CN106908598B - A kind of aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon and its preparation and detection method - Google Patents
A kind of aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon and its preparation and detection method Download PDFInfo
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Abstract
The invention belongs to field of nano biotechnology, specifically disclose a kind of aptamer-suspending chip based on single-walled carbon nanotube, and preparation method thereof and detection method.Object is detected using aptamer-suspending chip of single-walled carbon nanotube of the present invention, it detects obtained median fluorescent signal strength and testing concentration is negatively correlated, by establishing the standard curve of testing concentration and fluorescence signal, the testing concentration in measuring samples can be obtained.Detection method disclosed by the invention has wider detection range, higher detection sensitivity and specificity, has greater advantage compared with other detection techniques.
Description
Technical field
The present invention relates to field of nano biotechnology, and in particular to a kind of aptamer-suspension of single-walled carbon nanotube
Chip detecting method.
Background technology
Suspending chip, can be using a diameter of 5.6 μm of polystyrene or the magnetic fluorescent microsphere of doping as solid phase carrier
Exist in suspension free state in detection architecture, and flows passage in fluid circuit with the direction of exerting pressure of hydraulic pressure；Each is micro-
Ball makes it be defined as unique coded address so that numerous codings is micro- because it is coated with the special fluorescent dye of different proportion
Ball is aligned to the collection of illustrative plates similar to " array ".There are different group Molecules, such as carboxyl, hydroxyl in the solid surface modification of microballoon
Base, biotin, Streptavidin etc., and microsphere probe is formed by chemical reaction and specific biomolecule, these biologies point
Son can be capture antibody, nucleotide probe, polypeptide or receptor.To carry out Ag-Ab, enzyme-substrate, ligand-receptor
Association reaction or nucleic acid hybridization reaction.The technology is detected using the laser of 2 kinds of different wave lengths, wherein：Red laser excites
Be red classification dyestuff on microballoon, it is fluorescence-encoded according to the difference of microballoon, microballoon can be classified, to will be each
Different analysis reactions distinguish；Green laser excitation is reporter fluorescence molecule, it is therefore an objective to determine the report combined on microballoon
The quantity of announcement fluorescent molecular, the value, that is, median fluorescent intensity value quantified (Median Fluorescent Intensity,
MFI), up to 500 kinds of different biologicallies can be theoretically completed in a reacting hole.The technology technology is with fluorescence
Detection means based on coding microball, rapid sensitive, background signal are low, the high-throughput detection of multiple samples can be achieved, in life
Object and field of medical analysis show many advantages.Currently, traditional Suspension array technique is based on immunological response, such as：
Big Molecular Detection, generally use sandwich method；For small molecule, then detected using competition law.
Aptamer is a kind of single stranded nucleic acid molecule with specific recognition function, has and is mutually equal to monoclonal antibody
Beautiful affinity and specificity.Aptamer has further the advantage that compared with monoclonal antibody：It can screen in vitro, target molecule range
Extensively, molecular weight is relatively low, without immunogenicity and toxicity, can be prepared, be transformed and marked by chemical synthesis, and chemical stability is good,
Energy reversible denaturation and renaturation, can pass through enzymatic amplification, shearing etc..These advantages make it have wide application in biomedical sector
Foreground, the bioanalytical method based on aptamers are developed rapidly.Replace monoclonal antibody by aptamer, it can
It is effectively improved by antigen immunogenicity is limited, time-consuming and preparation process is complicated, monoclonal antibody is expensive, is not easy to obtain
Problem.In addition, traditional antigen-antibody is not strong to the characteristic group binding force of small molecule, in identification small-molecule chemical object space face
It has some limitations.So being detected to small molecule target using aptamer, become a kind of new and effective
Detection mode.
Carbon material has good characteristic, and wherein carbon nanotube has higher specific surface area, higher tensile strength, height
The excellent characteristics such as characteristic electron elastic, from metal to semiconductor.It is modified, can be made by the transformation of single pipe surface
Improve carbon nanotube and is difficult to compatible disadvantage with other substances.Single-walled carbon nanotube is fixed on microballoon, is formed skeleton, is utilized it
Abundant specific surface area, physical absorption aptamers chain so that the high-affinity of aptamers further amplifies.
Invention content
The purpose of the present invention includes：A kind of composite nucleic acid aptamers-suspension core suitable for field quick detection is provided
Piece；
The preparation method, detection method and purposes of the aptamer-suspending chip are provided.
Aptamer of the present invention based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, it is a kind of
Embodiment is that the aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon includes：
The single-walled carbon nanotube (1) of surface amination modification, the suspending chip magnetic microsphere (2) with carboxyl site are raw
Object element aptamer (3) is coupled fluorescein-labeled Streptavidin (4)；
The wherein described single-walled carbon nanotube (1) by coupling, is distributed in by amido bond and suspending chip magnetic microsphere (2)
The surface of suspending chip magnetic microsphere (2)；
The biotinylation aptamer (3) connect with single-walled carbon nanotube (1) physical absorption.
Preferably, the aptamer in the biotinylation aptamer (3) is single strand dna oligonucleotide；
Preferably, the fluorescein in the fluorescein-labeled Streptavidin (4) is phycoerythrin.
It is further preferred that the nucleic acid that the aptamer in the biotinylation aptamer (3) is bisphenol-A is adapted to
Body, nucleotides sequence are classified as：
5'-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-
3'；
Or the aptamer that the aptamer in the biotinylation aptamer (3) is estradiol, core
Nucleotide sequence is：
5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAA
GCGCGAAG-3'；
Or the aptamer that the aptamer in the biotinylation aptamer (3) is Polychlorinated biphenyls,
Nucleotides sequence is classified as：
5'-AGCAGCACAGAGGTCAGATGCACTCGGACCCCATTCTCCTTCCATCCCTCATCCGTCCACCCTAT
GCGTGCTACCGTGAA-3'。
Aptamer of the present invention based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, it is another
Kind embodiment is that the aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon is by following steps
It prepares, including：
1) single-walled carbon nanotube surface amination base group modification is transformed：
Two step acidizations handle single pipe surface, after so that its surface is produced carboxyl, then carry out amination transformation,
Make the single-walled carbon nanotube of carboxylated again by amination；
2) the carboxyl site of amido modified single-walled carbon nanotube and suspending chip magnetic microsphere is coupled：
The carboxyl site of the single-walled carbon nanotube and suspending chip magnetic microsphere of amination modification prepared by step 1) is logical
Amino condensation reaction is crossed to be coupled；
3) Streptavidin of biotinylation aptamer coupling fluorescent marker is prepared：
Respectively by biotin labeling in object aptamer, fluorescein is marked on Streptavidin, then makes life
The Streptavidin of object element aptamer and fluorescent marker is coupled；
4) aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon is prepared：
The Streptavidin of biotinylation aptamer-fluorescent marker prepared by step 3) is prepared with step 2)
Single-walled carbon nanotube-suspending chip magnetic microsphere is coupled, and the aptamer based on single-walled carbon nanotube-suspension core is obtained
Piece magnetic detection microballoon；
The wherein magnetic microsphere of coupling single-walled carbon nanotube in the mixing that is vortexed in vortex instrument, to after uniformly keep away by ultrasonic disperse
Striation part is added into the Streptavidin of biotinylation aptamer-fluorescent marker, and water-bath at room temperature is slowly shaken, and prepares
Obtain the aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon.
Preferably, the amination described in the step 1) is transform as：Made for ammoniacal substance with ethylenediamine, dicyclohexyl carbon two is sub-
Amine is heated to reflux at 120 DEG C, makes the single-walled carbon nanotube of carboxylated again by amination as condensing agent；The single wall carbon is received
The inventory of mitron is 0.01mg；
Preferably, the amino setting-up point described in step 2) is 120 DEG C, and the reaction time is 24 hours；The magnetism
The inventory of microballoon is 1.25 × 105It is a；
Preferably, fluorescent marker used in the fluorescent marker described in step 3) is phycoerythrin.
Preferably, the inventory of step 4) the biotinylation aptamer coupling phycoerythrin-Streptavidin is
0.2nmol, reaction time are 1.5h hours, and reaction temperature is 28 DEG C.
Aptamer of the present invention based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, it is a kind of
Enforceable mode is to mix three kinds of aptamers based on single-walled carbon nanotube-suspending chip magnetic detection microballoon
It arrives；
Three kinds of fingers：The nucleotides sequence of aptamer is classified as the single-walled carbon nanotube of sequence table SEQ NO ID.1
Aptamer-suspending chip magnetic detection microballoon, the nucleotides sequence of aptamer are classified as the list of sequence table SEQ NO ID.2
The nucleotides sequence of the aptamer of wall carbon nano tube-suspending chip magnetic detection microballoon and aptamer is classified as sequence
The aptamer of the single-walled carbon nanotube of table SEQ NO ID.3-suspending chip magnetic detection microballoon.
Preferably, the object of the described three kinds aptamers based on single-walled carbon nanotube-suspending chip magnetic detection microballoon
The amount ratio of matter is 1:1:1.
In addition, the present invention also provides above-mentioned all aptamers based on single-walled carbon nanotube-suspending chip magnetism
The detection method of microballoon is detected, step includes：Aptamer-the suspending chip is mixed with object to be measured object, in detection
Position fluorescence signal intensity, establishes examination criteria curve, obtains testing concentration；Wherein testing concentration and median fluorescent signal is strong
Spend negatively correlated relationship.
The present invention also provides the above-mentioned all aptamer based on single-walled carbon nanotube-suspending chip magnetic detections are micro-
Ball detects the purposes of nucleic acid, protein, microorganism and/or chemical pollutant；
Preferably, wherein being 1 × 10 to the detection range of the nucleic acid, protein, microorganism and/or chemical pollutant- 13G/mL~1 × 10-7G/mL, detection sensitivity >=1 × 10-15g/mL。
About the aptamer of the present invention based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, say
Bright book attached drawing 1 gives its structural model schematic diagram.Wherein, Fig. 1 only illustrates single-walled carbon nanotube (1) and magnetic detection microballoon
(2) connection relation, the quantity without limiting the single-walled carbon nanotube (1) that magnetic detection microballoon (2) is connected；Meanwhile Fig. 1 is only
Illustrate the connection relation of biotinylation aptamer (3) and single-walled carbon nanotube (1), single-walled carbon nanotube (1) is not inhaled
Attached biotinylation aptamer (3) quantity makes limitation.
By combining single-walled carbon nanotube, Suspension array technique to construct the novel nucleic acids aptamers detection of super sensitivity detection
Technology.The advantage for making full use of aptamer high specificity, affinity high so that detection probe is easy to operate, and detection is sensitive.
Magnetic polystyrene microsphere has big specific surface area, possesses up to 107-108Reaction site, the abundant ratio of single-walled carbon nanotube
The microballoon of carboxylated and amido modified single-walled carbon nanotube are coupled by surface area and excellent mechanics feature, can be in conjunction with more
Detection probe, further expand signal reaction.The single-walled carbon nanotube that amination is modified is coupled on microballoon first, is added
Probe through SA-PE fluorescent markers namely target molecule, meeting physical absorption on the carbon nanotubes, after adding target molecules,
The probe of fluorescent marker can be competed, fluorescent value is caused to reduce.The calibration curve equation of target analyte detection is established, the detection
Platform will provide technology and theoretic support for the field quick detection of more chemical pollutants, can be used for food hygiene,
The fields such as sanitary inspection and environmental monitoring have good theory significance and practical value.
The Avidin of fluorescent marker of the present invention, the fluorescent marker are phycoerythrin, gained fluorescent marker
Avidin is streptavidin-phycoerythrin, whole also to claim fluorescent reporter protein；Fluorescent marker of the present invention it is affine
Element can also use fluorescent marker commonly used in the art or labelled protein to carry out fluorescent marker, such as：Fluorescein isothiocynate,
5- carboxyls fluorescence, cyanine dye fluorescein 3 and the label of cyanine dye fluorescein 5 etc..
The preparation of aptamer of the present invention based on single-walled carbon nanotube-suspending chip magnetic detection microballoon walks
Suddenly：(1) single-walled carbon nanotube surface amination base group modification is transformed；(2) amido modified single-walled carbon nanotube and suspending chip
The carboxyl site of magnetic code microballoon is coupled；(3) biotinylation aptamer coupling fluorescent reporter molecule-strepto- is affine
Plain phycoerythrin；(4) biotinylation aptamer-Streptavidin phycoerythrin is in single-walled carbon nanotube-suspending chip magnetic
Property coding microball on be coupled, formed aptamer specific probe suspending chip microballoon, can refer to Luminex corporate networks
It stands technical manual
(https://www.luminexcorp.com/zh/research/reagents-and-accessories/
magplex-microspheres/product-details/)。
Suspending chip magnetic microsphere of the present invention is purchased from Luminex companies.
Advantageous effect
The present invention is based on the aptamer of single-walled carbon nanotube-suspending chip magnetic detection microballoons, for nucleic acid, albumen
The detection of matter, microorganism and/or chemical pollutant has wider detection range and higher detection sensitivity；Its detection range
It is 1 × 10-13~1 × 10-7G/mL, detection sensitivity is at fg grades (1 × 10-15G/mL more than).This and standard suspension chip technology
It is compared with greater advantage with classic ELISA technology.Through specific detection it is demonstrated experimentally that novel nucleic acids aptamers-suspending chip
Technology specificity is good.
Description of the drawings
The structural model of the hypersensitive aptamer of Fig. 1 single-walled carbon nanotubes-suspending chip magnetic detection microballoon is illustrated
Figure.
Wherein, (1) refers to single-walled carbon nanotube, and (2) refer to magnetic detection microballoon, and (3) refer to biological object element aptamer,
(4) refer to fluorescein-labeled Streptavidin phycoerythrin.
Fig. 2 is a kind of hypersensitive aptamer-suspending chip magnetic detection microballoon detection method of single-walled carbon nanotube
Establishment model figure.
Fig. 3 is the hypersensitive aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon detection method
Detect bisphenol-A (BPA), estradiol (E2) and Polychlorinated biphenyls (PCB) canonical plotting.
Specific implementation mode
Ground further instruction is made to the present invention with reference to specific attached drawing 2.
This research is with typical environment incretion interferent bisphenol-A (Bisphenol A, BPA), estradiol (17- β-
Estradiol, E2) and Polychlorinated biphenyls (PCB) etc. be used as research object, large specific surface area, controllable object based on carbon nanomaterial
Rationality adsorbs biomaterial feature, establishes novel nucleic acids aptamers-suspending chip skill of a variety of environment incretion interferent detections
Art.
Use ethylenediamine condensing agent method to carbon nanomaterial, that is, single-walled carbon nanotube (Single-walled first
Nanotubes, SWNTs) amination base group modification transformation is carried out, the carbon nanotube of amination modification is prepared, and utilize infrared
It is characterized with XPS, it is 2.46% to measure amido modified rate.
The carboxyl site of modified SWNTs and suspending chip magnetic code microballoon are coupled, and the ratio of microballoon is increased
Surface area, while playing signal amplification；
Based on Streptavidin-biotin reaction mechanism, by fluorescent reporter protein streptavidin-phycoerythrin
(Strepavidin-phycoerythrin, SA-PE) is connected to biotinylated BPA, PCB and E2On aptamer,
Using the property of aptamer single stranded DNA and the controllable physical absorptions of SWNTs, corresponding specific probe is prepared
Microballoon；
If waiting for that there are BPA, PCB and E in test sample2, then captured by probe microballoon, and with report molecule (i.e. BPA and E2's
Aptamer) specific binding, to change the three dimensions configuration of aptamers so that the compound of formation is from microballoon
It falls off, the median fluorescent intensity value (Median Fluorescence Intensity, MFI) that suspension chip system measures also phase
It should reduce.
This experiment is by optimizing reaction condition, obtaining best SWNT, SA-PE addition and incubation time and being incubated temperature
Degree etc..With this condition, BPA, E are established2With the examination criteria curve of PCB.Illustrate that this method has wider detection range
(the 5-6 order of magnitude) and higher detection sensitivity (pg grades sub-), with standard suspension chip technology and classic ELISA technology etc. its
His detection technique, which is compared, has greater advantage.Through specific detection it is demonstrated experimentally that novel nucleic acids aptamers-Suspension array technique is special
It is anisotropic good.
Embodiment
Embodiment one, the single-walled carbon nanotube for preparing surface amination modification
Carbon nanotube is handled using two step acidizations：The dense H of 100mL 98%2S04With 68% dense HN03According to 3:1
Volume ratio mixes, the addition 200mg SWNT in mixed acid, back flow reaction 4h under the conditions of 38 DEG C of supersonic oscillations.It is cooled to room temperature
Afterwards, deionized water dilution is added, cellulose filter membrane filtering is cleaned with deionized water until the pH of filtrate is in neutrality.Reaction product
It is placed in electric drying oven with forced convection, is dried in vacuum overnight at 60 DEG C.
The dense H of 100mL 98%2S04With 30% H202According to 4:1 volume ratio mixing, by the nitration mixture acidizing product in 1
It is placed in H2S04With H202Mixed solution in, flow back 2h at 70 DEG C.After being cooled to room temperature, deionized water dilution is added, uses filter membrane
Filtering, cleaning are in neutrality to being washed till filtrate pH.Product is placed in electric drying oven with forced convection, is dried overnight at 60 DEG C.
The coupling of embodiment two, amido modified single-walled carbon nanotube and suspending chip magnetic code microballoon
The SWNT of 3mg amination modifieds is taken, 3mL dH are added2In O, ultrasonic 5min, being repeated 6 times the cumulative time reaches
30min is disappeared heat with ice water halfway.800 μ L are taken, are dispersed in the distilled water of 1.5mL.Light protected environment takes out blank carboxylic fluorescent
The brown bottle (52#) of coded magnetic microsphere suspensions dries in the air to room temperature, water bath sonicator 30s, vortex mixing 1min.Take out 120 μ L suspensions
Into the 1.5mL centrifuge tubes coated with aluminium foil, 5min is placed on magnetic separator, it is adherent to suck supernatant.120μL 0.1M MES
(pH=4.5) it adds in microballoon and mixes well, clean microballoon.Magneto separate discards supernatant, and adds 100 μ L MES that microballoon, tinfoil paper is resuspended
Paper bag, which is lived centrifuge tube and is protected from light, to be put to 4 DEG C, spare.
Take 20 μ L SWNT that centrifuge tubes are added, shake mixing rapidly, then be separately added into a concentration of 10mg/mL NHS of 10 μ L and
30 μ L a concentration of 10mg/mL EDC, MES complement to 120 μ L systems, and room temperature, which is protected from light, is incubated 4h, 800r.Magneto separate abandons supernatant, adds
200 μ L Block buffers close unreacted carboxyl site 30min, room temperature, 800rpm.
Microballoon is cleaned with 1mL cleaning buffer solutions I and II respectively, 100 μ L TE buffer solutions is added after Magneto separate, microballoon is resuspended,
Water bath sonicator 30s after vortex mixing, is counted under an optical microscope with blood counting chamber.By probe microballoon be diluted to 2000/
μ L are kept in dark place in 4 DEG C.
Embodiment three prepares biotinylation aptamer coupling fluorescent reporter molecule-Streptavidin phycoerythrin
Biotinylated aptamer is taken, in centrifuge 12000rpm before uncapping, 60s is centrifuged, slowly opens pipe lid, add
Enter the PBS (MgCl containing 0.1M of 34 μ L2) buffer solution, it is configured to 100 μM of storage liquid, the fully shaking that closes the lid mixing, packing
(2 μ L/PCR pipes, 10 pipes) are spare, remaining -20 DEG C freeze；Take 1 pipe, the 2 biotinylated aptamers of μ L, 95 DEG C of water bath processings
5min is cooled to room temperature.It remakes 100 times of dilutions and obtains 1 μM, that is, 198 μ L Tris-HCI (20mM Tris-HCI, pH=are added
7.4,100mM NaCl, 2mM MgCl2) coupling buffer, totally 200 μ L are spare；It is protected from light the SA-PE for taking 2 μ L, uses PBS buffer solution
100 times of dilution is spare to 200 μ L；
Take 1 μM biotinylation aptamer and 100 × diluted SA-PE according to volume ratio 3:2 mixing, 37 DEG C
800rpm oscillation incubations 30min.
Example IV, the biotinylation aptamer-Streptavidin phycoerythrin-single for preparing bisphenol-A
Pipe suspending chip detects magnetic microsphere.
In the 10 μ L of fluorescence-encoded magnetic microsphere (2000/μ L) and 96 orifice plates that take out coupling SWNT with pipettor, per hole
1 μ L are added, by 96 orifice plates in vortex mixing 30s in vortex instrument, ultrasonic 1min, until being uniformly dispersed.It is protected from light each hole of condition and 2 μ L is added
(aptamer is biotinylation aptamer coupling fluorescent reporter molecule-Streptavidin phycoerythrin：Bisphenol-A
Aptamer, nucleotide sequence is as shown in sequence table SEQ ID NO.1), 50 μ L systems are supplied with coupling buffer, are incubated
28 DEG C of temperature, incubation time 1.5 hours obtain biotinylation aptamer-Streptavidin algae red egg of detection bisphenol-A
In vain-single-walled carbon nanotube suspending chip detects magnetic microsphere.
Embodiment five, the biotinylation aptamer-Streptavidin phycoerythrin-single for preparing estradiol
Pipe suspending chip detects magnetic microsphere.
Only it is that bisphenol-A aptamer is replaced with into estradiol aptamer with reference to the preparation method of example IV；Institute
The nucleotide sequence of estradiol aptamer is stated as shown in sequence table SEQ ID NO.2.
Embodiment six, the biotinylation aptamer-Streptavidin phycoerythrin-single wall carbon for preparing Polychlorinated biphenyls are received
Mitron suspending chip detects magnetic microsphere.
Only it is that bisphenol-A aptamer is replaced with into Polychlorinated biphenyls aptamer with reference to the preparation method of example IV；
The nucleotide sequence of the Polychlorinated biphenyls aptamer is as shown in sequence table SEQ ID NO.3.
Embodiment seven, the biotinylation core for preparing threefold ring border incretion interferent (bisphenol-A, estradiol and Polychlorinated biphenyls)
Sour aptamers-Streptavidin phycoerythrin-single-walled carbon nanotube suspending chip magnetic detection microballoon.
Example IV, five, the six biotinylation aptamer-Streptavidin phycoerythrin-single wall carbon prepared are received
Mitron suspending chip detects magnetic microsphere and carries out equimolar amount mixing, and the biotinylation of threefold ring border incretion interferent is made
Aptamer-Streptavidin phycoerythrin-single-walled carbon nanotube suspending chip detects magnetic microsphere.
The biotinylation aptamer-of threefold ring border incretion interferent (bisphenol-A, estradiol and Polychlorinated biphenyls)
Streptavidin phycoerythrin-single-walled carbon nanotube suspending chip magnetic detection microballoon, also referred to as based on the more of single-walled carbon nanotube
First aptamer-suspending chip magnetic detection microballoon.
Test case
The detection of test case one, bisphenol-A (Bisphenol A, BPA).
It using scheme best after condition optimizing, is incubated 2 hours under the conditions of 25 DEG C of 600rpm, measures various concentration BPA
MFI values under (0,0.256,1.28,6.4,32,160,800 and 4000pg/mL), parallel determination are averaged three times.Not have
MFI values when BPA detections are added as positive MFI0, establish concentration and MFI/MFI0Standard curve (standard curve is shown in
Bright book attached drawing 3).10 holes μ L/ samples are taken to be added in the reacting hole of 96-well microtiter plate when detection；PH is drawn respectively with pipettor
=7.4 37 μ L of PBS buffer solution, the sample that every hole is added carry out piping and druming mixing；
Test case two, estradiol (17- β-Estradiol, E2) detection.
Only it is that BPA is replaced with into E with reference to the preparation method of test case one2, measure various concentration E2(0、0.5、2、8、32、
128,512 and 2048pg/mL) under MFI values, establish standard curve (standard curve is shown in Figure of description 3).
The detection of test case three, Polychlorinated biphenyls (PCB).
Only be that BPA is replaced with into PCB with reference to the preparation method of test case one, measure various concentration PCB (0,0.064,
0.32,1.6,8,40,200 and 1000pg/mL) under MFI values, establishing standard curve, (standard curve is shown in Figure of description
3)。
Test case four, BPA, E2It is detected while with PCB.
It, can be with the detection target molecule of multichannel since suspending chip is a high-throughput detection platform.It is building
On the basis of high-affinity aptamers-suspending chip detection technique of vertical single channel BPA, E is increased2, PCB totally three kinds of indexs into
Common detection is gone.It is suitable respectively at the biotinylation nucleic acid that 3 μ L threefold rings border incretion interferents are added in every hole with pipettor
Ligand-Streptavidin phycoerythrin-single-walled carbon nanotube suspending chip detects magnetic microsphere, in vortex oscillator in 28 DEG C
Speed oscillation 1.5 hours；Suspension chip system reads 100 microballoon/holes, obtains median fluorescent intensity value；With three kinds of targets
A concentration of X-axis, it is Y-axis to obtain median fluorescent intensity value and the ratio of positive median fluorescent intensity value, and the standard for substituting into acquisition is bent
In line, BPA, E in 3 water samples are calculated2With the detected value of PCB, specific testing result is as shown in table 1.
BPA, E in 13 water samples of table2The testing result (n=5) of and PCB
The present invention is by optimizing reaction condition, obtaining best SWNT, SA-PE addition and incubation time and being incubated temperature
Degree etc..With this condition, BPA, E are established2With the examination criteria curve of PCB.Illustrate that this method has wider detection range
(1×10-13~1 × 10-7) and higher detection sensitivity (1 × 10 g/mL-15G/mL more than), with standard suspension chip technology and
Classic ELISA technology, which is compared, has greater advantage.Through specific detection it is demonstrated experimentally that novel nucleic acids aptamers-suspending chip skill
Art specificity is good.
Sequence table
<110>Guangzhou medical university
<120>A kind of aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon and its prepare and
Detection method
<130> 2017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 63
<212> DNA
<213>Artificial sequence
<400> 1
ccggtgggtg gtcaggtggg atagcgttcc gcgtatggcc cagcgcatca cgggttcgca 60
cca 63
<210> 2
<211> 74
<212> DNA
<213>Artificial sequence
<400> 2
gcttccagct tattgaatta cacgcagagg gtagcggctc tgcgcattca attgctgcgc 60
gctgaagcgc gaag 74
<210> 3
<211> 80
<212> DNA
<213>Artificial sequence
<400> 3
agcagcacag aggtcagatg cactcggacc ccattctcct tccatccctc atccgtccac 60
cctatgcgtg ctaccgtgaa 80
Claims (10)
1. a kind of aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, including：
The single-walled carbon nanotube (1) of surface amination modification, the suspending chip magnetic microsphere (2) with carboxyl site, biotin
Change aptamer (3) and is coupled fluorescein-labeled Streptavidin (4)；
The wherein described single-walled carbon nanotube (1) is coupled by amido bond and suspending chip magnetic microsphere (2), is distributed in suspending chip
The surface of magnetic microsphere (2)；
The biotinylation aptamer (3) connect with single-walled carbon nanotube (1) physical absorption.
2. the aptamer according to claim 1 based on single-walled carbon nanotube-suspending chip magnetic detection microballoon,
It is characterized in that, the aptamer in the biotinylation aptamer (3) is single strand dna oligonucleotide.
3. the aptamer according to claim 2 based on single-walled carbon nanotube-suspending chip magnetic detection microballoon,
The fluorescein being characterized in that in the fluorescein-labeled Streptavidin (4) is phycoerythrin.
4. the aptamer according to claim 3 based on single-walled carbon nanotube-suspending chip magnetic detection microballoon,
It is characterized in that, the aptamer in the biotinylation aptamer (3) is the aptamer of bisphenol-A, nucleotide
Sequence is：
5'-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCACGGGTTCGCACCA-3'；
Or the aptamer that the aptamer in the biotinylation aptamer (3) is estradiol, nucleotide
Sequence is：
5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGC
GCGAAG-3'；
Or the aptamer that the aptamer in the biotinylation aptamer (3) is Polychlorinated biphenyls, nucleosides
Acid sequence is：
5'-AGCAGCACAGAGGTCAGATGCACTCGGACCCCATTCTCCTTCCATCCCTCATCCGTCCACCCTATGCGT
GCTACCGTGAA-3'。
5. aptamer of the claim 1-4 any one of them based on single-walled carbon nanotube-suspending chip magnetic detection is micro-
Ball, which is characterized in that it is prepared by following steps, including：
1) single-walled carbon nanotube surface amination base group modification is transformed：
Two step acidizations handle single pipe surface, after so that its surface is produced carboxyl, then carry out amination transformation, make carboxylic
The single-walled carbon nanotube of base is again by amination；
2) the carboxyl site of amido modified single-walled carbon nanotube and suspending chip magnetic microsphere is coupled：
The carboxyl site of the single-walled carbon nanotube and suspending chip magnetic microsphere of amination modification prepared by step 1) is passed through into ammonia
Base condensation reaction is coupled；
3) Streptavidin of biotinylation aptamer coupling fluorescent marker is prepared：
Respectively by biotin labeling in object aptamer, fluorescein is marked on Streptavidin, then makes biotin
The Streptavidin for changing aptamer and fluorescent marker is coupled；
4) aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon is prepared：
The Streptavidin of biotinylation aptamer-fluorescent marker prepared by step 3), the single wall prepared with step 2)
Carbon nanotube-suspending chip magnetic microsphere is coupled, and the aptamer based on single-walled carbon nanotube-suspending chip magnetic is obtained
Property detection microballoon；
For the wherein magnetic microsphere of coupling single-walled carbon nanotube in the mixing that is vortexed in vortex instrument, ultrasonic disperse is protected from light item to after uniformly
Part is added into the Streptavidin of biotinylation aptamer-fluorescent marker, and water-bath at room temperature is slowly shaken, is prepared
Aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon.
6. the aptamer according to claim 5 based on single-walled carbon nanotube-suspending chip magnetic detection microballoon,
It is characterized in that,
Amination described in step 1) is transform as：Made for ammoniacal substance with ethylenediamine, dicyclohexylcarbodiimide as condensing agent,
It is heated to reflux at 120 DEG C, makes the single-walled carbon nanotube of carboxylated again by amination；The inventory of the single-walled carbon nanotube is
0.01mg；
Amino setting-up point described in step 2) is 120 DEG C, and the reaction time is 24 hours；The inventory of the magnetic microsphere
It is 1.25 × 105It is a；
Fluorescent marker used in fluorescent marker described in step 3) is phycoerythrin；
The inventory of step 4) the biotinylation aptamer coupling phycoerythrin-Streptavidin is 0.2nmol, reaction
Time is 1.5 hours, and reaction temperature is 28 DEG C.
7. a kind of polynary aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon, which is characterized in that will
The three kinds of aptamer based on single-walled carbon nanotube-suspending chip magnetic detections described in claim 4 or claim 6 are micro-
Ball is mixed to get.
8. the polynary aptamer of single-walled carbon nanotube according to claim 7-suspending chip magnetic detection microballoon,
It is characterized in that, the amount of the substance of the described three kinds aptamers based on single-walled carbon nanotube-suspending chip magnetic detection microballoon
Than being 1:1:1.
9. aptamer of the claim 1-8 any one of them based on single-walled carbon nanotube-suspending chip magnetic detection is micro-
The detection method of ball, includes the following steps, the aptamer-suspending chip magnetic detection microballoon is mixed with object to be measured object
It closes, detects median fluorescent signal strength, establish examination criteria curve, obtain testing concentration；Wherein testing concentration and middle position
The negatively correlated relationship of fluorescence signal intensity.
10. aptamer of the claim 1-8 any one of them based on single-walled carbon nanotube-suspending chip magnetic detection is micro-
Ball detects the purposes of chemical pollutant；It is wherein 1 × 10 to the detection range of the chemical pollutant-13G/mL~1 × 10-7g/
ML, detection sensitivity >=1 × 10-15g/mL。
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| CN108152256B (en) * | 2017-12-11 | 2020-06-12 | 环境保护部华南环境科学研究所 | Sensitive high-selectivity good method for detecting BPA in water body |
| CN112834469B (en) * | 2020-12-28 | 2022-07-22 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection method and application of environmental estrogen |
| CN114199969B (en) * | 2021-12-03 | 2023-04-28 | 清华大学 | Nano electrode biosensor based on nucleic acid aptamer and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101198707A (en) * | 2004-05-12 | 2008-06-11 | 内诺斯佩尔公司 | Bio-barcode based detection of target analytes |
| EP1981994A2 (en) * | 2006-02-08 | 2008-10-22 | Oxonica Inc. | Sers nanotag assays |
| KR20090038597A (en) * | 2007-10-16 | 2009-04-21 | 한국전자통신연구원 | Signal detecting method for trace of biomolecule using photolytic oligomer and nano particle |
| CN103926407A (en) * | 2013-01-15 | 2014-07-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Multi-element suspension array detecting method for prostate tumor markers |
| CN104034712A (en) * | 2014-06-30 | 2014-09-10 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Kit for detecting thrombin with adapters and suspension chip |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043045A (en) * | 2009-10-26 | 2011-05-04 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | High-flux detection method of various pesticide and veterinary drug residues |
| EP2513650A4 (en) * | 2009-12-17 | 2013-12-18 | Abay Sa | Novel assays for detecting analytes in samples and kits and compositions related thereto |
| CN101812528B (en) * | 2010-04-26 | 2012-07-04 | 湖南大学 | Switch mode aptamer probe and application thereof in tumor living cell and vital detection |
| KR101467938B1 (en) * | 2010-04-29 | 2014-12-15 | 차의과학대학교 산학협력단 | Bio imaging probe for detecting intracellular target molecules and curing a disease |
| GB2489504A (en) * | 2011-03-31 | 2012-10-03 | Sapient Sensors | A device for identifying the presence of a specific target molecule or biomarker by sensing an electrical property |
| CN104651501A (en) * | 2015-01-30 | 2015-05-27 | 湖南大学 | Cracking activation type nucleic acid adaptor diagnosis and treatment probe and application thereof |
| CN107796938B (en) * | 2017-10-17 | 2020-03-27 | 中国农业科学院农业质量标准与检测技术研究所 | Nucleic acid aptamer fluorescent test strip and preparation method and application thereof |
-
2017
- 2017-03-21 CN CN201710168299.5A patent/CN106908598B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101198707A (en) * | 2004-05-12 | 2008-06-11 | 内诺斯佩尔公司 | Bio-barcode based detection of target analytes |
| EP1981994A2 (en) * | 2006-02-08 | 2008-10-22 | Oxonica Inc. | Sers nanotag assays |
| KR20090038597A (en) * | 2007-10-16 | 2009-04-21 | 한국전자통신연구원 | Signal detecting method for trace of biomolecule using photolytic oligomer and nano particle |
| CN103926407A (en) * | 2013-01-15 | 2014-07-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Multi-element suspension array detecting method for prostate tumor markers |
| CN104034712A (en) * | 2014-06-30 | 2014-09-10 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Kit for detecting thrombin with adapters and suspension chip |
Non-Patent Citations (2)
| Title |
|---|
| Label-Free Protein Biosensor Based on Aptamer-Modified Carbon Nanotube Field-Effect Transistors;Kenzo Maehashi et al;《Analytical Chemistry》;20061209;第79卷(第2期);5257-5263 * |
| Suspension Array with Shape-Code Silica Nanotubes for Multiplexed Immunoassay;Bo He et al;《Analytical Chemistry》;20070607;第79卷(第14期);782-787 * |
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