CN109321577A - A kind of aptamer group for detecting exosome, lateral flow aptamer biosensor and preparation method thereof - Google Patents
A kind of aptamer group for detecting exosome, lateral flow aptamer biosensor and preparation method thereof Download PDFInfo
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- CN109321577A CN109321577A CN201811173676.5A CN201811173676A CN109321577A CN 109321577 A CN109321577 A CN 109321577A CN 201811173676 A CN201811173676 A CN 201811173676A CN 109321577 A CN109321577 A CN 109321577A
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- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims abstract description 69
- 101150084967 EPCAM gene Proteins 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 46
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 32
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G01—MEASURING; TESTING
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Abstract
本发明提供一种检测外泌体的适配体组、侧流式适配体生物传感器及其制备方法，属于外泌体检测技术领域。检测外泌体的适配体组包括CD63适配体和EpCam适配体，CD63适配体能特异性与外泌体的CD63蛋白结合，EpCam适配体能特异性与外泌体的EPCAM蛋白结合，两种适配体组不易受pH、温度等环境因素影响稳定性好。本发明还提供侧流式适配体生物传感器，主要基于层析试纸条原理将CD63适配体喷涂于结合垫上，将EpCam适配体喷涂形成检测线上，质控线上喷涂有能与纳米金标记的适配体的DNA探针通过碱基互补配对结合。测流式适配体生物传感器可以检测到500000个外泌体，为癌症快速筛查和诊断提供了革新的工具。
The invention provides an aptamer group for detecting exosomes, a lateral flow aptamer biosensor and a preparation method thereof, belonging to the technical field of exosome detection. The aptamer group for detecting exosomes includes CD63 aptamer and EpCam aptamer. The CD63 aptamer can specifically bind to the CD63 protein of exosomes, and the EpCam aptamer can specifically bind to the EPCAM protein of exosomes. The two aptamer groups are not easily affected by environmental factors such as pH and temperature, and have good stability. The invention also provides a lateral flow aptamer biosensor, mainly based on the principle of chromatographic test strips, the CD63 aptamer is sprayed on the binding pad, and the EpCam aptamer is sprayed to form a detection line, and the quality control line is sprayed with an aptamer capable of The DNA probe of the gold nano-labeled aptamer is bound by base complementary pairing. The cytometric aptamer biosensor can detect 500,000 exosomes, providing an innovative tool for rapid cancer screening and diagnosis.
Description
Technical field
The invention belongs to excretion body detection technique fields, and in particular to a kind of to detect the aptamers group of excretion body, lateral flow type
Aptamers biosensor and preparation method thereof.
Background technique
Excretion body (exosome) is the vesica corpusculum secreted by a variety of living cells, wherein a variety of containing protein and RNA etc.
Component.Generally existing nanoscale can participate in intercellular mass exchange and information interchange by membrane structure in this body,
It plays a significant role in a variety of physiology and pathologic process.Excretion body has in the body fluid such as peripheral blood, urine, saliva, ascites, amniotic fluid
Have a very high abundance, and the excretion body of different tissue sources form and function in terms of have differences, while this species diversity by
The dynamic regulation of extracellular matrix and microenvironment.Tumour source or the relevant excretion body of tumour are the weights of modulate tumor occurrence and development
Mechanism is wanted, the analysis and detection to tumour excretion body can be with the early diagnosis, therapeutic evaluation and prognostic analysis of adjuvant therapy.Excretion
Although body is still in infancy as biomarker, but with further research, the clinical application of excretion body will be had
Good prospect.
Currently, excretion volume morphing can be directly observed using Electronic Speculum, it can be according to excretion body using Western blot method
The transmembrane molecules such as marker CD9, the CD63 on surface detect excretion using CD9, CD63 monoclonal antibody in conjunction with corresponding antigens
Body protein expression quantity.Carrying out analysis to the hereditary information substance contained in excretion body using PCR or the method for sequencing is to answer at present
With more and more extensive detection method, there is also some identification technologies such as dynamic light scattering technique, flow cytometry is received
Rice particle follow-up analysis, electrochemistry, fluorescence, SPR, SPRI, color analysis etc..Upper method needs expensive large-scale instrument,
Or complicated operating procedure is needed, it is unfavorable for extensively sieving.The method for detecting excretion body further includes ELISA, immunoblotting
Analysis.Although higher detection sensitivity can be obtained using antigen-antibody immunoassay, large-scale instrument is not needed yet, is screened
Often step is complicated with the antibody of excretion body surface face marker protein specific binding, while antibody protein is vulnerable to pH, temperature etc.
Such environmental effects and be denaturalized and synthesize expensive.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of synthesis to be easier, the good detection excretion body of stability is fitted
Ligand and aptamers group, at the same present invention offer additionally provide a kind of detection sensitivity it is high, it is simple and efficient, do not need complicated hold high
Expensive instrument and equipment and cheap lateral flow type aptamers biosensor and preparation method thereof.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of CD63 aptamers for detecting excretion body, and the nucleotide sequence of the CD63 aptamers group is such as
In sequence table shown in SEQ ID No.1.
The present invention provides it is a kind of detect excretion body aptamers group, the aptamers group include CD63 aptamers and
EpCam aptamers, the nucleotide sequence of the EpCam aptamers is as shown in SEQ ID No.2 in sequence table.
The present invention provides a kind of lateral flow type aptamers biosensor, including bottom plate and it is adhered on the bottom plate under
Upward sequentially connected bonding pad, detecting pad and absorption pad, the detecting pad include detection line and nature controlling line, the bonding pad spray
The CD63 aptamers conjugates of nano gold mark are coated with, the detection line is coated with Streptavidin-biotin labeling EpCam
Aptamers conjugates, the nature controlling line are coated with Streptavidin-biotin labeling DNA probe, the nucleotides sequence of DNA probe
It arranges complementary with 3 ' terminal sequences of the nucleotide sequence of CD63 aptamers；
CD63 aptamers are that the CD63 of the detection excretion body is suitable in the CD63 aptamers conjugates of the nano gold mark
Ligand；
Preferably, in the DNA probe of the Streptavidin-biotin labeling DNA probe nucleotide sequence such as sequence
In table shown in SEQ ID No.2.
Preferably, the quantity for spray of the CD63 aptamers conjugates of the nano gold mark is 3 μ L/cm bonding pads.
Preferably, the spraying number of the EpCam aptamers conjugates of the Streptavidin-biotin labeling is 3 times；Institute
The spray volume for stating Streptavidin-biotin labeling EpCam aptamers conjugates is 3 μ l/cm detecting pads.
The present invention provides the preparation methods of lateral flow type aptamers biosensor, comprising the following steps:
(1) dATP solution, SDS solution and NaCl solution are sequentially added under the conditions of earthquake into nano-Au solution to carry out instead
It answers, obtained reaction solution and CD63 aptamers mixing reacts 3h at 60 DEG C, is separated by solid-liquid separation, gained is precipitated and dissolved in nanometer
Particle stores in liquid, obtains the CD63 aptamers conjugates solution spraying of nano gold mark on bonding pad, obtains being coated with and receive
The bonding pad of the CD63 aptamers conjugates of rice gold label；
(2) it after the EpCam aptamers and Streptavidin of biotin labeling being mixed 1h, is placed in ultra-filtration centrifuge tube
Ultrafiltration centrifugation, after washing 2 times to ultra-filtration centrifuge tube, the supernatant of collection is Streptavidin-biotin labeling EpCam adaptation
Liquid solution；
(3) the EpCam aptamers in the step (2) are replaced with DNA probe, repeats step (3) operation, obtain strepto- parent
With element-biotin labeling DNA probe solution；
(4) by Streptavidin-biotin labeling EpCam adaptation liquid solution in the step (2) and strepto- in (3)
The DNA probe solution of Avidin-Biotin label is successively crossed on detecting pad, and the inspection for being coated with detection line and nature controlling line is obtained
Survey pad；
(5) by the bonding pad of the CD63 aptamers conjugates for being coated with nano gold mark in the step (1), the step
Suddenly the detecting pad for being coated with detection line and nature controlling line in (4) and blotting paper are successively assembled in bottom plate after cutting respectively from bottom to top
On, slitting obtains lateral flow type aptamers biosensor；
There is no the limitation of time sequencing between the step (1) and (2)~(4), while between step (2) and step (3)
Also the not limitation of time sequencing.
Preferably, the volume ratio of nano-Au solution in the step (1), dATP solution, SDS solution and NaCl solution is
200:2:3:10；The concentration of the nano-Au solution is 15~20nmol/L, and the concentration of the dATP solution is 1mmol/L, institute
The mass concentration for stating SDS solution is 1%, and the concentration of the NaCl solution is 0.2mol/L.
Preferably, the component that liquid includes following content: 20mmol/LNa is stored in the step (1)3PO4·12H2O, matter
Measure concentration 5%BSA, 10% sucrose of mass concentration 0.25%Tween 20 and mass concentration.
Preferably, the mode being separated by solid-liquid separation in the step (2) includes centrifugation；The revolving speed of the centrifugation is 6000rpm, institute
The time for stating centrifugation is 20min, and the temperature of the centrifugation is 4 DEG C.
The present invention provides it is a kind of detect excretion body aptamers group, the aptamers group include CD63 aptamers and
EpCam aptamers, the nucleotide sequence of the CD63 aptamers is as shown in SEQ ID No.1 in sequence table, the EpCam adaptation
Body is as shown in SEQ ID No.2 in sequence table.The CD63 albumen of CD63 adaptation the physical efficiency specificity and excretion body surface expression
In conjunction with the EPCAM protein binding of EpCam adaptation the physical efficiency specificity and excretion body surface expression, the aptamers group is not easy
It is good by the such environmental effects such as pH, temperature stability, while aptamers are small in size, synthesis is easy, and substantially reduces the cost of preparation.
Simultaneously it is provided by the invention it is a kind of detect excretion body aptamers group, by with the dual protein-specific knot of excretion body
It closes, the visual detection concentration of aptamers group detection excretion body provided by the invention is 5 × 105, specificity with higher.Simultaneously
Aptamers group provided by the invention has been filled up in the prior art using the blank of aptamers detection excretion body, to utilize adaptation physical examination
The technology for surveying excretion body provides important information.
The present invention provides lateral flow type aptamers biosensors, will be specifically bound based on chromatograph test strip principle
The aptamers of the nano gold mark of excretion body surface protein are sprayed on bonding pad, another aptamers in above scheme are sprayed
Base complementrity can be passed through with the DNA probe of the aptamers of nano gold mark by being coated in the detection line of detecting pad, on nature controlling line
Pairing combines.When in sample contain target excretion body when, the aptamers of nano gold mark formed in conjunction with excretion body compound with
Chromatography moves to detection line in dampening flow direction, in conjunction with another aptamers in detection line, while not in conjunction with excretion body
Nano gold mark aptamers in conjunction with the DNA probe on nature controlling line, detection line and nature controlling line become red at this time.Work as sample
In there is no that the content of excretion body or excretion body is extremely low, be only displayed in red on nature controlling line, do not develop the color in detection line.The present invention provides
The lateral flow type aptamers biosensor can be realized qualitative and quantitative detection.Realization shows flow measurement provided by the invention
Formula aptamers biosensor can detecte to 500000 excretion bodies.It is simultaneously 0~1 × 10 in sample concentration6Concentration range
Interior, signal value and excretion bulk concentration are in a linear relationship, show in the concentration range content, the flow measurement formula aptamers bio-sensing
The testing result of device is more accurate.
Lateral flow type aptamers biosensor provided by the invention is easy to operate simultaneously, does not depend on the instrument of Large expensive,
A kind of tool of innovation is provided for the rapid screening of cancer and diagnosis.
The present invention also provides the preparation methods of lateral flow type aptamers biosensor, and the method is reproducible, operation
Easy to be quick, raw material is cheap and easy to get, greatly reduces the production cost of lateral flow type aptamers biosensor.
Detailed description of the invention
Fig. 1 is the package assembly of lateral flow type aptamers biosensor provided by the invention；
Fig. 2 is the testing principle of lateral flow type aptamers biosensor provided by the invention；
Fig. 3 is scanning electron microscope (SEM) magnetic nanoparticle-aptamers-excretion body: being to be adapted what body captured in circle
Excretion body；
Fig. 4 is the standard curve for detecting excretion body.
Specific embodiment
The present invention provides a kind of CD63 aptamers for detecting excretion body, and the nucleotide sequence of the CD63 aptamers group is such as
In sequence table shown in SEQ ID No.1.CD63 adaptation physical efficiency specifically with the CD63 albumen knot of excretion body surface expression
It closes, is based on this, the CD63 aptamers can accurately capture excretion body.
The present invention provides a kind of aptamers group for detecting excretion body, the aptamers group includes the CD63 of above scheme suitable
Ligand and EpCam aptamers, the nucleotide sequence of the EpCam aptamers is as shown in SEQ IDNo.2 in sequence table.
In the present invention, the source of the CD63 aptamers and EpCam aptamers is not particularly limited, according to aptamers
Nucleotide sequence uses synthetic method known in the art.
The characteristic of excretion body is able to detect based on aptamers group provided by the invention, it is suitable that the present invention provides a kind of lateral flow types
Ligand biosensor, including bottom plate and sequentially connected bonding pad, detecting pad and suction from bottom to top are adhered on the bottom plate
Pad is received, the detecting pad includes detection line and nature controlling line, and the bonding pad is coated with the CD63 aptamers coupling of nano gold mark
Object, the detection line are coated with Streptavidin-biotin labeling EpCam aptamers conjugates, and the nature controlling line is coated with
Streptavidin-biotin labeling DNA probe, the nucleotide sequence of the nucleotide sequence and CD63 aptamers of DNA probe
3 ' terminal sequences are complementary.
Lateral flow type aptamers biosensor provided by the invention includes bottom plate.The present invention does not have the material of the bottom plate
It is specifically limited, using the bottom plate known in the art for preparing immunity test strip.In embodiments of the present invention, the bottom plate
Material use PVC board.The present invention is not particularly limited the source of the PVC board, is using PVC board known in the art
It can.The bottom plate has the function of carrying bonding pad, detecting pad and absorption pad, its is made to form a complete chromatograph test strip.
Lateral flow type aptamers biosensor provided by the invention includes bonding pad.The bonding pad is coated with nanogold mark
The CD63 aptamers conjugates of note.The spraying number of the DNA probe of the Streptavidin-biotin labeling is preferably 3 times.Institute
The material for stating bonding pad includes polyester film or glass fibre.The present invention is not special to the source of the polyester film or glass fibre
Limitation, using polyester film known in the art or glass fibre.
Lateral flow type aptamers biosensor provided by the invention includes detecting pad.The detecting pad includes detection line and matter
Control line.The CD63 aptamers conjugates of nano gold mark are coated in the detection line.The CD63 of the nano gold mark is adapted to
The quantity for spray of body conjugates is preferably 3 μ L/20cm bonding pads.
In the present invention, Streptavidin-biotin labeling DNA probe is coated on the nature controlling line.The present invention couple
The sequence of the DNA probe is not particularly limited, by the nucleotide sequence of DNA probe and the nucleotide sequence of CD63 aptamers
3 ' terminal sequences are complementary.The nucleotide sequence of DNA probe is preferred in the DNA probe of the Streptavidin-biotin labeling
As shown in SEQ ID No.2 in sequence table.The spraying of the EpCam aptamers conjugates of the Streptavidin-biotin labeling
Number is preferably 3 times.Control line is this time to test whether for examining effectively, when showing macroscopic color on control line
When (marker is nanogold, then is red), illustrate that this test strips is effective, and then can be according to the macroscopic face in detection line
Color depth, which shallowly changes, to be used for quickly detecting.If not developing the color on control line, then it represents that this time test invalidation, the result on p-wire are not intended to
Justice.
In the present invention, the material of the detecting pad preferably uses nitrocellulose filter (NC film).The material of the bonding pad
Matter includes polyester film or glass fibre.The present invention is not particularly limited the source of the nitrocellulose filter, using this field
Known nitrocellulose filter.The NC film with Streptavidin interaction by realizing to Streptavidin-life
The EpCam aptamers conjugates of object element label and stablizing for Streptavidin-biotin labeling DNA probe are coated with.
Lateral flow type aptamers biosensor provided by the invention includes absorption pad.The material of the absorption pad is preferably inhaled
Water paper.The absorption pad is used to control prepare liquid and flows into uptake extra in test strips, and it is multiple to rinse the label to dissociate on NC film
Object is closed, background interference can be reduced to improve the sensitivity of detection.
The present invention provides the preparation methods of lateral flow type aptamers biosensor, comprising the following steps:
(1) dATP solution, SDS solution and NaCl solution are sequentially added under the conditions of earthquake into nano-Au solution to carry out instead
It answers, obtained reaction solution and CD63 aptamers mixing reacts 3h at 60 DEG C, is separated by solid-liquid separation, gained is precipitated and dissolved in nanometer
Particle stores in liquid, obtains the CD63 aptamers conjugates solution spraying of nano gold mark on bonding pad, obtains being coated with and receive
The bonding pad of the CD63 aptamers conjugates of rice gold label；
(2) it after the EpCam aptamers and Streptavidin of biotin labeling being mixed 1h, is placed in ultra-filtration centrifuge tube
Ultrafiltration centrifugation, after washing 2 times to ultra-filtration centrifuge tube, the supernatant of collection is Streptavidin-biotin labeling EpCam adaptation
Liquid solution；
(3) the EpCam aptamers in the step (2) are replaced with DNA probe, repeats step (3) operation, obtain strepto- parent
With element-biotin labeling DNA probe solution；
(4) by Streptavidin-biotin labeling EpCam adaptation liquid solution in the step (2) and strepto- in (3)
The DNA probe solution of Avidin-Biotin label is successively crossed on detecting pad, and the inspection for being coated with detection line and nature controlling line is obtained
Survey pad；
(5) by the bonding pad of the CD63 aptamers conjugates for being coated with nano gold mark in the step (1), the step
Suddenly the detecting pad for being coated with detection line and nature controlling line in (4) and blotting paper are successively assembled on bottom plate from bottom to top after cutting,
Slitting, obtains lateral flow type aptamers biosensor；
There is no the limitation of time sequencing between the step (1) and (2)~(4), while between step (2) and step (3)
Also the not limitation of time sequencing.
Sequentially added under the conditions of earthquake of the present invention into nano-Au solution dATP solution, SDS solution and NaCl solution into
Row reaction, obtained reaction solution and CD63 aptamers mixing, 3h is reacted at 60 DEG C, is separated by solid-liquid separation, gained is precipitated and dissolved in
Nanoparticle stores in liquid, obtains the CD63 aptamers conjugates solution spraying of nano gold mark on bonding pad, is sprayed
There is the bonding pad of the CD63 aptamers conjugates of nano gold mark；The CD63 aptamers are nucleosides in above scheme aptamers group
Acid sequence CD63 aptamers as shown in SEQ ID No.1 in sequence table.
In the present invention, the volume ratio of the nano-Au solution, dATP solution, SDS solution and NaCl solution is preferably
200:2:3:10.The concentration of the nano-Au solution is preferably 15~20nmol/L, more preferably 18nmol/L.The dATP is molten
The concentration of liquid is preferably 1mmol/L, and the mass concentration of the SDS solution is preferably 1%, and the concentration of the NaCl solution is preferably
0.2mol/L.The concussion time that dATP solution is added is preferably 20min.The concussion time that SDS solution is added is preferably 10min.
The concussion time that NaCl solution is added is preferably 10min.2 μ are added in every 2~3min in the addition speed control of the NaCl solution
l。
In the present invention, the volume ratio of the nano-Au solution and CD63 adaptation liquid solution is preferably 100:1.The CD63
The concentration of aptamers is 1OD.In the present invention, the revolving speed of the centrifugation is preferably 12,000rpm.The time of the centrifugation is preferred
For 10min.The model of centrifugation centrifuge is not particularly limited, and is using centrifuge well-known to those skilled in the art
It can.
In the present invention, preferably obtained precipitating is carried out before gained being precipitated and dissolved in nanoparticle storage liquid
Washing and centrifugation.The cleaning fluids are 0.01mol/LPBS solution.The number of the washing and centrifugation is preferably 2~3 times.
After washing and centrifugation, the present invention is precipitated and dissolved in what centrifugation obtained in nanoparticle storage liquid.
In the present invention, the storage liquid preferably includes the component of following content: 20mmol/LNa3PO4·12H2O, quality
Concentration 5%BSA, mass concentration 0.25%Tween 20 and 10% sucrose of mass concentration.It is even dissolved with nanogold-CD63 aptamers
The nanoparticle storage liquid for closing object preferably saves under the conditions of 4 DEG C.
In the present invention, the CD63 aptamers conjugates solution spraying method of nano gold mark preferably use gold spraying instrument into
Row.The spraying rate of the CD63 aptamers conjugates solution of the nano gold mark is preferably 50mm/s.The nano gold mark
CD63 aptamers conjugates solution quantity for spray be 3 μ L/cm bonding pads.The present invention will preferably be coated with nanogold mark after spraying
The bonding pad of the CD63 aptamers conjugates of note is dried.The temperature of the drying is preferably 37 DEG C, the time of the drying
Preferably 30min.
The EpCam aptamers and Streptavidin of biotin labeling are mixed, and are separated by solid-liquid separation, and it is washed to collect supernatant
After separation of solid and liquid, collecting supernatant is that Streptavidin-biotin labeling EpCam is adapted to liquid solution.
The source of the EpCam aptamers of the biotin labeling make a living work company synthesis.The EpCam of the biotin labeling
The volume ratio of aptamers and solution of streptavidin is 1:4.The concentration of the EpCam adaptation liquid solution of the biotin labeling is preferred
For 50nmol/L.The concentration of the solution of streptavidin is preferably 2.5mg/ml.It is preferably buffered in PBS when the mixed culture
It is carried out in liquid.The concentration of the PBS buffer solution is preferably 0.01mol/L.The time of the mixed culture is preferably 55~
70min, more preferably 60min.
In the present invention, the separation of solid and liquid is preferably centrifuged.Centrifugation pipe is preferably ultra-filtration centrifuge tube.It is described
The molecular cut off for analysing pipe is preferably 30000.In the embodiment of the present invention, the ultra-filtration centrifuge tube is the super of Millipore Corp.'s production
Filter dialysis tubing.The revolving speed of the centrifugation is preferably 6000rpm.The time of the centrifugation is preferably 20min.The temperature of the centrifugation
Preferably 4 DEG C.The purpose of the centrifugation is the unbonded aptamers probe of removal.Being centrifuged obtained supernatant is that chain enzyme is affine
PBS buffer solution washing is added in element-biotin EpCam aptamers, and washing repeats above step twice, after finally collecting centrifugation
Solution is settled to 600 μ l.
The EpCam aptamers of the DNA probe replacement of the present invention biotin labeling biotin labeling, repeat step
(3) it operates, obtains Streptavidin-biotin labeling DNA probe solution.The DNA probe of the biotin labeling is by giving birth to work
Co., Ltd's synthesis.
Obtain Streptavidin-biotin labeling EpCam adaptation liquid solution and Streptavidin-biotin labeling
The EpCam of the Streptavidin-biotin labeling is adapted to liquid solution and Streptavidin-life by DNA probe solution, the present invention
The DNA probe solution of object element label is successively crossed on detecting pad, and the detecting pad for being coated with detection line and nature controlling line is obtained.
The present invention is not particularly limited the method for the spraying, using spraying method well-known to those skilled in the art
?.The speed of the spraying is preferably 50mm/s.The number of the spraying is preferably stood alone as 3 times.Streptavidin-the life
The EpCam aptamers conjugates of object element label or the spray volume of Streptavidin-biotin labeling DNA probe solution are 3 μ
L/cm detecting pad.
The present invention by the bonding pad of the CD63 aptamers conjugates for being coated with nano gold mark, described be coated with detection
The detecting pad and water absorption pad of line and nature controlling line are assembled on bottom plate from bottom to top after cutting, and are cut, and it is raw to obtain lateral flow type aptamers
Object sensor.
In the present invention, the overlap length of bonding pad or water absorption pad and detecting pad is preferably 2mm when the assembling.When assembling
Detecting pad is first pasted, then pastes bonding pad or water absorption pad, to guarantee that solution to be measured can be moved smoothly in test strips.
In the present invention, the slitting is preferably carried out using cutting machine.The width of the slitting preferably 2~
4mm。
Present invention preferably provides a kind of, and the lateral flow type aptamers biosensor based on above-mentioned preparation detects excretion body
Method preferably includes following steps:
The 100 μ l sample liquid for containing excretion body is slowly added dropwise in the bonding pad of lateral flow type aptamers biosensor
On, 20 μ l buffer solution for cleaning are added after 5min, the face of detection line is visually observed according to the difference of excretion bulk concentration in sample
Color depth shallowly changes, and finally test strips are placed in a machine of reading and read signal, calculates excretion body in sample according to standard curve
Content.
In the present invention, the buffer is the Tris-HCl solution of the 1%BSA containing mass concentration.The standard curve is
Signal value and excretion bulk concentration are in a linear relationship, and the method for drafting of the standard curve uses conventional method known in the art
?.The normal equation of the standard curve is y=0.0007x+6.3364, R2=0.981, wherein y indicates signal strength, institute
Stating x indicates the concentration of excretion body.
It is biological to a kind of aptamers group, the lateral flow type aptamers of detection excretion body provided by the invention below with reference to embodiment
Sensor and preparation method thereof is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Magnetic nano-particle (the raw work number NO.D110557 in Shanghai) and 10 μ L, 100 μ for taking 10 μ L streptavidins to mark
The biotin modification CD63 aptamers (the raw work in Shanghai) of mol/L mix 1h in 0.01mol/LPBS buffer, after magnet separation
Twice with 0.01mol/LPBS buffer solution for cleaning, 20 μ L blood plasma (containing excretion body in blood plasma) is then added, after cultivating 2h, uses magnetic
Iron separation.Magnetic nano-particle-aptamers-excretion body conjugates are observed at SEM scanning electron microscope (karr Zeiss EVO-18)
(Fig. 3), as a result, it has been found that the part that circle marks is the excretion body for being adapted body capture.This illustrates that CD63 provided by the invention is suitable
With physical efficiency specificity in conjunction with excretion body.
Embodiment 2
1. the preparation of nanogold-CD63 aptamers conjugates
Nanogold (15~20nm) solution for taking ten times of 1ml concentrations, is added the dATP of 10 microlitres of 1mM, at room temperature, with vibration
Device oscillation 20min is swung, 15 μ l mass concentration 1%SDS are subsequently added into, shake culture ten minutes later, is added 50 μ l 0.2mol/L's
NaCl (2 μ l are added in every 2~3min in speed control), is then added 10 μ l 1OD CD63 aptamers probes, reacts at 60 DEG C
3h is centrifuged with centrifuge (revolving speed 12,000rpm, 10min), is removed supernatant, is cleaned 3 with PBS buffer solution (pH7.2~7.4)
It is secondary, precipitating is finally dissolved in 1mL nanoparticle storage liquid (20mmol/LNa3PO4·12H2O, mass concentration 5%BSA, volume are dense
Spend 0.25%Tween20, mass concentration 10%sucrose) in, conjugates solution is placed on 4 DEG C of refrigerators and saves for use.
2. the EpCam aptamers of biotin labeling and the combination of Streptavidin
By the EpCam aptamers of 50nmol/L biotin labeling
(the raw work in the Shanghai 5 '-Bio-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3 ' is closed
At) be mixed 1h in 0.01mol/LPBS with the Streptavidin of 200 μ l 2.5mg/ml (purchased from the raw work in Shanghai) after, will
Mixed liquor is transferred in dialysis tubing (molecular cut off 30000), and (6000rpm, 20min, 4 DEG C) is centrifuged in refrigerated centrifuge and is gone
Except unbonded aptamers probe.0.01mol/LPBS buffer is added and repeats above step twice, after finally collecting centrifugation
Solution is settled to 600 μ l.Finally the mixture solution that the EpCam aptamers and Streptavidin of biotin labeling combine is being tried
It is sprayed three times in the detection line of paper slip.
Streptavidin-biotin labeling DNA probe is prepared according to the method described above.The strepto- being prepared is affine
Element-biotin labeling DNA probe sprays three times in the detection line of test strips.
3. the nanometer gold test paper strip assembling of aptamers label
The mixture that nanogold is connect with CD63 aptamers is sprayed on bonding pad with gold spraying instrument, Streptavidin and biology
The EpCam aptamers of element label are sprayed in the detection line of NC film with gold spraying instrument.Finally by test strips each section according to certain suitable
Sequence is assembled on bottom plate, each overlapped 2mm in part, to guarantee that solution to be measured can be moved smoothly in test strips.With sanction item
Assembled bottom plate is planted the test strips for being cut into proper width by machine, is placed in 4 DEG C of refrigerators and is saved for use.
Embodiment 3
0~1 × 10 are drawn according to the detection method of excretion body in embodiment 26Standard curve in concentration range, research letter
Number value is in a linear relationship with excretion bulk concentration.As a result see Fig. 4.Detection sensitivity is 5 × 105。
Different samples are detected according to the detection method of excretion body in embodiment 2, wherein different samples include tumour
Clinical samples, healthy person sample and blank control.Discovery is visually observed, the test strips generation two of detection tumor patient sample is red
The test strips of line, healthy person sample and dialogue empty map sample generate a red line on nature controlling line.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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cactacagag gttgcgtctg tcccacgttg tcatgggggg ttggcctg 48
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Claims (10)
1. a kind of CD63 aptamers for detecting excretion body, which is characterized in that the nucleotide sequence such as sequence of the CD63 aptamers group
In list shown in SEQ ID No.1.
2. a kind of aptamers group for detecting excretion body, which is characterized in that the aptamers group includes CD63 described in claim 1
Aptamers and EpCam aptamers, the nucleotide sequence of the EpCam aptamers is as shown in SEQ ID No.2 in sequence table.
3. a kind of lateral flow type aptamers biosensor, including bottom plate and it is adhered to sequentially connected from bottom to top on the bottom plate
Bonding pad, detecting pad and absorption pad, the detecting pad include detection line and nature controlling line, which is characterized in that the bonding pad spraying
There are the CD63 aptamers conjugates of nano gold mark, it is suitable that the detection line is coated with Streptavidin-biotin labeling EpCam
Ligand conjugates, the nature controlling line are coated with Streptavidin-biotin labeling DNA probe, the nucleotide sequence of DNA probe
It is complementary with 3 ' terminal sequences of the nucleotide sequence of CD63 aptamers；
CD63 aptamers are detection excretion body described in claim 1 in the CD63 aptamers conjugates of the nano gold mark
CD63 aptamers；
EpCam aptamers in the EpCam aptamers conjugates of the Streptavidin-biotin labeling are that right benefit requires 2
EpCam aptamers in the aptamers group.
4. lateral flow type aptamers biosensor according to claim 3, which is characterized in that the Streptavidin-life
The nucleotide sequence of DNA probe is as shown in SEQ ID No.2 in sequence table in the DNA probe of object element label.
5. lateral flow type aptamers biosensor according to claim 3 or 4, which is characterized in that the nano gold mark
CD63 spray volume be 3 μ l/cm bonding pads.
6. lateral flow type aptamers biosensor according to claim 3, which is characterized in that the Streptavidin-life
The spraying spraying number of the EpCam aptamers conjugates of object element label is 3 times, the Streptavidin-biotin labeling
The spray volume of EpCam aptamers conjugates is 3 μ l/cm detecting pads.
7. the preparation method of lateral flow type aptamers biosensor described in claim 3~6 any one, which is characterized in that packet
Include following steps:
(1) dATP solution, SDS solution and NaCl solution are sequentially added under the conditions of earthquake into nano-Au solution to be reacted,
Obtained reaction solution and CD63 aptamers mixing, 3h is reacted at 60 DEG C, is separated by solid-liquid separation, gained is precipitated and dissolved in nanoparticle
It stores in liquid, obtains the CD63 aptamers conjugates solution spraying of nano gold mark on bonding pad, obtain being coated with nanogold
The bonding pad of the CD63 aptamers conjugates of label；
(2) after the EpCam aptamers and Streptavidin of biotin labeling being mixed 1h, it is placed in ultrafiltration in ultra-filtration centrifuge tube
Centrifugation, after washing 2 times to ultra-filtration centrifuge tube, the supernatant of collection is that Streptavidin-biotin labeling EpCam aptamers are molten
Liquid；
(3) the EpCam aptamers in the step (2) are replaced with DNA probe, repeats step (3) operation, it is affine obtains strepto-
Element-biotin labeling DNA probe solution；
(4) Streptavidin-biotin labeling EpCam in the step (2) is adapted to liquid solution and step (3) strepto- is affine
Element-biotin-labelled DNA probe solution is successively crossed on detecting pad, and the detecting pad for being coated with detection line and nature controlling line is obtained；
(5) by the bonding pad of the CD63 aptamers conjugates for being coated with nano gold mark in the step (1), the step
(4) detecting pad for being coated with detection line and nature controlling line and blotting paper in are successively assembled in bottom plate after cutting respectively from bottom to top
On, slitting obtains lateral flow type aptamers biosensor；
There is no the limitation of time sequencing between the step (1) and (2)~(4), while not having between step (2) and step (3) yet
The limitation of having time sequence.
8. preparation method according to claim 7, which is characterized in that nano-Au solution, dATP are molten in the step (1)
The volume ratio of liquid, SDS solution and NaCl solution is 200:2:3:10；The concentration of the nano-Au solution is 15~20nmol/L,
The concentration of the dATP solution is 1mmol/L, and the mass concentration of the SDS solution is 1%, and the concentration of the NaCl solution is
0.2mol/L。
9. preparation method according to claim 7, which is characterized in that storage liquid includes following content in the step (1)
Component: 20mmol/LNa3PO4·12H2O, mass concentration 5%BSA, mass concentration 0.25%Tween20 and mass concentration
10% sucrose.
10. according to preparation method described in claim 7~9 any one, which is characterized in that solid-liquid point in the step (2)
From mode include centrifugation；The revolving speed of the centrifugation is 6000rpm, and the time of the centrifugation is 20min, the temperature of the centrifugation
It is 4 DEG C.
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