CN110082531A - A kind of tumour excretion body nano fluorescent detection kit and its application - Google Patents

A kind of tumour excretion body nano fluorescent detection kit and its application Download PDF

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CN110082531A
CN110082531A CN201910289902.4A CN201910289902A CN110082531A CN 110082531 A CN110082531 A CN 110082531A CN 201910289902 A CN201910289902 A CN 201910289902A CN 110082531 A CN110082531 A CN 110082531A
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excretion body
psma
prostate cancer
fluorescence
aptamer
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CN110082531B (en
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郑磊
李博
潘炜伦
刘春辰
司徒博
张晔
安泰学
张涵
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Southern Hospital Southern Medical University
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57434—Specifically defined cancers of prostate

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Abstract

It includes recognition component, fluorescent reporter group, fluorescent quenching group that the present invention, which provides a kind of tumour excretion body nano fluorescent detection kit and its application, the kit,.The present invention not only confirms that PSMA can be used as the biomarker for identifying health volunteer and patients with prostate cancer on the excretion body of human plasma source, and the method for further proving us can effectively avoid influence of the normal cell source excretion body to its testing result in clinical blood sample, with good anti-interference ability, there are biggish clinical application potentiality in terms of carrying out the body subgroup analysis of prostate cancer PSMA (+) excretion.

Description

A kind of tumour excretion body nano fluorescent detection kit and its application
Technical field
The present invention relates to field of biotechnology, are based on aptamer and gathering induced luminescence material more particularly to one kind " Turn-on " type tumour excretion body nano fluorescent detection kit and its application.
Background technique
In recent years, China's prostate-cancer incidence increases year by year, and most new patient diagnosed's middle and advanced stage ratios in area are higher than Europe Man, the U.S., and prostate cancer has the characteristics that quickly grow, easily shift, and diagnoses and controls in time before sings and symptoms appearance It treats, prognosis can be obviously improved, extend patient survival.Therefore sensitive, special, noninvasive, convenient, quick detection method is established For the early screening of prostate cancer, diagnosis or monitoring, seem outstanding to control prostate cancer development and raising life in patients It is important.
Liquid biopsy (Liquid Biopsy) obtains tumor information in body fluid by the sampling mode of Noninvasive, mainly Be discharged into including tumour or transfer stove circulating tumor cell (Circulating Tumor Cell, CTC) in extracellular environment, Circulating tumor DNA (ctDNA) and excretion body (Exosome) are that " precisely medical treatment " is representative to assist cancer diagnosis and treatment Diagnostic techniques.Compared with traditional tissue biopsy, liquid biopsy has rapid, convenient, the damaging many merits such as small.Substantially Degree shortens cancer while make a definite diagnosis the time, can the treatment condition to cancer patient tracked at any time, really realize that " individuation is smart Quasi- treatment ".Therefore liquid biopsy is expected to the ideal method as early prostate cancer screening, diagnosis or monitoring.
Early-stage study confirm as the excretion body of one of liquid biopsy " carriage drawn by a team of three horses " and the generation of prostate cancer, development, Transfer and drug resistance have certain correlation, it is considered to be the prostate cancer liquid biopsy biology with wide application prospect Marker.Biosensor technique is to realize sensitive, spy due to integrating the advantages that efficient, sensitive, special, small and exquisite, economic The important technological platform of different, noninvasive, convenient, quick prostate cancer liquid biopsy.With the research day of nanotechnology in recent years Gradually deeply, nano material is gradually introduced into the design and application of biosensor technique, and the nanometer designed on this basis is glimmering Optical biosensor because having many advantages, such as strong interference immunity, without reference device, amount of samples is few, easy to operate gradually causes people Concern therefore construct a kind of easy to operate based on nano fluorescent biosensor technique and there is highly sensitive and specificity Excretion body rapid detection method, be the urgent need of prostate cancer liquid biopsy.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of tumour excretion body nano fluorescents Detection kit, for solve in the prior art it is complicated for operation to the detection method of prostate cancer, time-consuming, anti-interference is weak etc. Problem.
In order to achieve the above objects and other related objects, the present invention provides a kind of for the detection of tumour excretion body nano fluorescent Kit, including recognition component, fluorescent reporter group, fluorescent quenching group.
Optionally, the recognition component is selected from aptamer.
Optionally, the nucleotide sequence of the aptamer contains the sequence as shown in SEQ ID NO.1-SEQ ID NO.4 Any one of column.
Optionally, the fluorescent reporter group is selected from aggregation-induced emission molecule (AIE molecule).
Optionally, the aggregation-induced emission molecule is selected from TTAPE (derivative of tetraphenylethylene).
Optionally, the TTAPE is selected from TPE-TA (tetraphenyl ethylene containing tertiary amine), [12] aneN3modified tetraphenylethene(TPE)compounds([12]aneN3Modified tetraphenyl ethylene (TPE) compound derives from DOI: 10.1021/acsami.6b01949), TPE-Py molecule (derive from DOI:10.1021/acs.analchem.5b04756), TPE-Z molecule (derives from DOI:10.1016/j.talanta.2017.02.064).
Above-mentioned AIE molecule is only partially enumerated, as long as the present invention can be suitable for the DNA AIE molecule combined.
Optionally, the fluorescent quenching group is selected from graphene oxide.
Optionally, the graphene oxide can be from market with a thickness of 0.8-1.2nm selected from single-layer graphene oxide On be commercially available, form can be sheet, powder, dispersion liquid, preferably powder.
Single-layer graphene oxide sheet, powder piece diameter be 500nm-5 μm.
It optionally, further include buffer.
Optionally, the buffer is selected from 1X PBS buffer solution.
Optionally, the concentration of the fluorescent reporter group is 0.4 μM, which is system concentration.
Optionally, the volume of the fluorescent reporter group is 0.5 μ L.
Optionally, the concentration of the fluorescent quenching group is 1mg/mL, which is system concentration.
Optionally, the volume of the fluorescent quenching group is 2 μ L.
Optionally, the volume of the buffer is 48.5 μ L.
Optionally, the total volume of the kit is 60 μ L.
Optionally, the concentration of the recognition component is 10 μM, and volume is 6 μ L.
The targeted excretion body of mentioned reagent box can derive from a variety of prostate gland cancer cells, including but not limited to LNCaP, PC3, DU145 cell specifically can be prostate cancer patient's blood plasma source excretion body.
The concentration of excretion body can be 4.07 × 105-1.83×107Particles/ μ L, volume are 3 μ L.
The present invention also provides above-mentioned fluorescence detection reagent kit prepare or screen prostate cancer preparation for diagnose and/or Application in the medicament of prevention and/or treatment prostate cancer, the kit are used for the in-vitro diagnosis and/or danger of prostate cancer Layering.
As described above, a kind of tumour excretion body nano fluorescent detection kit of the invention and its application, having following has Beneficial effect: the present invention not only confirms that PSMA can be used as identification health volunteer and prostate on the excretion body of human plasma source The biomarker of cancer patient, and the method for further proving us can effectively avoid normal cell in clinical blood sample and come Influence of the source excretion body to its testing result has good anti-interference ability, sub- carrying out prostate cancer PSMA (+) excretion body There are biggish clinical application potentiality in terms of cluster analysis.
Detailed description of the invention
Fig. 1 is shown as in the embodiment of the present invention sensing of " Turn-on " type excretion body nano fluorescent based on aptamer Technical schematic diagram.
Fig. 2 is shown as the LNCaP cell origin excretion body phenogram of the embodiment of the present invention, wherein (a) transmission electron microscopy Mirror figure, (b) NTA phenogram, (c) immunoblotting phenogram.
Fig. 3 is shown as in the embodiment of the present invention using binary channels super-resolution imaging technology in LNCap cell culture medium points From excretion body in CD63 and PSMA phenogram.(a) CD63 contaminates excretion body memebrane protein CD63；(b) Alexa Fluor 647 is glimmering Signal PSMA；(c) binary channels super-resolution blending image.
Fig. 4 is shown as in the embodiment of the present invention sensing of " Turn-on " type excretion body nano fluorescent based on aptamer Technology testing result figure, wherein (a) P1+TPE-TA；(b)exosome+P1+TPE-TA+GO；(c)P1+TPE-TA+GO；(d) The fluorescence spectra of TPE-TA.
Fig. 5 is shown as the second level of four kinds of PSMA aptamer:P1 (a), P2 (b), P3 (c), P4 (d) in the embodiment of the present invention Structure and physical parameter figure.
Fig. 6 is shown as the fluorescence signal response statistical chart and PAGE electrophoretogram of the embodiment of the present invention, wherein (a) ratio Best aptamer is screened compared with four kinds of PSMA aptamer sequence fluorescence signal responses；(b) PAGE electrophoresis result shows PSMA Aptamer P1 can be in conjunction with prostate cancer LNCaP excretion body, and swimming lane (1) to (6) is respectively (1) DNA ladder (20- 500bp)；(2)P1；(3)P1+107particles/μL exosome；(4)P1+108particles/μL exosome；(5)P1 +109particles/μL exosome；(6)109particles/μL exosome。
Fig. 7 is shown as TPE-TA concentration in the embodiment of the present invention, GO concentration, reaction temperature and reaction time to fluorescence response The influence diagram of value.(a) TPE-TA reaction density is 1.6nM, 3.2nM, 4.8nM, the fluorescence signal response under the conditions of 6.4nM； (b) GO reaction density is the fluorescence signal response under the conditions of 16 μ g/mL, 24 μ g/mL, 32 μ g/mL, 40 μ g/mL；(c) it reacts Temperature be 25 DEG C, 37 DEG C, 42 DEG C of fluorescence response value signal-to-noise ratio；(d) reaction time be 10min, 15min, 20min, 30min, The fluorescence response value signal-to-noise ratio of 45min, 60min, 75min.
Fig. 8 is shown as in the embodiment of the present invention " Turn-on " type prostate cancer excretion body nanometer based on aptamer Fluorescence sense technology detects specific statistical chart.
The sensitivity and linear detection range analysis chart, wherein (a) that Fig. 9 is shown as in the embodiment of the present invention are based on nucleic acid " Turn-on " type prostate cancer excretion body nano fluorescent sensing technology sensitivity of aptamers and linear detection range analysis chart. (a) various concentration PSMA (+) excretion body fluorescence spectra collected, from the bottom up along ordinate, the song of different colours are detected Excretion bulk concentration corresponding to line successively increases；(b) standard curve of various concentration PSMA (+) excretion body is detected.
Figure 10 is shown as in the embodiment of the present invention " Turn-on " type prostate cancer excretion body nanometer based on aptamer Fluorescence sense technology clinical samples testing result figure.(a) 7 Healthy Peoples and 20 prostate cancer patient plasma specimen PSMA (+) Fluorescence response value is surveyed in excretion physical examination；(b) scatter plot shows that Healthy People group and patients with prostate cancer group PSMA (+) excretion bulk concentration have Significant difference.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.
The present invention it is quasi- using prostate-specific membrane antigen (Prostate Specific Membrane Antigen, PSMA) aptamer (aptamer) specific recognition prostate cancer PSMA (+) excretion body subgroup, aggregation-induced emission molecule TPE-TA fluorescent reporter group and graphene oxide (Graphene Oxide, GO) fluorescent quenching group collectively form label-free " Turn-on " type fluorescence report system, and the inspection of prostate cancer PSMA (+) excretion body subgroup nano fluorescent bio-sensing is constructed with this Survey technology.On this basis, optimize analysis condition, verify clinical diagnosis performance, it is intended to for the prostate cancer of building quickly, easy Liquid biopsy new method provides experiment basis and theoretical foundation.
Sensitivity and specificity are had both for building, and excretion body rapid detection method, this seminar are close easily to operate Pay close attention to the progress of two elements necessary to excretion body nano fluorescent biosensor technique constructs.
(1) recognition component
Excretion body membrane structure is mainly by the bases group such as phospholipid bilayer, memebrane protein, skelemin and chaperone At, and specific membrane protein ingredient may mediate the specific function of different cell origin excretion bodies, also become different cell origins The major target that excretion physical examination is surveyed.Current research result is proved in addition to the highly expressed transmembrane protein of separate sources excretion body (such as CD63, CD9, CD81), outside the memebrane proteins such as MHC class Ⅱmolecule, excretion body surface face is special in early prostate cancer blood samples of patients circulation Property height expression memebrane protein prostate-specific membrane antigen (Prostate Specific Membrane Antigen, PSMA), therefore The excretion body subgroup of PSMA memebrane protein specificity overexpression, abbreviation PSMA (+) excretion body subgroup can be used as early prostate cancer sieve It looks into and is applied to clinical detection with the target of diagnosis.
Traditional memebrane protein recognition component is mainly memebrane protein specific antibody, in recent years, with in-vitro screening skill Art --- index concentration Fas lignand system evolution (Systematic Evolution Of Ligands By Exponential Enrichment, SELEX) research gradually deeply, people screen it is some can be with the aptamer of memebrane protein specific bond (Aptamer).Compared with antibody, aptamer is stable with chemical property, immunogenicity is low and easily flexible by various groups The advantages that modification, is widely used in the step of target identifies in biosensor technique.Such as Zhou, Q. etc. will CD63aptamer is coated on the excretion body constructed in electrochemical biosensor system quantitative detection serum on gold electrode, detection Sensitivity is 100 times of 3 antibody excretion body detection kit of anti-CD 6, and detects linear relationship and stablize.Therefore, this seminar is quasi- PSMA aptamer is screened as the ideal recognition component of prostate cancer excretion body for clinical application.
(2) fluorescence signal system
Since all cells of body can secrete excretion body, from specific tumors tissue or cell in Peripheral Circulation Excretion body will be diluted by a large amount of normal cell source excretion bodies, therefore it is glimmering to need that labeling effciency is high, fluorescence intensity is strong, background is low Light reporter group faces for sensitive instruction PSMA aptamer to the specific recognition of PSMA (+) excretion body subgroup to realize The quantitative detection of extremely low abundance PSMA (+) excretion body subgroup in bed sample.Currently, nano fluorescent biosensor technique only relies on biography System fluorescent dye and nano material (such as: quantum dot, upper transfer point, the nanoparticle for encapsulating organic fluorescent molecule) label biology Substance, that there may be labeling effciencies is not high for the above material, fluorescence intensity is low, background is high, is easy to happen photobleaching phenomenon, and highly concentrated It will appear aggregation Fluorescence-quenching when spending, be unable to satisfy the quantitative detection demand of low abundance excretion body.
Aggregation-induced emission (Aggregation-Induced Emission, AIE) molecule is that a kind of fluorescence signal-to-noise ratio is high And the novel nano fluorescent molecule of conventional fluorescent material aggregation fluorescent quenching disadvantage can be overcome, so far in chemistry, material The fields such as material science, life science attract extensive attention.Especially in biosensor technique field, because its solubility is good, biological Compatibility is good and its characteristic with aggregation-induced emission, and AIE nano molecular is enabled to be suitable for liquid phase to construct and detect " Turn-on " type fluorescence sense technology of system, this Technology design is easy, practical, operates particularly suitable for exploitation Easy highly sensitive tumour excretion body fluid body biopsy new method.Hong Y etc. reports specific binding DNA/RNA points a kind of The AIE nano molecular TTAPE (Tetraphenylethene Derivatives) of son, the basic unstressed configuration hair under free state It penetrates, and when there is DNA/RNA molecule to occur in solution, a large amount of TTAPE are gathered on DNA/RNA chain and start aggregation-induced emission There is fluorescence emission peak at 480nm in effect.And aptamer is substantially exactly single stranded DNA (ssDNA) molecule, therefore TTAPE Can and the aggregation-induced emission effect on PSMA aptamer quantitative detection PSMA (+) excretion body subgroup.
However, due to the free aptamer that there is unbonded excretion body in detection architecture, these aptamer can equally gather Collection TTAPE issues fluorescence in turn, to interfere the detection signal of PSMA (+) excretion body subgroup.Graphene oxide (Graphene Oxide, GO) be graphene a kind of derivative, containing there are many oxygen containing functionalizing group, show excellent hydrophily and plus Work performance.Other than high oxidation area, on surface there is also non-oxidizing graphite-like region, this region keeps graphene oxide To the strong adsorption ability of biomolecule, and organic fluorescence molecule is effectively adsorbed and fluorescence quenching.There is research to demonstrate,prove Real GO can effectively adsorb ssDNA molecule and AIE nano molecular.It therefore is the detection specificity for improving fluorescence signal system, this class Topic group proposed adoption nano molecular TTAPE and GO establish " Turn-on " type fluorescence report system, when in detection architecture without prostate cancer When PSMA (+) excretion body subgroup, free PS MA aptamer is adsorbed by GO, and quenches the TTAPE fluorescence signal assembled thereon；When In the presence of having prostate cancer PSMA (+) excretion body subgroup, PSMAaptamer falls off and makes in conjunction with target excretion body, from the surface GO The TTAPE that assembles thereon shines, and " Turn-on " type fluorescent assay signal occurs, and PSMA (+) is outside in the signal strength or weakness and solution Secrete the concentration correlation of body subgroup.
Based on previous research, and refering to amount of literature data, this project is quasi- to utilize PSMAaptamer specific recognition Prostate cancer PSMA (+) excretion body subgroup, AIE molecule TTAPE fluorescent reporter group is collectively formed with GO fluorescent quenching group exempts from " Turn-on " type fluorescence report system is marked, and prostate cancer PSMA (+) excretion body subgroup nano fluorescent biology is constructed with this and is passed Feel detection technique.On this basis, optimize analysis condition, verify clinical diagnosis performance, it is intended to for the forefront of building quickly, easy Gland cancer liquid biopsy new method provides experiment basis and theoretical foundation.
The present invention utilizes PSMAaptamer specific recognition prostate cancer PSMA (+) excretion body subgroup, and for the first time by AIE Molecule TPE-TA fluorescent reporter group and GO fluorescent quenching group collectively form label-free " Turn-on " type fluorescence report system, And prostate cancer PSMA (+) excretion body subgroup nano fluorescent bio-sensing detection technique is constructed with this, to realize prostate cancer The quantitative detection of PSMA (+) excretion body subgroup.This part experimental result confirms that PSMAaptamer identifies prostate cancer excretion body Specificity, and the feasibility of " Turn-on " type fluorescence report system based on TPE-TA and GO is certain density being added After the excretion body of prostate cancer cell line LNCaP source, the fluorescence response value of the nano fluorescent bio-sensing detection technique is obviously increased, Fluorescence response value signal-to-noise ratio (S/N) is about 6 times or so compared with control group (c).Based on this, we are related to this experiment important Reaction condition (mainly including TPE-TA concentration, GO concentration, reaction temperature and reaction time) is studied, according to experimental result point Analysis is as can be seen that when the optimum response concentration of fluorescent reporter group TPE-TA is 3.2nM, the optimum response of fluorescent quenching group GO Concentration is 32 μ g/mL, and under 37 DEG C of mild reaction conditions, and only by the reaction time of 15min, fluorescence signal is i.e. reachable Maximum value, this reaction condition can be rapidly completed on portable constant temperature instrument, and result interpretation can be carried out in the short time, thus into One step proves the easy and efficient of this detection method.
Further, we carry out elemental method to the quantitative detecting method of the prostate cancer PSMA (+) excretion body subgroup Evaluation.Firstly, we evaluate this nano fluorescent sensing technology to prostate gland cancer cell source PSMA (+) excretion body subsets counts Specificity.The excretion body subsets counts method established as the result is shown based on nano fluorescent sensing technology can effectively distinguish prostate Cancer LNCaP cell origin PSMA (+) excretion body subgroup and other tumor cell lines or normal cell source excretion body, and be avoided that Interference of the normal cell source excretion body to its testing result, has good specificity in blood plasma.Secondly, we are best anti- It is detected, and analyzed with a series of prostate cancer cell line LNCaP source excretion body of this method to various concentrations under the conditions of answering Fluorescence intensity at most by force transmitting light 480nm, it is sub- with PSMA (+) the excretion body for assessing this method detection prostate gland cancer cell source The range of linearity and sensitivity of group.The results show that the fluorescence signal of this method and prostate cancer cell line LNCaP source excretion body are dense Degree is positively correlated, and linear detection range is 4.07 × 105-1.83×107/ μ L, lowest detection is limited to 3.43 × 105Particles/ μ L, compared to the excretion body Fast Detection Technique that other are established based on aptamer, operation is easier, Reaction time is shorter, and sensitivity is also similar, has apparent application advantage.
In addition, we are in clinic in order to further verify the clinical application potentiality of constructed nano fluorescent sensing technology On have collected 20 prostate cancer patients and 7 human normal plasma samples, under optimum reaction condition, with method of the invention Fluorescence detection is carried out with Healthy People group blood plasma source excretion body to prostate cancer patient's group, to assess the clinical detection of this method Energy.PSMA (+) excretion bulk concentration of patients with prostate cancer is significantly higher than Healthy People as the result is shown, this experiment not only confirms human body PSMA can be used as the biomarker for identifying health volunteer and patients with prostate cancer on excretion body in blood, and illustrate me Method can effectively avoid influence of the normal cell source excretion body to its testing result in clinical blood sample, before progress Column gland cancer PSMA (+) excretion body subgroup analysis aspect has clinical application potentiality.
1, principle is illustrated
In the present invention, we use ssDNAPSMAaptamer as PSMA (+) excretion body recognition component, AIE molecule TPE-TA constructs label-free " Turn-on " type fluorescence report as fluorescent quenching group as fluorescent reporter group and GO jointly System is to realize the quantitative detection of PSMA (+) excretion body.Testing principle is as shown in Figure 1, ssDNAPSMAaptamer and TPE-TA After reaction, TPE-TA is gathered in formation aptamer/TPE-TA compound, aggregation inducing on PSMAaptamer single strand nucleotide sequence Luminescent effect leads to the rapid enhancing of fluorescence intensity in solution.Then, GO is added in reaction solution, because between aptamer and GO High strength bond power, aptamer/TPE-TA compound is adsorbed to the surface GO, and the fluorescent energy between AIE molecule and GO is total Vibration transferance quenches the fluorescence signal of aptamer/TPE-TA effectively.When in solution there are when PSMA (+) excretion body, Aptamer/TPE-TA compound can effectively be combined with PSMA (+) excretion body, thus weaken aptamer/TPE-TA compound with Combination between GO, and then GO is reduced to the fluorescence quenching of AIE molecule, so that being incorporated into PSMA (+) excretion body surface face Aptamer/TPE-TA complex fluorescence can be gradually recovered.
2, materials and methods
2.1 material
2.1.1 cell line
Human prostate cancer cell line: LNCaP is purchased from Shanghai Inst. of Life Science, CAS cell resource center, It is stored in liquid nitrogen.
2.1.2 main agents consumptive material
1 reagent consumptive material summary sheet of table
The bibliography of TPE-TA molecule are as follows: Dual-Mode Ultrasensitive Detection of Nucleic Acids via an Aqueous“Seesaw”Strategy by Combining Aggregation-Induced Emissionand Plasmonic Colorimetry, Jianlei Shen, Yiru Zhang, Rong Hu, Ryan T.K.Kwok,Zhiming Wang,Anjun Qin,and Ben Zhong Tang,ACS Appl.Nano Mater.2019, 2,163-169。
It is special that the specific structure of TPE-TA molecule reference may also be made to the Chinese invention that patent application publication number is CN 108949919A Sharp " a kind of aggregation-induced emission/surface plasma colorimetric analysis double mode nucleic acid detection method ", specification [0038- 0039] section elaborates molecular structure, preparation method bibliography " Hong, Y.；Xiong,H.；Lam,J.W.Y.； Ha uβler,M.；Liu,J.；Yu,Y.；Zhong,Y.；Sung,H.H.Y.；Williams,I.D.；Wong,K.S.；Tang, B.Z.Fluorescent Bioprobes:Structural Matching in the Docking Processes of Aggregation-Induced Emission Fluorogens on DNASurfaces.Chem.-Eur.J.2010,16, 1232-1245”。
2.1.3 key instrument
2 instrument title of table and producer's summary sheet
Nucleic acid sequence table involved in 3 the present embodiment of table
2.2 method
2.2.1 prostate cancer cell line LNCaP culture is isolated and purified with excretion body in cell supernatant
(1) cell culture
1) cell recovery: the prostate cancer LNCaP cell strain cryopreservation tube in liquid nitrogen container will be frozen first and is taken out, is turned rapidly It moves in 37 DEG C of constant water bath box and is allowed to quickly dissolve, shaking cryopreservation tube during being incubated for from time to time is heated evenly it.To It after cell is completely dissolved, takes 15mL centrifuge tube that 5mL RPMI-1640 culture solution is added, the cell suspension in cryopreservation tube is blown and beaten mixed Even to move in centrifuge tube, 1200rpm is centrifuged 2min, discards the supernatant in centrifuge tube, and 5mL complete medium is then added (10% fetal calf serum and 90%RPMI-1640 culture solution) is resuspended to mix and move back to 25cm2In Tissue Culture Flask, be placed in 37 DEG C, 5%CO2Wet constant incubator in cultivate 2-3 days after, cell state and density case are observed, to cell growth and breeding to covering Lid culture bottle or when 70-80% of culture dish floor space, pass on.
2) cell passes on: culture medium old in culture bottle or ware is discarded using liquid-transfering gun, PBS cleans culture bottle or ware 2-3 It is secondary, 1mL pancreatin is added, jog culture bottle or ware make pancreatin be uniformly distributed in cell surface, trypsin digestion cell 1min or so, Microscopically observation is set, if space between cells significantly increases, cellular morphology starts to be rounded, then 200 μ L serum are added and terminate digestion, add Enter appropriate PBS and gently blow and beat cell, after after cell detachment culture bottle or ware bottom and mixing, is transferred to 15mL centrifuge tube, 1200rpm is centrifuged 3min, inhales RPMI-1640 culture solution of the addition containing 10% fetal calf serum after abandoning supernatant and is resuspended, 1:3 or 1:4 Passage is placed on 37 DEG C, 5%CO2Wet constant incubator in cultivate.
3) cell supernatant collect and pre-treatment: observation LNCaP cell growth status, to cell growth and breeding to cover training It supports bottle or when ware floor space 60-70%, discards culture medium old in bottle or ware, PBS washer bottle or ware 2-3 time, addition serum-free RPMI-1640 culture solution, cultivate 12h in the incubator under the conditions of then same and (remove remaining serum secretion excretion body Influence), then culture medium old in bottle or ware is discarded, PBS washer bottle or ware 2-3 time are added containing 1-2%Exo-FBSTM(go excretion Body fetal calf serum) RPMI-1640 culture solution, continue to cultivate 48h, so that cell secretes a certain number of excretion bodies to culture solution In.LNCaP cell supernatant is collected after 48h and carries out pre-treatment, specific steps are as follows: 300g is centrifuged 10min and removes culture supernatant In residual cells, abandon precipitating collect supernatant；Cell fragment in 2,000g centrifugation 20min removal supernatants, abandons precipitating and receives Collect supernatant；10,000g centrifugation 30min, remove biggish vesica in supernatant, and it is spare to abandon precipitating collection supernatant.
(2) excretion body isolates and purifies in cell supernatant
With low temperature Ultracentrifuge L-80XP135,000g centrifugation 70min acquisition is precipitated as excretion body, is added suitable PBS continues 135,000g centrifugation 70min after mixing is resuspended to remove the impurity protein being dissolved in PBS, and final gained precipitating is It for purer excretion body, is finally resuspended and is precipitated with 100 μ L PBS, it is standby to be stored in -80 DEG C of refrigerators for the excretion body as isolated and purified With.
2.2.2 the morphosis of transmission electron microscope observation prostate cancer cell line LNCaP source excretion body
The excretion body isolated and purified is subjected to different multiples dilution, then therefrom draws the excretion body sample drop of 15 μ L in electricity On the dedicated copper mesh of mirror, the static 2min of room temperature blots liquid from the other side of copper mesh with filter paper, 20 μ L is then added dropwise on copper mesh 2% phosphotungstic acid dye liquor, static 10min, blots extra phosphotungstic acid under room temperature, 1-2 times wash with distilled water, uses filter paper Distilled water is blotted, 10min is stored at room temperature, it is dry to copper mesh.The copper mesh of load sample is taken to be put into sample installation with fine-pointed forceps sub-folder In slot, load onto gasket, fix, then by sample cell alignment transmission electron microscope injection port, at bayonet with thumb by it gently Push-in, after hearing sound, red light brightens.Lower switch to be opened, green light is seen and brightens, dextrorotation sample feeding pipe, sample will be automatically drawn into, Then 5 DEG C or so of convolution makes it suck bottommost, excretion volume morphing can be observed at transmission electron microscope H-7650 and clapped According to.
2.2.3NanoSight NS300 analyzes the concentration and its particle diameter distribution of prostate cancer cell line LNCaP source excretion body
Since the ideally machine particle concentration of NanoSight NS300 analyzer is 1 × 108-1×109particles/ ML, therefore need to be diluted the excretion body of separating-purifying before upper machine makes its concentration in ideal detection range, and upper machine Sample size is 1mL.Before analyzing sample, uses PBS as blank control, choose 405nm laser, observe sensing chamber's background value, use PBS cleans pipeline and sensing chamber until background occurs without particle bright spot.Then the dilute sample of 1mL is drawn with the syringe of 1mL Solution is slowly pushed into pipeline, carries out video capture after liquid stream is stablized, shoots the video for continuing 60s (30frames/s) every time. To guarantee that result is accurate, it is typically repeated sample introduction three times and detection, and the stability of operating table surface is kept during shooting.So Afterwards, threshold value is arranged to the particle in shooting video, reduces the counting of ion false positive and false negative.Then it is based on NTAsoftware (Version 3.2, NanoSight) software carries out calculating analysis to the particle of Brownian movement in video, in conjunction with The scattering strength and Einstein equation of particle obtain the concentration and Hydrodynamic diameter of detection particle, draw particle scattering The three-dimensional map of intensity, concentration and particle diameter distribution intensity.Finally, the sample that will test in room is withdrawn into syringe, disassembly swashs After light device and sense channel component, the remaining sample on laser is dried with lens wiping paper, and wash with water sense channel, after drying It places spare.
2.2.4Western Blot verifies CD63 albumen and PSMA egg on the excretion body of prostate cancer cell line LNCaP source White content
(1) main solution is prepared
10% Ammonium Persulfate 98.5 (AP): ammonium persulfate 0.1g adds deionized water 1mL to dissolve, and dissolution is placed on 4 DEG C of refrigerators and protects It deposits.
10%SDS: being added deionized water constant volume by SDS weight (g)/solution final volume (mL)=1:10 in SDS powder, It is filtered after mixing with filter paper, is placed in room temperature preservation.
5 × Tris- glycine electrophoresis liquid buffer solution
Room temperature preservation after dissolution, solution reusable 3~5 times.
1 × transfering buffering liquid
Room temperature preservation after dissolution, solution reusable 3~5 times.
TBST buffer
Confining liquid (the TBST buffer containing 5% skimmed milk power): in 1g skimmed milk power plus TBST constant volume is 20mL, is placed in Room temperature preservation.
10% separation gel and 5% concentration glue, TEMED are added before encapsulating.
4 separation gel of table, concentration glue component table
(2) excretion body protein is extracted and is quantified
1) extraction of excretion body protein
4%SDS, 100mM Tris-HCl, 1mM DTT is taken to be configured to the SDT lysate of pH 7.6, by 100 μ L lysates It is added in excretion body suspension, sufficiently after cracking, 14000g is centrifuged 15min, and taking supernatant is excretion body protein extract.
2) excretion body protein concentration quantifies
Sample determination of protein concentration is carried out using the micro BCA protein quantification kit of ThermoFisher.Utilize kit Middle protein standard solution configuration respective concentration protein liquid and sample to be tested, draw the sample of 25 μ L into 96 orifice plates respectively, then 200 μ L working solution l (by volume, A:B=1:50 are all added respectively；A liquid: 1%BCA, 2%Na2CO3·H2O, 0.16% Na2C4H4O6·2H2O, 0.4%NaOH, 0.95%NaHCO3, pH:11.25；B liquid: 4%CuSO4·5H2O.), 30s is shaken, it is thorough Bottom is sealed after mixing, and is incubated for 30min in 37 DEG C of water baths, is cooled to room temperature, then measures sample under microplate reader 562nm wavelength Product absorbance.With protein content (μ g) for abscissa, light absorption value is that ordinate draws standard curve.Sample to be tested according to survey inhale Shading value checks in corresponding protein content (μ g) on standard curve, according to suspension volume conversion protein concentration and applied sample amount.
3) protein sample is denaturalized: the volume ratio of protein sample and Loading buffer 4:1 is pressed in protein extract After addition Loading buffer, boiling water bath 10min, -20 DEG C of refrigerator storages.
(3) SDS-PAGE electrophoresis
1) two blocks of clean glass plates of cleaning treatment are stacked into bottom alignment, be fixed on bracket, distilled water is added extremely Glass plate upper limb waits 20-30min, and assessment liquid level declines situation, if decline > 5mm is retightened, outwells after determining sealing Distilled water simultaneously blots (a small amount of residual aqueous does not influence encapsulating) with filter paper.
2) encapsulating
10% separation gel is prepared by formula, is eventually adding AP and TEMED, mixes encapsulating immediately after TEMED is added；Separation gel Add to small glass plate 2/3 it is wide at, about (can suitably be added according to leak adhesive situation) at 1cm below the lower edge of insertion stripping fork, so Distilled water is added immediately afterwards to small glass plate edge, stands 30min (summer can foreshorten to 20 minutes), it is seen that apparent boundary Line illustrates that separation gel has solidified, and outwells the distilled water on glue, and blotted remaining water with filter paper；5% concentration glue is added to small Glass plate upper limb, is immediately inserted into loading stripping fork, and stripping fork is inserted in parallel into and avoids generating bubble by when insertion, in being stored at room temperature 30-45min is to be solidified.
3) loading
Stripping fork carefully to be extracted after gelling, and offset plate is removed from glue frame, two pieces of platelets are relatively fixed in electrophoresis tank, It is added at SDS electrophoretic buffer to platelet upper limb 5mm between two pieces of glass plates, it is slow that electrophoresis appropriate is poured between electrophoresis tank and glass plate Fliud flushing；It is loaded using micro syringe, draws 5 μ L of pre-dyed albumen Marker, 10 μ g of sample, when sample-adding reaches needle point Well bottom, is slowly carefully added into.
4) electrophoresis
Glue part is concentrated and uses low-voltage 80V, 20min, makes sample concentration at a narrowband, is moved to separation gel to band When, high voltage constant-voltage power supply 120V is selected, until bromophenol blue reaches bottom and stops electrophoresis.
(4) transferring film
It cuts glue: removing offset plate, carefully separate size glass plate, drawing is avoided to destroy glue, and prevent glue dry, according to mesh It marks molecular weight of albumen and cuts required part according to molecular size range shown in Marker, cut according to the size for cutting glue same big Small 6 filter paper and a pvdf membrane (shear angle is to indicate front and back sides).Pvdf membrane is impregnated into about 10s in 100% methanol, then It is put into distilled water rinsing 2-3min, transfers in transferring film buffer and balances 5min, filter paper is put into transferring film buffer and is impregnated 3-5min。
Transferring film folder (sandwich) production: transferring film folder, which is opened, keeps black one side holding horizontal, is padding a sea above Silk floss pad removes bubble removing with glass bar, 4 layers of filter paper is padded on cushion, fixed filter paper removes bubble removing with glass bar on the other hand on the other hand.Cutting Glue it is smooth be placed on filter paper, by membrane cover on glue, covering completely entire glue (can not move again after under membrane cover) and remove bubble removing.? Film upper cover 4 opens filter paper and removes bubble.Other side foam-rubber cushion is covered, after the bubble with glass bar removal the inside, closes clip.It is whole A operation carries out in transfer liquid, constantly rolls bubble.Transferring film folder is closed, transferring film is pressed from both sides black, white to red by black It is put into transferring film slot, transferring film buffer is filled it up in transferring film slot, be put into ice bag, homochromy electrode is opposite to connect power supply；Transferring film slot is put Enter in cold water and be put into ice bag, 200A constant current electrotransfer about 1h (wet turn of Bio-rad).
(5) it closes
Transferring film folder is opened, film is taken out and sees that marker is turned on pvdf membrane, indirect proof albumen electricity changes into function.It takes the film out It is placed in confining liquid, under room temperature, shaking table 80rpm/min closes 2h, and the test poor for antibody specificity can set film It is incubated overnight in 4 DEG C of refrigerators, with the non-specific sites in close membrane.
(6) primary antibody, secondary antibody are incubated for
It is separately added into the primary antibody dilution of CD63 and PSMA different proportion, plastic foil is closed, and is incubated on 4 DEG C of refrigerator shaking tables Night；Next day TBST washes film 3 times, each 5min；It is separately added into the anti-rabbit secondary antibody diluent of the horseradish peroxidase label diluted, Shaking table is incubated for 1-2h under room temperature, and TBST is cleaned film 3 times, each 10min.
(7) chemiluminescence is developed
Liquid A, B liquid are exposed by 1:1 (volume ratio) mixing, and each 1mL or so is placed in plate that (A liquid refers to that hydrogen peroxide is molten Liquid, B liquid refer to that luminol solution, A liquid, B liquid are purchased from Hangzhou Fu De Biotechnology Co., Ltd, and product information is as follows: FD Fdbio science,size:100ml；Fdbio-Dura ECL Kit；Enhanced Chemiluminescence kit HRP；Store at:4℃；Cat No:FD8020；Lot No:20190120)；Film is gently filtered dry on filter paper, is put into mixing A, B liquid in；Using ChemiDocXRS imaging system scan stripes band, the protein expression level that each band is shown is analyzed.
2.2.5 the expression of the verifying of super-resolution microscope imaging CD63 antibody and PSMA on excretion body film
(1) excretion body super-resolution imaging operating procedure
1) PLL-coated coverslip is covered on shooting ware, often with the Poly-L-lysine of 50 μ L 1mg/mL Warm 30min is incubated for, and appropriate PBS is washed 3 times.
2) (setting various concentration) in+40 μ L PBS of 10 μ L sample, 50 μ L are added in shooting ware, and room temperature is incubated for 30min.
3) 50 μ L Dilution C and 0.25 μ L PKH67 are added in the EP pipe of shading, mix.
4) the PKH67 suspension of mixing is added in shooting ware, is incubated at room temperature 4min, jog.
5) it sucks and the 1%BSA of 100 μ L is added after solution is incubated for 2min, appropriate PBS cleaning is three times.
6) the CD63 antibody being commercially available is diluted by the volume ratio of 1:400, the CD63 antibody incubation after taking 50 μ L to dilute 1h, appropriate PBS cleaning is three times.
7) it is incubated for 40min using the 647 mark fluorescent secondary antibody of goat-anti rabbit Alexa Fluor of 1:2000 dilution (volume), PBS is cleaned 3 times.
8) PBS covers sample area, 4 DEG C of preservations.
9) PBS is inhaled after abandoning, changes super-resolution imaging buffer into and covers entire sample area.
10) after being irradiated under 488nm and 647nm wavelength laser using Nikon N-SIM super-resolution microscope system Capture image.
2.2.6 " Turn-on " type excretion body nano fluorescent sensing technology feasibility confirmatory experiment based on aptamer
PSMA aptamer P4 nucleic acid sequence (being shown in Table 3) synthesis PSMA aptamer according to the literature is (with reference to text It offers: Development of a Single Stranded DNA Aptamer as a Molecular Probe for LNCap CellsUsing Cell-SELEX, Faezeh Almasi, Seyed Latif Mousavi Gargari, Fatemeh Bitaraf, and Samaneh Rasoulinejad, Avicenna Journal of Medical Biotechnology, Vol.8, No.3, July-September 2016), the pure solution of 60 μ L 3.2nM TPE-TA, 60 μ L are contained into 1 μM of PSMA respectively The 3.2nM TPE-TA of 3.2nM TPE-TA solution, 60 μ L containing 1 μM of PSMA aptamer and 32 μ g/mL GO of aptamer is molten Liquid and 60 μ L and concentration are 1 × 108A/μ L prostate cancer cell line LNCaP source excretion body be incubated for altogether after PSMA Aptamer/GO/TPE-TA solution carries out fluorescence signal detection, verifying PSMA aptamer identification on sepectrophotofluorometer After prostate cancer cell line LNCaP source PSMA (+) excretion body subgroup, " Turn-on " type fluorescence report based on TPE-TA and GO Aggregation-induced emission phenomenon caused by system.
2.2.7 fluorescence signal response detects
After opening sepectrophotofluorometer and software, " Status " is selected in " Application " menu, optical viewer State (including light source, light path system, sample cell and signal detection).In the case where instrument is in correct status, selection " Validation " fills deionized water using matched ware, confirms to instrument state.First it will test ware with 95% alcohol It cleans up, then is cleaned repeatedly with deionized water three times afterwards with being dried with nitrogen.Fluorescence signal response testing conditions are set: scanning Wave-length coverage is 370nm-650nm, and exciting light 350nm, exciting light and transmitting optical slits are 10nm, scanning speed 700nm/ Fluorescence intensity at min, analysis most by force transmitting light 480nm.With this condition, 60 μ L deionized waters are detected, it is glimmering in scanning range When luminous intensity is respectively less than 1, Samples detection can be carried out；Conversely, continuing cleaning until detection ware is qualified.
2.2.8PSMA the screening and verifying of (+) excretion body subgroup specificity PSMA aptamer
(1) PSMAaptamer is screened
PSMAaptamer pertinent literature is searched in PubMed database, finds out four kinds of PSMA having been reported Aptamer, respectively P1, P2, P3 and P4.The DNA of four kinds of PSMA aptamer is checked in aptamer database Aptagen Sequence (is shown in Table 3), and the secondary structure of four aptamer is predicted with online software Unpack.
The DNA sequence dna for synthesizing four kinds of PSMA aptamer, with 1 μM of four kinds of PSMA aptamer solution (solvent is DEPC water) Respectively with 1 × 108A/μ L prostate cancer cell line LNCaP source excretion body (being dissolved with PBS buffer solution) is incubated for altogether, and 3.2nM is added After TPE-TA molecule is protected from light, adds 32 μ g/mL GO and adsorb free aptamer and TPE-TA molecule, finally in fluorescence point Fluorescence signal detection is carried out on light photometer, and the PSMA of this test is most suitable for according to fluorescence signal-to-noise ratio (S/N) height selection aptamer。
5 reaction reagent table of table
(2) best PSMA aptamer identification PSMA (+) excretion body Asia is verified with non denatured polyacrylamide gel electrophoresis The ability of group
1) required plate glass (1mm edge strip), short glass plate, glue comb, glue bracket and glue frame are cleaned repeatedly, so It is rinsed well afterwards with deionized water, required apparatus is dried.
2) it will be inserted into the groove of vertical glue frame after clean glass plate alignment, pay attention to arrow upward, two sides folder Sub-folder fastens, and bottom, which is adjusted, ensures that lower end is concordant, and glue frame is fixed on glue bracket.
3) to deionized water is filled between plate glass and short glass plate in gap, observation 10min leaks hunting, guarantees to match Glue again after colloid system no leakage.
4) water in glue frame is poured out in inclination, and sufficiently absorbs residual water at each angle of glue frame with filter paper, spare.
5) 5 × tbe buffer liquid: weighing 54g Tris-base, 27.5g boric acid, 4.65g EDTA with electronic analytical balance, 500mL deionized water is added, and is stirred with glass bar to solution and is completely dissolved, then be settled to 1L, normal temperature storage.
The preparation process of 0.5 × tbe buffer liquid is as follows: graduated cylinder accurately measures 5 × tbe buffer liquid of 50mL, and is added The dilution of 450mL deionized water, and sufficiently shaken up in using preceding.
6) 10% AP: 1g ammonium persulfate powder is weighed with electronic analytical balance, 5mL deionized water dissolving is added, is used in combination Glass bar is stirred to solution and is completely dissolved, then is settled to 10mL, is placed in 4 DEG C of preservations.
7) NaCl solution (1M): weighing 58.44g NaCl powder with electronic analytical balance, and 500mL deionized water is added, and It is stirred with glass bar to solution and is completely dissolved, then be settled to 1L, normal temperature storage.
8) 3 × nucleic acid staining liquid: 45mL deionized water, 5mL 1M NaCl solution are taken, and the 4S Red of 15 μ L is added Plus nucleic acid staining agent (10 000 ×) mixes well and is placed on 4 DEG C and is kept in dark place.It needs to be balanced to room temperature before use, this Nucleic acid staining liquid is recyclable, reuses 3 times.
9) gel is prepared
According to PSMA aptamer molecular size range, 12% glue is selected, concrete composition ingredient such as table 1.4:
6 PAGE gel of table prepares formula table (12%Gel)
10) injecting glue
It after gel solution is configured by volume in step 9), mixes well, rapidly with sample loading gun by prepared gel Gap of the solution between plate glass and short glass plate slowly fills, and the generation of bubble is avoided in adition process.Then, close to Heavy sheet glass stick is gently combed glue in the gel solution between the gap of two pieces of glass plates of insertion, careful operation, avoids generating gas Bubble, need to exclude completely if any bubble.Gum-making rack is placed in 37 DEG C of 20~40min of water bath to solution solidification again.It is solidifying to gel Gu after completely, carefully extract glue comb vertically upward, the glue of solidification is placed in 0.5 × tbe buffer liquid with glass plate save it is standby With.
11) loading
According to the direction of short glass plate inwardly, two pieces of glue are carefully stuck in electrode cores with glass plate, then electrode cores are pressed Be fixed on electrophoresis tank according to corresponding electrode, toward (inside groove) between two pieces of glue be full of 0.5 × tbe buffer liquid, with plate glass Top edge is concordantly advisable.By the volume ratio of 5:1, by PSMA aptamer and various concentration (1 × 106,1×107,1×108 A/μ L) incubation altogether of 37 DEG C of prostate cancer LNCaP excretion body, it is incubated for product and is mixed well with 6 × loading buffer, carefully It is added in sample well, loading total volume is about 6 μ L, and the 20bp DNA of a 5 μ L of sample well addition is stayed in every piece of glue ladder marker。
12) electrophoresis
According to the quantity of electrode used therein core, 0.5 × tbe buffer solution is poured into graduation mark position toward electrophoresis tank outer groove.It connects Top electrode opens power supply, voltage is adjusted to 150V, electrophoresis time about 30min, is advisable with instruction band swimming to complete 2/3 position of glue. It after electrophoresis to required position, cuts off the power, recycles buffer, unload and open glass plate, carefully take out gel, use deionized water It rinses well.
13) nucleic acid staining
3 × 4S Red Plus nucleic acid staining the agent diluted is poured into clean container, gel is put into, uses aluminium-foil paper Covering container is protected from light coloring agent, is placed on shaking table, and 60rpm is mixed, and dyeing time is about 30min.
14) it images
Gel is placed in ultraviolet gel imaging system, is opened " TRANS UVI ", " Gel Doc XR " scanning is selected Device, and " Auto Exposure " mode is used, when nucleic acid bands shows clear, click " Freeze " freeze frame, and by result It is saved and is exported.
2.2.9 reaction condition influences test
TPE-TA concentration, GO concentration, reaction temperature and reaction time condition are studied respectively to the shadow of fluorescence signal response It rings.Successively detection TPE-TA reaction density is respectively 1.6nM, 3.2nM, 4.8nM, 6.4nM, and GO reaction density is respectively 16 μ g/ ML, 24 μ g/mL, 32 μ g/mL, 40 μ g/mL, reaction temperature are respectively 25 DEG C, 37 DEG C, 42 DEG C, the reaction time be respectively 10min, Under the conditions of 15min, 20min, 30min, 45min, 60min, 75min etc., " Turn-on " type excretion body based on aptamer The fluorescence signal response of nano fluorescent sensing detection method calculates fluorescence response value signal-to-noise ratio, finds the best of each parameter Reaction condition.
2.2.10 specificity analysis
Prostate cancer cell line LNCaP cells and supernatant is collected, ultracentrifugation method extracts excretion body, uses same method The breast cancer cell line MDA-MB-231 of extraction, normal liver cell system HL7702, the training of normal macrophages system RAW264.7 cell It supports supernatant source excretion body and human normal plasma source excretion body as a control group, observes its particle diameter distribution with NanoSight, Concentration is unified to 1 × 108A/μ L is detected.It is passed with " Turn-on " type excretion body nano fluorescent based on aptamer Sense detection method detects above five kinds of excretions body sample, records every group of fluorescence signal response, calculates fluorescence and rings Signal-to-noise ratio should be worth, detection is repeated three times to every kind of excretion body sample, and evaluate the detection specificity of this method.
2.2.11 sensitivity analysis
Under the optimum experimental condition optimized, respectively to " Turn-on " type excretion body nanometer based on aptamer The LNCaP cell origin excretion body that 3 μ L various concentrations are added in fluorescence sense detection architecture is reacted, after reaction with glimmering Light spectrophotometer detects fluorescence signal response, calculates fluorescence response value signal-to-noise ratio.To the excretion body sample of each concentration It repeats detection three times, records testing result, and calculate analysis and obtain detection prostate cancer cell line LNCaP source PSMA (+) excretion The range of linearity and Monitoring lower-cut of body subgroup, to explore the detection sensitivity of this method.
2.2.12 clinical is assessed
20 prostate cancer patients and 7 human normal plasma marks are collected in Hospital of Southern Medical University clinical laboratory This, take 500 μ L plasma specimens, through 300g, 2,000g, 10,000g centrifugation after, with micro ultracentrifuge with 54,000rpm from Heart 2h, PBS is resuspended after collecting precipitating, with identical pelleted by centrifugation, collects blood plasma source excretion body.Blood plasma sample is measured using NTA The concentration of this excretion body takes identical excretion bulk concentration to carry out subsequent experimental.Under optimum reaction condition, we are glimmering with this nanometer Light sensing detection method carries out fluorescence detection to prostate cancer patient's group and Healthy People group blood plasma source excretion body, this experiment repeats It carries out three times, to evaluate " Turn-on " type excretion body nano fluorescent sensing technology clinical samples inspection based on aptamer Survey ability.
1) TEM, NanoSight and Western Blot identify the excretion body in ultracentrifugation method purification cell supernatant
Using the excretion body in ultracentrifugation method purification prostate cancer cell line LNCaP cell line source.Such as Fig. 2 (a) institute Show, TEM image shows that the excretion body extracted through ultracentrifugation has the structure of complete film property lipid bilayer, and straight Diameter is in 100nm or so, with document report (Shao Huilin, Im Hyungsoon, Castro Cesar M et al.New Technologies for Analysis of Extracellular Vesicles.[J].Chem.Rev.,2018,118: Point out that excretion body magnitude range is 30-200nm in 1917-1950. document at the 2.1.Vesicle Formation of page 1918) It is consistent；Then this research is again using the excretion body particle diameter distribution and concentration in NanoSight analysis sample, if Fig. 2 (b) is shown, The average grain diameter of excretion body is 106.7 ± 2.1nm, is consistent with the result of TEM, and concentration is 2.22 × 109±2.39×108A/ mL；Utilize significant PROTEIN C D63 and the detection with prostate cancer diagnosis value in Western blot detection excretion body sample Property albumen PSMA expression such as Fig. 2 (c) shown in, the LNCaP cell protein of selection and withdrawal is used as positive control, based on BCA Albuminimetry makes each sample well applied sample amount be 20 μ g, it can be seen that the excretion body in LNCaP prostate cancer cell line source Both albumen are expressed, provide experimental basis for next research.
2) expression of the super-resolution microscope imaging verifying CD63 and PSMA on excretion body film
According to the literature, significant PROTEIN C D63 and the detection with prostate cancer diagnosis value in excretion body sample Albumen PSMA is memebrane protein, further to confirm this conclusion, this project using super-resolution imaging characterized by techniques CD63 and The common location of PSMA and excretion body film, to verify CD63 and PSMA on the excretion body film of LNCaP prostate cancer cell line source Expression.As shown in Fig. 3 (a), green fluorescence group is the excretion body handled by CD36-FITC, and CD63 can be with excretion body Memebrane protein CD63 is combined, and excretion body (green group) diameter is shown in figure in 100nm or so, the excretion body for meeting NTA characterization is straight Diameter size, Fig. 3 (b) are handled excretion body memebrane protein PSMA with red fluorescence dyestuff Alexa Fluor 647, Fig. 3 c be by Excretion body membrane channels and the fused effect picture of excretion body memebrane protein channel image, excretion body film and excretion body memebrane protein (green Fluorescence and red fluorescence) common location confirm CD63 and PSMA on the excretion body film in LNCaP prostate cancer cell line source Expression.
3) " Turn-on " type excretion body nano fluorescent sensing technology feasibility verifying based on aptamer
In order to verify the feasibility of this nano fluorescent biosensor technique, we are by 1 μM of PSMA aptamer and prostate After 37 DEG C of cancer LNCaP cell origin excretion body is incubated for 15min altogether, after addition 3.2nM TPE-TA molecule is protected from light 10min, add Enter 32 μ g/mL GO and adsorb free aptamer and TPE-TA molecule, fluorescence signal detection is carried out on sepectrophotofluorometer, together When by the pure solution of 3.2nM TPE-TA, be added 1 μM of PSMA aptamer 3.2nM TPE-TA solution and 1 μM of PSMA The 3.2nM TPE-TA solution of aptamer and 32 μ g/mL GO are carried out at 480nm wavelength with sepectrophotofluorometer glimmering respectively Light detection.As a result as shown in figure 4, the pure solution of TPE-TA (the fluorescence response value of the curve d) of Fig. 4 is minimum；In the pure solution of TPE-TA (the curve a) of Fig. 4, fluorescence response value sharply increase figure after PSMA aptamer is added；(Fig. 4 after addition fluorescent quenching group GO Curve c), fluorescence response value drops to close to baseline level；When there are (the curves of Fig. 4 when PSMA (+) excretion body in solution B), it is effectively combined due to aptamer with excretion body, the TPE-TA compound assembled thereon cannot be quenched by GO, fluorescence response Value obviously increases, and with control group, (fluorescence response value signal-to-noise ratio (S/N) is about 6 times or so compared with the curve c) of Fig. 4.
4) PSMA (+) excretion body subgroup specificity PSMA aptamer screening and verifying
Four kinds of PSMA aptamer are compared in " Turn-on " type fluorescence report system based on TPE-TA and GO, and it is preceding Fluorescence signal-to-noise ratio (S/N) data after column gland cancer LNCaP cell origin excretion precursor reactant, as shown in Fig. 5 (a)-(d), Ke Yifa Now fluorescence signal-to-noise ratio (S/N) highest (Fig. 6 (a)) of the LNCaP excretion body of PSMA aptamer P1 identification under the same conditions.With Online software NUPACK predicts the secondary structure and melting temperature of four PSMA aptamer (P1, P2, P3, P4).Four kinds of analysis DNA sequence dna and the structure of PSMA aptamer is it is found that the DNA base number of PSMA aptamer P1 is most, and secondary structure is the most Complexity, therefore more TPE-TA molecules can be accommodated and be gathered in aptamer sequence, the fluorescence signal intensity launched is higher. Therefore, this experiment is quasi- selects P1 as best PSMA aptamer.
With 1 μM of best PSMA aptamer P1 and various concentration (1 × 107,1×108,1×109Particles/ μ L) before The reaction product of column gland cancer LNCaP excretion body carries out the verifying of PAGE electrophoresis, as shown in Fig. 6 (a), Fig. 6 (b), as the result is shown P1 and Various concentration (1 × 107,1×108,1×109Particles/ μ L) after prostate cancer LNCaP excretion precursor reactant, residual ionization P1 Amount it is different, and as LNCaP excretion body quantity increases, the amount of free P1 is constantly reduced, thus can also indirect proof PSMA The ability of aptamer P1 identification prostate cancer cell line LNCaP source excretion body.
5) reaction condition development test
The important reaction condition that this experiment is related to mainly includes TPE-TA concentration, GO concentration, reaction temperature and reaction time. In this regard, we devise a series of experiments to observe detection effect of this method under the conditions of differential responses, to grope to reach The optimum reaction conditions of optimum detection performance.
For AIE nano molecular TPE-TA as fluorescence signal group, reaction density directly affects this nano fluorescent biology biography The fluorescence response value signal-to-noise ratio of sensor.To inquire into TPE-TA optimum response concentration, experimental setup blank control group (excretion is not added Body) and experimental group (1 × 108Particles/ μ L excretion body), in the case where other reaction conditions are constant, observation is a series of not Influence with TPE-TA reaction density (1.6nM, 3.2nM, 4.8nM, 6.4nM) to two groups of fluorescence response values and signal-to-noise ratio.Such as Fig. 7 (a) shown in, as TPE-TA concentration increases, the fluorescence response value of control group and experimental group increases, this is because TPE-TA points Son have multiple positive charge, can by electrostatic attraction in solution dissociate PSMA aptamer combine after sending high intensity it is glimmering Light in the case that other conditions are constant, leaves the TPE-TA combined on the free PS MA on the surface GO in fluorescence response value and solution Molecular amounts are directly proportional, therefore the fluorescence response value of control group and experimental group is increased as the concentration of TPE-TA increases；Fluorescence Response signal-to-noise ratio is the ratio between experimental group and control group fluorescence response value, the energy of reflection this method difference target excretion body presence or absence Power, when TPE-TA concentration is 3.2nM, fluorescence response value signal-to-noise ratio reaches maximum value, illustrates to be greater than when TPE-TA concentration 3.2nM, although experimental group fluorescence response value still increases as the concentration of TPE-TA increases, because of control group solution middle reaches It is more from TPE-TA aggregation on PSMA aptamer, and high concentration TPE-TA may cause excessive TPE-TA reunite in the solution and It shines, background signal increase is caused to become apparent from, instead decline fluorescence response value signal-to-noise ratio.Therefore, select 3.2nM for TPE-TA Optimum response concentration.
For GO as fluorescent quenching group, reaction density in this experiment also directly affects this nano fluorescent biosensor Fluorescence response value signal-to-noise ratio.Best GO concentration is reacted to inquire into, experimental setup blank control (excretion body is not added) and experiment Group (is added 108A prostate cancer excretion body), in the case where other reaction conditions are constant, it is dense to observe a series of difference GO reactions Spend the influence of (16 μ g/mL, 24 μ g/mL, 32 μ g/mL, 40 μ g/mL) to fluorescence response value and signal-to-noise ratio.As shown in Fig. 7 (b), with GO concentration increase, the fluorescence response value of control group and experimental group reduces, this is because GO has very various fluorogens High quenching ability, in the case that other conditions are constant, fluorescence response value is inversely proportional with GO concentration in solution, thus control group and The fluorescence response value of experimental group is reduced as the concentration of GO increases；But when GO concentration is 32 μ g/mL, fluorescence response value noise Than reaching maximum value, illustrate when GO concentration is greater than 32 μ g/mL, although experimental group fluorescence response value is still with the concentration liter of GO It is high and reduce, but the quenching ability of control group GO cannot increase with concentration, cause background signal that cannot continue to reduce, instead Decline fluorescence response value signal-to-noise ratio.Therefore, select 32 μ g/mL that can further decrease background signal simultaneously for GO optimum response concentration Improve the sensitivity of measurement.
Temperature can change the structure, recognition capability and reaction rate of aptamer, and then to the sensitivity of reaction and specifically Property affects.To probe into influence of the differential responses temperature to reaction result, experimental setup blank control (target excretion is not added Body) and experimental group (addition 108A target excretion body), in the case where other reaction conditions are constant, observe a series of different temperatures The influence of (25 DEG C, 37 DEG C, 42 DEG C) to fluorescence response value and signal-to-noise ratio.As shown in Fig. 7 (c), when reacted between be 37 DEG C when, it is glimmering Photoresponse value signal-to-noise ratio reaches maximum value, this result may indicate that under the too low environment of temperature, influences because of energy deficiency The recognition capability and reflection efficiency of aptamer；When environment temperature is excessively high, the aptamer for participating in reacting is likely to form unstable Secondary structure, to influence fluorescence response value and stability.Therefore, the optimal reaction temperature of the cancer excretion body detecting method It is determined as 37 DEG C.
Optimum reacting time can not only make detection method make effective response to target, farthest method for improving Sensitivity, and can utmostly shorten detection time.To investigate influence of the differential responses time to reaction result, experiment is set It sets blank control (target excretion body is not added) and experimental group (is added 108A target excretion body), in the feelings that other reaction conditions are constant Under condition, a series of differential responses times (10min, 15min, 20min, 30min, 45min, 60min, 75min) are observed to fluorescence The influence of response and signal-to-noise ratio.As shown in Fig. 7 (d), when reacted between be 15min when, fluorescence response value reaches maximum value.It Afterwards, as the reaction time increases, fluorescence response value does not rise anti-drop, this may be with the exciting light of TPE-TA through a long time in the solution The lower generation fluorescent quenching of irradiation is related.Therefore, 15min is selected both to can guarantee fluorescence response as the optimum reacting time of this method Value maximizes, and detection efficiency can be improved, and realizes the rapid sensitive detection of PSMA (+) excretion body subgroup.
6) specific test
The diversity of excretion body subgroup protein biomarkers becomes more complicated single subgroup excretion body counting.? In this research, the analysis detection to excretion body subgroup is the specific recognition based on PSMA aptamer to PSMA (+) excretion body, Therefore, this method has excellent specificity.In order to evaluate this nano fluorescent sensing technology to prostate gland cancer cell PSMA (+) outside The specificity for secreting body subsets counts, under optimum reaction condition, we carry out fluorescence inspection to LNCaP exo with this detection method It surveys, while recording breast cancer cell excretion body (MDA-MB-231exo), normal liver cell excretion body (HL-7702exo), mouse The fluorescence of normal macrophages excretion body (RAW264.7exo) and human normal plasma's excretion body (Plasma exo) four control groups Response signal-to-noise ratio, this experiment repeat three times, the specificity surveyed to evaluate the detection method to PSMA (+) excretion physical examination (experimental results are shown in figure 8).Before the experiments, using NTA measure LNCaP exo, MDA-MB-231exo, HL-7702exo, The concentration of RAW264.7exo and Plasma exo is respectively 3.66 × 108、2.22×108、4.22×108、2.86×108、 1.08×108particles/μL.After appropriate dilution, so that the excretion bulk concentration in each sample is 1 × 108particles/μ L.Result of study shows that four groups of control group fluorescence detection response signal-to-noise ratio are respectively less than 1, and LNCaP excretion body fluorescence detection responds It is worth signal-to-noise ratio and is greater than 5, is significantly higher than other four groups of fluorescent assay signals.It is noted that human normal plasma source excretion body Fluorescence detection response signal-to-noise ratio be only 1/10th of prostate gland cancer cell source excretion body fluorescence response value signal-to-noise ratio.It should The result shows that can effectively distinguish prostate cancer based on the excretion body subsets counts method that nano fluorescent sensing technology is established LNCaP cell origin PSMA (+) excretion body subgroup and other tumour cells or normal cell source excretion body, and can effectively avoid Interference of the normal cell source excretion body to its testing result, has good specificity in blood plasma.
7) sensitivity test
To assess the detection method based on the foundation of nano fluorescent sensing technology for the PSMA in prostate gland cancer cell source The sensitivity of (+) excretion body subsets counts, we are with this method to a series of prostate cancer cell line LNCaP source of various concentrations PSMA (+) excretion body subgroup is detected.We by the excretion body in prostate gland cancer cell source carry out gradient dilution (1.83 × 107,1.53×107,1.22×107,6.10×106,3.66×106,1.22×106, 6.10 × 105,4.07× 105Particles/ μ L), as the target substance of the system, under optimum reaction condition (37 DEG C, 15min), with the detection side Method analyzes the excretion body (3 groups of parallel tests) of these various concentrations.It can be seen that from fluorescence spectra collected With the increase of excretion bulk concentration, most the transmitting collected fluorescence intensity in light place accordingly enhances by force, i.e., acquires within this range To fluorescence intensity be positively correlated with excretion bulk concentration.According to fluorescence spectrum Fig. 9 (a), each experimental group is acquired at 480nm Fluorescence signal is for statistical analysis and linear fit obtains Fig. 9 (b): being 4.07 × 10 in excretion bulk concentration5-1.83× 107Within the scope of particles/ μ L, fluorescence intensity is directly proportional to tumour cell quantity；By linear fit can be obtained this method into The mathematical model of PSMA (+) excretion body subsets counts in row prostate gland cancer cell source, linear equation y=1.139x+51 (y is fluorescence intensity, and x is the concentration of PSMA (+) excretion body), related coefficient 0.994；3 times of standards are added according to blank signal Signal value corresponding to difference estimates detection limit, and the lowest detection that this method is calculated is limited to 3.43 × 105particles/μL。
2.2.12 clinical detection Performance Evaluation
In detection method building and method benchmark test, the excretion body sample that we use is both from cell Culture supernatant, in order to further verify whether constructed method has analysis feasibility and clinical application potentiality, we are facing 20 prostate cancer patients and 7 human normal plasma samples are had collected on bed, this research is cured by Southern Medical University The examination & approval of Ethics Committee, institute, all subjects and family members obtain and sufficiently inform and sign informed consent form.Prostate cancer is suffered from The diagnosis of person meets " Chinese urological diseases diagnoses and treatment guide (2014 editions) " standard.
After taking 500 μ L blood plasma first with 10,000g centrifugation 30min pretreatment, with ultracentrifuge with 54,000rpm centrifugation 2h, PBS is resuspended after collecting precipitating, with identical pelleted by centrifugation, collects blood plasma source excretion body.Blood plasma excretion is measured using NTA The concentration of body sample takes identical excretion bulk concentration to carry out subsequent experimental after diluting.Under optimum reaction condition, we are with originally Detection method carries out fluorescence detection with Healthy People group blood plasma source excretion body to prostate cancer patient's group, this experiment repeats three It is secondary, to evaluate the detection method clinical detection performance.The results are shown in Figure 10, according to the detection method in prostate cancer patient PSMA (+) excretion of group and patients with prostate cancer known to the fluorescence intensity comparing result of Healthy People group blood plasma source excretion body Bulk concentration is significantly higher than Healthy People.
In conclusion the present invention not only confirms that it is tested to can be used as identification health by PSMA on the excretion body of human plasma source The biomarker of person and patients with prostate cancer, and the method for further proving us can effectively avoid in clinical blood sample Influence of the normal cell source excretion body to its testing result has good anti-interference ability, is carrying out prostate cancer PSMA (+) excretion body subgroup analysis aspect has biggish clinical application potentiality.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.
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Claims (10)

1. a kind of kit for the detection of tumour excretion body nano fluorescent, it is characterised in that: including recognition component, fluorescence report Group, fluorescent quenching group.
2. kit according to claim 1, it is characterised in that: the recognition component is selected from aptamer.
3. detection kit according to claim 2, it is characterised in that: the nucleotide sequence of the aptamer contains Any one of sequence as shown in SEQ ID NO.1-SEQ ID NO.4.
4. kit according to claim 1, it is characterised in that: the fluorescent reporter group is selected from aggregation-induced emission point Son.
5. kit according to claim 4, it is characterised in that: the aggregation-induced emission molecule is selected from tetraphenylethylene Derivative, it is preferable that the derivative of the tetraphenylethylene be selected from TPE-TA, [12] aneN3Modified tetraphenyl ethylene chemical combination At least one of object, TPE-Py, TPE-Z.
6. kit according to claim 1, it is characterised in that: the fluorescent quenching group is selected from graphene oxide.
7. kit according to claim 6, it is characterised in that: the graphene oxide is selected from single-layer graphene oxide.
8. kit according to claim 7, it is characterised in that: the single-layer graphene oxide is with a thickness of 0.8-1.2nm.
9. kit according to claim 1, it is characterised in that: it further include buffer, preferably PBS buffer solution.
10. kit described in -9 any one is being prepared or is being screened for diagnosing and/or preventing and/or control according to claim 1 The application in the medicament of prostate cancer is treated, the kit is used for the in-vitro diagnosis and/or risk stratification of prostate cancer.
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