CN107233941A - 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 PDFInfo
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- CN107233941A CN107233941A CN201710271597.7A CN201710271597A CN107233941A CN 107233941 A CN107233941 A CN 107233941A CN 201710271597 A CN201710271597 A CN 201710271597A CN 107233941 A CN107233941 A CN 107233941A
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Abstract
The present invention proposes a kind of technology for enrichment cycles tumour cell.The core of this technology is the micro fluidic device based on plasma Fluorescence Increasing chip.The device includes：The fluid circulation space of upper opening is limited in body, the body；Injection port, the injection port is arranged on the bottom of the body；Outlet, the outlet is arranged on the bottom of the body；Plasma Fluorescence Increasing chip, the plasma Fluorescence Increasing chip is arranged on the upper opening of the fluid circulation space, and the lower surface of the plasma Fluorescence Increasing chip is loaded with antibody；Magnetic field generating assembly, the magnetic field generating assembly is arranged on the upper surface of the plasma Fluorescence Increasing chip.Utilize micro fluidic device according to embodiments of the present invention, it is possible to achieve the simple separation of circulating tumor cell and efficient capture and enrichment, and the highly sensitive detection of fluorescence can be strengthened to the progress NIR for the enrichment cycles tumour cell being enriched to.
Description
Technical field
The present invention relates to field of biological detection, in particular it relates to which the enhancing screening circulation of multiple near-infrared fluorescent is swollen
Oncocyte is thin more particularly, the present invention relate to the micro fluidic device of enrichment cycles tumour cell, for enrichment cycles tumour
The kit of born of the same parents, the method for enrichment cycles tumour cell, the system detected to circulating tumor cell and to circulating tumor
The method that cell is detected.
Background technology
The characteristics of liquid biopsy is due to its Noninvasive and Precise Diagnosis, it has in clinical research and biomedical detection
There is huge prospect.Especially, the analysis to the circulating tumor cell (CTC) in blood samples of patients is preferably managing kinds cancer
In play vital effect.Include biopsy from conventional method and imaging method is different, 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
It is very challenging to swim analysis, because their abundance in blood are extremely low, a usual CTC is blended in billions of
In other haemocytes.In order to build high-performance CTC assay methods, it is necessary to consider following aspect in clinic, including：I) capture
CTC enrichment method；II CTC analysis method) is detected；III) practical application processing blood samples of patients；IV) laboratory automation is big
Scale is used.The new tool for solving above-mentioned aspect is in demand, and needs the material and device that rationally design.
Plasma material is commonly referred to as noble metal (such as golden) and its compound, its light in particular range of wavelengths
There is unique surface plasmon resonance effect according to lower.Because with specific structure and surface chemical property, this grade 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 medicals 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 Detections success, another challenge is cell detection on chip, work before
Merely due to the enhancing effect of pdp body chip in itself, but be a lack of being used for operation that rare cell (such as CTC) analyzes and
Multiple NIR Fluorescence Increasings step, it can only provide limited enhancing effect (2-30 times).
Therefore, exploitation design device makes it be integrated with NIR-FE cell detections on chip, and overcomes mentioned in this area
Major obstacle have great importance.
The content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.
In this application, inventor illustrate for the first time the multiple enhancing NIR fluorescent applications of the golden chip of plasma in
CTCs is screened.Inventor combines NIR-FE detections on CTC microfluid immunomagnetic enrichment and chip.Pass through cell enrichment
Operation and its morphologic change, inventor observe about 50-122 times of multiple NIR Fluorescence Increasings, and the detection better than other chips is imitated
Really.Inventor is detected to 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 senior CTC analyses clinically
Management, and contribute to the bioanalysis application design at the interface of plasma material/between equipment and cell.
In the first aspect of the present invention, the present invention proposes a kind of micro fluidic device for enrichment cycles tumour cell.
Embodiments in accordance with the present invention, described device includes：The fluid circulation space of upper opening is limited in body, the body；
Injection port, the injection port is arranged on the bottom of the body；Outlet, the outlet is arranged on the bottom of the body；
Plasma Fluorescence Increasing chip, the plasma Fluorescence Increasing chip is arranged on the top of the fluid circulation space
Opening, the lower surface of the plasma Fluorescence Increasing chip is loaded with antibody；Magnetic field generating assembly, the magnetic field generating assembly
It is arranged on the upper surface of the plasma Fluorescence Increasing chip., can be with using micro fluidic device according to embodiments 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 that is enriched to
Progress NIR strengthen fluorescence highly sensitive detection.Inventor has found, under conditions of having CTC microfluid immunomagnetic enrichment
About 50-122 times of the multiple NIR Fluorescence Increasings of plasma Fluorescence Increasing chip (pGold), two detected far above other chips
The optimal report of the order of magnitude and prior art.
Embodiments in accordance with the present invention, above-mentioned micro fluidic device can further include following additional technical feature at least
One of：
Embodiments in accordance with the present invention, the magnetic field generating assembly is magnet.
Embodiments in accordance with the present invention, the plasma Fluorescence Increasing chip is removably disposed in the flow of fluid
The upper opening in space.
In the second aspect of the present invention, the present invention proposes a kind of kit for enrichment cycles tumour cell.According to
Embodiments of the invention, the kit includes：Magnetic nano-particle, the magnetic nano-particle is suitable to catch from the blood
Obtain circulating tumor cell；It is described previously for the micro fluidic device of enrichment cycles tumour cell.Using according to present invention implementation
The kit of example, it is possible to achieve the simple separation of circulating tumor cell and efficient capture and enrichment, and can be to being enriched to
The progress NIR of enrichment cycles tumour cell strengthens the highly sensitive detection of fluorescence.Inventor has found, immune in the microfluid for having CTC
About 50-122 times of the multiple NIR Fluorescence Increasings of plasma Fluorescence Increasing chip (pGold) under conditions of magnetite gathering, far above it
Two orders of magnitude of his chip detection and the optimal report of prior art.
Embodiments in accordance with the present invention, mentioned reagent box can further include following additional technical feature at least it
One：
Embodiments in accordance with the present 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 improved.
Embodiments in accordance with the present 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 gravity and magnetic field force (MNPs) compound action selective enrichment from
The surface of daughter Fluorescence Increasing chip.
In the third aspect of the present invention, the present invention proposes a kind of thin using foregoing kit enrichment cycles tumour
The method of born of the same parents.Embodiments in accordance with the present invention, methods described includes：(1) by the sample containing circulating tumor cell and the magnetic
Property nano-particle 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 discharges the fluid circulation space from the outlet,
Wherein, the magnetic nano-particle-circulating tumor cell complex is enriched in described in the presence of the magnetic field generating assembly
The lower surface of plasma Fluorescence Increasing chip.Utilize method according to embodiments of the present invention, it is possible to achieve circulating tumor cell
Simple separation and efficient capture and enrichment, and can be glimmering to the carry out NIR of the enrichment cycles tumour cell being enriched to enhancing
The highly sensitive detection of light, the plasma Fluorescence Increasing chip (pGold) under conditions of the microfluid immunomagnetic enrichment for having CTC
Multiple about 50-122 times of NIR Fluorescence Increasings, two orders of magnitude and the optimal report of prior art detected far above other chips.
In the fourth aspect of the present invention, the present invention proposes a kind of system detected to circulating tumor cell.According to
Embodiments of the invention, the system includes：Foregoing device；And fluorescence detection device.Using according to present invention implementation
The system of example, it is possible to achieve the simple separation of circulating tumor cell and efficient capture and enrichment, and can be to the richness that is enriched to
Collect the highly sensitive detection of the progress enhancing fluorescence of circulating tumor cell.
Embodiments in accordance with the present invention, said system can further include at least one following additional technical feature：
Embodiments in accordance with the present invention, the fluorescence detection device is NIR fluorescence detection devices.Inventor has found, is having
The multiple NIR Fluorescence Increasings of plasma Fluorescence Increasing chip (pGold) under conditions of CTC microfluid immunomagnetic enrichment are about
50-122 times, using NIR fluorescence detection devices, sensitivity of the system to CTC detection is significantly improved.
In the fifth aspect of the present invention, the present invention proposes a kind of method detected to circulating tumor cell.According to
Embodiments of the invention, methods described includes：Method as described above, is carried out rich to the circulating tumor cell in sample
Collection, to obtain the plasma Fluorescence Increasing chip for being enriched with the circulating tumor cell；Utilize NIR fluorescence detection devices pair
The circulating tumor cell of the plasma Fluorescence Increasing chip enrichment carries out target molecule detection.Using according to the present invention
The method of embodiment, it is possible to achieve the NIR of enrichment cycles tumour cell strengthens the highly sensitive detection of fluorescence, is there is CTC miniflow
About 50-122 times of the multiple NIR Fluorescence Increasings of plasma Fluorescence Increasing chip (pGold) under conditions of body immunomagnetic enrichment,
Two orders of magnitude and the optimal report of prior art detected far above other chips.
The technology for enrichment cycles tumour cell that the application is proposed, the core of this technology is glimmering based on plasma
Light strengthens the above-mentioned micro fluidic device of chip.
Brief description of the drawings
Fig. 1 is the micro fluidic device schematic diagram of enrichment cycles tumour cell according to embodiments of the present invention；
Fig. 2 is the system schematic according to embodiments of the present invention detected to circulating tumor cell；
Fig. 3 is CTC screening installation figures according to embodiments of the present invention,
Wherein, a be screening process and device schematic diagram (on) and viewgraph of cross-section (under)；B shows pGold chips and mould
The digital picture of the integration of block (left side) and the SEM image of pGold chips (right side)；C shows the biological mark of CTC on pGold chips
The schematic diagram of the NIR fluoroscopic examinations of will thing albumen, RBC refers to red blood cell, and for the SEM image in b illustrations, engineer's scale is
500nm；
Fig. 4 is MNP according to embodiments of the present invention characterization result,
Wherein, a SEM, the B-H loop that b TEM and c are MNP.D is (from colloidal suspension by magnet Magnetic Isolation
Liquid) MNP, engineer's scale is respectively a) 200nm and b) 100nm；
Fig. 5 is the extinction spectra of pGold chips according to embodiments of the present invention and exciting for IRDye680 and IRDye800
(line) and transmitting (shadow region) area coincidence；
Fig. 6 is multiple NIR Fluorescence Increasings result figure according to embodiments of the present invention,
Wherein, enrichment CTC NIR fluorescence (marks) image (top, illustration display light field cyclogram by IRDye800
Picture) and average fluorescent strength (bottom), including a) MCF-7 on chip, b) SKBR-3 and c) COLO-205, used chip
Including (i) glass-chip, (ii) sGold chips, the pGold chips of (iii) without immunomagnetic enrichment and (iv) are with immune
The pGold chips of magnetite gathering.For a-c) in all fluoroscopic images, engineer's scale be 10 μm；
Fig. 7 is CTC according to embodiments of the present invention scanning fluoroscopic image,
Wherein, a) MCF-7, b) SKBR-3 and c) COLO-205 NIR fluorescence (IRDye800) figure of the record on chip
Picture, used chip includes (i) glass-chip, (ii) sGold chips, the pGold chips of (iii) without immunomagnetic enrichment
(iv) has the pGold chips of immunomagnetic enrichment.For a-c) in all fluoroscopic images, engineer's scale be 100 μm；
Fig. 8 is the size of the CTC according to embodiments of the present invention without and with magnetite gathering,
Wherein, the average-size of the CTC on the pGold chips without and with magnetite gathering, including MCF-7, SKBR-
3 and COLO-205.Black and grey represent the average-size of the CTC without and with magnetite gathering respectively, analyze three cells
Standard deviation (s.d.) to calculate as error bars, data display is average value ± s.d.(n=3)；
Fig. 9 is multiple enhanced mechanism results figure according to embodiments of the present invention,
Wherein, a) schematic diagram, display (i) under no immunomagnetic enrichment, CTC (MCF-7) enhanced NIR fluorescence and
(ii) there is the CTC of immunomagnetic enrichment multiple enhancing NIR fluorescence on pGold chips.B) CTC side view and c) top view
SEM image, (i) has immunomagnetic enrichment without immunomagnetic enrichment and (ii) on pGold chips.Engineer's scale is b) and c)
In be 5 μm；
Figure 10 is the multiple analysis result figure of biomarker according to embodiments of the present invention,
Wherein, a) glass-chip and b) fluorescence microscopy images of the CTC (MCF-7) on pGold chips, c) glass-chip
And d) CTC (MCF-7) scan image on the fluorescence of pGold chips.It is left：Anti- EGFR IRDye680 passages；It is middle：Anti- CK's
IRDye800 passages；It is right：The amalgamation result of two passages.A) illustration on right side shows bright field image.In a) and b), ratio
Chi is respectively 10 μm；
Figure 11 is CTC according to embodiments of the present invention capture rate result figure,
Wherein, a) CTC of capture identification, it shows the CTC (MCF-7) of capture (i) light field, and (ii) DAPI is marked,
(iii) anti-CK marks, and the fluoroscopic image that (iv) three merges, the b in (i) PBS solution and (ii) whole blood) MCF-7, c)
SKBR-3 and d) COLO-205 capture rate.Five independent experiments are carried out to calculate standard deviation, and the data shown are led to
Cross 5 times calculating average value ± s.d show (n=5) on the diagram, a) in engineer's scale be 10 μm；
Figure 12 is the scanning fluorescence analysis knot of two kinds of biomarkers according to embodiments of the present invention from multiple cells
Fruit is schemed,
Wherein, the multiple CTC (MCF-7) captured by NIR Fluorescence Scanners on a) glass-chip and b) pGold chips
Multi-biological analysis of markers.It is left：Anti- EGFR IRDye680 passages；It is middle：IRDye800 passages are anti-CK；It is right：Two
The amalgamation result of passage, engineer's scale is 20 μm in a) and b).
Figure 13 is capture CTC according to embodiments of the present invention qualification result figure,
Wherein, a) SKBR-3 that is captured on pGold chips and b) COLO-205 (i) light field, (ii) DAPI marks,
(iii) anti-CK mark, engineer's scale is 10 μm in a) and b)；
Figure 14 is the selection result figure of CTC in cancer patient according to embodiments of the present invention,
Wherein, a is the CTC captured in the 5mL whole blood chips from 11 cancer patients quantity；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 fluoroscopic image, engineer's scale in b) be 5 μm；And
Figure 15 is the qualification result figure of CTC in cancer patient according to embodiments of the present invention,
Wherein, three fluorescence method shows a leucocyte (upper left, CD45+/DAPI+/CK-) fluorescence from patient 5
Image and CTC (bottom right, CD45-/DAPI+/CK+) fluoroscopic image of a capture：A) anti-CD45 FITC passages, b) anti-CK
IRDye800 passages, c) the DAPI passages of nucleus and d) merge passage, figure a-d in engineer's scale be 10 μm.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings.Below with reference to
The embodiment of accompanying drawing description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.
On the one hand, the present invention proposes a kind of micro fluidic device for enrichment cycles tumour cell.According to the present invention's
Embodiment, with reference to Fig. 1, the device 1000 includes：The fluid circulation space of upper opening is limited in body 100, body 100；
Injection port 200, injection port 200 is arranged on the bottom of body 100；Outlet 300, outlet 300 is arranged on the bottom of body；Deng
Gas ions Fluorescence Increasing chip 400, plasma Fluorescence Increasing chip 400 is arranged on the upper opening of fluid circulation space, etc.
The lower surface of gas ions Fluorescence Increasing chip 400 is loaded with antibody；Magnetic field generating assembly 500, the magnetic field generating assembly is set
In the upper surface of plasma Fluorescence Increasing 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 present invention proposes a kind of system detected to circulating tumor cell.According to the reality of the present invention
Example is applied, with reference to Fig. 2, the system includes：Foregoing device 1000；With fluorescence detection device 2000.Specifically, fluoroscopic examination
Equipment 2000 is NIR fluorescence detection devices.
Utilize micro fluidic device and system according to embodiments of the present invention, it is possible to achieve the simple separation of circulating tumor cell
With efficient capture and enrichment, and the highly sensitive of fluorescence can be strengthened to the progress NIR of the enrichment cycles tumour cell being enriched to
Detection.Plasma Fluorescence Increasing chip (pGold) multiple NIR is glimmering under conditions of the microfluid immunomagnetic enrichment for having CTC
Light strengthens about 50-122 times, two orders of magnitude and the optimal report of prior art detected far above other chips.
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this reality
With new, and it is not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according in the art
Document described by technology or condition or carried out according to product description.Agents useful for same or the unreceipted production firm of instrument
Person, being can be by the conventional products of acquisition purchased in market.
In following experiment, specific method and material used is as described below：
It is prepared by chip
Prepare the chip of three types, including golden (pGold) chip of plasma, golden (sGold) chip of sputtering and glass
Chip.The pGold chips with abundant nano island are synthesized by the seed law in the solution of optimization.In short, slide is existed
It is stirred vigorously lower immersion HAuCl4(5mM) and NH420 minutes in OH (20 μ L/mL, 0.6%, V%) solution.By the slide of acquisition
Immerse in water-bath twice and wash successively.After washing, it is molten that the slide that Au ions are inoculated with is put into sodium borohydride (1mM) at room temperature
In liquid 1 minute to be reduced, and washed by immersing successively in water-bath twice.These slides are transferred to mol ratio
For 1:1 another HAuCl4And NH4In OH mixed solutions, the continued growth for Au films.Growth course is vigorously shaken at room temperature
(100RPM) 20 minutes, and wash to obtain final pGold chips.PGold chips are stored at room temperature sealable
In container.
SGold chips prepare that (GSL-1100X-SPC-16M, Shenyang section crystalline substance automation equipment has by magnetically controlled sputter method
Limit company).Gold target (99.99%) is purchased from Shanghai Daheng Optical Fine Mechinery Co., Ltd.Sputter procedure is under vacuum (10Pa)
Carried out 2 minutes with 20mA.SGold chips are stored in sealable container at room temperature.
Sheet glass is bought from Chinese Chemical Reagent Co., Ltd., Sinopharm Group.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,
With pure water three times and it is maintained at room temperature.
Micro fluidic device is built
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 tubes, 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 mould of integrated chip is made and used with polypropylene
The size of optimization is designed with being laid out.The air stream in pipeline is driven to adjust flow rate of liquid (1mL/h) with miniflow pump.
It is prepared by magnetic nanoparticle
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 companies (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)s (PBS, pH=7.4,50mM), by MNP and antibody at 37 DEG C
Mixture vibrates 1.5 hours, makes itself and biotinylated EpCAM antibody (Abcam Inc., Cambridge, USA) (100:1,
W/W) fully reaction.After being washed twice again with 50mM PBS (pH=7.4), remove and swim by centrifuging (200G, 5 minutes) and magnetic force
From EpCAM antibody.Gained feature MNP is resuspended to the 50mM PBS containing 3% bovine serum albumin(BSA) (BSA, Sigma)
(pH=7.4) in buffer solution, for closing and being stored at 4 DEG C.
Material and facility is characterized
The sign of material and device include transmission electron microscope (TEM), SEM (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 lead to
Cross AuCy UV1901PC spectrophotometer measurements.TEM image is shot under 10kV by JEOL JEM-2100F TEM instruments.
10 μ L ethanol (0.1 μ g/mL) particle suspension liquid is dripped on copper mesh, treats that sample carries out tem observation after air-drying.Pass through under 10kV
Hitachi S-4800 carry out sem analysis.By particle suspension liquid drop on silicon and after being dried at room temperature for, in SEM devices
In directly observe.Use vibrating specimen magnetometer (Quantum Design, Physical Property Measurement
System) measurement hysteresis curve (making magnetic field be circulated between -50 and 50kOe).
Cell culture
Use three kinds of cells of scheme culture of report, including MCF-7 (human breast cancer cell line, EpCAM is positive), SKBR-3
(human breast cancer cell line, EpCAM is positive) and COLO-205 (human colon cancer cell line, EpCAM is positive).During all cells come from
The academy of sciences of state (Shanghai cell resource center research institute), and moist 5%CO at 37 DEG C2Cultivated in incubator.MCF-7 is thin
Born of the same parents are cultivated in Eagle (Sigma) culture medium.The Eagle culture mediums that SKBR-3 cell culture is improved in Dulbecco
In (DMEM, Sigma).COLO-205 cell culture is in Roswell Park Memorial Institute culture mediums (RPMI-
1640) in.In order to obtain free cell, 1mL trypsase (0.25%, V/V) is instilled in Tissue Culture Flask, in CO2Culture
Digested 5 minutes in case, and the cell of acquisition is stored in PBS (pH=7.4,50mM) buffer solution.
Standard addition experiment
Above-mentioned three kinds of cell line (MCF-7, SKBR-3 and COLO-205) is respectively added to 50mM PBS (pH=7.4) molten
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, capture rate (n/N) is calculated by immunomagnetic enrichment and counting fluorescence analysis.
CTC immunomagnetic enrichment
In order to carry out immunomagnetic enrichment, by 30 μ g functionalization MNP and biological mixture (5mL mark-ons sample or patient
Blood) 1.5 hours are incubated at 37 DEG C with optionally labeling CT C.Said mixture is added to the sample of microfluidic device
Guan Zhong, it is connected with chip load-on module.Flow velocity is 1mL/h, it is necessary to which~5h completes enrichment.By labeled as CTC MNP in magnetic field
In be attached to the surface of chip (from magnet), and continuously with containing 0.05%tween-20 (PBST) PBS (pH=7.4,
50mM) wash three times.Gained chip is handled 15 minutes with ice acetone soln (99%, V%) lucifuge, and stored at 4 DEG C.
Near infrared fluorescent dye labelled antibody
With fluorescent dye (IRDye800/680) mark CK detection antibody and EGFR detection antibody.Antibody passes through EDC/NHS
(1- ethyls -3- (3- dimethylaminopropyls) carbodiimide/n-hydroxysuccinimide) is reacted to mark with IRDye800/680.
IRDye680-NHS/IRDye800cw-NHS esters (Licor Biosciences) are dissolved in anhydrous dimethyl sulphoxide, and made
It is kept in dark place with preceding at -20 DEG C.By antibody and dyestuff with 1:4 mixed in molar ratio 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 posts (GE Healthcare).After EDC/NHS is reacted
Raw mixture be added in the post with 10mL PBS (pH=7.4,50mM).The eluent containing labelled antibody is collected,
And stored at -20 DEG C under the conditions of lucifuge.
Chip is dyed online
In order to be dyed to the biomarker protein for being attached to cell surface, chip is cleaned with PBST three times, then
Detection antibody (250 μ L, 5 μ g/mL) in the dark with dye marker at 37 DEG C is incubated 1.5 hours.Then, by the core of dyeing
Piece is washed three times with PBST, to remove impurity.For staining cell core, with 4', 6- diamidinos -2-phenylindone (DAPI, 5mg/
ML) dye 15 minutes, then washed three times with PBST (pH=7.4,50mM, 0.05%Tween-20).The chip obtained exists
4 DEG C are stored in before near infrared imaging and scanning.
ESEM and microscope imaging
The cell on chip is detected and identified by scanning and microscope imaging.Innopsys scanners
(InnoScan 710) is used to scan chip, so as to cell imaging.The scanning resolution of all experiments is set to 3 μm/pixel.
Pass through NIS-Elements BR 4.51.00 (ECLIPSE Ti-E related softwares, 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) it is used to record the microscope imaging of cell.
The CTCs analyses of cancer patient
Collect the 5mL whole bloods from cancer patient and for chip CTC captures.Knowing for patient has been obtained during project initiation
And agreement.According to comprehensive cancer network (NCCN) guide diagnosis cancer patient of country.Eliminate the medical science 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 numbers are counted.All research approaches of this work are through Ningbo the second hospital institution Ethics Committee and Shanghai Communications University
Ratify School of Biomedical Engineering.
Experimentation of the invention described in detail below and result：
The design of CTC screening installations
Inventor devises a kind of microfluidic device to carry out the immunomagnetic enrichment of CTC on chip, as shown in Figure 3 a.Hair
A person of good sense is using positive enrichment method, with the magnetic nano-particle of epithelial cell adhesion molecule antibody (anti-EpCAM) functionalization
(MNPs) CTCs in blood samples of patients or addition standard cell lines is marked.According to Fig. 4 a, SEM (SEM) in 4b and
Transmission electron microscope (TEM) is analyzed, and the MNP of functionalization has~25-40nm average-size, saturation magnetisation value for~
0.9emu/g (cyclical field between -50 and 50kOe, Fig. 4 c) can use magnet quick separating without remaining magnetic in 1 minute
(Fig. 4 d).Then inventor makees biological mixture by being pumped into microfluidic channel, and by the combination of gravity and magnetic field force
Selective enrichment CTC is used, 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 institutes
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 observes the unique form gold nano island that distance between island (Fig. 3 b, right) about 10nm is dispersed with chip.For on chip
Detection, be that can provide plasma in the NIR region in its absorption/extinction spectra (Fig. 5) to be total to the characteristics of pGold chips
Shake.Inventor is marked with NIR fluorescent dyes (IRDye800/680) is enriched with CTC biomarker protein for being analyzed on chip
(Fig. 3 c).
CTC NIR-FE detections 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 detections, including glass-chip, golden (sGold) chip of sputtering and pGold chips (have and without immune magnetic
Property microfluid enrichment).As shown in Figure 6 a, for MCF-7, glass-chip and sGold provide very weak signal, and mean fluorecence is strong
Degree is respectively 637 and 1456, and this is due to there is fluorescent quenching on no NIR-FE detections or chip surface.In order to compare, hair
A person of good sense realizes that the NIR-FE of cell is detected using the pGold chips without magnetite gathering, average fluorescent strength 5136 is produced, with glass
Fluorescence intensity on glass chip, which is compared, adds 8.1 times.
It is worth noting that, for magnetite gathering, inventor observes that cell size adds 30.4% (from~136.4
To~177.9 μm2, Fig. 8), and further enhanced CT C NIR fluorescence signals, compared with glass-chip, it is possible to provide mean fluorecence
Intensity 32540 and 51.1 times of enhancer (strengthening 55.3 times compared with subtracting background signal).Inventor using SKBR-3 and
When COLO-205 is detected, it has been found that similar result, they distinguish 62.2 times, and (background signal subtracted is 69.1 times, figure
Enhancer 6b) and 95.3 times of enhancers (background signal subtracted is 122.0 times, Fig. 6 c).Inventor is total in table 1-3
These results have been tied, and have confirmed (51.1-122.0 times) detection of multiple NIR Fluorescence Increasings on pGold chips.
Table 1, MCF-7 average fluorescent strength analysis：
Table 2, SKBR-3 average fluorescent strength analysis：
Table 3, COLO-205 average fluorescent strength analysis：
Multiple enhanced mechanism
The mechanism of multiple NIR Fluorescence Increasings detection is inventors herein proposed, and has carried out SEM sign (Fig. 9).Conventional is thin
Born of the same parents NIR-FE detections lack the optimization of the average distance between the operation of cellular morphology and cell and pGold chip surfaces.Compare
Under, inventor compresses cell in immune magnetic microfluid enrichment process by magnetic force, and reduces cell and pGold chip lists
Average distance between face, its result causes multiple enhancement effect (Fig. 9 a).As shown in figure 9b, the side view SEM figures of pGold chips
As not respectively illustrating not and the cell with Magnetic Isolation normal and the form of compression (thickness of reduction is about 2 μm).This
Outside, in SEM is overlooked, inventor also found due to compression process, cell size increase, 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 CTC multiplexed protein matter biomarker analysis by IRDye680 and IRDye800 passages
(Figure 10).Inventor detects two kinds of protein biological markers of cancer cell on IRDye800 and IRDye680 passages respectively
Thing, including cytokeratin (CK) and EGF-R ELISA (EGFR).Inventor is seen by the microscope on pGold chips
Observe in clearly cell image (many cells analysis in the single cell analysis and Figure 11 in Figure 10 a), better than two passages
Result on glass-chip.It is worth noting that, inventor is also imaged by micro scanning to the CTC being enriched with chip
(multiple cell analysis in the single cell analysis and Figure 12 in Figure 10 b), it is consistent that its result and microscope are observed, and sends out
This technology of a person of good sense may have bigger detection high flux during actual rare cell analysis and easily operated etc. have
Beneficial to the advantage of extensive automation application.
Capture rate and identification
Inventor passes through capture rate of the equipment to CTC of experimental studies for adding standard cell lines inventor a series of
(Figure 11).Inventor identifies the CTC of enrichment by the fluorescent staining in Figure 11 a, and illustrates CTC typical image.
Inventor mixes 6-200MCF-7 in standard liquid (Figure 11 b-i) or whole blood (Figure 11 b-ii), and counts the CTC of enrichment number
Amount.Capture rate reaches 86.9% in standard liquid, 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)), the similar knot that inventor also observes
Really.Because sample complexity is high, the capture rate in whole blood is suitable or is slightly below the capture rate of (~5%) standard liquid.Cause
This, the method for inventor is illustrated can meet the capture rate of demand under the conditions of various low concentration CTC.
Diagnostic application in cancer patient
Method is applied to capture CTC from cancer patient's blood by inventor, to prove the reality of pGold chips and equipment
Using.Using the scheme set up, 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 bloods 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 fluoroscopic image CD45-/DAPI+/CK+).Inventor also passes through polychrome
Fluorescent method shows that typical many cells fluoroscopic image is used for CTC identification in fig .15.Inventor is cancer patient's
CTC multiple NIR Fluorescence Increasings screening is realized on pGold chips.
Table 4：
A large amount of CTC analysis and research work for being directed to cancer diagnosis have been carried out.Due to the identification of effective specific antibody,
Chemical method provides the selectivity and sensitivity of height in enrichment CTC, and it clinically has larger advantage, this clinic
Diagnostic method is ratified by FDA.It is worth noting that, concentrating platforms and detected downstream are analyzed to pass for the CTC based on antibody
It is important.Different from former CTC research and designs, concentrating platforms and detected downstream by pGold chips our reasonable combinations.
In the work of the application, microfluid system provides high capture rate on the premise of simple separation, it is often more important that, Neng Gouji
Into various chips in order to easy operation and highly sensitive detection, and it is applied into further detection application.
Plasma resonance material and facility is applied can solve biomedical applications field in near-infrared fluorescent enhancing detection
Low and small throughput the problem of interior sensitivity.It is most of current although having achieved substantial progress in CTC diagnostic fields
Method mainly still concentrates on the structure optimization of material and the selection of particular organisms mark.To analysis target (such as CTC)
Operation is still difficult, and awaits further exploitation, and the research of this technology may provide the new visual field.Inventor has found
The change of cytomorphology on pGold chips, and react the pGold under conditions of the microfluid immunomagnetic enrichment for having CTC
Multiple about 50-122 times of NIR Fluorescence Increasings, two orders of magnitude and optimal report are detected higher than other chips.The research table of inventor
Bright nanometer/microtechnique is in the meaning of field of bioanalysis, its even quilt that generally got the brush-off 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 work, the multiple analysis that the application illustrates CK in two kinds of NIR fluorescence channels and EGFR is used for potential cell and divided
Type.Meanwhile, the micro scanning that inventor is fast and automatically changed to the CTC being enriched with 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 analyses of the patient of the quasi- cell experiment of mark-on and clinical various cancer types.In view of above-mentioned advantage, hair
A person of good sense will be estimated to be managed and rare cell analysis in large-scale cancer in hospital application.
In a word, inventor reports the CTC of cancer patient multiple NIR Fluorescence Increasings screening.The work of inventor is not only
Rare cell analysis is advanced, including but not limited to CTC and cancer are illustrated with plasma resonance material in biomedical sector
Material or equipment are main body and with the design that cell etc. is the interface between object, so that following to produce preferably application flat not far
Platform and detection technique.
In the description of the invention, it is to be understood that term " " center ", " longitudinal direction ", " transverse direction ", " length ", " width ",
" thickness ", " on ", " under ", "front", "rear", "left", "right", " vertical ", " level ", " top ", " bottom " " interior ", " outer ", " up time
The orientation or position relationship of the instruction such as pin ", " counterclockwise ", " axial direction ", " radial direction ", " circumference " be based on orientation shown in the drawings or
Position relationship, is for only for ease of the description present invention and simplifies description, rather than indicate or imply that the device or element of meaning must
There must be specific orientation, with specific azimuth configuration and operation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying relative importance
Or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can express or
Implicitly include at least one this feature.In the description of the invention, " multiple " are meant that at least two, such as two, three
It is individual etc., unless otherwise specifically defined.
In the present invention, unless otherwise clearly defined and limited, term " installation ", " connected ", " connection ", " fixation " etc.
Term should be interpreted broadly, for example, it may be fixedly connected or be detachably connected, or integrally；Can be that machinery connects
Connect or electrically connect or can communicate each other；Can be joined directly together, can also be indirectly connected to by intermediary, can be with
It is connection or the interaction relationship of two elements of two element internals, unless otherwise clear and definite restriction.For this area
For those of ordinary skill, the concrete meaning of above-mentioned term in the present invention can be understood as the case may be.
In the present invention, unless otherwise clearly defined and limited, fisrt feature can be with "above" or "below" second feature
It is that the first and second features are directly contacted, or the first and second features pass through intermediary mediate contact.Moreover, fisrt feature exists
Second feature " on ", " top " and " above " but fisrt feature are directly over second feature or oblique upper, or be merely representative of
Fisrt feature level height is higher than second feature.Fisrt feature second feature " under ", " lower section " and " below " can be
One feature is immediately below second feature or obliquely downward, or is merely representative of fisrt feature level 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 to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the not be the same as Example or the feature of example and non-be the same as Example or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (10)
1. a kind of micro fluidic device for enrichment cycles tumour cell, it is characterised in that including：
The fluid circulation space of upper opening is limited in body, the body；
Injection port, the injection port is arranged on the bottom of the body；
Outlet, the outlet is arranged on the bottom of the body；
Plasma Fluorescence Increasing chip, the plasma Fluorescence Increasing chip is arranged on the described of the fluid circulation space
Upper opening, the lower surface of the plasma Fluorescence Increasing chip is loaded with antibody；
Magnetic field generating assembly, the magnetic field generating assembly is arranged on the upper surface of the plasma Fluorescence Increasing chip.
2. micro fluidic device according to claim 1, it is characterised in that the magnetic field generating assembly is magnet.
3. micro fluidic device according to claim 1, it is characterised in that the plasma Fluorescence Increasing chip is detachable
Ground is arranged on the upper opening of the fluid circulation space.
4. a kind of kit for enrichment cycles tumour cell, it is characterised in that including：
Magnetic nano-particle, the magnetic nano-particle is suitable to capture circulating tumor cell from the blood；
The micro fluidic device for enrichment cycles tumour cell described in any one of claims 1 to 3.
5. kit according to claim 4, it is characterised in that the magnetic nano-particle area load has circulating tumor
Cell specific marker molecules.
6. kit according to claim 5, it is characterised in that the specific marker molecule is anti-EpCAM.
7. a kind of method of the kit enrichment cycles tumour cell described in any one of utilization claim 4~6, its feature exists
In, including：
(1) sample containing circulating tumor cell is mixed with the magnetic nano-particle, to form magnetic nano-particle-follow
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 sample mouthful discharge fluid circulation space, wherein, the magnetic nano-particle-circulating tumor cell complex is sent out in the magnetic field
The lower surface of the plasma Fluorescence Increasing chip is enriched in the presence of raw component.
8. a kind of system detected to circulating tumor cell, it is characterised in that including：
Micro fluidic device described in any one of claims 1 to 3；With
Fluorescence detection device.
9. system according to claim 8, it is characterised in that the fluorescence detection device is NIR fluorescence detection devices.
10. a kind of method detected to circulating tumor cell, it is characterised in that including：
In accordance with the method for claim 7, the circulating tumor cell in sample is enriched with, so as to obtain be enriched with it is described
The plasma Fluorescence Increasing chip of circulating tumor cell；
The circulating tumor cell being enriched with using NIR fluorescence detection devices to the plasma Fluorescence Increasing chip carries out mesh
Mark Molecular Detection.
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| WO2018196335A1 (en) * | 2017-04-24 | 2018-11-01 | Wwhs Biotech, Inc | Method for screening circulating tumor cells with multi-enhanced near-infrared fluorescence biochip |
| CN109060756A (en) * | 2018-06-01 | 2018-12-21 | 宁波美晶医疗技术有限公司 | Rare cell detection method in a kind of blood based on surface enhanced effect |
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| CN110142068B (en) * | 2019-06-12 | 2024-02-02 | 杭州华得森生物技术有限公司 | Kit and method for detecting epithelial and mesenchymal mixed circulating tumor cells |
| CN111896512B (en) * | 2020-08-10 | 2022-09-13 | 中南大学湘雅医院 | Lung cancer circulating tumor cell detection equipment |
| CN112067589A (en) * | 2020-09-07 | 2020-12-11 | 湖南时代基因医学检验技术有限公司 | Malignant tumor recurrence and metastasis risk assessment system based on circulating tumor cell detection |
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| CN103131634A (en) * | 2013-02-25 | 2013-06-05 | 宁波美晶医疗技术有限公司 | Immune tumor cell screening system based on micro-magnetic field |
| CN103837676A (en) * | 2014-03-20 | 2014-06-04 | 苏州纳达生物科技有限公司 | Metal nano island carrier and preparation method thereof as well as application of metal nano island carrier in immunodetection |
| CN204111767U (en) * | 2014-09-29 | 2015-01-21 | 颜卫山 | A kind of novel tumor cell electromagnetic sieve screening device |
| CN104990905A (en) * | 2015-06-30 | 2015-10-21 | 曲凯 | Kit for diagnosis of hepatocellular carcinoma metastasis based on solid-phase enzyme immunoassay fluorescence spots |
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| WO2018196335A1 (en) * | 2017-04-24 | 2018-11-01 | Wwhs Biotech, Inc | Method for screening circulating tumor cells with multi-enhanced near-infrared fluorescence biochip |
| CN109060756A (en) * | 2018-06-01 | 2018-12-21 | 宁波美晶医疗技术有限公司 | Rare cell detection method in a kind of blood based on surface enhanced effect |
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| CN107233941B (en) | 2019-11-26 |
| WO2018196335A1 (en) | 2018-11-01 |
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