WO2018196335A1

WO2018196335A1 - Method for screening circulating tumor cells with multi-enhanced near-infrared fluorescence biochip

Info

Publication number: WO2018196335A1
Application number: PCT/CN2017/110453
Authority: WO
Inventors: Guoxin Wang; Tao LIAO; Kun QIAN; Meijie TANG
Original assignee: Wwhs Biotech, Inc; Nirmidas Biotech, Inc.
Priority date: 2017-04-24
Filing date: 2017-11-10
Publication date: 2018-11-01
Also published as: CN107233941B; CN107233941A

Abstract

Provided is a micro-fluidic device (1000) for enriching circulating tumor cells including a body (100), defining a fluid flowing chamber, the fluid flowing chamber having an opening at an upper side thereof; an inlet (200), disposed at a bottom of the body (100); an outlet (300), disposed at the bottom of the body (100); a plasma fluorescence enhancement chip (400), disposed at the opening of the fluid flowing chamber, and loaded with antibodies at a lower surface thereof; and a magnetic field generating component (500), disposed at an upper surface of the plasma fluorescence enhancement chip (400). Also provided are a kit and a system including the micro-fluidic device (100), a method for enriching circulating tumor cells, and a method for detecting circulating tumor cells.

Description

METHOD FOR SCREENING CIRCULATING TUMOR CELLS WITH MULTI-ENHANCED NEAR-INFRARED FLUORESCENCE BIOCHIP

CROSS-REFERENCE TO RELATED APPLICATION

This application claims a priority to and benefits of Chinese Patent Application Serial No. 201710271597.7, filed with the State Intellectual Property Office of P. R. China on April 24, 2017, the entire content of which is incorporated herein by reference.

FIELD

The present disclosure relates to the field of biological detection, particularly to screening of circulating tumor cells with multi-enhanced near-infrared fluorescence, and more particularly to a micro-fluidic device for enriching circulating tumor cells, a kit for enriching circulating tumor cells, a method for enriching circulating tumor cells, a system for detecting circulating tumor cells and a method for detecting circulating tumor cells.

BACKGROUND

Liquid biopsy promises the clinical research and biomedical practice due to its non-invasiveness and precision for diagnostic purpose. Particularly, analysis of circulating tumor cells (CTCs) in patient blood plays a paramount role towards the better management of diverse cancer phenotypes. Distinct from conventional approaches including biopsy and imaging methods, CTCs offer new insights and opportunities during precise screening, curative assessment, patients staging, metastasis/recurrence studies of cancer diseases. However, it is very challenging for enrichment and downstream analysis of CTCs, owing to their low abundance in nature usually with one CTC mixing in billions of other blood cells. In order to construct a high performance CTCs assay in clinics, following aspects need to be considered including: I) enriching method to capture CTCs; II) analytical approach to detect CTCs; III) practical application dealing with patient blood; and IV) laboratory automation for large-scale use. Novel tools that addressed the above aspects are highly desirable and rationally designed materials and device are also desirable.

Plasmonic materials are typically referred to noble metals (e.g. gold) and their hybrids, which enjoy unique surface plasmon resonance under light irradiation within specific wavelength ranges. With tailored structural parameters and surface chemistry, plasmonic materials engaged with near-infrared fluorescence enhanced (NIR-FE, 650-1700 nm) applications and achieved diagnosis of diseases based on the NIR-FE detection of biomarkers through micro-printing of selected probes on surface in the chip format. Notably, current plasmonic chips are only integrated with the microarrays technology and have not been introduced in other micro-/nano-device for advanced diagnostics. Meanwhile, despite the success of NIR-FE molecular detection on-chip, a further challenge is the cellular detection on-chip, which affords limited enhancement factors (2-30 folds) by the plasmonic chip itself and lack of manipulation of cells on-chip for both rare cell (e.g. CTCs) analysis and multi-enhanced NIR fluorescence.

Therefore, it is of significance to develop and design a device to couple with on-chip NIR-FE cellular detection and overcome the as-mentioned major obstacles in the field.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent.

In one aspect, there is provided in embodiments a micro-fluidic device for enriching circulating tumor cells. In an embodiment, the micro-fluidic device includes a body, defining a fluid flowing chamber, the fluid flowing chamber having an opening at an upper side thereof; an inlet, disposed at a bottom of the body; an outlet, disposed at the bottom of the body; a plasma fluorescence enhancement chip, disposed at the opening of the fluid flowing chamber, and loaded with antibodies at a lower surface thereof; and a magnetic field generating component, disposed at an upper surface of the plasma fluorescence enhancement chip. With the micro-fluidic device according to embodiments of the present disclosure, it is possible to achieve the simple separation and efficient capture and enrichment of the circulating tumor cells and also to perform NIR enhanced fluorescence detection of the enriched circulating tumor cells in high sensitivity. The inventors found that the plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports in related art.

In an embodiment of the present disclosure, the magnetic field generating component is a magnet.

In an embodiment of the present disclosure, the plasma fluorescence enhancement chip is detachably disposed at the opening of the fluid flowing chamber.

In an embodiment of the present disclosure, the plasma fluorescence enhancement chip is a plasma gold chip.

In an embodiment of the present disclosure, the plasma gold chip comprises gold nano islands.

In an embodiment of the present disclosure, the plasma gold chip is of an inter-islands distance less than 100nm.

In an embodiment of the present disclosure, the plasma gold chip is of an inter-islands distance of about 10 nm.

In another aspect, there is provided in embodiments a kit for enriching circulating tumor cells. In an embodiment, the kit includes magnetic nanoparticles for capturing the circulating tumor cells from a blood sample; and a micro-fluidic device described above. With the kit according to embodiments of the present disclosure, it is possible to achieve the simple separation and efficient capture and enrichment of the circulating tumor cells and also to perform NIR enhanced fluorescence detection of the enriched circulating tumor cells in high sensitivity. The inventors found that the plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports in related art.

In an embodiment of the present disclosure, the magnetic nanoparticles are loaded with specific markers for the circulating tumor cells on surfaces thereof, thereby achieving a mark specific for circulating tumor cells contained in a sample (such as blood sample) . With the kit according to an embodiment of the present disclosure, the enrichment efficiency of circulating tumor cells is further improved.

In an embodiment of the present disclosure, the marker is anti-EpCAM. Specifically, the magnetic nanoparticles (MNPs) are such magnetic nanoparticles (MNPs) that are functionalized by epithelial cell adhesion molecule antibodies (anti-EpCAM) . Therefore, the circulating tumor cells entering the fluid flowing chamber will be selectively enriched on the surface of the plasma fluorescence enhancement chip under a combined action of gravity and magnetic forces.

In a further aspect, there is provided a method for enriching circulating tumor cells with a kit described above. In an embodiment, the method includes mixing a sample containing the circulating tumor cells with magnetic nanoparticles to form a mixture containing magnetic nanoparticle-circulating tumor cell complexes; and allowing the mixture to enter the fluid flowing chamber through the inlet of the micro-fluidic device and out of the fluid flowing chamber through the outlet, wherein the magnetic nanoparticle-circulating tumor cell complexes are enriched on the lower surface of the plasma fluorescence enhancement chip under the action of magnetic field generating component. With the method according to embodiments of the present disclosure, it is possible to achieve the simple separation and efficient capture and enrichment of the circulating tumor cells and also to perform NIR enhanced fluorescence detection of the enriched circulating tumor cells in high sensitivity. The plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports in related art.

In still a further aspect, there is provided in embodiments a system for detecting circulating tumor cells. In an embodiment, the system includes a micro-fluidic device described above and a fluorescence detection device. With the system according to embodiments of the present disclosure, it is possible to achieve the simple separation and efficient capture and enrichment of the circulating tumor cells and also to perform NIR enhanced fluorescence detection of the enriched circulating tumor cells in high sensitivity.

In an embodiment of the present disclosure, the fluorescence detection device is a near-infrared (NIR) fluorescence detection device. The inventors found that the plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs. Using the NIR fluorescence detection device, the detection sensitivity of the system to CTCs is significantly improved.

In still a further aspect, there is provided in embodiments a method for detecting circulating tumor cells. In an embodiment, the method includes: enriching circulating tumor cells contained in a sample according to the method described above thereby allowing the circulating tumor cells to be enriched on a plasma fluorescence enhancement chip; detecting the circulating tumor cells enriched on the plasma fluorescence enhancement chip by a near-infrared fluorescence detection device. With the method according to embodiments of the present disclosure, it is possible to achieve the NIR enhanced fluorescence detection of the circulating tumor cells in high sensitivity. The plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports in related art.

The core of the technology for enriching circulating tumor cells provided in embodiments of the present disclosure is the micro-fluidic device based on the plasma fluorescence enhancement chip, as described above.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the drawings, in which:

Fig. 1 is a schematic diagram of a micro-fluidic device for enriching circulating tumor cells according to an embodiment of the present disclosure.

Fig. 2 is a schematic diagram of a system for detecting circulating tumor cells according to an embodiment of the present disclosure.

Fig. 3 is a schematic diagram of a device for screening CTCs according to an embodiment of the present disclosure, where a) shows a screening process and a schematic diagram (upper panel) and a cross sectional view (lower panel) of the device; b) shows an integrated digital image of a pGold chip and a module (left) and a SEM image of the pGold chip with a scale bar of 500 nm (right) ; c) shows a schematic diagram of NIR fluorescence detections of biomarker proteins of the CTCs on the pGold chip; RBC refers to red cells.

Fig. 4 shows characterization results of MNPs according to an embodiment of the present disclosure, where a) is a SEM; b) is a TEM; c) shows a hysteresis curve of MNPs; d) shows a magnetic separation of MNPs (from colloidal suspension) by a magnet, a scale bar is 200 nm for a) and 100 nm for b) .

Fig. 5 shows an extinction spectrum of a pGold chip according to an embodiment of the present disclosure, which coincides with excitations (line) and emission regions (shaded area) of IRDye680 and IRDye800.

Fig. 6 shows results of multi-enhanced NIR fluorescence according to an embodiment of the present disclosure, including NIR fluorescence (labeled by IRDye800) images of enriched CTCs (upper, illustration shows bright field image) and mean fluorescence intensities (lower) , where CTCs includes a) MCF-7, b) SKBR-3 and c) COLO-205, chips used include (i) glass chip, (ii) sGold chip, (iii) pGold chip without immuno-magnetic enrichment, and (iv) pGold chip with immuno-magnetic enrichment, and for all the fluorescence images in a-c, the scale bar is 10 μm.

Fig. 7 shows scanning fluorescence images of CTCs according to an embodiment of the present disclosure, where NIR fluorescence (IRDye800) images of a) MCF-7, b) SKBR-3 and c) COLO-205 on chips are recorded, chips used include (i) glass chip, (ii) sGold chip, (iii) pGold chip without immuno-magnetic enrichment, and (iv) pGold chip with immuno-magnetic enrichment, and for all the fluorescence images in a-c, the scale bar is 100 μm.

Fig. 8 is a graph showing mean sizes of CTCs including MCF-7, SKBR-3 and COLO-205, with magnetic enrichment or without magnetic enrichment, on pGold chips, according to an embodiment of the present disclosure, where black represents mean sizes of the CTCs without magnetic enrichment and gray represents mean sizes of the CTCs with magnetic enrichment, the three cells are analyzed to calculate a standard deviation (s. d. ) as an error bar, and the data are shown as mean ± s. d. (N = 3) .

Fig. 9 shows diagrams for illustrating a mechanism and results of multi-enhancement according to an embodiment of the present disclosure,

where a) shows schematic diagrams illustrating (i) enhanced NIR fluorescence of CTCs (MCF-7) without immuno-magnetic enrichment and (ii) multi-enhanced NIR fluorescence of CTCs on pGold chip with magnetic enrichment; b) and c) respectively shows side-views SEM images and top-view SEM images of CTCs (i) without immuno-magnetic enrichment and (ii) on pGold chip with magnetic enrichment, for the images both in b and c, the scale bar is 5 μm.

Fig. 10 shows diagrams for illustrating results of multi-analysis of biomarkers according to an embodiment of the present disclosure,

where a) shows fluorescence microscope images of CTCs (MCF-7) on glass chip; b) shows fluorescence microscope images of CTCs (MCF-7) on pGold chip; c) shows fluorescence scan images of CTCs (MCF-7) on glass chip; d) shows fluorescence scan images of CTCs (MCF-7) on pGold chip; Left: IRDye680 channel of anti-EGFR; Middle: IRDye800 channel of anti-CK; Right: merged result of two channels, the illustration on the right of a) shows a bright field image, in both a) and b) , the scale bar is 10 μm.

Fig. 11 shows the capturing efficiency of CTCs, according to an embodiment of the present disclosure, where a) shows the identification of captured CTCs displaying (i) bright field, (ii) DAPI labelled, (iii) Anti-CK labelled, and (iv) merged fluorescence images of a captured CTC (MCF-7) ; b) -d) show capture efficiencies of b) MCF-7, c) SKBR-3, and d) COLO-205 in (i) PBS solution and (ii) whole blood; five independent experiments were performed to calculate the standard deviation (s.d. ) , and the data shown in figures are record as mean of 5 calculations ± s.d. (n=5) , the scale bar in a) is 10 μm.

Fig. 12 shows the scanning fluorescence analysis results of two biomarkers from several cells, according to an embodiment of the present disclosure, where a) and b) show multiplexed protein marker analysis of several CTCs (MCF-7) captured on a) glass chips and b) pGold chips by NIR fluorescence scanners. Left: IRDye680 channel for anti-EGFR; Middle: IRDye800 channel for anti-CK; Right: merge of two channels; the scale bar in a) and b) is 20 μm.

Fig. 13 shows diagrams of identification results of the captured CTCs, according to an embodiment of the present disclosure, displaying (i) bright field, (ii) DAPI labelled, (iii) Anti-CK labelled of captured a) SKBR-3 and b) COLO-205 on pGold chips, where the scale bar in a) and b) is 10 μm.

Fig. 14 shows the screening results of CTCs in cancer patients, according to an embodiment of the present disclosure, where a) shows the number of CTCs captured on-chip from 5 mL of whole blood obtained from 11 cancer patients; b) shows fluorescence images displaying the identification of a leukocyte (CD45+/DAPI+/CK-, up) and a captured CTC (CD45-/DAPI+/CK+, down) from Patient 1 (lung cancer) , in which the scale bar is 5 μm.

Fig. 15 shows diagrams displaying identification results of CTCs in cancer patients, according to an embodiment of the present disclosure, where fluorescence images of a leukocyte (upper left, CD45+/DAPI+/CK-, ) and a captured CTC (lower right, CD45-/DAPI+/CK+) from Patient 5 are shown by tricolour fluorescent method: a) FITC channel for anti-CD45, b) IRDye800 channel for anti-CK, c) DAPI channel for cell nucleus and d) merged channel, and the scale bar in a) -d) is 10 μm.

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

In the present disclosure, the inventors have for the first time demonstrated multi-enhanced NIR fluorescence screening of CTCs on the plasmonic gold chip. The inventors combined microfluidic immuno-magnetic enrichment of CTCs with on-chip NIR-FE detection. Owing the manipulation of cells and their morphology through enrichment, the inventors observed multi-enhanced NIR fluorescence by about 50-122 folds, superior to other on-chip protocols. The inventors conducted multiplexed protein biomarkers detection of CTCs and applied the method in spiked experiments and the detection of blood samples of cancer patients. The technical solutions of the present disclosure not only contributes to the advanced CTCs analysis towards efficient cancer management in clinics, but also to the design of interface between plasmonic materials/device and cells for bio-analytical applications.

In an aspect, the present disclosure provides in embodiments a micro-fluidic device for enriching circulating tumor cells. In an embodiment, with reference to Fig. 1, the micro-fluidic device 1000 includes: a body 100, defining a fluid flowing chamber, the fluid flowing chamber having an opening at an upper side thereof; an inlet 200, disposed at a bottom of the body 100; an outlet 300, disposed at the bottom of the body 100; a plasma fluorescence enhancement chip 400, disposed at the opening of the fluid flowing chamber, and loaded with antibodies at a lower surface thereof; and a magnetic field generating component 500, disposed at an upper surface of the plasma fluorescence enhancement chip 400. Specifically, the magnetic field generating component 500 is a magnet. The plasma fluorescence enhancement chip 400 is detachably disposed at the opening of the fluid flowing chamber.

In another aspect, the present disclosure provides in embodiments a system for detecting circulating tumor cells. In an embodiment, with reference to Fig. 2, the system includes a micro-fluidic device 1000 described above; and a fluorescence detection device 2000. Specifically, the fluorescence detection device 2000 is a near-infrared fluorescence detection device.

With the micro-fluidic device and system according to embodiments of the present disclosure, it is possible to achieve the simple separation and efficient capture and enrichment of the circulating tumor cells, and also to perform NIR enhanced fluorescence detection of the enriched circulating tumor cells in high sensitivity. The plasma fluorescence enhancement chip (pGold) can achieve multi-enhanced NIR fluorescence by about 50-122 folds under the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports in related art.

In the following, the technical solution of the present disclosure will be described in detail in combination with examples. It should be appreciated to those skilled in the art that, the examples described below are explanatory, illustrative, and used to generally understand the present disclosure, and shall not be construed to limit the present disclosure. Examples which do not indicate specific techniques or conditions are carried out either in accordance with the techniques or conditions described in the literatures in the related art or in accordance with the product specifications. Reagents or instruments whose manufacturers are not indicated are all conventional products, which are commercially available.

Methods and materials used in the following experiments are as follows.

Preparation of chips

Three types of chips were prepared, including a plasma gold (pGold) chip, a sputter gold (sGold) chip and a glass chip. The pGold chips with abundant nano islands were synthesized in an optimized solution by a seeding process. Briefly, slides were immersed in a solution of HAuCl₄ (5 mM) and NH₄OH (20 μL/mL, 0.6%, V%) for 20 minutes under vigorous stirring. The resulted slides were washed twice by sequentially immerging into two water baths. After washing, the obtained slides inoculated with Au ions were placed in a solution of sodium borohydride (1 mM) for 1 min at room temperature to be reduced and then washed twice by sequentially immersing into two water baths. These slides were transferred to another solution of HAuCl₄ and NH₄OH at a molar ratio of 1: 1 for the continued growth of Au films for 20 min at room temperature under vigorous shaking (100 RPM) and then washed to obtain the final pGold chips. The pGold chips were stored in a sealable container at room temperature.

The sGold chips were prepared by a magnetron sputtering method (GSL-1100X-SPC-16M, Shenyang Kejing Auto-instrument Co., Ltd. ) . Gold target (99.99%) was purchased from Shanghai Daheng Optical Precision Machinery Co., Ltd. The sputtering process was carried out at 20 mA for 2 min under vacuum (10 Pa) . The sGold chips were stored in a sealable container at room temperature.

Glass was purchased from Sinopharm Chemical Reagent Co., Ltd. The slides were immersed into 15 mL Piranha solution (3/1, V/V, concentrated sulfuric acid (95%by mass) and hydrogen peroxide (30%by mass) ) for 5 min to remove the organic residue, and washed for three times with pure water, then stored at room temperature.

Construction of micro-fluidic device

Key components of the micro-fluidic device include a chip module, a fluid pump (19*13*6.2mm, 350-400 mT, 42.8*42.3*48.3mm) , a sample tube (15 mL centrifuge tube, BD Company in United States) , a magnetic bar (19*13*6.2mm, 350-400 mT) and a control system, all provided by Nanolife systems Co., Ltd., Ningbo, China. The die for integrating chip is made of polypropylene and is designed with optimized size and layout. Liquid flow rate (1 mL/h) is adjusted by driving the air in the pipe with a micro-fluidic pump.

Preparation of magnetic nanoparticles

Magnetic nanoparticles (MNPs) were functionalized by epithelial cell adhesion molecule antibodies (EpCAM antibodies) so as to facilitate the immuno-magnetic enrichment of cells. MNPs were streptavidin-labeled MNPs purchased from MicroMod (Rostock, Germany) . Immuno-magnetic nanoparticles functionalized by EpCAM antibodies were synthesized through the specific interaction between the streptavidin and biotin. Briefly, streptavidin-labeled MNPs were washed twice with phosphate buffered saline (PBS, pH=7.4, 50 mM) , afterwards, a mixture of streptavidin-labeled MNPs and biotinylated EpCAM antibodies (Abcam Inc., Cambridge, USA) (100: 1, W/W) was shaken at 37 ℃ for 1.5 h so as to allow the streptavidin-labeled MNPs to sufficiently react with the biotinylated EpCAM antibodies, and then washed twice with 50 mM PBS (pH = 7.4) , subsequently, the free EpCAM antibodies were removed by centrifugation (200 G, 5 min) and magnetic force. The resulting functionalized MNPs were re-suspended in 50 mM PBS (pH = 7.4) buffer containing 3%bovine serum albumin (BSA, Sigma) for blocking the functionalized MNPs and stored at 4 ℃.

Materials and device characterizations

Materials and device characterizations include transmission electron microscopy (TEM) , scanning electron microscopy (SEM) , hysteresis analysis, UV-visible (UV) spectroscopy, digital images and video. Digital images and videos were taken by Nikon d7100. UV spectra were measured by AuCy UV1901PC spectrophotometer. The TEM image was taken at 10 kV via the JEOL JEM-2100F TEM instrument. 10 μL particle suspension in ethanol (0.1 μg/mL) was dropped onto a copper mesh, and TEM observation was performed after the sample was air dried. SEM analysis was performed at 10 kV via Hitachi S-4800. The particle suspension was dropped on a silicon wafer and dried at room temperature, then observed directly in an SEM apparatus. The hysteresis loop is measured using a vibrating sample magnetometer (Quantum Design, Physical Property Measurement System) (circulating the magnetic field between -50 and 50 kOe) .

Cell culture

Three kinds of cells, including MCF-7 (human breast cancer cell line, EpCAM positive) , SKBR-3 (human breast cancer cell line, EpCAM positive) and COLO-205 (human colon cancer cell line, EpCAM positive) , were cultured with the reported protocol. All cells were provided by Chinese Academy of Sciences (Shanghai Institute of Cell Resource Center) and cultured in a humid 5%CO₂ incubator at 37 ℃. SKBR-3 cells were cultured in the Dulbecco modified Eagle medium (DMEM, Sigma) . COLO-205 cells were cultured in Roswell Park Memorial Institute medium (RPMI-1640) . To obtain free cells, 1 mL trypsin (0.25%, V/V) was dropped into a cell culture flask and digested in a CO₂ incubator for 5 min, and the obtained cells were preserved in PBS (pH=7.4, 50mM) buffer.

Standard addition test

The three cell lines (MCF-7, SKBR-3 and COLO-205) mentioned above each were added to 50 mM PBS (pH=7.4) solution and whole blood respectively to measure the capture efficiency of the method of the present disclosure. The whole blood samples were donated by healthy volunteers. Briefly, 6-200 cells were added to 5 mL PBS (pH=7.4, 50 mM) solution and the whole blood. The capture efficiency (n/N) was calculated by immuno-magnetic enrichment and counting fluorescence analysis based on the number of captured cells (n) and spiked cells (N) .

Immuno-magnetic enrichment of CTCs

For immuno-magnetic enrichment, 30 μg functionalized MNPs was incubated with a bio-mixture (5 mL spiked sample or patient blood) at 37 ℃ for 1.5 h to selectively label CTCs. The mixture is added to the sample tube of the microfluidic device, which is connected to the chip module. Flow rate was 1mL/h, and about 5h is required to complete the enrichment. MNP labeled CTCs were attached to the surface of the chip in a magnetic field (from the magnet) and continuously washed for three times with PBS (pH = 7.4, 50 mM) containing 0.05%tween-20 (PBST) . The resulting chip was treated away from light with ice acetone solution (99%, V%) for about 15 min and stored at 4 ℃.

Near infrared fluorescent dye labeled antibody

CK detection antibodies and EGFR detection antibodies were labelled with fluorescent dyes (IRDye800/680) . Antibodies were labelled with IRDye800/680 through reaction of EDD/NHS (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) . The IRDye680-NHS/IRDye800cw-NHS ester (Licor Biosciences) was dissolved in anhydrous dimethylsulfoxide and stored in the dark at -20 ℃ before use. The antibodies and dyes were mixed at a molar ratio of 1: 4 and placed on the shaker for 1.5 h in the dark. NAP-5 column (GE Healthcare) was used to remove uncoupled dyes and purify the labelled antibodies. The raw material mixture after the EDC/NHS reaction was added to a column having 10 mL PBS (pH = 7.4, 50 mM) . The eluate containing the labelled antibodies was collected and stored away from light at -20 ℃.

Chip online staining

In order to stain the biomarker protein attached to the cell surface, chips were washed with PBST for three times and then incubated with dye-labelled detection antibodies (250 μL, 5 μg/mL) in the dark for 1.5 h at 37 ℃. Afterwards, the dyed chips were washed with PBST for three times to remove impurities. In order to stain the cell nucleus, the chips were stained for 15 min with 4', 6-diamidino-2-phenylindole (DAPI, 5 mg/mL) and then washed for three times with PBST (pH = 7.4, 50 mM, 0.05%Tween-20) . The resulting chips were stored at 4 ℃ before near infrared imaging and scanning.

Scanning electron microscopy and microscopic imaging

Cells on chip were detected and identified by scanning and microscopic imaging. Innopsys scanner (InnoScan 710) was used to scan the chip and thereby imaging the cells. The scan resolution of all experiments was set to 3 μm/pixels. Fluorescence scanning images were analysed by NIS-Elements BR 4.51.00 (ECLIPSE Ti-E related software, Nikon) , and the fluorescence intensity was up to 65536. ECLIPSE Ti-E (Nikon) fluorescence microscope with a specific colour filter (for DAPI, fluorescein isothiocyanate (FITC) , IRDye680/800) was used to record the microscopic imaging of the cells.

CTCs analysis of cancer patient

5 mL whole blood was collected from a cancer patient and used for CTC capture on chip. Informed consents of the patients were obtained at the start of the project. Cancer patients are diagnosed according to the national comprehensive cancer network (NCCN) guide. Medical conditions, such as active bleeding, were excluded. CTCs were identified by fluorescence staining results (CD45-/DAPI+/CK+) with morphological analysis, and the number of CTCs in the cancer patient was counted. All the research projects have been approved by the institutional ethics committee of Ningbo second hospital and the school of biomedical engineering of Shanghai Jiao Tong University.

Experiment process and results according to embodiments of the present disclosure will be described in detail as follows.

Design of CTCs screening device

The inventors designed a micro-fluidic device to perform the immuno-magnetic enrichment of CTCs on-chip, as shown in Fig. 3. The inventors used a positive enrichment method and labelled CTCs in patient blood or spiked cells by epithelial cell adhesion molecule antibodies (anti-EPCAM) functionalized magnetic nanoparticles (MNPs) . The functionalized MNPs have an average size of about 25-40 nm according to scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis shown in Figs. 4a and 4b, with a saturation magnetization value of about 0.9 emu/g (circulating the magnetic field between -50 and 50 kOe, Fig. 4c) without remanence for quick separation by magnet within 1 min (Fig. 4d) . Then bio-mixture was pumped into the micro-fluidic channel. CTCs were selectively enriched under the combination of gravity and magnetic forces for final detection.

Integration and detection on chips

An important feature of the device according to the present disclosure is the easy integration and detection using various chips, as shown in Fig. 3b. For integration with chips, the inventors developed the module allowing the seal of micro-fluids without leakage, attached on the surface of the chip in a clickable (detachable) manner (Fig. 3b, Left) . The inventors prepared the plasmonic gold (pGold) chip for use in a controlled solution seeding process and observed the gold nano-islands with unique morphology and inter-islands distances less than 100nm, such as about10 nm by SEM (Fig. 3b, Right) . For detection on chips, the characteristic morphology of pGold chip afforded plasmon resonances in the NIR region in the absorption/extinction spectrum (Fig. 5) . The inventors labelled the biomarker proteins of enriched CTCs with NIR fluorescence dyes (IRDye800/680) for on-chip analysis (Fig. 3c) .

NIR-FE detection of CTCs on-chip

The inventors performed NIR-FE detection of three types of CTCs (MCF-7, SKBR-3, and COLO-205) on selected chips (Figs. 6 and 7) , including glass chip, sputter gold (sGold) chip, and pGold chip (with and without immuno-magnetic micro-fluidic enrichment) . As shown in Fig. 6a, for MCF-7, glass chip and sGold chip afforded very weak signals with mean fluorescent intensities of 637 and 1456, respectively, due to none NIR-FE detection or quenching of fluorescence on surface. For comparison, the inventors enabled NIR-FE detection of cells using pGold chips without magnetic enrichment, yielding mean fluorescent intensities of 5136 and enhancement factors of 8.1 folds over glass chips.

Notably, with magnetic enrichment, the inventors observed increased cell size by 30.4%(from about 136.4 to about 177.9 μm², Fig. 8) and further enhanced NIR fluorescence signals of CTCs, affording mean fluorescence intensities of 32540 and enhancement factors of 51.1 folds (55.3 folds with subtracted background) over glass chips. The inventors found similar results using SKBR-3 with enhancement factors of 62.2 folds (69.1 folds with subtracted background, Fig. 6b) and COLO-205 with enhancement factors of 95.3 folds (122.0 folds with subtracted background, Fig. 6c) . Results are summarized in Tables 1-3 and demonstrate the multi-enhanced NIR fluorescence (51.1-122.0 folds) detection on the pGold chip.

Table 1 Mean fluorescence intensity analysis of MCF-7

Table 2 Mean fluorescence intensity analysis of SKBR-3

Table 3 Mean fluorescence intensity analysis of COLO-205

Mechanism of multi-enhancement

The inventors proposed the mechanism of multi-enhanced NIR fluorescence detection and performed SEM for demonstration (Fig. 9) . Normal NIR-FE detection of cells lacks manipulation of cellular morphology and optimization of mean distance between the cells and pGold chip surface. In contrast, the inventors compressed the cells by magnetic force during immuno-magnetic microfluidic enrichment and reduced the mean distance between the cells and pGold chip surface, which led to the multi-enhancement effect (Fig. 9a) . As demonstrated in Fig. 9b, the side-view SEM images of pGold chips demonstrate the normal and compressed cellular morphologies (with reduced thickness by about 2 μm) , for the cells without and with magnetic separation, respectively. Moreover, in the top-view SEM, The inventors also found the increased cell sizes due to the compressing process, which was consistent with side-view SEM and previous fluorescence imaging results (Fig. 9 and Fig. 8) , validating the proposed mechanism of multi-enhancement.

Multiplexed protein marker analysis

The inventors demonstrated multiplexed proteins biomarker analysis of CTCs through both IRDye800 and IRDye680 channels (Fig. 10) . The inventors detected two proteins biomarkers for cancer cells including cytokeratin (CK) and epidermal growth factor receptor (EGFR) , on the IRDye800 and IRDye680 channels, respectively. The inventors observed clear cellular images with enhanced NIR fluorescence by microscopy on the pGold chip (single cell analysis in Fig. 10a and multiple cells analysis in Fig. 11) , superior to the results on the glass chip in both channels. Notably, the inventors also imaged the enriched CTCs on-chip through micro-scanning (single cell analysis in Fig. 10b and multiple cells analysis in Fig. 12) , which afforded consistent results with microscopy and can be advantageous towards rare cells analysis in real case with enhanced throughput and easy operation for automation applications in large-scale.

Capture efficiency and identification

The inventors investigated the capture efficiency of the device according to the present disclosure on CTCs through series of parallel spiked experiments (Fig. 11) . The inventors identified the enriched CTCs through fluorescence staining in Fig. 11, showing the typical images of a CTC. The inventors spiked 6-200 MCF-7 into either standard solutions (Fig. 11b-i) or whole blood (Fig. 11b-ii) and counted the number of enriched CTCs. The capture efficiency reached 86.9%in standard solutions and 81.4%in whole blood. The inventors observed similar results dealing with another two types of cells (including SKBR-3 (Figs. 11c, 13a) and COLO-205 (Figs. 11d, 13b) ) . The capture efficiency in whole blood is comparable and slightly lower (about 5%) , compared with that in standard solution, due to the high sample complexity. Thus, the method of the present disclosure displayed desirable capture efficiency of various low concentrated CTCs.

Diagnostic application in cancer patients

The inventors applied the method according to the present disclosure to capture CTCs contained in blood from cancer patients to demonstrate the practical application of the pGold chip and the device of the present disclosure. Using the as-established protocol, 1-20 CTCs was/were captured from 5 mL of whole blood obtained from 11 patients (Fig. 14a and Table 4) with various phenotypes, including breast, lung, pancreatic, and colorectal cancer. Based on the biomarker analysis of CD45 and CK, and 4′, 6-diamidino-2-phenylindole (DAPI) fluorescence staining, Fig. 14b displays the typical single cell fluorescence images of the leukocyte (CD45+/DAPI+/CK-) and identified CTC (CD45-/DAPI+/CK+) . The inventors also showed the typical multiple cells fluorescence images by the multi-colour fluorescence method for CTC identification in Fig. 15. The inventors achieved multi-enhanced NIR fluorescence screening of CTCs on the pGold chip for cancer patients.

Table 4

There have been intense research efforts focusing on the analysis of CTCs for cancer diagnostics. Due to the efficient antibody recognition, chemical methods afford high selectivity and sensitivity in the enrichment of CTCs, which provides great advantages in clinics and has been approved by FDA. Notably, both the enrichment platform and down-stream detection are critical for the antibody based methods towards analysis of CTCs. The design of the present disclosure is distinct from previous CTC studies and demonstrates the rational combination of enrichment platform and down-stream detection by the pGold chip. In embodiments of the present disclosure, the micro-fluidic system provides high capture efficiency in facile separation, and more importantly, enables the integration of diverse chips for easy operation and sensitive detection for further application tests.

NIR-FE (near infrared fluorescence enhancement) detection by plasmonic materials and device addresses key issues (such as low sensitivity and low throughput) for biomedical applications. Despites the substantial progress in the CTC diagnostic use, most of current approaches focuses on the structural optimization of materials and selection of specific biomarkers. The manipulation of analytical targets (e.g. CTCs) is still difficult and remains to be further explored in analysis, which may provide new insights in the field. The inventors have found that the change of cellular morphology on the pGold chip results in multi-enhanced NIR fluorescence by ～50-122 folds, during the microfluidic immuno-magnetic enrichment of CTCs, which is two orders of magnitudes higher compared to other chips and best reports. The technical solutions according to embodiments of the present disclosure indicate the significance of nano-/micro-scaled manipulation of targets in bio-analytical filed, which is usually underlined or even neglected in current research and practice.

In the cellular imaging and applications, multiplexed analysis and laboratory automation are crucial for applications in large-scale. In this work, the present disclosure demonstrates the multiplexed analysis of CK and EGFR in two NIR fluorescence channels for potential cell typing. Meanwhile, the inventors performed fast and automated micro-scanning of enriched CTCs on-chip and the as-recorded images can be subject to digital signal processing towards none-microscope cellular analysis. Notably, the inventors validated the established protocol not only in spiked experiments, but also in clinics CTCs analysis of patients with various cancer types. Considering the merits as demonstrated above, the inventors anticipate that the method of the present disclosure will be applied in hospitals for cancer management and rare cell analysis in large-scale.

In summary, embodiments of the present disclosure report multi-enhanced NIR fluorescence screening of CTCs for cancer patients. The present disclosure not only advances the rare cells analysis including but not limited to CTCs and cancer in the biomedical field, but also shed lights on the design of interface between plasmonic hosts (e.g. plasma resonance materials or devices) and guests (e.g. cells) to produce better application platforms and detection techniques in near future.

In the specification, it is to be understood that terms such as “central, ” “longitudinal, ” “lateral, ” “length, ” “width, ” “thickness, ” “upper, ” “lower, ” “front, ” “rear, ” “left, ” “right, ” “vertical, ” “horizontal, ” “top, ” “bottom, ” “inner, ” “outer, ” “clockwise, ” and “counterclockwise” should be construed to refer to the orientation as then described or as shown in the drawings under discussion. These relative terms are for convenience of description and do not require that the present disclosure be constructed or operated in a particular orientation.

In addition, terms such as “first” and “second” are used herein for purposes of description and are not intended to indicate or imply relative importance or significance or to imply the number of indicated technical features. Thus, the feature defined with “first” and “second” may comprise one or more of this feature. In the description of the present disclosure, “a plurality of” means two or more than two, unless specified otherwise.

In the present disclosure, unless specified or limited otherwise, the terms “mounted, ” “connected, ” “coupled, ” “fixed” and the like are used broadly, and may be, for example, fixed connections, detachable connections, or integral connections; may also be mechanical or electrical connections; may also be direct connections or indirect connections via intervening structures; may also be inner communications of two elements, which can be understood by those skilled in the art according to specific situations.

In the present disclosure, unless specified or limited otherwise, a structure in which a first feature is “on” or “below” a second feature may include an embodiment in which the first feature is in direct contact with the second feature, and may also include an embodiment in which the first feature and the second feature are not in direct contact with each other, but are contacted via an additional feature formed therebetween. Furthermore, a first feature “on, ” “above, ” or “on top of” a second feature may include an embodiment in which the first feature is right or obliquely “on, ” “above, ” or “on top of” the second feature, or just means that the first feature is at a height higher than that of the second feature; while a first feature “below, ” “under, ” or “on bottom of” a second feature may include an embodiment in which the first feature is right or obliquely “below, ” “under, ” or “on bottom of” the second feature, or just means that the first feature is at a height lower than that of the second feature.

Reference throughout this specification to “an embodiment, ” “some embodiments, ” “one embodiment” , “another example, ” “an example, ” “a specific example, ” or “some examples, ” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments, ” “in one embodiment” , “in an embodiment” , “in another example, ” “in an example, ” “in a specific example, ” or “in some examples, ” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A micro-fluidic device for enriching circulating tumor cells, comprising:

a body, defining a fluid flowing chamber, the fluid flowing chamber having an opening at an upper side thereof;

an inlet, disposed at a bottom of the body;

an outlet, disposed at the bottom of the body;

a plasma fluorescence enhancement chip, disposed at the opening of the fluid flowing chamber, and loaded with antibodies at a lower surface thereof; and

a magnetic field generating component, disposed at an upper surface of the plasma fluorescence enhancement chip.
The micro-fluidic device according to claim 1, wherein the magnetic field generating component is a magnet.
The micro-fluidic device according to claim 1 or 2, wherein the plasma fluorescence enhancement chip is detachably disposed at the opening of the fluid flowing chamber.
The micro-fluidic device according to any one of claims 1 to 3, wherein the plasma fluorescence enhancement chip is a plasma gold chip.
The micro-fluidic device according to claim 4, wherein the plasma gold chip comprises gold nano islands.
The micro-fluidic device according to claim 5, wherein the plasma gold chip is of an inter-islands distance less than 100 nm.
The micro-fluidic device according to claim 6, wherein the plasma gold chip is of an inter-islands distance of about 10 nm.
A kit for enriching circulating tumor cells, comprising:

magnetic nanoparticles for capturing the circulating tumor cells from a blood sample; and

a micro-fluidic device according to any one of claims 1 to 7.
The kit according to claim 8, wherein the magnetic nanoparticles are loaded with a marker specific for the circulating tumor cells on surfaces thereof.
The kit according to claim 9, wherein the marker is anti-EpCAM.
A method for enriching circulating tumor cells with a kit according to any one of claims 8 to 10, comprising:

mixing a sample containing the circulating tumor cells with magnetic nanoparticles to form a mixture containing magnetic nanoparticle-circulating tumor cell complexes; and

allowing the mixture to enter the fluid flowing chamber through the inlet of the micro-fluidic device and out of the fluid flowing chamber through the outlet, wherein the magnetic nanoparticle-circulating tumor cell complexes are enriched on the lower surface of the plasma fluorescence enhancement chip under an action of the magnetic field generating component.
A system for detecting circulating tumor cells, comprising:

a micro-fluidic device according to any one of claims 1 to 7; and

a fluorescence detection device.
The system according to claim 12, wherein the fluorescence detection device is a near-infrared fluorescence detection device.
A method for detecting circulating tumor cells, comprising:

enriching circulating tumor cells contained in a sample according to a method according to claim 11, thereby allowing the circulating tumor cells to be enriched on a plasma fluorescence enhancement chip; and

detecting the circulating tumor cells enriched on the plasma fluorescence enhancement chip by a near-infrared fluorescence detection device.