Rhipsalis (Cactaceae)-like Hierarchical Structure Based Microfluidic

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Rhipsalis (Cactaceae)-like Hierarchical Structure based Microfluidic Chip for High Efficient Isolation of Rare Cancer Cells Shuangqian Yan, Xian Zhang, Xiaofang Dai, Xiaojun Feng, Wei Du, and Bi-Feng Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11673 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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ACS Applied Materials & Interfaces

Rhipsalis (Cactaceae)-like Hierarchical Structure based Microfluidic Chip for High Efficient Isolation of Rare Cancer Cells

Shuangqian Yana†, Xian Zhanga†, Xiaofang Daib, Xiaojun Fenga, Wei Dua and Bi-Feng Liua*

a

The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics – Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China b

Cancer Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430074, China

† These authors contributed equally to this work. * Corresponding author Add:

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

Tel: 86-27-8779-2203 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT The Circulating tumor cells (CTCs), originating from the primary tumor, play a vital role in the cancer diagnosis, prognosis, disease monitoring and precise therapy. However, the CTCs are extremely rare in the peripheral bloodstream and hard to be isolated. To overcome current limitations associated with CTC capture and analysis, the strategy incorporating nanostructures with microfluidic devices receives wide attention. Here, we demonstrated a three-dimensional microfluidic device (Rm-chip) for capturing cancer cells with high efficiency by integrating a novel hierarchical structure, the “Rhipsalis (Cactaceae)”-like micropillar array into the Rm-chip. The PDMS micropillar array was fabricated by soft-lithography and rapid prototyping method, which was then conformally plated with a thin gold layer through electroless plating. EpCAM antibody was modified onto the surface of the micropillars through the thiol-oligonucleotide linkers in order to release captured cancer cells by DNase I treatment. The antibody-functionalized device achieved an average capture efficiency of 88% in PBS and 83.7% in whole blood samples. We believe the Rm-chip provided a convenient, economical and versatile approach for cell analysis with wide potential applications.

KEYWORDS: rare cancer cell; three-dimensional capture; micropillar array; electroless plating; hierarchical structure


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1. INTRODUCTION The Circulating tumor cell (CTC) is an important indicator in cancer diagnosis, prognosis and disease monitoring.1-4 However, efficient isolation of CTCs from the peripheral bloodstream has been a persistent challenge due to the rarity of CTCs.5 Cellsearch, the only FDA-approved CTC enumeration system, uses antibody-coated magnetic beads for CTC isolation, dominating the clinical field. However, the capture efficiency of current methods still needs improvement. Recently, research has been carried out for overcoming this issue. Among literatures, microfluidics and nanotechnology are two emerging approaches for improving the capture efficiency of cancer cells.6,7 Two most significant advantages of microfluidics are convenient and precise flow control,8-12 Currently, two types of microfluidic methods have been reported for the isolation of CTCs, including size-based and immunoaffinity-based microfluidics. The size-based microfluidics has poor separation efficiency and purity due to the heterogeneity of the CTCs.13,14 The immunoaffinity-based microfluidics has better performance in comparison to the size-based methods, especially in terms of purity. However, there is still a large room for improving the capture efficiency.14-18 It is a potentially effective to increase the number of binding sites for target cells in microchannels to improve the capture efficiency. As is well known, nanostructures can offer high specific surface area allowing more immunoaffinity molecules to be immobilized onto their surface. Moreover, the hierarchical structures provided extra physical cues to interact with the cancer cells. Therefore, taking advantages of both from nanostructures and microfluidics is becoming a logical



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choice and several groups have attempted to integrate nanostructures into microfluidics channels previously.19,20 For instance, three-dimensional nanopillars,21 nanowires,18 nanofiber network,22-24 silicon-nanopillar (SiNP) array,25 rough glass surfaces26 and other nanomaterials or arrays19,27-37 were incorporated into the microfluidics. However, aforementioned hierarchical structures require complicated fabrication process and most of them are based on silicon, resulting in poor optical transparency.18,32 In this work, we reported a “Rhipsalis (Cactaceae)” like micropillar array-based device with high optical transparency for efficiently capturing cancer cells in three dimensions (XY and Z planes). The PDMS micropillar array was fabricated by soft-lithography and rapid prototyping method. The micropillar array was plated with a thin gold layer through electroless plating. EpCAM antibody was then modified onto the surface of the micropillars through the thiol-oligonucleotide linkers.40 Hierarchical structures originating from the gold layer are both on the surface of the micropillars and the microchannel. The immune-affinity molecules (EpCAM antibodies) could be modified on the gold-coated micropillars and microchannel. Thus, capture of cancer cells could be realized simultaneously on the surface of the micropillars and the microchannel, which was different from those reported previously41-46 The device in this study demonstrated a high capture efficiency for different kinds of cancer cells (i.e., MCF-7, NCI-H1650), with potential applications in clinical field.


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2. EXPERIMENTAL SECTION 2.1. Reagents and Instruments. Avidin, Biotin-FITC, Calcein-AM, KHCO3 4,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA) and TCEP (tris(2-carboxyethyl)phosphine) were purchased from Sigma-Aldrich , Inc. (U.S.A.). HAuCl4·3H2O and glucose were obtained from Sinopharm Chemical Reagent (Shanghai, China). Anti-CD45 (FITC) and Anti-Cytokeratin (PE) were bought from BD Biosciences (U.S.A). Anti-EpCAM (Biotin) was purchased from abcam. Oligonucleotide and DNase I were obtained from sangon Biotech (shanghai). Human breast cancer cells (MCF-7) was a gift from the Dr. Shuai Xia. Human cervical carcinoma cells (HeLa) and human non-small cell lung cancer cells (NCI-H1650) were purchased from Wuhan finetest biotech (China). Sylgard 184 including polydimethylsiloxane (PDMS) monomer and curing agent was purchased from Dow Corning (Midland, MI, U.S.A.). All the media for cell culture were bought from Gibco Corp. Human blood samples were supplied by Wuhan union hospital. Ultrapure water (18 MΩ·cm) was made by a Millipore Milli-Q system. 2.2. Microchip design, fabrication and modification. The PDMS micropillar array was fabricated by conventional soft lithography. SU8-1075 (Gersteltec Sarls, Switzerland) was spinning-coated on silicon substrate and then covered with a photomask for subsequent lithography. After obtaining the expected micropattern (microwell) on the silicon substrate, PDMS prepolymer solution (A and B in 10 to 1 ratio) was poured onto it. To prevent the cured PDMS from sticking to the silicon master,

TFOCS

(tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane)



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(Sigma-Aldrich) was vacuum vaporized to the surface of the master. After curing at 65 ºC for 2h, the PDMS was peeled off from the silicon substrate by a tweezer. After fabricating PDMS micropillar (diameter, 100 µm; dimension of the channel was 40 mm in length and 20 mm in width), we conformally plated gold layer on the array. The procedure of electroless plating was performed according to the previous literature (Fig 1b).38 Briefly, the solution for gold plating containing 0.5% (w/v) HAuCl4, 0.05 g mL-1 KHCO3, and 5 mg mL-1 glucose was prepared prior to use. The mixing solution was let sit for one hour to eliminate bubbles before plating. The PDMS micropillar array was hydrophilizated by oxygen plasma treatment for 10 s. The hydrophilic PDMS micropillar array was then gently placed onto a PDMS frame that contained the plating solution, allowing the array in contact with the plating solution for gold deposition in dark room at ambient temperature for about 6 hours. The edges of the PDMS microarray were protected with thin PDMS slices to prevent gold deposition, which might result in difficulty in sealing the array with glass slide by plasma treatment. After inlet and outlet was punched at the two ends of the PDMS microarray, it was bonded onto a glass slide by plasma treatment to form the final Rm-chip (Fig S4). 2.3.

Device

modification

and

characterization.

Oligonucleotide

(5ʾ

-HS-TTTTTTTTTT-biotin-3ʾ) was dissolved in TE buffer (10 mM Tris, 5 mM EDTA, 100 mM NaCl, pH 8.0) with a final concentration of 100 mM and mixed with 2 mg/ml TCEP solution (volume ratio 1:1) for 1 hour to reduce the disulfide bridges. After reaction, TE buffer was added to the mixture (volume ratio 9:1) to obtain the


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final immobilization solution. The Rm-chip was treated with oxygen plasma and then filled with the immobilization solution. The immobilization solution of 400 µL was perfused into the microchip with a 1mL-syringe at a slow rate at room temperature for overnight reaction. After removing the excessive DNA with PBS (50 µl/min, 0.5 ml), Avidin (0.5 ml, 50 µg/ml in pH 7.4 PBS) was perfused through the microchip at 20 µl/min. After 1 h incubation, the microchannel was rinsed with PBS at 50 µl/min to remove excessive avidin. Finally, biotinylated EpCAM antibody with a concentration of 20 µg/ml containing 1% (wt/vol) BSA in PBS was perfused through the device for 10 min at 20 µl/min. After another hour’s incubation, PBS containing 1% BSA (0.3 ml) was perfused through the microchannel at 50 µl /min, which was then allowed to incubate for 30 min. The modified Rm-chip was stored at 4 ºC with high humidity. FITC-biotin was used to confirm whether avidin was successfully conjugated onto the surface of the micropillar array. 2.4. Cell culture. MCF-7 and NCI-H1650 were cultured in RPMI medium 1640 (FisherScientific). HeLa was cultured in DMEM medium. All cell culture medium were supplemented with 10% fetal bovine serum (FBS; heat-inactivated; Gibco) and 100 units/mL penicillin streptomycin (Gibco). All cultures were incubated at 37 ºC under a 5% CO2 atmosphere. Dulbecco's phosphate-buffered saline with calcium and magnesium (FisherScientific, USA) was used to wash cells. 2.5. Cell capture assay in Rm-chip. When cells reached more than 70–80% confluence, they were collected and labelled with Calcein-AM. Subsequently, these labelled cells were used to perform the capture efficiency experiments. To obtain a



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given number of cells, the cells in culture dish were transferred to a 1.5 ml tube after treatment with trypsinization solution. The collected cells were stained with fluorescent dye and then were diluted properly. A cell suspension of 1 µL was transferred to a glass slide under fluorescence microscope for cell counting, which was repeated for at least 5 times. The average cell number was calculated with standard deviation. Then, 1 µl cell suspension was pipetted to the 1ml of PBS or whole blood in 1mL-syringe and mixed at least 3 min by a tiny magnetic stirring bar placed inside the syringe before introduced into the Rm-chip by a Micro4 syringe pump (World Precision Instruments, Sarasota, FL, USA) that could well control the flow rate. The magnetic stirring bar was kept working to prevent the cell precipitation while the cell mixture was being pumped through the chip. For the blood samples, the captured cells were washed with PBS, fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton-X and incubated for 30 min followed by a PBS wash and blocking the chip with 3% BSA. Anti-cytokeratin (PE), anti-CD45 (FITC) and DAPI were used to stain the cancer cells, leukocytes and nuclei respectively. To determine cell number, the Rm-chip was placed on the stage of a fluorescence microscope (Zeiss Axio Zoom. V16 or Olympus America, Melville, NY, USA) to capture images at different fluorescence channel. The microchip was scanned automatically under an inverted microscope (Objective lens, 60×). Cancer cell counting was conducted according to a previous literature10 Briefly, the cell debris and nonspecific cells were excluded according to size, shape and the nuclear size. The images were analyzed by image-pro plus to count the cell number. Cells with positive


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staining for cytokeratin (cytokeratin+, DAPI+ and CD45-) and matching the phenotypic morphological characteristics were scored as cancer cells. For cell release, the DNase I was used to hydrolyse the DNA linker. After cell capture and rinsing with PBS, we used the fluorescence microscope (Zeiss Axio Zoom. V16) to image an area on the Rm-chip that was premarked. And then 200 µl DNase I (0.1 mg/ml) solution was pumped into the chip. After incubating the device setup for 50 min at 37 °C in FC-40 oil bath, the chip was rinsed with PBS. The same area on the chip was imaged by the fluorescence microscope for evaluating the release efficiency.

3. RESULTS AND DISCUSSION 3.1. Devices fabrication and characterization. The Rm-chip is made up of a hierarchical structured micropillar array and a glass slide. The conventional soft-lithography was employed for the fabrication of PDMS micropillar array and the electroless plating method was then applied to conformally plate a thin gold layer onto the whole array (Fig 1a and 1b) which formed the hierarchical structures on the micorpillars as shown in Fig 1c and 1d. For clarity, the magnified image of selected area from Fig. 1d was shown in Fig 1e where yellow arrows indicate the microstructures, as more clearly illustrated from the close-up image in Fig S1. Nanostructures were also observed on the micropillars as shown in the AFM image of Fig 1f. Both the microstructures and nanostructures on the side of the micropillar form the thorn-like structure just as the ones on the stem of the Rhipsalis (Cactaceae) (inset



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of Fig.1e). Apart from the micorpillar side, the top and bottom also possess hierarchical structures (Fig 1e, Fig S2).

Those hierarchical structured biointerface

has the potential capability of interacting with cellular pseudopods, improving the cell capture efficiency. In addition, the gold layer was only about 1.7 µm (Fig S3), so it will not affect the optical transparency of the whole Rm-chip (Fig 1g, Fig S4), allowing directly to monitor cancer cells under microscope. 3.2.

Modification

and

cancer-cell

capture

principle

of

the

Rm-chip.

Oligonucleotides are versatile linkers for the modification of nanoparticles and microfluidic channels. Here, the mercapto single-stranded DNA-biotin (ssSHDNA-B) and biotin-avidin interaction system were used to conjugate the EpCAM antibodies to the microchannel of the Rm-chip (Fig 2a). After the modification with ssSHDNA-B and avidin in the channel, biotin-FITC was used to verify the above-mentioned conjugations. The confocal image of the micropillar array in Fig S5 indicates the biotin–FITC

has

been

successfully

assembled

to

the

"ssSHDNA-B

and

avidin"-modified Rm-chip channel, which reveals the feasibility of subsequent modification with biotin-antibody. The whole conjugation chemistry and the cell capture schematic were showed in Fig 2b. In contrast with conventional nanostructured-substrate and micropillar array which could only capture cells in two dimensions (Fig 2c), Our Rm-chip possess both the nanostructured-substrate (here the PDMS bottom of the chip) and micropillar array with topological structures, offering the capability of capturing cancer cells in three dimensions (XY and Z planes). Fig S6 and Fig 2d present the fluorescent and SEM images of captured cells respectively,


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clearly revealing that cancer cells could be well captured both by the micropillars and the substrate. 3.3. Enhanced efficiency of Cancer Cell Capture by Rm-chip. To determine the efficiency of capture, the human breast cancer cell line (MCF-7), non-small-cell lung cancer (NSCLC) cells (NCI-H1650) and cervical cancer cell line (HeLa) prelabeled with fluorescence were spiked into phosphate buffered saline (PBS) at varying concentrations and then flowed through the Rm-chip respectively. With increasing the flow rates, the capture efficiency of spiked MCF-7 cells was reduced (Fig 3a). To ensure the best capture efficiency, the rate of 1ml/h was applied in the following experiments. As seen in Fig 3b, different number of MCF-7 cells (70-300) spiked in PBS can be efficiently captured by the Rm-chip. Apart from the MCF-7 cells, the NCI-H1650 cells also could be captured with high efficiency, while for the non-EpCAM expressing cell line, HeLa cells, the capture efficiency was less than 10% (Fig 3c) which was rational since no specific binding occurs. At the rate of 1ml/h, the capture efficiency of Rm-chip for MCF-7 is 88% as seen in Fig 3d, which is much higher than the one in following two cases: microarray sputtered with gold layer which is supposed to have no hierarchical structures (66%) and flat PDMS plated with gold layer of hierarchical structures (21%). The high capture yield of Rm-chip mainly originates from the thorn-like topological structures on the micropillars and the substrate (bottom). Those hierarchical structures enhance the interaction between the cells and Rm-chip, leading to high efficient capture. However, the sputtered gold layer on micropillars was flat



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without cellular pseudopod fitting nanostructures (Fig S7a) and the hierarchical structured-PDMS lack of micropillars (Fig S7b) could not perform cell-solution mixing efficiently, resulting in the poor capture yield. 3.4. Efficient Isolation of Cancer Cells from Whole Blood Using Rm-chip. Varying numbers of NCI-H1650 cells (5-20 cells, 30-50 cells, 70-160 cells) were spiked into 1 ml of whole blood.

After flowing through the Rm-chip with

above-prepared cell-solutions, the capture efficiency of our device for human blood was investigated.

As illustrated in Fig 4a, the average recovery rates of 30–50 and

70-160 spiked cells per 1 ml were 82.7% and 83.7% respectively. In the case of 5–20 spiked cells per 1 ml, the average recovery rate was 76.5%. These results demonstrated that the Rm-chip was suitable for clinical samples. 3.5. Release of captured cells from Rm-chip.

Release of cancer cells from

the device channel may pave the way for implementing subsequent molecular and functional analyses. In this work, exonuclease I was used to hydrolyse DNA linkers for the cell-release. The schematic of the cell release assay was shown in Fig 5a. As can be clearly seen in Figure 5b, the number of captured NCI-H1650 cells labeled by fluorescent dye was pretty high before DNase I treatment. As a contrast, Figure 5c shows few cells in the same area after treatment with DNase I at 37 °C for 50 min. The high release efficiency (92 ± 2%) indicates that our Rm-chip was suitable for the subsequent bioanalysis. CONCLUSION In summary, we have successfully used a “Rhipsalis (Cactaceae)”-like micropillar


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array-based chip (Rm-chip) to efficiently capture and release cancer cells. The average capture efficiency of 88% was achieved in PBS. The thorn-like structure on the micropillar and substrate was proved to be the dominating factor for the high capture efficiency. The high transparency of the whole device allows for direct monitoring captured cells under microscopy. Since the anti-EpCAM antibody was immobilized through DNA linker, the captured cancer cells could be easily released by the exonuclease I with high efficiency. The Rm-chip successfully demonstrated the cancer-cell capture capability with average efficiency of 83.7% in whole blood samples, showing its potential applications in CTC studies and clinical diagnosis.

Acknowledgements We gratefully acknowledge the financial supports from National Natural Science Foundation of China (21475049, 31471257 and 21275060) and National Key R&D Program of China (2016YFF0100801). Supporting Information Supporting Information is available free from the Internet at http://pubs.acs.org. Information of the SEM images of miciropillar and characterization of chip, Figure S1-S7.



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Figure 1. Design and characterization of Rm-chip. (a, b) Schematic of the gold depositing on micropillar array and its bottom by the electroless plating. (c) SEM image of the micropillar array with gold layer. (d) SEM image of the hierarchical structures on the top, side and bottom of the micorpillar. (e) Close-up of side view of the micropillar in b, yellow arrows indicating the micro-protrude and thorn-like structure on the side of the micorpillar. The inset showing the real picture of Rhipsalis (Cactaceae). (f) AFM image of nano-topological structure on micropillar after gold deposition. (g) Photo of microarray chip against the background of a pH test strip suggesting its high optical transparency. Inset showing the Rm-chip filled with blood.



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Figure 2. Schematic of the micropillar functionalization and cancer-cell capture principle of the Rm-chip. (a) Modification procedures of micropillar array. (b) Schematic of the conjugation chemistry and the cell capture. (c) Schematic of cancercell capture principle of the nanostructured-substrate, conventional micropillar and our micro/nanostructured-micropillar. (d) SEM image of the captured MCF-7 cell on the micropillar and bottom. Inset: magnified SEM image of the captured MCF-7 cell on the bottom.


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Figure 3. Characterization of the Rm-chip with cells in buffer solution. (a) Dependence of capture efficiency of MCF-7 cells on flow rate. (b) Capture efficiency of MCF-7 cells at 1 ml/ h. The red solid line indicating the linear fitting to the data. (c) Capture efficiency of different cell lines. (d) Cell recovery of MCF-7 cells compared with the results of micropillar sputtered with gold and flat PDMS plated with gold layer. Error bars representing the standard deviation of at least three replicates.



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Figure 4. Characterization of the Rm-chip with NCI-H1650 cells spiked into whole blood. a) Cell recovery for NCI-H1650 cells spiked into 1 ml of whole blood at spike concentrations from 5 to 160 cells per ml. b, c) Fluorescence microscope images of NCI-H1650 and white blood cells stained with DAPI (blue), Anti-cytokeratin (PE, red) and anti-CD45 (FITC, green).

Figure 5. Efficient release of captured NCI-H1650 cells. a) Schematic description of the cell release assay using exonuclease I to break down DNA linker. b) Fluorescence microscopy image of captured cells prestained with calcein-AM before treatment with exonuclease. c) Fluorescence microscopy image of the same area after treatment with exonuclease I at 37 °C for 50 min.


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Rhipsalis (Cactaceae)-like Hierarchical Structure Based Microfluidic

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