CN110261359A - A kind of cancer markers imaging method based on laser confocal microscope - Google Patents

A kind of cancer markers imaging method based on laser confocal microscope Download PDF

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CN110261359A
CN110261359A CN201910565325.7A CN201910565325A CN110261359A CN 110261359 A CN110261359 A CN 110261359A CN 201910565325 A CN201910565325 A CN 201910565325A CN 110261359 A CN110261359 A CN 110261359A
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confocal microscope
cancer markers
laser confocal
aptamer
imaging method
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CN110261359B (en
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朱丽娜
刘芸
乔亮
刘宝红
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Fudan University
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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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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Abstract

The invention discloses a kind of cancer markers imaging method based on laser confocal microscope.This method is specifically to identify cancer markers by aptamer, after incorporating quantum point, can be used for the research being imaged.With this method, using the signal transduction factor on circulating tumor cell surface as object, the imaging under laser confocal microscope is realized.This method is quick, sensitive, and specificity is excellent, and imaging clearly is accurate, is a kind of method with potential application foreground.

Description

A kind of cancer markers imaging method based on laser confocal microscope
Technical field
The invention belongs to microscopic study technical fields, and in particular to a kind of cancer mark based on laser confocal microscope Will object imaging method.
Background technique
Cancer is to endanger the maximum killer of human health (Brindle K. New approaches for imaging Tumour responses to treatment [J] Nature Reviews Cancer, 2008,8 (2): 94-107). Related data shows have within 2007 13% people to die of cancer, and this data can rise to 1,200,000,000 person-times in the year two thousand thirty (Scheinberg, D. A.; Villa, C. H.; Escorcia, F. E.; McDevitt, M. R., Conscripts of the infinite armada: systemic cancer therapy using Nanomaterials. Nat Rev Clin Oncol 2010,7 (5), 266-276.).Therefore, it is early to establish effective cancer Phase detection and diagnostic method are particularly important.According to it has been reported that more and more scientists pay close attention to low concentration cell Super sensitivity detection can detect the protein of cell surface as important biomarker, the number of these protein The change of amount and state can prompt the generation and development of our diseases.
Laser confocal microscope is a kind of high-precision laser source microscope, light source is used a laser as, in traditional light The principle of confocal is used on the basis of microscope, and observed analysis object is carried out at digital picture using computer The a set of observation and analysis system of reason.It can greatly beyond tradition microscope, in recent years in multi-field research saliency advantage.
Cancer markers are observed using laser confocal microscopy, it will are brought in terms of medical diagnosis on disease, clinical application new Enlightenment.
Summary of the invention
The purpose of the present invention is to provide a kind of cancer markers imaging method based on laser confocal microscope.
The present invention is using signal transduction factor (EpCAM) as goals research object, using aptamer (aptamer) It specifically identifies signal transduction factor, and then marks quantum dot probe, to realize in laser confocal microscope Lower imaging.
A kind of cancer markers imaging method based on laser confocal microscope proposed by the present invention, specific steps are such as Under:
(1) in the end modified biotin of nucleic acid aptamer sequence, aptamer-biotin (aptamer- is obtained Biotin)；
(2) Streptavidin (SA) is modified on Quantum dots CdS e, obtains QDs-SA；
(3) nanogold particle that 13nm is modified on the ito glass cleaned up, is added dropwise aptamer, is incubated at room temperature 14 hours, It adds mercaptoethanol to close 30 minutes, is eventually adding cancer markers.Each step uses buffer solution (50mM after combining Tris-HCl it) cleans, is dried with nitrogen.
(4) aptamer-biotin that step (1) obtains is added into the substrate of step (3), is incubated at room temperature 1 hour, uses Buffer solution (50mM Tris-HCl) cleaning, is dried with nitrogen.
(5) QDs-SA that step (2) obtain is added into step (4), is incubated at room temperature 1 hour, with buffer solution (50mM Tris-HCl it) cleans, is dried with nitrogen.
(6) under laser confocal microscope, setting wavelength 458nm is excited, and picture number is acquired within the scope of 500-600nm According to
In the present invention, step (3) cancer markers are signal transduction factor (EpCAM).
In the present invention, the concentration of step (3) described signal transduction factor is 10aM-500nM, and volume is 5 μ L-10 μ L。
In the present invention, the volume of aptamer-biotin described in step (3) is 1 μ L to 5 μ L, 5 μM -10 μM of concentration.
In the present invention, QDs-SA volume described in step (3) be 1 μ L to 5 μ L, 5 μM -10 μM of concentration.
In the present invention, the setting of microscope parameter described in step (6) is 405-488nm in exciting light.
What a kind of preparation method proposed by the present invention obtained is used for imaging of the cancer markers under laser co-focusing Application.
The present invention uses aptamer identification cancer markers to be imaged under laser confocal microscope for the first time.Nucleic acid is suitable Ligand has excellent specificity, can sensitively capture cancer markers.The fluorescence efficiency of quantum dot is visited than general fluorescence Needle wants high, facilitates the imaging of object under the microscope.
Aptamer (aptamer) be screened for some target molecule is special, therefore have high specific and The characteristics of high-affinity.Aptamer can be in conjunction with different kinds of molecules, from small molecule to protein or even cell, so being based on The application range of the biosensor of aptamer is very extensive.Aptamer once screens success, so that it may with commercialization Raw material carry out high duplication, the synthesis of high-purity.Moreover, aptamer is usually highly stable compared with antibody and enzyme , this point, be other sensors identification original part be expected it is untouchable.Aptamer and the combination of target molecule after usually all It experienced specific structure to change.This makes the sensor based on aptamer when detecting target molecule, high sensitivity, specifically Property is good.Aptamer is easy to modify.Since it is substantially oligonucleotide chain, thus it is easy to be chemically modified.And antigen is anti- Body is protein, and in contrast, modification group just seems more difficult above it.It is anti-that Aptamer is typically considered chemistry Body.It is a bit of using in-vitro screening SELEX technology (systematic evolution of ligands by Exponential enrichment, i.e. exponential enrichment ligand evolution techniques) obtained from oligonucleotide chain, by can be with Corresponding ligand target molecule carry out the oligonucleotide sequence of the combination of high-affinity and strong specificity in artificial synthesized DNA or RNA in hangar with screening.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention visualizes the signal transduction factor of one of cancer markers,
(2) present invention compared with the prior art, has preparation simple, detects quick advantage.
(3) present invention can be extended to kinds cancer detection field, promote the early diagnosis of cancer.
Detailed description of the invention
Fig. 1 is imaging of 3 signal transduction factor of embodiment under laser confocal microscope.
Specific embodiment
Below in conjunction with drawings and examples, the invention will be further described.
A kind of cancer markers imaging method based on laser confocal microscope of the invention, comprising the following steps:
The first step designs nucleic acid aptamer sequence, in end modified biotin, i.e. aptamer-biotin；
Second step modifies Streptavidin, i.e. QDs-SA in CdSe quantum dot；
Third step modifies the nanogold particle of 13nm on the ito glass cleaned up, and aptamer, incubation at room temperature 14 is added dropwise Hour, it adds mercaptoethanol and closes 30 minutes, be eventually adding 5 μ L, 100 fM cancer markers, be incubated at room temperature 1 hour.It is each Step is cleaned with buffer solution (50mM Tris-HCl) after combining, and is dried with nitrogen；
The aptamer-biotin that step 1 obtains is added into the substrate of step 3, is incubated at room temperature 1 hour, with buffering for 4th step Solution (50mM Tris-HCl) cleaning, is dried with nitrogen.
5th step is incubated at room temperature 1 hour, to the QDs-SA that addition step 2 obtains in step 4 with buffer solution (50mM Tris-HCl it) cleans, is dried with nitrogen.
6th step, under laser confocal microscope, setting wavelength 458nm is excited, and figure is acquired within the scope of 500-600nm As data.
Embodiment 1:
(1) preparation of aptamer and biotin composite:
Taking 20 μ L concentration is 10 μM of aptamer, 5 μ L biotins is added, concussion mixing 4 hours, take out, be placed under the conditions of 4 degree It places 12 hours and is combined under the conditions of 4 degree.The aptamer of available end modified biotin.
(2) preparation of observing samples:
The nanogold particle that 13nm is modified on the ito glass cleaned up, is added dropwise aptamer (5 μ L, 5 mM), room temperature is incubated It educates 14 hours, adds mercaptoethanol (50 μ L, 10 mM) and close 30 minutes, be eventually adding 5 μ L, 100 fM cancer markers, Incubation at room temperature 1 hour.10 μM of the aptamer-biotin(1 μ L that step 1 obtains is added), it is incubated at room temperature 1 hour, is eventually adding 8 μM of the QDs-SA(1 μ L that step 2 obtains), it is incubated at room temperature 1 hour.Each step uses buffer solution (50mM Tris- after combining HCl it) cleans, is dried with nitrogen.
(3) laser confocal microscope acquires image data:
The slide made is placed under Lycra sp5 laser confocal microscope, 458nm excitation wavelength is set, in 500- Data are acquired within the scope of 600nm.It can clearly be observed that obvious fluorescence signal is presented in signal transduction factor.Such as Fig. 1 institute Show.

Claims (6)

1. a kind of cancer markers imaging method based on laser confocal microscope, it is characterised in that: specific step is as follows:
(1) in the end modified biotin of nucleic acid aptamer sequence, aptamer-biotin (aptamer- is obtained Biotin)；
(2) Streptavidin (SA) is modified on Quantum dots CdS e, obtains QDs-SA；
(3) nanogold particle that 13nm is modified on the ito glass cleaned up, is added dropwise aptamer, is incubated at room temperature 14 hours, It adds mercaptoethanol to close 30 minutes, is eventually adding cancer markers, each step uses buffer solution (50mM after combining Tris-HCl it) cleans, is dried with nitrogen；
(4) aptamer-biotin that step (1) obtains is added into the substrate of step (3), is incubated at room temperature 1 hour, with buffering Solution (50mM Tris-HCl) cleaning, is dried with nitrogen；
(5) QDs-SA that step (2) obtain is added into step (4), is incubated at room temperature 1 hour, with buffer solution (50mM Tris- HCl it) cleans, is dried with nitrogen；
(6) under laser confocal microscope, setting wavelength 458nm is excited, and image data is acquired within the scope of 500-600nm.
2. the cancer markers imaging method according to claim 1 based on laser confocal microscope, which is characterized in that Step (3) cancer markers are signal transduction factor (EpCAM).
3. the cancer markers imaging method according to claim 2 based on laser confocal microscope, it is characterised in that: The concentration of step (3) described signal transduction factor is 10aM-500nM, and volume is 5 μ L-10 μ L.
4. the cancer markers imaging method according to claim 1 based on laser confocal microscope, it is characterised in that: The volume of aptamer-biotin described in step (3) be 1 μ L to 5 μ L, 5 μM -10 μM of concentration.
5. the cancer markers imaging method according to claim 1 based on laser confocal microscope, it is characterised in that: QDs-SA volume described in step (3) be 1 μ L to 5 μ L, 5 μM -10 μM of concentration.
6. the cancer markers imaging method according to claim 1 based on laser confocal microscope, it is characterised in that: The excitation wavelength of laser confocal microscope described in step (6) is within the scope of 405-488nm.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2211178A1 (en) * 2009-01-22 2010-07-28 Universität Potsdam Method for detecting an analyte in a sample by multiplexing FRET analysis and kit
WO2011046842A1 (en) * 2009-10-12 2011-04-21 The Regents Of The University Of California Targeted nanoclusters and methods of their use
CN104854243A (en) * 2012-08-02 2015-08-19 迪肯大学 Epcam aptamer for detection of cancer stem cells
CN107607501A (en) * 2017-08-21 2018-01-19 樊之雄 A kind of biomarker multiple detection method based on fluorescent quenching
CN109554369A (en) * 2018-02-02 2019-04-02 中国科学院化学研究所 Aptamer is identifying and is combining the application in alkaline phosphatase heterodimer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2211178A1 (en) * 2009-01-22 2010-07-28 Universität Potsdam Method for detecting an analyte in a sample by multiplexing FRET analysis and kit
WO2011046842A1 (en) * 2009-10-12 2011-04-21 The Regents Of The University Of California Targeted nanoclusters and methods of their use
CN104854243A (en) * 2012-08-02 2015-08-19 迪肯大学 Epcam aptamer for detection of cancer stem cells
CN107607501A (en) * 2017-08-21 2018-01-19 樊之雄 A kind of biomarker multiple detection method based on fluorescent quenching
CN109554369A (en) * 2018-02-02 2019-04-02 中国科学院化学研究所 Aptamer is identifying and is combining the application in alkaline phosphatase heterodimer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIMIN CHUN等: "Electrochemical detection of HER2 using single stranded DNA aptamer modified gold nanoparticles electrode", 《SENSORS AND ACTUATORS B: CHEMICAL》 *
RUIJUN DU等: "Ultrasensitive Detection of Low-Abundance Protein Biomarkers by Mass Spectrometry Signal Amplification Assay", 《ANAL. CHEM.》 *
WANMING LI等: "An ssDNA aptamer selected by Cell-SELEX for the targeted imaging of poorly differentiated gastric cancer tissue", 《TALANTA》 *
武春雷: "基于量子点和核酸适体的肺癌细胞识别与检测", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

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