixed solution reacts 1 h at 25 oC；
The ingredient of buffer solution is the sodium chloride that concentration is 0.75 M and the disodium hydrogen phosphate of 50 mM, pH value 7.4；
(3) background quenches
Concentration is added for 0.5 mg/mL graphene oxide solution, under 25oC to mixed solution obtained in (2) described step
It is incubated for 0.5 h；
(4) fluorescence detection
The experiment parameter of fluorescence detection is as follows: excitation wavelength is 480 nm, and launch wavelength range is 500-650 nm.
Beneficial effects of the present invention
(1) using adaptation body technique, it can be realized the specific recognition of object small peptide MUC1；
(2) using the cross chain reaction between hair fastener probe 1 and hair fastener probe 2, realize that fluorescence signal quickly amplifies；
(3) the method for the invention is easy to operate, rapid reaction.
Detailed description of the invention
Fig. 1 is the experimental principle figure of methods described herein.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment and attached drawing content that the present invention is furture elucidated, but this
The content of invention is not limited solely to following implementation.
Embodiment 1
A kind of method of quick detection tumour correlation small peptide MUC1, it is characterized in that the following steps are included:
(1) probe initial denaturation
Firstly, the hair fastener probe 1 and hair fastener probe 2 and aptamers, English name of Fluoresceincarboxylic acid FAM label are
Aptamer is incubated for 0.5 h respectively at 95 oC；These three solution are placed into 1 h at 25oC；
(2) hybrid reaction
The overall reaction mixeding liquid volume that following steps carry out is 200 μ L, and the Mucin1 and concentration of various concentration are 10
The aptamers that the hair fastener probe 2 and concentration that the hair fastener probe 1 of nM, concentration are 10 nM are 10 nM are mixed in buffer solution, are mixed
It closes solution and reacts 0.5 h at 25 oC；
The ingredient of buffer solution is the sodium chloride that concentration is 0.75 M and the disodium hydrogen phosphate of 50 mM, pH value 7.4；
(3) background quenches
It is 0.5 mg/mL graphene oxide solution that concentration, which is added, to mixed solution obtained in (2) described step, at room temperature
It is incubated for 0.5 h；
(4) fluorescence detection
The experiment parameter of fluorescence detection is as follows: excitation wavelength is 480 nm, and launch wavelength range is 500-650 nm.
Embodiment 2
Detecting step is a difference in that with example 1: 1 concentration of hair fastener probe is 50 nM in step 2, and 2 concentration of hair fastener probe is
50 nM, sequence be 1 h in the reaction time.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>a kind of method of quickly detection tumour correlation small peptide MUC1
<130> 2016
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 69
<212> DNA
<213>artificial sequence
<400> 1
tgaggtagta ggttgtatag ttgcagttga tcctttggat accctggaac tatacaacct 60
actacctca 69
<210> 2
<211> 44
<212> DNA
<213>artificial sequence
<400> 2
agtaggttgt atagttcaaa gtaactatac aacctactac ctca 44
<210> 3
<211> 44
<212> DNA
<213>artificial sequence
<400> 3
actttgaact atacaaccta cttgaggtag taggttgtat agtt 44
Claims (1)
1. a kind of application of aptamers sequence in preparation small peptide MUC1 quick detection reagent, it is characterized in that the following steps are included:
(1) probe initial denaturation
Firstly, hair fastener probe 1, the hair fastener probe 2 of Fluoresceincarboxylic acid FAM label, aptamers are incubated for 0.5 h respectively at 95 oC；
These three solution are placed into 1 h at room temperature；The wherein hair fastener probe 1 of Fluoresceincarboxylic acid FAM label, sequence AGTAG
GTTGTATAGTTCAAAGTAACTATACAACCTACTACCTCA；The hair fastener probe 2 of Fluoresceincarboxylic acid FAM label, sequence A
CTTTGAACTATACAACCTACTTGAGGTAGTAGGTTGTATAGTT；Aptamers, English name aptamer, sequence T
GAGGTAGTAGGTTGTATAGTTGCAGTTGATCCTTTGGATACCCTGGAACTATACAACCTACTACCTCA；
(2) hybrid reaction
The overall reaction mixeding liquid volume that following steps carry out is 200 μ L, the Mucin1 of various concentration and certain density hair
The aptamers that card probe 1 and hair fastener probe 2 and concentration are 5 nM are mixed in buffer solution, and mixed solution is anti-at 25 oC
Answer 1 h；
The ingredient of buffer solution is the sodium chloride that concentration is 0.75 M and the disodium hydrogen phosphate of 50 mM, pH value 7.4；
(3) background quenches
It is 0.5 mg/mL graphene oxide solution that concentration, which is added, to mixed solution obtained in the step (2), at room temperature
It is incubated for 0.5 h；
(4) fluorescence detection
The experiment parameter of fluorescence detection is as follows: excitation wavelength is 480 nm, and launch wavelength range is 500-650 nm.
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| CN201610184950.3A CN105784658B (en) | 2016-03-29 | 2016-03-29 | A method for rapid detection of tumor-associated small peptide MUC1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107245488B (en) * | 2017-06-07 | 2021-01-08 | 中山火炬职业技术学院 | Test strip and method for detecting aflatoxin B1 |
| CN111819288B (en) * | 2018-01-26 | 2023-07-14 | 北京生命科学研究所 | Immune Signal Amplification Method Based on Hybridization Chain Reaction |
| CN110734960B (en) * | 2019-09-19 | 2023-03-14 | 中国科学院苏州生物医学工程技术研究所 | Trace MUC1 fluorescence detection method based on chain type hybridization reaction and fluorescent carbon quantum dots |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703594A (en) * | 2012-06-12 | 2012-10-03 | 华南师范大学 | Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform |
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN104020198A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for detecting DNA by electrochemical transducer with signal amplification technology |
| CN104764790A (en) * | 2015-03-26 | 2015-07-08 | 济南大学 | Biosensor for detecting streptomycin based on aptamer and preparation method of biosensor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2573092T3 (en) * | 2009-07-10 | 2016-06-06 | Perkinelmer Health Sciences, Inc. | Multinucleotide Repeat Detection |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703594A (en) * | 2012-06-12 | 2012-10-03 | 华南师范大学 | Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform |
| CN104007152A (en) * | 2014-06-18 | 2014-08-27 | 青岛科技大学 | DNA determining electrochemical sensor and method based on platinum nano particle catalysis electrochemistry circulation signal amplification technology |
| CN104020198A (en) * | 2014-06-18 | 2014-09-03 | 青岛科技大学 | Method for detecting DNA by electrochemical transducer with signal amplification technology |
| CN104764790A (en) * | 2015-03-26 | 2015-07-08 | 济南大学 | Biosensor for detecting streptomycin based on aptamer and preparation method of biosensor |
Non-Patent Citations (3)
| Title |
|---|
| A hairpin aptamer-based electrochemical biosensing platform for the sensitive detection of proteins;Zai-Sheng Wu 等;《Biomaterials》;20090228;第30卷;第2950–2955页 |
| Electrochemical immunoassay for the protein biomarker mucin 1 and for MCF-7 cancer cells based on signal enhancement by silver nanoclusters;Qingjun Guo 等;《Microchim Acta》;20150325;第182卷;第1483–1489页 |
| Immobilization of redox-labeled hairpin DNA aptamers on gold: Electrochemicalquantitation of epithelial tumor marker mucin 1;Fen Ma 等;《Electrochimica Acta》;20130226;第110卷;第139–145页 |
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