CN107233941B - A kind of multiple near-infrared fluorescent enhancing biochip screening circulating tumor cell method - Google Patents

A kind of multiple near-infrared fluorescent enhancing biochip screening circulating tumor cell method Download PDF

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CN107233941B
CN107233941B CN201710271597.7A CN201710271597A CN107233941B CN 107233941 B CN107233941 B CN 107233941B CN 201710271597 A CN201710271597 A CN 201710271597A CN 107233941 B CN107233941 B CN 107233941B
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chip
cell
ctc
circulating tumor
fluorescence enhancement
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CN107233941A (en
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王国新
廖滔
钱昆
唐梅杰
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Nada Biotechnology Co
Shenzhen Micro Wentz Biotechnology Co Ltd
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Nada Biotechnology Co
Shenzhen Micro Wentz Biotechnology Co Ltd
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Priority to PCT/CN2017/110453 priority patent/WO2018196335A1/en
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Abstract

The invention proposes a kind of technologies for enrichment cycles tumour cell.The core of this technology is the micro fluidic device based on plasma fluorescence enhancement chip.The device includes: ontology, and the fluid circulation space of upper opening is limited in the ontology；The bottom of the ontology is arranged in injection port, the injection port；The bottom of the ontology is arranged in outlet, the outlet；The upper opening of the fluid circulation space is arranged in plasma fluorescence enhancement chip, the plasma fluorescence enhancement chip, and the lower surface load of the plasma fluorescence enhancement chip has antibody；The upper surface of the plasma fluorescence enhancement chip is arranged in magnetic field generating assembly, the magnetic field generating assembly.Using micro fluidic device according to an embodiment of the present invention, the simple separation and efficient capture and enrichment of circulating tumor cell may be implemented, and can be to the highly sensitive detection for carrying out NIR enhancing fluorescence for the enrichment cycles tumour cell being enriched to.

Description

A kind of multiple near-infrared fluorescent enhancing biochip screening circulating tumor cell method
Technical field
The present invention relates to field of biological detection, in particular it relates to which multiple near-infrared fluorescent enhancing screening circulation is swollen Oncocyte, more particularly, the present invention relate to the micro fluidic device of enrichment cycles tumour cell, for enrichment cycles tumour it is thin The kit of born of the same parents, the method for enrichment cycles tumour cell, the system that circulating tumor cell is detected and to circulating tumor The method that cell is detected.
Background technique
The characteristics of liquid biopsy is due to its Noninvasive and Precise Diagnosis has in clinical research and biomedical detection There is huge prospect.Particularly, kinds cancer is preferably being managed to the analysis of the circulating tumor cell (CTC) in blood samples of patients In play a crucial role.It include that biopsy and imaging method are different from conventional method, CTC can be commented in accurate screening, treatment Estimate, patient provides new opinion and opportunity by stages, in the transfer of Cancerous disease/recurrence research.However, enrichment to CTC and under Trip analysis is challenging, because their abundance in blood are extremely low, a usual CTC is blended in billions of In other haemocytes.In order to construct high-performance CTC measuring method in clinic, need to consider following aspect, comprising: I) capture The enrichment method of CTC；II the analysis method of CTC) is detected；III) practical application handles blood samples of patients；IV) laboratory automation is big Scale uses.It is in demand for solving the new tool of above-mentioned aspect, and the material and device for needing to rationally design.
Plasma material is commonly referred to as noble metal (such as gold) and its compound, the light in particular range of wavelengths There is unique surface plasmon resonance effect according to lower.Because having specific structure and surface chemical property, it is this it is equal from Daughter material has enhancement effect of fluorescence near infrared region (NIR-FE, 650-1700nm), by micro-printing technology in this material Specific albumen probe is fixed on material, it may be achieved the NIR-FE medical diagnosis on disease of biomarker.It is worth noting that, current etc. Gas ions excimer chip is only integrated with microarray technology, and is not yet introduced into other micro-/ nano devices for high level diagnostics. Meanwhile although on chip NIR-FE Molecular Detection success, another challenge is cell detection on chip, work before Merely due to the reinforcing effect of pdp body chip itself, but be a lack of for rare cell (such as CTC) analysis operation and Multiple NIR fluorescence enhancement step only can provide limited reinforcing effect (2-30 times).
Therefore, exploitation design device integrates it with NIR-FE cell detection on chip, and overcomes mentioned in this field Major obstacle have great importance.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.
In this application, inventor illustrate for the first time the multiple enhancing NIR fluorescent applications of plasma gold chip in CTCs screening.Inventor is combined the microfluid immunomagnetic enrichment of CTC with NIR-FE detection on chip.Pass through cell enrichment Operation and its morphologic change, inventor observe about 50-122 times of multiple NIR fluorescence enhancement, and the detection better than other chips is imitated Fruit.Inventor detects multiple protein biomarkers on the CTC of capture, and this method is applied in addition standard The detection of cell and cancer patient's blood sample.The work of the application not only facilitates the effective cancer of advanced CTC analysis clinically Management, and facilitate the bioanalysis application design at the interface of plasma material/between equipment and cell.
In the first aspect of the present invention, the invention proposes a kind of micro fluidic devices for enrichment cycles tumour cell. According to an embodiment of the invention, described device includes: ontology, the fluid circulation space of upper opening is limited in the ontology； The bottom of the ontology is arranged in injection port, the injection port；The bottom of the ontology is arranged in outlet, the outlet； The top of the fluid circulation space is arranged in plasma fluorescence enhancement chip, the plasma fluorescence enhancement chip The lower surface load of opening, the plasma fluorescence enhancement chip has antibody；Magnetic field generating assembly, the magnetic field generating assembly The upper surface of the plasma fluorescence enhancement chip is set.It, can be with using micro fluidic device according to an embodiment of the present invention Realize the simple separation and efficient capture and enrichment of circulating tumor cell, and can be to the enrichment cycles tumour cell being enriched to Carry out NIR enhancing fluorescence highly sensitive detection.Inventors have found that under conditions of having the microfluid immunomagnetic enrichment of CTC About 50-122 times of the multiple NIR fluorescence enhancement of plasma fluorescence enhancement chip (pGold), much higher than two of the detection of other chips The best report of the order of magnitude and the prior art.
According to an embodiment of the invention, above-mentioned micro fluidic device can further include following additional technical feature at least One of:
According to an embodiment of the invention, the magnetic field generating assembly is magnet.
According to an embodiment of the invention, the plasma fluorescence enhancement chip is removably disposed in the fluid flowing The upper opening in space.
In the second aspect of the present invention, the invention proposes a kind of kits for enrichment cycles tumour cell.According to The embodiment of the present invention, the kit include: magnetic nano-particle, and the magnetic nano-particle is suitable for catching from the blood Obtain circulating tumor cell；It is described previously for the micro fluidic device of enrichment cycles tumour cell.Using implementing according to the present invention The kit of example, may be implemented the simple separation and efficient capture and enrichment of circulating tumor cell, and can be to being enriched to The highly sensitive detection for carrying out NIR enhancing fluorescence of enrichment cycles tumour cell.Inventors have found that immune in the microfluid for having CTC About 50-122 times of the multiple NIR fluorescence enhancement of plasma fluorescence enhancement chip (pGold) under conditions of magnetite gathering is much higher than it Two orders of magnitude of his chip detection and the best report of the prior art.
According to an embodiment of the invention, mentioned reagent box can further include following additional technical feature at least it One:
According to an embodiment of the invention, the magnetic nano-particle area load has circulating tumor cell specific marker point Son.And then can realize the specific marker of circulating tumor cell in sample (such as blood sample), using according to embodiments of the present invention Kit the bioaccumulation efficiency of circulating tumor cell is further increased.
According to an embodiment of the invention, the specific marker molecule is anti-EpCAM.Specifically, the magnetic nano particle Sub (MNPs) is the magnetic nano-particle (MNPs) with epithelial cell adhesion molecule antibody (anti-EpCAM) functionalization, and then is entered The circulating tumor cell of the fluid circulation space can the compound action selective enrichment of gravity and magnetic field force (MNPs) from The surface of daughter fluorescence enhancement chip.
In the third aspect of the present invention, it is thin using mentioned-above kit enrichment cycles tumour that the invention proposes a kind of The method of born of the same parents.According to an embodiment of the invention, the described method includes: (1) is by the sample containing circulating tumor cell and the magnetic Property nanoparticle mixing, to form magnetic nano-particle-circulating tumor cell complex；(2) by the mixture via institute The injection port for stating micro fluidic device enters the fluid circulation space, and the fluid circulation space is discharged from the outlet, Wherein, the magnetic nano-particle-circulating tumor cell complex is enriched in described under the action of the magnetic field generating assembly The lower surface of plasma fluorescence enhancement chip.Using according to the method for the embodiment of the present invention, circulating tumor cell may be implemented It is simple separation and efficient capture and enrichment, and can to the carry out NIR for the enrichment cycles tumour cell being enriched to enhance it is glimmering The highly sensitive detection of light, the plasma fluorescence enhancement chip (pGold) under conditions of having the microfluid immunomagnetic enrichment of CTC About 50-122 times of multiple NIR fluorescence enhancement, much higher than two orders of magnitude of other chips detection and the best report of the prior art.
In the fourth aspect of the present invention, the invention proposes the systems that a kind of pair of circulating tumor cell is detected.According to The embodiment of the present invention, the system comprises: mentioned-above device；And fluorescence detection device.Using implementing according to the present invention The simple separation and efficient capture and enrichment of circulating tumor cell may be implemented in the system of example, and can be to the richness being enriched to The highly sensitive detection for carrying out enhancing fluorescence of collection circulating tumor cell.
According to an embodiment of the invention, above system can further include at least one following additional technical feature:
According to an embodiment of the invention, the fluorescence detection device is NIR fluorescence detection device.Inventors have found that having The multiple NIR fluorescence enhancement of plasma fluorescence enhancement chip (pGold) under conditions of the microfluid immunomagnetic enrichment of CTC is about 50-122 times, using NIR fluorescence detection device, system significantly improves the sensitivity of the detection of CTC.
In the fifth aspect of the invention, the invention proposes the methods that a kind of pair of circulating tumor cell is detected.According to The embodiment of the present invention, which comprises method as described above carries out the circulating tumor cell in sample rich Collection, to obtain the plasma fluorescence enhancement chip for being enriched with the circulating tumor cell；Utilize NIR fluorescence detection device pair The circulating tumor cell of the plasma fluorescence enhancement chip enrichment carries out target molecule detection.Using according to the present invention The highly sensitive detection of the NIR enhancing fluorescence of enrichment cycles tumour cell may be implemented, in the miniflow for having CTC in the method for embodiment About 50-122 times of the multiple NIR fluorescence enhancement of plasma fluorescence enhancement chip (pGold) under conditions of body immunomagnetic enrichment, Much higher than two orders of magnitude of other chips detection and the best report of the prior art.
The technology for enrichment cycles tumour cell that the application is proposed, it is glimmering that the core of this technology is based on plasma The above-mentioned micro fluidic device of light enhancing chip.
Detailed description of the invention
Fig. 1 is the micro fluidic device schematic diagram of enrichment cycles tumour cell according to an embodiment of the present invention；
Fig. 2 is the system schematic according to an embodiment of the present invention detected to circulating tumor cell；
Fig. 3 is CTC screening installation figure according to an embodiment of the present invention,
Wherein, a be screening process and device schematic diagram (on) and viewgraph of cross-section (under)；B shows pGold chip and mould The digital picture of the integration of block (left side) and the SEM image of pGold chip (right side)；C shows the biology mark of CTC on pGold chip The schematic diagram of the NIR fluorescence detection of will object albumen, RBC refer to red blood cell, and for the SEM image in b illustration, scale bar is 500nm；
Fig. 4 is the characterization result of MNP according to an embodiment of the present invention,
Wherein, a SEM, b TEM and c are the B-H loop of MNP.D is by magnet Magnetic Isolation (from colloidal suspension Liquid) MNP, scale bar is respectively a) 200nm and b) 100nm；
Fig. 5 is the extinction spectra of pGold chip according to an embodiment of the present invention and the excitation of IRDye680 and IRDye800 (line) and transmitting (shadow region) area coincidence；
Fig. 6 is multiple NIR fluorescence enhancement result figure according to an embodiment of the present invention,
Wherein, the NIR fluorescence for being enriched with CTC (marks) image (top, illustration display light field cyclogram by IRDye800 Picture) and average fluorescent strength (lower part), including a) MCF-7 on chip, b) and SKBR-3 and c) COLO-205, used chip Including (i) glass-chip, (ii) sGold chip, (iii) not the pGold chip of immunomagnetic enrichment and (iv) have it is immune The pGold chip of magnetite gathering.For a-c) in all fluorescent images, scale bar be 10 μm；
Fig. 7 is the scanning fluorescent image of CTC according to an embodiment of the present invention,
Wherein, a) MCF-7 being recorded on chip, b) SKBR-3 and c) NIR fluorescence (IRDye800) figure of COLO-205 Picture, used chip include (i) glass-chip, (ii) sGold chip, the pGold chip of (iii) not no immunomagnetic enrichment (iv) has the pGold chip of immunomagnetic enrichment.For a-c) in all fluorescent images, scale bar be 100 μm；
Fig. 8 be it is according to an embodiment of the present invention without and with magnetite gathering CTC size,
Wherein, the average-size of the CTC on the pGold chip without and with magnetite gathering, including MCF-7, SKBR- 3 and COLO-205.Black and grey do not respectively indicate not and the average-size of the CTC with magnetite gathering, analyze three cells To calculate the standard deviation (s.d.) as error bars, data are shown as average value ± s.d.(n=3)；
Fig. 9 is the mechanism results figure of multiple enhancing according to an embodiment of the present invention,
Wherein, a) schematic diagram, show (i) under no immunomagnetic enrichment, the NIR fluorescence of the enhancing of CTC (MCF-7) and (ii) the multiple enhancing NIR fluorescence of the CTC on pGold chip with immunomagnetic enrichment.B) side view of CTC and c) top view SEM image (i) has immunomagnetic enrichment without immunomagnetic enrichment and (ii) on pGold chip.Scale bar is b) and c) In be 5 μm；
Figure 10 is the multiple analysis result figure of biomarker according to an embodiment of the present invention,
Wherein, a) glass-chip and b) fluorescence microscopy images of the CTC (MCF-7) on pGold chip, c) glass-chip And d) CTC (MCF-7) scan image on the fluorescence of pGold chip.It is left: the channel IRDye680 of anti-EGFR；It is intermediate: anti-CK's The channel IRDye800；It is right: the amalgamation result in two channels.A) illustration on right side shows bright field image.In a) and b), ratio Ruler is respectively 10 μm；
Figure 11 is the capture rate result figure of CTC according to an embodiment of the present invention,
Wherein, a) identification of the CTC of capture shows that (i) light field of the CTC (MCF-7) of capture, (ii) DAPI mark, (iii) anti-CK label, and the fluorescent image that (iv) three merges, the b in (i) PBS solution and (ii) whole blood) MCF-7, c) The capture rate of SKBR-3 and d) COLO-205.Five independent experiments are carried out to calculate standard deviation, and the data shown are logical Cross 5 times calculating average value ± s.d show (n=5) on the diagram, a) in scale bar be 10 μm；
Figure 12 is the scanning fluorescence analysis knot of two from multiple cells kind biomarker according to an embodiment of the present invention Fruit figure,
Wherein, the multiple CTC (MCF-7) captured on a) glass-chip and b) pGold chip by NIR Fluorescence Scanner Multi-biological analysis of markers.It is left: the channel IRDye680 of anti-EGFR；Intermediate: the channel IRDye800 is anti-CK；It is right: two The amalgamation result in channel, scale bar are 20 μm in a) and b).
Figure 13 is the qualification result figure of capture CTC according to an embodiment of the present invention,
Wherein, a) SKBR-3 that is captured on pGold chip and b) (i) light field of COLO-205, (ii) DAPI label, (iii) anti-CK label, scale bar are 10 μm in a) and b)；
Figure 14 is the selection result figure of CTC in cancer patient according to an embodiment of the present invention,
Wherein, a is the quantity of the CTC of the 5mL whole blood core on piece capture from 11 cancer patients；B is shown from trouble The leucocyte (CD45+/DAPI+/CK-, on) of person 1 (lung cancer) and the CTC of a capture (CD45-/DAPI+/CK+, under) Identification fluorescent image, scale bar is 5 μm in b)；And
Figure 15 is the qualification result figure of CTC in cancer patient according to an embodiment of the present invention,
Wherein, three fluorescence method shows a leucocyte (upper left, CD45+/DAPI+/CK-) fluorescence from patient 5 CTC (bottom right, CD45-/DAPI+/CK+) fluorescent image of image and a capture: a) channel FITC of anti-CD45, b) anti-CK The channel IRDye800, c) nucleus the channel DAPI and d) merge channel, scheme a-d in scale bar be 10 μm.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
On the one hand, the invention proposes a kind of micro fluidic devices for enrichment cycles tumour cell.It is according to the present invention Embodiment, with reference to Fig. 1, which includes: ontology 100, and the fluid circulation space of upper opening is limited in ontology 100； The bottom of ontology 100 is arranged in injection port 200, injection port 200；The bottom of ontology is arranged in outlet 300, outlet 300；Deng The upper opening of fluid circulation space is arranged in gas ions fluorescence enhancement chip 400, plasma fluorescence enhancement chip 400, etc. The lower surface load of gas ions fluorescence enhancement chip 400 has antibody；Magnetic field generating assembly 500, the magnetic field generating assembly setting In the upper surface of plasma fluorescence enhancement chip 400.Specifically, magnetic field generating assembly 500 is magnet.Plasma fluorescence increases Strong chip 400 is removably disposed in the upper opening of fluid circulation space.
On the other hand, the invention proposes the systems that a kind of pair of circulating tumor cell is detected.Reality according to the present invention Example is applied, with reference to Fig. 2, which includes: mentioned-above device 1000；With fluorescence detection device 2000.Specifically, fluorescence detection Equipment 2000 is NIR fluorescence detection device.
Using micro fluidic device according to an embodiment of the present invention and system, the simple separation of circulating tumor cell may be implemented With efficient capture and enrichment, and can to the enrichment cycles tumour cell being enriched to carry out NIR enhancing fluorescence it is highly sensitive Detection.Plasma fluorescence enhancement chip (pGold) multiple NIR is glimmering under conditions of having the microfluid immunomagnetic enrichment of CTC Light enhances about 50-122 times, much higher than two orders of magnitude of other chips detection and the best report of the prior art.
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this reality With novel, and it is not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according in the art Document described in technology or conditions or carried out according to product description.Production firm is not specified in agents useful for same or instrument Person, being can be with conventional products that are commercially available.
In following experiment, specific method used and material are as described below:
Chip preparation
The chip of three types, including golden (pGold) chip of plasma are prepared, golden (sGold) chip and glass are sputtered Chip.There is in the solution of optimization by the seed law synthesis pGold chip of abundant nano island.In short, glass slide is existed It is vigorously stirred lower immersion HAuCl4(5mM) and NH420 minutes in OH (20 μ L/mL, 0.6%, V%) solution.By the glass slide of acquisition It successively immerses in water-bath twice and washs.After washing, it is molten that the glass slide that Au ion is inoculated with is put into sodium borohydride (1mM) at room temperature In liquid 1 minute to be restored, and washed by successively immersing in water-bath twice.These glass slides are transferred to molar ratio For another HAuCl of 1:14And NH4Continued growth in OH mixed solution, for Au film.Growth course is vigorously shaken at room temperature (100RPM) 20 minutes, and wash to obtain final pGold chip.PGold chip is stored at room temperature sealable In container.
SGold chip prepares that (GSL-1100X-SPC-16M, Shenyang section crystalline substance automation equipment have by magnetically controlled sputter method Limit company).Gold target (99.99%) is purchased from Shanghai Daheng Optical Fine Mechinery Co., Ltd.Sputtering process is at vacuum (10Pa) With 20mA progress 2 minutes.SGold chip is stored at room temperature in sealable container.
Sheet glass is bought from Chinese Sinopharm Chemical Reagent Co., Ltd..Glass slide is immersed into 15mL Piranha solution (3/1, V/V, in the concentrated sulfuric acid (mass fraction 95%) and hydrogen peroxide (mass fraction 30%) 5 minutes to remove organic residue, Three times and room temperature is maintained at pure water.
Micro fluidic device building
The critical component of microfluidic device includes chip module, fluid pump (19*13*6.2mm, 350-400mT, 42.8* 42.3*48.3mm), sample cell (15mL centrifuge tube, U.S. company BD), bar magnet (19*13*6.2mm, 350-400mT) and control System is provided by ningbo of china Mei Jing medical technology Co., Ltd is unified.The mold of integrated chip is made and is used with polypropylene The size of optimization is designed with layout.The air stream in driving pipeline is pumped with miniflow to adjust flow rate of liquid (1mL/h).
Magnetic nanoparticle preparation
Magnetic nanoparticle (MNP) epithelial cell adhesion molecule antibody (EpCAM antibody) functionalization, in order to cell Immunomagnetic enrichment.The MNP of marked by streptavidin is purchased from MicroMod company (Rostock, Germany).We pass through strepto- Specificity interaction between Avidin and biotin has synthesized the antibody functionalized immunomagnetic nanoparticles of EpCAM.Letter Yan Zhi, MNP are washed twice with 50mM phosphate buffered saline (PBS) (PBS, pH=7.4,50mM), by MNP and antibody at 37 DEG C Mixture vibrates 1.5 hours, make its with biotinylated EpCAM antibody (Abcam Inc., Cambridge, USA) (100:1, W/W) sufficiently reaction.After being washed twice again with 50mM PBS (pH=7.4), trip is removed by centrifugation (200G, 5 minutes) and magnetic force From EpCAM antibody.Gained functionality MNP is resuspended to the 50mM PBS containing 3% bovine serum albumin(BSA) (BSA, Sigma) (pH=7.4) in buffer, for closing and being stored at 4 DEG C.
Material and facility characterization
The characterization of material and device include transmission electron microscope (TEM), scanning electron microscope (SEM), magnetic hysteresis analysis, UV, visible light (UV) spectrum, digital picture and video.Digital picture and video are shot by Nikon d7100 to be obtained.UV spectrum is logical Cross AuCy UV1901PC spectrophotometer measurement.TEM image is shot at 10kV by JEOL JEM-2100F TEM instrument. 10 μ L ethyl alcohol (0.1 μ g/mL) particle suspension liquid is dripped on copper mesh, carries out tem observation after air-drying to sample.Pass through under 10kV Hitachi S-4800 carries out sem analysis.By particle suspension liquid drop on silicon and after being dried at room temperature for, in SEM device In directly observe.Use vibrating specimen magnetometer (Quantum Design, Physical Property Measurement System) hysteresis loop (recycling magnetic field between -50 and 50kOe) is measured.
Cell culture
Use three kinds of cells of scheme culture of report, including MCF-7 (human breast cancer cell line, EpCAM are positive), SKBR-3 (human breast cancer cell line, EpCAM are positive) and COLO-205 (human colon cancer cell line, EpCAM are positive).During all cells come from The academy of sciences, state (Shanghai cell resource center research institute), and moist 5%CO at 37 DEG C2It is cultivated in incubator.MCF-7 is thin Born of the same parents cultivate in Eagle (Sigma) culture medium.The Eagle culture medium that SKBR-3 cell culture is improved in Dulbecco In (DMEM, Sigma).COLO-205 cell culture is in Roswell Park Memorial Institute culture medium (RPMI- 1640) in.In order to obtain free cell, 1mL trypsase (0.25%, V/V) is instilled in Tissue Culture Flask, in CO2Culture It is digested 5 minutes in case, and the cell of acquisition is stored in PBS (pH=7.4,50mM) buffer.
Standard addition experiment
It is molten that above-mentioned three kinds of cell line (MCF-7, SKBR-3 and COLO-205) is respectively added to 50mM PBS (pH=7.4) In liquid and whole blood, to measure the capture rate of our methods.Whole blood sample is contributed by healthy volunteer.In short, by 6-200 Cell is added in 5mL PBS (pH=7.4,50mM) solution and whole blood.Number based on capture cell (n) and mark-on cell (N) Amount by immunomagnetic enrichment and counts fluorescence analysis calculating capture rate (n/N).
The immunomagnetic enrichment of CTC
In order to carry out immunomagnetic enrichment, by the functionalization MNP of 30 μ g and biological mixture (5mL mark-on sample or patient Blood) 1.5 hours are incubated at 37 DEG C with selectively labeling CT C.Said mixture is added to the sample of microfluidic device Guan Zhong is connect with chip loading module.Flow velocity is 1mL/h, needs~5h completion enrichment.The MNP of CTC will be labeled as in magnetic field In (from magnet) be attached to the surface of chip, and continuously with containing 0.05%tween-20 (PBST) PBS (pH=7.4, It 50mM) washs three times.Gained chip ice acetone soln (99%, V%) is protected from light processing 15 minutes, and is stored at 4 DEG C.
Near infrared fluorescent dye labelled antibody
Antibody is detected with fluorescent dye (IRDye800/680) label CK detection antibody and EGFR.Antibody passes through EDC/NHS It reacts (1- ethyl -3- (3- dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide) and IRDye800/680 is marked. IRDye680-NHS/IRDye800cw-NHS ester (Licor Biosciences) is dissolved in anhydrous dimethyl sulphoxide, and is made It is kept in dark place with preceding at -20 DEG C.Antibody and dyestuff are mixed with the molar ratio of 1:4 and to be placed on oscillator 1.5 in the dark small When.The antibody of the dyestuff not being coupled and purification tag is removed using NAP-5 column (GE Healthcare).After EDC/NHS is reacted Raw mixture be added in the column with 10mL PBS (pH=7.4,50mM).The eluent containing labelled antibody is collected, And it is stored under the conditions of being protected from light at -20 DEG C.
Chip dyes online
In order to dye to the biomarker protein for being attached to cell surface, chip is cleaned three times with PBST, then It is incubated for 1.5 hours with the detection antibody of dye marker (250 μ L, 5 μ g/mL) in the dark at 37 DEG C.Then, by the core of dyeing Piece is washed three times with PBST, to remove impurity.For staining cell core, with 4', 6- diamidino -2-phenylindone (DAPI, 5mg/ ML it) dyes 15 minutes, is then washed three times with PBST (pH=7.4,50mM, 0.05%Tween-20).Chip obtained exists 4 DEG C are stored in front of near infrared imaging and scanning.
Scanning electron microscope and microscope imaging
Detection and identification is carried out to the cell on chip with microscope imaging by scanning.Innopsys scanner (InnoScan 710) is used to scan chip, thus to cell imaging.The scanning resolution of all experiments is set as 3 μm/pixel. Pass through NIS-Elements BR 4.51.00 (ECLIPSE Ti-E related software, Nikon) analysis of fluorescence scan image, fluorescence Intensity is up to 65536.With specific colour filter ECLIPSE Ti-E (Nikon) fluorescence microscope (for DAPI, different sulphur cyanogen Sour fluorescein (FITC), IRDye680/800) for recording the microscope imaging of cell.
The CTCs of cancer patient is analyzed
It collects the 5mL whole blood from cancer patient and is captured for chip CTC.Knowing for patient has been obtained when project initiation And agreement.Cancer patient is diagnosed according to comprehensive cancer network (NCCN) guide of country.Eliminate the medicine such as active hemorrhage Situation.CTC is identified by the fluorescent staining result (CD45-/DAPI+/CK+) with morphological analysis, and to cancer patient's CTCs number counts.All research approaches of this work are through Ningbo the second hospital institution Ethics Committee and Shanghai Communications University School of Biomedical Engineering's approval.
Experimentation and result of the invention described in detail below:
The design of CTC screening installation
Inventor devises a kind of microfluidic device to carry out the immunomagnetic enrichment of CTC on chip, as shown in Figure 3a.Hair Bright people is using positive enrichment method, with the magnetic nano-particle of epithelial cell adhesion molecule antibody (anti-EpCAM) functionalization (MNPs) it marks blood samples of patients or adds the CTCs in standard cell.According to Fig. 4 a, scanning electron microscope (SEM) in 4b and Transmission electron microscope (TEM) analysis, the MNP of functionalization has~average-size of 25-40nm, and saturation magnetisation value is~ The remaining magnetism of 0.9emu/g (cyclical field between -50 and 50kOe, Fig. 4 c), can use magnet quick separating in 1 minute (Fig. 4 d).Then inventor is by biological mixture by being pumped into microfluidic channel, and the group cooperation for passing through gravity and magnetic field force With selective enrichment CTC, and for finally detecting.
Integrated and detection on chip
One important feature of the device of inventor is to be easily integrated and detected using various chips, such as Fig. 3 b institute Show in order to integrated chip, inventor develops the microfluid seal modules of No leakage, is removably attached to chip On surface (Fig. 3 b, left).Inventor is prepared for golden (pGold) core of plasma by a kind of controllable solution seed mediated growth method Piece, SEM is observed island is dispersed on chip between distance (Fig. 3 b, right) about 10nm the unique form gold nano island.For on chip Detection, the characteristics of pGold chip is that can provide plasma in the NIR region in its absorption/extinction spectra (Fig. 5) total Vibration.Inventor marks the biomarker protein of enrichment CTC for analyzing on chip with NIR fluorescent dye (IRDye800/680) (Fig. 3 c).
The NIR-FE detection of CTC on chip
Inventor has carried out CTC (MCF-7, SKBR-3 and COLO- of three types on selected chip (Fig. 6,7) 205) NIR-FE detection, including glass-chip, golden (sGold) chip of sputtering and pGold chip are (with and without immune magnetic Property microfluid enrichment).As shown in Figure 6 a, very weak signal is provided for MCF-7, glass-chip and sGold, mean fluorecence is strong Degree is respectively 637 and 1456, this is because that there are fluorescent quenchings on NIR-FE detection or chip surface.In order to compare, send out Bright people realizes that the NIR-FE of cell is detected using the pGold chip of not magnetite gathering, average fluorescent strength 5136 is generated, with glass Fluorescence intensity on glass chip, which is compared, increases 8.1 times.
It is worth noting that, inventor observes that cell size increases 30.4% (from~136.4 for magnetite gathering To~177.9 μm2, Fig. 8), and the further NIR fluorescence signal of enhanced CT C, compared with glass-chip, it is possible to provide mean fluorecence Intensity 32540 and 51.1 times of enhancement factor (enhancing 55.3 times compared with subtracting background signal).Inventor using SKBR-3 and When COLO-205 is detected, it has been found that similar as a result, they distinguish 62.2 times, (background signal subtracted is 69.1 times, figure Enhancement factor and 95.3 times of enhancement factors 6b) (background signal subtracted is 122.0 times, Fig. 6 c).Inventor is total in table 1-3 These have been tied as a result, and confirming (51.1-122.0 times) of multiple NIR fluorescence enhancement on pGold chip detection.
Table 1, the average fluorescent strength analysis of MCF-7:
Table 2, the average fluorescent strength analysis of SKBR-3:
Table 3, the average fluorescent strength analysis of COLO-205:
The mechanism of multiple enhancing
The mechanism of multiple NIR fluorescence enhancement detection is inventors herein proposed, and has carried out the characterization (Fig. 9) of SEM.Conventional is thin Born of the same parents NIR-FE detection lacks the optimization of the average distance between the operation and cell and pGold chip surface of cellular morphology.It compares Under, inventor compresses cell by magnetic force in immune magnetic microfluid enrichment process, and reduces cell and pGold chip list Average distance between face, result lead to multiple enhancement effect (Fig. 9 a).As shown in figure 9b, the side view SEM figure of pGold chip As not respectively illustrating not and the form of the cell with Magnetic Isolation normal and compression (thickness of reduction is about 2 μm).This Outside, in overlooking SEM, inventor be also found due to compression process, and cell size increases, this and side view SEM and previous fluorescence Imaging results (Fig. 9, Fig. 8) unanimously, demonstrate the multiple enhancing mechanism proposed.
Multiplexed protein labeled analysis
Inventor illustrates the multiplexed protein matter biomarker analysis of CTC by the channel IRDye680 and IRDye800 (Figure 10).Inventor detects two kinds of protein biological markers of cancer cell on the channel IRDye800 and IRDye680 respectively Object, including cytokeratin (CK) and EGF-R ELISA (EGFR).Inventor is seen by the microscope on pGold chip It observes clearly cell image (many cells analysis in the single cell analysis and Figure 11 in Figure 10 a), in better than two channels Result on glass-chip.The CTC being enriched on chip is imaged it is worth noting that, inventor also passes through micro scanning (multiple cell analysis in the single cell analysis and Figure 12 in Figure 10 b), result are observed consistent with microscope, and send out Bright this technology of people with bigger detection high throughput and easily operated etc. may have during actual rare cell analysis The advantages of conducive to extensive automation application.
Capture rate and identification
Inventor is by the equipment of the experimental studies inventor of addition standard cells a series of to the capture rate of CTC (Figure 11).Inventor identifies the CTC of enrichment by the fluorescent staining in Figure 11 a, and illustrates the typical image of a CTC. Inventor mixes 6-200MCF-7 in standard solution (Figure 11 b-i) or whole blood (Figure 11 b-ii), and counts the number of the CTC of enrichment Amount.Capture rate reaches 86.9% in standard solution, and 81.4% is reached in whole blood.In the cell of detection other two type During (including SKBR-3 (Figure 11 c, 13a) and COLO-205 (Figure 11 d, 13b)), similar knot that inventor also observes Fruit.Since sample complexity is high, the capture rate in whole blood is suitable or is slightly below the capture rate of (~5%) standard solution.Cause This, the method for inventor illustrate under the conditions of various low concentration CTC can meet demand capture rate.
Diagnostic application in cancer patient
Method is applied to capture CTC from cancer patient's blood by inventor, to prove the reality of pGold chip and equipment Using.Using established scheme, inventor is from various phenotypes (including breast cancer, lung cancer, cancer of pancreas and colorectum Cancer) 11 patients (Figure 14 a and table 4) 5mL whole blood in capture 1~20 CTC.Biomarker based on CD45 and CK Analysis and 4', 6- diamidino -2-phenylindone (DAPI) fluorescent staining, Figure 14 b show a leucocyte (CD45+/DAPI +/CK-) and an identified CTC model unicellular fluorescent image CD45-/DAPI+/CK+).Inventor also passes through polychrome Fluorescent method shows identification of the typical many cells fluorescent image for CTC in Figure 15.Inventor is cancer patient's The multiple NIR fluorescence enhancement screening of CTC is realized on pGold chip.
Table 4:
The CTC analysis and research work for being largely dedicated to cancer diagnosis has been carried out.Since effective specific antibody identifies, Chemical method provides the selectivity and sensitivity of height in enrichment CTC, clinically has great advantages, this clinic Diagnostic method is ratified by FDA.It is worth noting that, concentrating platforms and detected downstream analyze to pass the CTC based on antibody It is important.Different from pervious CTC researching and designing, pass through pGold chip our reasonable combinations concentrating platforms and detected downstream.In In the work of the application, microfluidic system provides high capture rate under the premise of simple separation, it is often more important that, Neng Gouji At various chips in order to easily operate with highly sensitive detection, and be applied into further detection application.
Plasma resonance material and facility, which is applied, can solve biomedical applications field in near-infrared fluorescent enhancing detection The low problem with small throughput of interior sensitivity.It is most of current although having achieved substantive progress in CTC diagnostic field Method mainly still concentrates on the structure optimization of material and the selection of particular organisms marker.To analysis target (such as CTC) Operation is still difficult, and up for further developing, the research of this technology may provide the new visual field.Inventor's discovery The variation of cytomorphology on pGold chip, and react pGold under conditions of having the microfluid immunomagnetic enrichment of CTC About 50-122 times of multiple NIR fluorescence enhancement is higher than other chips and detects two orders of magnitude and best report.The research table of inventor Bright nanometer/microtechnique is in the meaning of field of bioanalysis, and usually get the brush-off even quilt in current research and practice Ignore.
In cell imaging and application, multiple analysis and laboratory automation are most important for large-scale application.At this In item work, the application illustrates the multiple analysis of CK and EGFR in two kinds of NIR fluorescence channels for potential cell point Type.Meanwhile the micro scanning that inventor fast and automatically changes the CTC being enriched on chip, and record image can be carried out Digital Signal Processing, to carry out non-microscopic cell analysis.It is worth noting that, the method that inventor demonstrates the foundation is adding It can be applied in the CTCs analysis of the patient of the quasi- cell experiment of mark-on and clinical various cancer types.In view of above-mentioned advantage, hair Bright people is estimated will be in hospital application in large-scale cancer management and rare cell analysis.
In short, inventor reports the multiple NIR fluorescence enhancement screening of the CTC of cancer patient.The work of inventor is not only Rare cell analysis is advanced, including but not limited to CTC and cancer are illustrated in field of biomedicine with plasma resonance material The design of interface based on material or equipment and with cell etc. between object, with preferably application is flat in the not far following generation Platform and detection technique.
In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", " length ", " width ", " thickness ", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside", " up time The orientation or positional relationship of the instructions such as needle ", " counterclockwise ", " axial direction ", " radial direction ", " circumferential direction " be orientation based on the figure or Positional relationship is merely for convenience of description of the present invention and simplification of the description, rather than the device or element of indication or suggestion meaning must There must be specific orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or Implicitly include at least one this feature.In the description of the present invention, the meaning of " plurality " is at least two, such as two, three It is a etc., unless otherwise specifically defined.
In the present invention unless specifically defined or limited otherwise, term " installation ", " connected ", " connection ", " fixation " etc. Term shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integral；It can be mechanical connect It connects, is also possible to be electrically connected or can communicate each other；It can be directly connected, can also indirectly connected through an intermediary, it can be with It is the interaction relationship of the connection or two elements inside two elements, unless otherwise restricted clearly.For this field For those of ordinary skill, the specific meanings of the above terms in the present invention can be understood according to specific conditions.
In the present invention unless specifically defined or limited otherwise, fisrt feature in the second feature " on " or " down " can be with It is that the first and second features directly contact or the first and second features pass through intermediary mediate contact.Moreover, fisrt feature exists Second feature " on ", " top " and " above " but fisrt feature be directly above or diagonally above the second feature, or be merely representative of First feature horizontal height is higher than second feature.Fisrt feature can be under the second feature " below ", " below " and " below " One feature is directly under or diagonally below the second feature, or is merely representative of first feature horizontal height less than second feature.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

Claims (9)

1. a kind of micro fluidic device for enrichment cycles tumour cell characterized by comprising
Ontology limits the fluid circulation space of upper opening in the ontology；
The bottom of the ontology is arranged in injection port, the injection port；
The bottom of the ontology is arranged in outlet, the outlet；
Plasma fluorescence enhancement chip, the plasma fluorescence enhancement chip are arranged in described in the fluid circulation space The lower surface load of upper opening, the plasma fluorescence enhancement chip has antibody；
The upper surface of the plasma fluorescence enhancement chip is arranged in magnetic field generating assembly, the magnetic field generating assembly.
2. micro fluidic device according to claim 1, which is characterized in that the magnetic field generating assembly is magnet.
3. micro fluidic device according to claim 1, which is characterized in that the plasma fluorescence enhancement chip is detachable The upper opening of the fluid circulation space is arranged in ground.
4. a kind of kit for enrichment cycles tumour cell characterized by comprising
Magnetic nano-particle, the magnetic nano-particle are suitable for capturing circulating tumor cell from blood；
The described in any item micro fluidic devices for enrichment cycles tumour cell of claims 1 to 3.
5. kit according to claim 4, which is characterized in that the magnetic nano-particle area load has circulating tumor Cell specific marker molecules.
6. kit according to claim 5, which is characterized in that the specific marker molecule is anti-EpCAM.
7. a kind of method using the described in any item kit enrichment cycles tumour cells of claim 4~6, feature exist In, comprising:
(1) sample containing circulating tumor cell is mixed with the magnetic nano-particle, is followed to form magnetic nano-particle- Ring tumour cell complex；
(2) by the mixture via the micro fluidic device injection port enter the fluid circulation space, and from it is described go out The fluid circulation space is discharged in sample mouth, wherein the magnetic nano-particle-circulating tumor cell complex is sent out in the magnetic field The lower surface of the plasma fluorescence enhancement chip is enriched under the action of raw component.
8. the system that a kind of pair of circulating tumor cell is detected characterized by comprising
The described in any item micro fluidic devices of claims 1 to 3；With
Fluorescence detection device.
9. system according to claim 8, which is characterized in that the fluorescence detection device is NIR fluorescence detection device.
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