ZnO nanowire-integrated bio-microchips for specific capture and non-destructive release of circulating tumor cells

Heng Cui a, Qing Liu a, Rui Li a, Xiaoyun Wei a, Yue Sun a, Zixiang Wang a, Lingling Zhang a, Xing-Zhong Zhao a, Bo Hua *b and Shi-Shang Guo *a
aKey Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, 430072, Hubei, P. R. China. E-mail: gssyhx@whu.edu.cn
bThe State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, 430072, Hubei, P. R. China. E-mail: kq000511@whu.edu.cn

Received 26th August 2019 , Accepted 26th November 2019

First published on 27th November 2019

Circulating tumor cells (CTCs) are one type of significant biomarker in cancer patients’ blood that have been attracting attention from researchers for decades, and their efficient and viable isolation is of vital importance in cancer prevention and treatment. However, the development of efficient and low-cost bio-microchips still faces significant challenges. In this paper, we construct a novel three-dimensional micro-nano bio-microchip that has dual functions of specifically capturing and non-destructively releasing cancer cells. ZnO nanowire arrays were vertically grown on the surface of a polydimethylsiloxane (PDMS) pillar substrate with a gear structure (ZnO-coated G-PDMS pillar microchips). The gear structure provides more binding sites for antibodies and target cancer cells, while ZnO nanowires provide a rough surface for CTC attachment and size-specific effects for retaining CTCs. For subsequent culture and bioanalysis, the captured CTCs can be non-destructively released with high efficiency and good viability using a mild acidic solution treatment. Furthermore, the manufacturing process of the G-PDMS pillar microchips is convenient and low-cost, and the preparation approach of the ZnO nanowire is mature and simple to operate. In particular, the bio-microchips showed high capture efficiency (91.11% ± 5.53%) and excellent cell viability (96%) using a spiked cell sample. Moreover, we successfully achieved the specific fluorescent labeling of CTCs in 9 clinical breast cancer patients’ samples. The ZnO-coated G-PDMS pillar microchips not only have great potential for new target drug development for cancer stem cells but also open up new opportunities for individualized treatment.

1. Introduction

Tumor metastasis is the leading cause of cancer deaths, and the spread of cancer in the body is caused by circulating tumor cells (CTCs). These cancer cells are detached from the primary tumor, then circulated in the blood and transferred to a secondary site. These CTCs in peripheral blood carry information about the primary tumor and may be valuable biomarkers for early cancer diagnosis, and they may alone facilitate the monitoring of the effectiveness of drug treatments.1–4 Given that CTCs are extremely rare in the blood, researchers are committed to developing fast, efficient, and non-destructive technologies for CTC isolation.5–7 Furthermore, the downstream analysis of CTCs relies on high efficiency release and good cell viability. To this end, the establishment of a cell-friendly intelligent response platform will open up broad prospects for clinical applications. Of course, high stability and low fabricating costs are also quite necessary. The current situations of the field show that various approaches and devices have been developed for CTC capture and identification, such as density gradient centrifugation,8,9 immunomagnetic bead capture technology,10,11 size-based methods,12 filtration-based methods,13 microfluidic chips14,15 and flow cytometry.16 However, for example, in the approach of immunomagnetic beads to capture CTCs, the efficient release of cells is an obstacle, and in the size-based sorting method, the purity of CTCs after separation is a great challenge. In contrast to the existing technologies, microfluidic chips are easy to integrate with other functional units and have adjustable features, which are more sensitive to the enrichment of CTCs, especially when coupled with specific molecules. However, considering the scarcity of CTCs, the capture efficiency of microfluidic chips has yet to be optimized.

In recent years, with the continuous development of nanotechnology, a variety of nanomaterials have been used for CTC capture and detection.17,18 Nanomaterials have great value in solving the problem of capture efficiency due to their high surface area to volume ratio and excellent affinity for biomolecules.19 Meanwhile, the size of the nanostructures provides an excellent opportunity for the attachment, extension and differentiation of biomolecules. In particular, magnetic nanoparticles,20 Si nanopillars,21,22 MnO2 nanospheres, chitosan nanofibers23,24 and graphene oxide25,26 have been used to trap CTCs with high capture efficiency. However, some nanostructures are non-degradable, such as nanotubes, Au nanomaterials27,28 and nanoparticles,29,30 thus cell release cannot be achieved and subsequent operation and analysis cannot be carried out. Among these materials, ZnO nanomaterials31 showed satisfactory performances for cell capture and release, such as simple synthesis methods, excellent biocompatibility and degradability.32,33 Considering the advantages of microfluidic chips and nanostructures, we can build a three-dimensional micro/nanostructure substrate that combines the outstanding properties of microfluidic devices with nanomaterials.

From this perspective, we constructed ZnO-coated gear structure PDMS pillar (G-PDMS pillars) microchips that simultaneously combine the advantages of a micro/nano structure (Fig. 1). The microchips shown here are primarily designed to sensitively capture and non-destructively release CTCs using functionalized ZnO nanowires on G-PDMS substrates. There are 1968 gear structure PDMS micropillars on the substrate, each with a diameter of 150 μm and a height of 70 μm. The distance between each structure in the pillars is 100 μm, and the overall size of the microchip is 10 mm × 10 mm. The micro-structured G-PDMS greatly increases the specific surface area to provide more binding sites for CTCs, while nano-sized ZnO nanowires match the pseudopod size of cancer cells to facilitate cancer cell attachment. Compared with other CTC capture chips, the substrate with a micro/nano structure can improve the capture selectivity, especially when combined with specific antibodies (such as anti-epithelial adhesion molecules, anti-EpCAM), which has exciting results on sensitive and effective CTC capture. Moreover, ZnO is sensitive to pH, and CTCs captured by ZnO nanowires can be detached from the pillars quickly, efficiently, and with minimal damage under weakly acidic conditions. The isolated cells maintain good viability and proliferative capacity and can be used for downstream analysis and basic research. It is worth mentioning that other advantages of our microchips are their low cost, easy fabrication and good biocompatibility. The micro-nanostructured chip successfully captured CTCs from the blood of cancer patients, providing a new technology with high sensitivity and high reliability for early cancer diagnosis and long-term monitoring. We hope that this ZnO-coated G-PDMS pillar microchip can be a candidate for clinical diagnosis of CTCs.

image file: c9nr07349c-f1.tif
Fig. 1 Diagram showing a ZnO-coated G-PDMS pillar microchip for capture and release of cancer tumor cells.

2. Results and discussion

2.1 Characterization of the ZnO-coated G-PDMS pillar microchip

In this study, the gear structure PDMS pillar microchip was fabricated by soft lithography technology. The photograph of the functional part is shown in Fig. 2a. The morphology of G-PDMS pillars (Fig. 2b) and the growth of the ZnO nanowire arrays synthesised by a hydrothermal method on the G-PDMS pillar microchip were characterized by SEM. Fig. 2c and d show the SEM image of a ZnO-coated G-PDMS pillar microchip with a diameter of each pillar of approximately 150 μm and a height of approximately 70 μm. Compared with a smooth G-PDMS microchip, the ZnO nanowires did not influence the chip gear structure but provided more antibody modification sites that increase the attachment between the nanostructure substrates and a specific cell, thereby increasing the capture efficiency. It can be clearly seen that the ZnO nanowire array is vertically grown on the chip, and the ZnO nanowire is about ∼200 nm in diameter and 1 μm in height (Fig. 2e and f). The SEM images with different magnifications indicate that the ZnO-coated G-PDMS pillar microchip was successfully prepared.
image file: c9nr07349c-f2.tif
Fig. 2 (a) Photograph of a ZnO-coated G-PDMS pillar microchip. (b) SEM image of G-PDMS pillars. (c and d) SEM images of ZnO-coated G-PDMS pillars with different magnifications. (e and f) SEM images of the ZnO nanowires.

Furthermore, we tried to identify the element distribution through EDS element mapping to verify the existence of ZnO. Zn and O elements are evenly distributed on the G-PDMS pillar surface except the Si elements, which indicates the uniformity of the ZnO nanowires prepared on the microchip (Fig. S1a–d). The FT-IR spectrum showed all the characteristic peaks of the PDMS pillars, and there was a strong and wide peak corresponding to –OH at 3000.3 cm−1, which indicated that the groups can be modified on the chip surfaces (Fig. S2).

2.2 Cell capture performance of the chip

A human breast cancer cell line (MCF-7), primary colorectal cancer cell line (SW480) and human cervical cancer cell line (HeLa) were used as model samples to evaluate the cell capture performance of the microchip and optimize the experiment. MCF-7 and SW480 are EpCAM positive cell lines and HeLa is an EpCAM negative cell line. Before the cell capture assay, FDA-labeled cell lines for cell capture were counted using a blood counting chamber under a microscope. The captured images of the cells are shown in Fig. 3a-i and b-i. The gear structure provided a high surface area-to-volume ratio and most of the cells are successfully attached to the slot of the gear structure regardless of the presence of ZnO nanowires. Fig. 3a-ii and b-ii are the SEM images of CTCs captured using a single G-PDMS pillar, which also indicates that most of the cells were attached to the surface of the pillars and the gear structure. It is a good illustration that our gear-structured micropillars provide a very suitable location for CTC adhesion. Furthermore, to directly observe the morphologies of cells on the ZnO-coated G-PDMS pillar microchip, the G-PDMS pillar microchip without ZnO nanowires was tested as the control. The cells were fixed with glutaraldehyde, and then the cell and microchip interactions were observed using SEM images (Fig. 3a-iii and b-iii). In particular, target cells immobilized on the ZnO-coated G-PDMS pillar microchip had fully extended pseudopodia and were captured with the ZnO nanowires, while target cells with fewer outspread pseudopodia were observed on the bare G-PDMS pillar microchip. This observation is consistent with the literature that surface roughness may be a key factor in enhancing cell-to-substrate interactions.
image file: c9nr07349c-f3.tif
Fig. 3 (a) Image of G-PDMS pillar microchip without ZnO. (b) Image of the ZnO-coated G-PDMS pillar microchip. (a-i) and (b-i) Fluorescence images of the CTC captured microchip. (a-ii) and (b-ii) SEM images of a single G-PDMS pillar of CTCs captured on the microchip. (a-iii) and (b-iii) The SEM images of CTCs captured under high magnification.

Notably, more cells were on the ZnO-coated G-PDMS pillar microchip than on the non-ZnO-coated microchip (Fig. 3a-ii and b-ii). The ZnO nanowires make the surface of the G-PDMS pillar microchips rougher. Indeed, one of the factors affecting cell capture efficiency is the thickness of the nanowires on the substrate, which is related to the hydrothermal reaction time. We achieved three thicknesses of ZnO nanowires at different reaction times (8 h, 10 h, and 12 h). As shown in Fig. S3, it can be seen that the obtained ZnO nanowires are sparse when the reaction time is 8 h, relatively uniform nanowires are obtained when the reaction time is 10 h, and the nanowires are thicker when the time is 12 h. We then investigated the capture efficiency of the three kinds of substrates for CTCs. As shown in Fig. 4a, the capture efficiencies were 68.84 ± 4.02% for 8 h, 93.9 ± 2.96% for 10 h, and 95.98 ± 3.55% for 12 h. The results indicate that the capture efficiency at 8 h is the lowest because the thinner ZnO nanowires lead to poor connectivity of the cells to the substrate. In addition, thicker ZnO nanowires did not achieve an efficient improved performance, which may be due to the saturation of antibody binding and cellular interactions. We used all the ZnO-coated G-PDMS pillar microchips that were hydrothermally reacted for 10 hours in the following experiment.

image file: c9nr07349c-f4.tif
Fig. 4 (a) Capture efficiencies for different hydrothermal reaction times. (b) Three types of cell capture efficiencies using different microchips. (c) Capture efficiency for cancer cells at different incubation times (30 min, 60 min, 90 min, 120 min, and 180 min). (d) Cell capture using the ZnO-coated G-PDMS pillar microchip under different quantities of cancer cells (1000, 3000, 5000, 7000, and 10[thin space (1/6-em)]000) suspended in 1 ml DMEM and 1 ml healthy blood.

Next, we added the three cell lines to different microchips (i.e., flat microchip, G-PDMS pillar microchip and ZnO-coated G-PDMS pillar microchip). The G-PDMS pillar microchip was functionalized with an anti-EpCAM antibody for tumor cell capture. The ZnO-coated G-PDMS pillar microchips are more efficient at capturing than the G-PDMS pillars without ZnO nanowires due to the increased surface roughness under the same conditions (capture efficiencies shown in Fig. S4). Meanwhile, controlled experiments without the G-PDMS pillars and ZnO coating illustrated that non-specific adsorption of CTCs on the surface of the microchip is low (17.84% ± 3.52% for HeLa cells, 26.9% ± 3.36% for MCF-7 cells and 34.74% ± 3.81% for SW480 cells). The results clearly showed that cells were specifically captured on the gear structure with the ZnO nanowires. Furthermore, the capture yields of MCF-7 cells and SW480 cells (high EpCAM expression cells) were relatively high, whereas those of negative EpCAM expression cells (HeLa) were relatively low, suggesting the necessity of the combination of a microchip and a specific antibody capture (Fig. 4b).

Furthermore, we observed the CTC capture efficiency when two essential parameters, the incubation time of CTCs and the number of CTCs, were changed. Incubation time is one of the important factors influencing the cell capture efficiency. We added 5000 SW480 cells to the two different microchips (G-PDMS pillar microchip and ZnO-coated G-PDMS pillar microchip) and incubated for 30 min, 60 min, 90 min, 120 min, and 150 min. After washing with DI water, we observed and counted the cells under an IX81 microscope (Olympus, Japan). Regardless of the substrate types, the cell capture efficiency increased significantly with the incubation time and reached its maximum value (91.11% ± 5.53% for ZnO-coated G-PDMS pillar microchips and 71.84% ± 2.47% for G-PDMS pillar microchips), and then growth slowed, as shown in Fig. 4c.

To further demonstrate the effect of the number of cells on the capture efficiency, we carried out cell capture experiments based on an artificial blood sample. A range of cell numbers was spiked into the mononuclear layer of healthy human blood and DMEM, followed by incubation for 90 min (Fig. 4d). The capture efficiencies were all over 75% (Table S1 for specific values) and the captured cells were mainly linear to the spiked cells. The results showed that the ZnO-coated G-PDMS pillar microchip combined with anti-EpCAM and had sensitive and specific performance for capturing EpCAM positive cells. Our result suggests that the ZnO-coated G-PDMS pillar microchips may offer a capture platform to obtain EpCAM-positive cells for further CTC biomedical studies or gene analysis.

2.3 Cell release and viability assay

The ZnO nanowires are sensitive to pH and can be degraded with low pH to achieve a non-destructive release of the captured cells.34 A substrate-sacrificed method was applied to break down the bonds between the CTCs and the microchip by reacting ZnO nanowires with sodium citrate at room temperature. Fig. 5a and b show the captured cells under a bright and fluorescence field before and after sodium citrate solution (pH = 5.5) treatment, respectively. We explored the release efficiency of cells at different concentrations of sodium citrate solution (50 mM, 100 mM, 150 mM, 200 mM, and 250 mM) and at different times (Fig. 5c). It is worth noting that almost 90% of captured cells were successfully released from the ZnO-coated G-PDMS pillar microchips. The release efficiency was the highest for the 200 mM sodium citrate solution treated within 30 min. The fluorescence images of the released cells at different times and different concentrations of sodium citrate solution are shown in Fig. S5. The average release efficiency (93.95% for MCF-7 and 91.01% for SW480) was obtained with cell counting and the results are shown in Fig. 5d.
image file: c9nr07349c-f5.tif
Fig. 5 (a and b) Representative images of CTCs on the microchip (a) before and (b) 30 min after sodium citrate treatment. (c) Comparison of the release efficiency for different concentrations of sodium citrate at the degradation time ranging from 10 to 40 min. (d) Release efficiency of ZnO nanowires for different kinds of cancer cells. (e) Cell viability of released cells after different re-culture times. (f–h) Corresponding FDA/PI fluorescence microscopy images of re-cultured cells. Scale bar: 50 μm.

The viability experiments of the released cells were carried out to confirm the non-destructive properties of our platform. After degrading with sodium citrate, the released cells were collected and re-cultured with DMEM for several days to investigate their proliferative capacity. The viability of cells just released from the G-PDMS pillar microchips was approximately 68%, and then it gradually increased with the re-culture time, and finally reached up to 96% after a re-culture time of 48 h (Fig. 5e). Fig. 5f–h show the fluorescence microscopy images of re-cultured cells, which exhibited excellent proliferative capacity. This indicates that ZnO-coated G-PDMS pillar microchips have been successfully prepared for efficient capture and friendly release of rare tumor cells due to the functionalizable and biodegradable characteristics of ZnO nanowires.

2.4 Capture and identification of CTCs from cancer patients’ blood samples

Under optimal capture conditions, the cell capture experiments were carried out with patients’ blood samples. Blood samples from nine breast cancer (MCF-7 cell) patients and three healthy individuals were used to study the clinical application value of our platform (Table S2). After the capture experiment, the common three-color immunofluorescence method was used to identify cells, which included phycoerythrin (PE) labeled anti-CK (a marker for epithelial cells), FITC-labeled anti-CD45 (a marker for leukocytes), and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (a marker for nuclear DNA). Indeed, CTCs showed high CK expression and the absence of CD45 expression (DAPI+, CK+, and CD45−), whereas leukocytes exhibited positive CD45 expression and negative CK expression (DAPI+, CK−, and CD45+). Moreover, the size of CTCs (10–30 μm) is larger than the leukocytes (<15 μm) (Fig. 6a). The results showed that the number of cells from the breast patients ranged from 3 to 14 per milliliter while no CTCs were detected in the healthy individuals (Fig. 6b).
image file: c9nr07349c-f6.tif
Fig. 6 (a) Fluorescence micrographs of CTCs isolated from breast cancer patients and healthy individuals. (b) Number of CTCs isolated from a 1 ml blood sample from breast cancer patients. Scale bar: 20 μm.

3. Conclusions

In summary, we have successfully implemented a ZnO-coated G-PDMS pillar microchip that combines a micro-structure and nano-structure, which is proposed as a convenient and cost-effective method for specific capture and non-destructive release of CTCs. The synergistic effects between the gear structure and ZnO nanowires greatly improved the cell-to-microchip interaction, while the destructible nature of the ZnO nanowires also enables high efficiency and high activity of released CTCs. Specific cell capture experiments show that the capture efficiency of CTCs can reach more than 90%, and the final release efficiency and viability can reach up to 93.95% and 96%, respectively. Furthermore, anti-EpCAM-modified ZnO-coated G-PDMS pillar microchips have been successfully used to capture CTCs from peripheral blood samples to demonstrate the ability of cancer diagnosis and therapeutic monitoring. Thus, we foresee that ZnO-coated G-PDMS pillar microchips may have potential application value in CTC-related detection and clinical analysis.

4. Experimental section

4.1 Materials and instruments

Poly(dimethylsiloxane) (PDMS, RTV615) was purchased from GE Toshiba Silicone Corporation Ltd, (USA). Zinc acetate, sodium hydroxide (NaOH), methanol, zinc nitrate, hexamethylenetetramine (HMTA), polyetherimide and sodium citrate were purchased from Sinopharm Chemical Reagent Co., Ltd, China. Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), streptavidin (SA) and Tween-20 were purchased from Invitrogen. Epithelial cell adhesion molecules (EpCAM), Triton X-100, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Sigma-Aldrich. The circulating tumor cells were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin at 37 °C under a 5% CO2 atmosphere in a cell incubator (Thermo Forma Series II, Thermo Scientific). Scanning electron microscopy (SEM) images were taken using a scanning Hitachi S4800 FEG electron microscope. The concentration of the captured cells was measured using a fluorescence microscope (IX81, Olympus, Japan). A low-speed centrifuge (RJ-TDL-40C) was purchased from Wuxi Ruijiang Analytical Instrument Co., Ltd (Wuxi, China).

4.2 Fabrication of the ZnO-coated G-PDMS pillar microchip

The G-PDMS pillar microchip was fabricated using the soft lithography technique. A negative photoresist (SU-8 2050) was spin-coated on the surface of a 4-inch silicon wafer. The photoresist was subjected to baking, exposure, and a development process to obtain a photoresist groove with a depth of 70 μm. The PDMS part A agent and part B agent were mixed in a ratio of 10[thin space (1/6-em)]:[thin space (1/6-em)]1, poured onto the photoresist mold and placed in an 80 °C oven for 2 h. Through the above steps, the template with a size of 1 × 1 cm2 was obtained, and the diameter of the G-PDMS pillar was 150 μm and the distance between each G-PDMS structure pillar was 100 μm. The G-PDMS pillar microchip was ultrasonically washed with DI water before growing ZnO nanowires. The ZnO nanocrystal solution was prepared with 0.01 M sodium hydroxide and 0.03 M zinc acetate dissolved in 25 ml methanol and was stirred at 60 °C for 1 h. Subsequently, the solution was dropped onto the G-PDMS pillar microchip and was annealed at 200 °C to obtain a seed layer. The ZnO nanowires were synthesized by a hydrothermal method. 0.05 M zinc nitrate, 0.05 M hexamethylenetetramine (HMTA) and 2 mM poly-etherimide (PEI) were dissolved in 50 ml water solution and then transferred to a Teflon-lined autoclave. The G-PDMS pillar microchip was placed top-down and immersed in the solution at 100 °C for 10 h. Finally, the G-PDMS pillar microchip was rinsed with DI water and dried at 65 °C for 1 h.

4.3 Functionalization of the ZnO-coated G-PDMS pillar microchip

To chemically modify the microchip, the G-PDMS pillar microchip was treated with O2 plasma. First, the prepared microchips were immersed in the 3-mercaptopropyl trimethoxysilane (MPTMS, 4% in ethanol) solution for 60 min, which introduced a –SH group. The –SH group of MPTMS and the –NH2 group of SA are combined using a protein coupling agent, N-maleimidobutyryloxy succinimide ester (GABS, 1 mg ml−1 in DMSO). After washing with the PBS solution, 50 μl streptavidin (SA, 100 μg ml−1 in PBS) was introduced onto the microchip surfaces overnight at 4 °C. Furthermore, the microchips were processed with 50 μl biotinylated anti-EpCAM (1 mg ml−1 in PBS) and incubated for 1 h before performing the cell capture experiments.

4.4 Cell capture from cell lines and spiked blood samples

In the cell line capture experiment, we used two kinds of target cells: breast cancer cell line (MCF-7) and primary colorectal cancer cell line (SW480). These cell lines were obtained from the Hubei Provincial Cancer Clinical Research Center of Zhongnan Hospital of Wuhan University. For ease of observation, CTCs were pre-stained with FDA solution for 10 min and then added to the microchip. The cells were counted using a cell counting plate under an inverted microscope (IX81, Olympus, Japan). Subsequently, different experimental variables were controlled, cancer cells were introduced to the chip surface and incubated, and then the fluorescence of the captured cells was observed under a microscope. Finally, artificial blood simulation experiments were carried out to suspend a range of CTCs (1000, 3000, 5000, 7000, and 10[thin space (1/6-em)]000 ml−1) in the 1 ml healthy whole blood sample.

4.5 Cell release and viability assay

After we added 2000 CTCs onto the microchip and incubated for 120 min, 200 mM sodium citrate solution (pH = 5.5) was added onto the chip at room temperature to degrade the ZnO nanowires for cell release. The released cells were collected and suspended in the DMEM solution. The cell viability was investigated by using fluorescein diacetate (FDA) and propidium iodide (PI). The mixed solution consisted of 8 μg ml−1 FDA and 20 μg ml−1 PI in the DMEM solution. The collected cells were stained with PDA/PI solution for 10 min at room temperature: the live cells were labeled with FDA and appeared green, and the dead cells were labeled with PI and appeared red.

4.6 CTC capture from clinical samples and immunofluorescence staining

The ethylenediaminetetraacetic acid (EDTA) anti-coagulated breast cancer patients’ and healthy donors’ blood specimens were obtained from the Zhongnan Hospital of Wuhan University, Hubei Cancer Clinical Study Center. Mononuclear layer containing CTCs were obtained by density gradient centrifugation using a lymphocyte separation solution as the medium. The collected cells were rinsed with PBS and introduced into the microchip for the capture procedure. The CTCs were fixed with a 4% formaldehyde solution (PFA) in PBS and perforated with 0.1% Triton X-100, followed by a blocking solution (3% BSA, 5% NGS and 0.1% Tween 20) in PBS. Moreover, CTCs were distinguished from WBCs through an immunofluorescence staining method in which the microchips were immersed in a mixed solution of FITC-labeled anti-CD45 and PE-labeled anti-CK overnight at 4 °C. The CTCs that we have captured from the blood have been identified by three-color immunofluorescence, so the identified cells can be directly counted under a fluorescence microscope. We counted the number of circulating tumor cells from the blood samples of 12 patients.

Conflicts of interest

The authors declare no competing financial interest.


This work was supported by the National Key R&D Program of China [No. 2017YFF0108600] and the National Natural Science Foundation of China [No. 81572860].


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Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr07349c

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