Highly Efficient Capture and Electrochemical Release of Circulating

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Highly Efficient Capture and Electrochemical Release of Circulating Tumor Cells by using Aptamers Modified Gold Nanowire Arrays Ting-Ting Zhai, Dekai Ye, Qian-Wen Zhang, Zeng-Qiang Wu, and Xing-Hua Xia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11107 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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ACS Applied Materials & Interfaces

Highly Efficient Capture and Electrochemical Release of Circulating Tumor Cells by using Aptamers Modified Gold Nanowire Arrays Ting-Ting Zhai, ‡ Dekai Ye‡, Qian-Wen Zhang, Zeng-Qiang Wu, and Xing-Hua Xia* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

ACS Paragon Plus Environment


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ABSTRACT The effectively capturing and releasing of circulating tumor cells (CTCs) is of significant importance in cancer prognose and treatment. Here we report a highly efficient method to capture and release human leukemic lymphoblasts (CCRF-CEM) using aptamers modified gold nanowire arrays (AuNWs). The gold nanowires, showing tunable morphologies from relatively random pillar deposit to relatively uniform arrays, were fabricated by electrochemical deposition using anodic aluminum oxide (AAO) as template. Upon simply modified with aptamers by Au-S chemistry, the AuNWs exhibit higher specificity to target cells. Also compared to flat gold substrate, the AuNWs with nanostructure can capture target cells with much higher capture yield. Moreover, the captured CCRF-CEM cells can be released from AuNWs efficiently with little damage through an electrochemical desorption process. We prognosis that our strategy has great potential in providing a simple and economical platform for CTCs isolation, cancer diagnosis and therapy. Keywords: AuNWs, circulating tumor cells, capture, electrochemical release, topographical interaction


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INTRODUCTION Recently, tumor metastasis has been the leading cause of death in cancer patients.1-4 After the tumor cells transferred into peripheral blood, they become circulating tumor cells (CTCs). Therefore, the detection of CTCs in peripheral blood is of significant importance in the early diagnosis of metastasis and treatment of cancer.5,6 However, CTCs are extremely rare in peripheral blood of early-stage cancer patients (about one CTC in 106-107 leukocytes), which makes the enrichment, isolation and detection of CTCs tremendously difficult. Based on the physical and chemical properties of the tumor cells, many methods have been developed to enrich and isolate CTCs, such as MACS,7 ISET,8 dielectrophoresis9 and micropore chip.10 However, these methods are poorly applicable in isolation and detection of CTCs in the blood of cancer patients due to the ultralow concentration and cell heterogeneity of CTCs.11 Many studies suggested that cells with nanosurface (microvilli etc) tend to adhere on nanostructured materials duo to enhanced topographical interactions effect.12-14 In addition, nanostructured materials of larger surface area can be easily modified with large amount of affinity molecules such as antibodies and aptamers.15-17 These properties have been used for effective capture of CTCs via promoting formation of more filopodia.18-20 To date, various nanomaterials with different structure have been applied for CTCs capture, including silicon nanowires/nanopillars array,15 nanowire or nanofiber arrays of inorganic oxides,21-25 polystyrene and carbon nanotube,26,27 nanospheres and microspheres of metal oxides and ploymers,28-31 nanoroughened surfaces,4,32-33 hydrogel,34 and grapheme oxide nanosheets.35 However, most of these nanostructured



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materials have uncontrollable structure and poor stability, while the fabrication process is complicated and uneconomic. A simple method to fabricate controllable and stable nanostructured surface could be an economic and effective approach for CTCs capture. Meanwhile, in consideration of further therapy research and molecular biological mechanism study of CTCs, it is full of significance to release the captured CTCs without cell damage. Currently, a series of methods based on DNA hybridization,34 electrochemistry,36-38 light,39 pH,40 heat,41 thermodynamic,42 chemical degradation43 have been developed for cell release. The long releasing time and change in surrounding environment of cells could lead to CTCs loss and viability decreased. Therefore, it is challenging and necessary to develop a simple strategy for efficient capture and release of CTCs. With high stability and biocompatibility, gold based materials have always been the preferential choice in nanostructure construction, electrochemical analysis and biomedical application.44 By chemical reaction and electrochemical deposition, many gold

nanostructures

have

been

constructed

on

surfaces.45

Although

the

micro/nanostructures can be tuned to some extent, the random nucleation growth makes the assembled structure on surface uncontrollable. Fabrication by template directed electrochemical deposition,46 AuNWs can be more regularly assembled on surface, making them applicable in the research of nonlinear optics47 and surface enhanced Raman scattering.48 Compared to silicon and quartz nanowires, AuNWs with well biological compatibility have unique advantages for CTCs capture. The morphology of AuNWs can be easily controlled by changing the deposition parameter and template


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structure. Besides, the surface property of the gold surface can be easily regulated by specific modification. In addition, the previous research confirmed that electrochemical desorption technique is effective approach for cleaning of gold surface and breaking the Au-S bonds. 49,50 DNA aptamer has been widely applied in biosensing and CTCs isolation since it has comparable specificity, higher stability and easier synthesis method comparing to antibody.51-54 Herein, we propose a highly efficient strategy for CTCs capture and release based on aptamers modified AuNWs. The fabrication steps of AuNWs and isolation process of target cells are illustrated in Figure 1. The AuNWs are prepared electrochemically using AAO as the template (Figure 1a). The AuNWs with different morphology are prepared by adjusting the deposition current and time. In addition, the prepared AuNWs are modified with cell aptamers55,56 for specifically capture of target cells. To reduce the nonspecific adsorption on nanostructured surface, mPEG modification and bovine serum albumin (BSA) passivation are also applied.57-59 Results reveal that the AuNWs exhibit much higher capture efficiency to target cells than the flat gold substrate does. Moreover, by electrochemical reduction desorption, we achieve a release efficiency of 96.2% in 30 s, which could greatly help the further research on CTCs and regeneration of AuNWs.



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Figure 1. Schematic illustration of the AuNWs for cell capture and release. (a) Fabrication process of AuNWs. (b) Representative process of capturing and releasing CCRF-CEM on aptamer-sgc8c modified AuNWs. EXPERIMENTAL SECTION Materials. Anodic alumina oxide membrane (200 nm pore diameter and 60 µm height) was purchased from Whatman International Ltd. (Maidstone). mPEG (O-(2Mercaptoethyl)-O′-methyl-hexa(ethylene glycol)) and BSA were purchased from Sigma-Aldrich (St. Louis, MO). Chloroauric acid was from Alfa Aesar. Acridine Orange (AO), Propidium iodide (PI) and ssDNA: 5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT TTT-3’-(CH2)3–SH were purchased from Shanghai Shenggong Biotechnology Co. (Shanghai). The flat gold substrate was from Haoyue Quartz Co. (Lianyungang). Poly(dimethylsiloxane) (PDMS) precursor and curing agent were from Sylgard 184, (Dow Corning, Midland, MI). The CCRF-CEM (CCL-119, T cell line, human ALL) cell line was obtained from Shanghai Cell Center. K562 cells (CCL-243, human CML) were obtained from Beijing Xiehe Hospital. RPMI-1640 culture medium, fetal bovine serum (FBS) and penicillinstreptomycin solution were purchased from Corning Co (Manassas). All other reagents


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were of analytical grade. All solutions were prepared using ultrapure water (18.3 M Ω·cm) from the Millipore Elix 5 Pure Water System.

Cell Culture. RPMI-1640 culture medium (500 mL) was supplemented with 1.25 g of D -glucose, 0.06 g of sodium pyruvate, 0.15 g of L-glutamin, 10% FBS, and 5 mL of penicillin-streptomycin solution (10 000 μg/mL). Both the two types of cells were routinely cultured in 25 cm2 cell culture flasks at 37 oC with 5% CO2 in air atmosphere.

Fabrication of AuNWs. The AAO template was ultrasonicated in acetone for 1 min and then immersed in acetone for another 1 h. After drying under a stream of nitrogen, one side of the AAO template was sputtered with about 60 nm gold film by using a current of 15 mA in a vacuum chamber with a pressure of 5x10-4 mbar (Ar plasma). PDMS monomer and curing agent were thoroughly mixed in a 10:1 weight ratio and vacuumed for 10 min to remove air bubbles. Then, the PDMS mixture was poured on a flat glass with a thickness of about 1 mm and heated at 80 oC for 20 min to form a PDMS film which could be easily peeled off from the glass. Electrochemical deposition of AuNWs was performed in a three-electrode configuration using a CHI660E electrochemical workstation (Chenhua Instrument Co., Ltd.). The gold sputtered AAO template was used as the working electrode, with a Pt wire as the counter electrode and an Ag/AgCl wire as the reference. The area of the working electrode exposed to solution was controlled by a hole (10 mm in diameter) punched on a PDMS film. The electrochemical deposition was performed with a current density of 0.1 mA in 24 mM HAuCl4 for 2 h at room temperature. For obtainging



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different length of gold nanowires, deposition time of 0.5 h, 1 h, 2 h, 3 h and 5 h was performed, respectively. Then, the AAO template electrode was rinsed with ultrapure water followed by drying with N2. The AAO template was chemically removed using HF for 20 min. (Caution: the chemical etching experiment must be done in a stink cupboard for the high corrosivity of HF). The AuNWs were obtained after further rising and drying with nitrogen.

Modification of AuNWs. The AuNWs were rinsed with ultrapure water and ethanol subsequently and dried with N2. Then, the AuNWs were further cleaned in UV-ozone cleaner (NOVASCAN PSD, Shenzhen Wisbay M&E Co., Ltd) for 15 min. After that, 100 μL of aptamer-sgc8c (10 μM in DPBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 and 5 mM MgCl2 )) was dropped on the AuNWs surface and vacuumization was performed to remove the air among gold nanowires to make the AuNWs completely immersed in the aptamer solution. After modification overnight, the AuNWs were rinsed with ultrapure water. Then, the AuNWs were immersed with 1 mM mPEG and 1mg/mL BSA solution in DPBS for 30 min, respectively. The mPEG modification was achieved by Au-S covalent bond and the BSA was modified by direct physical adsorption. These two reagents have been commonly used for surface blocking in proteins and cells interfacial biosensing research.

Cell capture. Cells were collected from the medium by centrifugation at 1000 rpm for 5 min and resuspended to a density of 1×105 cells/mL in DPBS with BSA (1mg/mL). The influence of cell concentration on cell capture efficiency was studied with cell


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concentration of 50 cells/mL, 500 cells/mL, 5000 cells/mL and 1×105 cells/mL, respectively. Before the blood sample analysis, CCRF-CEM cells were dyed with CellTracker™ Green CMFDA (Thermo Fisher Scientific) and the whole blood cells (drew from New Zealand Rabbits before experiments) were dyed with CellTracker™ Deep Red (Thermo Fisher Scientific). A concentration of 5000 cells/mL CCRF-CEM cells was spiked in whole blood sample (1×109 cells/mL). The concentration of cells was determined by a hemacytometer. A 200 μL droplet of CCRF-CEM cells suspension was dropped on the aptamer/mPEG/BSA modified AuNWs surface and incubated at 37 o

C. After incubation, the AuNWs were carefully washed with DPBS. A 10 μM AO

solution in DPBS was used to stain the captured cells for 20 min at 37 oC. After washing with DPBS for three times, the captured cells were observed and counted using a Fluorescence Inversion Microscope System (Nikon, TI-U). The cell capture in blood cells sample was observed and counted on a Leica TCS SP5 confocal microscope. As a control, a 200 μL of 1×105 cells/mL CCRF-CEM cells suspension was dropped onto the mPEG/BSA modified AuNWs and aptamer/mPEG/BSA modified flat gold substrate for incubating. Before aptamer modification, the gold substrate was annealed in acetylene flame for 5 min to smooth the gold substrate surface. A 200 μL of 1×105 cells/mL K562 cells suspension, as the control cell line, was dropped onto the aptamer/mPEG/BSA modified AuNWs and aptamer/mPEG/BSA modified flat gold substrate for incubating. The cell concentration was the same as CCRF-CEM cells.

Electrochemical release of captured cells from aptamers modified AuNWs. Electrochemical release of the captured cells was performed in a three-electrode



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configuration using a CHI660E electrochemical workstation (Chenhua Instument Co., Ltd.). The scg8c modified AuNWs with captured CCRF-CEM were as the working electrode, with Ag/AgCl as the reference electrode and Pt wire as the counter electrode. The release process was performed with -1.2 V for 30 s. After washed with DPBS gently, captured cells on sgc8c modified Au NWs before and after release were observed and counted using a Fluorescence Inversion Microscope System (Nikon, TI-U).

Scanning electron microscopy (SEM) observation. The cells were fixed with 4% glutaraldehyde buffered in DPBS for 5 h after incubation on the substrate and then dehydrated through a series of alcohol concentrations (30%, 50%, 70% and 90%) for 10 min subsequently before stained in 0.5% uranyl acetate for 1 h. Then, the cells were further dehydrated with alcohol (95%, 100% and 100%) and hexamethyldisilazane (HMDS). After air drying, the samples were sputtered with a thin gold film (15 mA, 100s) before examination with a Hitachi S480 field emission SEM.

Cell viability analysis. The viability of CCRF-CEM captured and released from sgc8c modified AuNWs were determined by AO/PI staining assay. AO is a membranepermeable dye with green fluorescence when binding with DNA in cells. Propidium iodide (PI) is a red fluorescent dye that cannot pass through intact cell membranes but can easily bind with DNA when the cell membranes are damaged. By AO/PI staining, dead cells and viable cells can be easily differentiated by fluorescent images under a microscope. For cells before captured, the cells were resuspended in AO/PI solution (1 μg/mL AO and 1 μg/mL PI in DPBS) and incubated for 5 min. After centrifuged at


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1000 rpm for 5 min, the cells were resuspended in DPBS for fluorescence imaging. The captured cells on AuNWs were immersed into an AO/PI solution consisted of 1 μg/mL AO and 1 μg/mL PI in DPBS for 5 min. Then, the cells were rinsed with DPBS and observed on a microscope. For cells after released from the substrates, the cells were collected, stained by AO/PI solution and observed using Fluorescence Inversion Microscope System (Nikon, TI-U).

RESULTS AND DISCUSSIONS Fabrication of AuNWs. Electrochemical deposition is commonly applied in fabricating metal nanostructure on conducting interface. By changing the composition of

electrolyte,

electrochemical

parameters and

template

structure, various

nanostructured surface can be farbicated. In our experiment, commercial AAO with relatively homogeneous pore size and distribution was chosen as the template. As shown in Figure 2a, the pores of AAO template are relatively uniformly distributed with pore diameter of about 200 nm. To realize the electrochemical deposition in nanopores, a thin gold film was sputtered on one side of AAO. After sputtered with 600s under a current of 15 mA, the structure of AAO template (Figure 2b) shows an obvious change with SEM characterization, indicating that a gold layer has been adhered on the AAO surface. The pore becomes smaller but is still open ended.



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Figure 2. SEM characterization of the AAO template. (a) Top view of the AAO template; (b) Top view of the AAO template sputtered with a thin gold film. The scale bar represents 1 μm. (c) Cyclic Voltammogram on AAO electrode sputtered with thin layer gold film in 24 mM HAuCl4 solution at a scan rate of 0.1 V/s. (d) SEM image of the AuNWs fabricated at -100 μA for 2 h. The scale bar represents 1 μm. Before electrochemical deposition of AuNWs in the AAO template, a cyclic voltammogram was collected in 24 mM chloroauric acid solution to optimize the deposition condition. As shown in Figure 2c, the deposition of gold starts at 0.80 V, and the cathodic current reaches a peak at 0.55 V. Oxidation of the deposited gold starts at 0.95 V and its current reaches a peak at 1.15 V. Electrochemical deposition of AuNWs was performed galvanostatically at -190 μA (cathodic peak current in Figure 2c) and 100 μA in 24 mM HAuCl4 solution for 1, 2 and 5 h, respectively. By SEM


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characterization we find that the AuNWs at -190 μA are mainly consisted of frangible nanotubes, which are easy to fall down with increasing deposition time (Figure S 1a-c). On the contrary, the AuNWs synthesized at -100 μA are much more uniform and solid. However, the structure also becomes disordered with increasing deposition time (Figure S1 d-f)). Based on the above results, we can find that in the electrochemical deposition process in the arrayed nanopores, current and time for deposition can both affect the finally formed structure. We speculate that high deposition current drives fast electrochemical reaction which results in longer gold nanowires, while long deposition time will result in the formation of nanowires with high length-to-diameter ratio. Nanowires with larger length-to-diameter aspect ratio are hard to keep rigid structure after removal of the template. As needed, in our experiment a current of -100 μA for 2 h was chosen as the optimized condition to obtain the relatively uniform AuNWs. Through the SEM characterization and statistical analysis, we find that the average diameter of formed gold nanowires is 208 ± 27 nm (Figure S2b), which is slight larger than the average diameter of nanopores in AAO template. The spacing distance between neighboring nanowires mainly distribute from 110 nm to 130 nm (Figure S2c), indicating the relatively uniform distribution of AuNWs.

Cell capture. To further study the capture ability of our AuNWs to CTCs, we chose CCRF-CEM cells and K562 cells as the model cells since they are both suspension cancer cells in peripheral blood. To selectively capture target cells, the AuNWs were modified with cell aptamer-sgc8c. Sgc8c can specifically bind protein tyrosine kinase 7 which is highly expressed on the cell membrane of CCRF-CEM cells but much lesser



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on the surface of K562 cells. Thus, in this experiment we chose CCRF-CEM cells as the target cells and K562 cells as the control cells, which should have different combining ability to aptamer-sgc8c. To avoid the false positive results, the AuNWs were passivated with mPEG and BSA, both of which could inhibit the nonspecific cells adsorption. First, to investigate the capture ability of the AuNWs, we compared the cell capture ability of aptamer/mPEG/BSA modified AuNWs with different lengths of 1.3 μm, 2.6 μm, 5.7 μm, 7.7 μm and 14.2 μm (Figure S3). As showing in Figure S4, with increase of the gold nanowires length, the cell capture yield increases accordingly and reaches the optimum yield when the average length is about 5.7 μm. When the gold nanowires becomes longer, the cell capture efficiency slightly decreases which could due to flattening of the longer gold nanowires. Further to verify the capture ability of aptamer modified AuNWs to target cells, we compared the number of cell captured on AuNWs

under

different

modification

conditions

(aptamer/mPEG/BSA

and

mPEG/BSA). A 200 μL of cell suspension (105 cells/mL) was incubated on the AuNWs for 60 min. As shown in Figure 3a and b, compared to the mPEG/BSA modified AuNWs without aptamer, the aptamer/mPEG/BSA modified AuNWs bind much more CCRF-CEM cells, proving the anti-nonspecific cells binding ability of mPEG/BSA modification and binding ability of aptamer. Further on, to investigate the specificity of this method, K562 cells were also incubated on the aptamer/mPEG/BSA modified AuNWs and flat gold substrate under the same conditions. As shown in Figure 3a and c, the number of captured K562 cells on AuNWs is also much lesser than the number of captured CCRF-CEM cells, indicating the specificity of aptamer-sgc8c to CCRF-


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CEM cells. On a flat gold substrate (Figure 3c and d), the number of captured K562 cells is obviously less than that on AuNWs, indicating that AuNWs itself promote the nonspecific adsorption of cells. Taken together, the results indicated that the aptamer/mPEG/BSA modified AuNWs can specifically and effectively capture target cells.

Figure 3. Fluorescent microscope imaging of the captured cells on AuNWs. (a) captured CCRF-CEM cells on the aptamer/mPEG/BSA modified AuNWs; (b) captured CCRF-CEM cells on the mPEG/BSA modified AuNWs; (c) captured K562 cells on the aptamer/mPEG/BSA modified

AuNWs;

(d)

captured

K562

cells

on

the

aptamer/mPEG/BSA modified flat gold substrate. The scale bars represent 200 μm. (e) Quantitative capture yield of AuNWs for cells under different conditions. To further investigate the improved capture ability of the modified AuNWs, we incubated the AuNWs in cell suspension for different time and then counted the number of captured cells. A 200 μL of cell suspension (105 cells/mL) was added on the surface of aptamer/mPEG/BSA modified AuNWs for a serial of incubating time. As shown in Figure 4, the number of target cells captured on aptamer/mPEG/BSA modified AuNWs



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increases with incubating time and the capture yield grows over 83±4.7% within 60 min. On the contrary, cell capture on the a flat gold substrate with the same modification conditions was also performed. As characterized by SEM (Figure S5), the flat gold substrate surface became smoother after annealing, which decreases cell capture. In our results, we find that the number of target cells captured on flat gold substrate is much less and the capture yield increases slowly with incubating time (about 9% at 60 min). Our results demonstrate that the cell capture ability using AuNWs can be improved about 9 folds, which could greatly help the isolation of CTCs in clinical sample analysis. By comparing the capture yield of K562 cells on AuNWs, we could find that only about 6% of the control cells were captured on AuNWs at 60 min, which suggests the excellent capture specificity under long incubating time.

Figure 4. Analysis for cell-capture performance. (a) Fluorescent microscope images of captured cells at different incubating times. The three columns represent CCRF-CEM cells captured on AuNWs (left), flat gold substrate (middle) and K562 cells captured on AuNWs (right), respectively. From top to bottom the incubating time are 10 min, 20 min, 40 min and 60 min, respectively. The scale bars represent 200 μm. (b) The target


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cell capture yield on AuNWs (red), flat gold substrate (green) and control cell capture yield on AuNWs (blue) at 10 min, 20 min, 40 min and 60 min, respectively. By comparing the structure of AuNWs and flat gold substrate, we infer that the AuNWs with high surface to volume ratio could bind more aptamer than flat gold substrate, which might be one of the reasons of improved cell capture ability on AuNWs. In addition, the nanostructured surface of AuNWs, which is similar to the nanostructured surface of cancer cells, might help their interaction to cells and promote formation of cells filopodia protruding. By SEM characterizations (Figure 5), we could find that cells can bind both on AuNWs and flat gold substrates modified with aptamer. However, on AuNWs the captured cells stretched out more filopodia to attach on the gold nanowires. On flat gold substrate, few filopodia were found around the cells. The results further prove that the AuNWs structure can better promote cells binding. Based on the above results, it is clear that the AuNWs might improve the cell capture ability from multiple aspects. To further evaluate the applicability of AuNWs in practical sample analysis, we analyzed the cell capture yield with rare target cells number and in blood samples, respectively. We find that the target cell capture yield decreases with decreasing the cell number from 1000 cells to 10 cells (Figure 5d). Even with only 10 cells added on the AuNWs, a capture yield of about 50% can be achieved. In addition, we added 1000 CCRF-CEM cells in blood sample to investigate the influence of blood cells’ nonspecific adsorption on target cell capture. Fluorescence imaging analysis shows that target cells can still be effectively captured by the AuNWs although adsorption of blood



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cells cannot be totally avoided (Figure 5c). As shown in Figure 5d, existence of huge number of blood cells in the sample will slightly decrease the target cell capture yield from 74% (pure target cells) to 61% (target cells in blood sample). Both the experiments with rare cell sample and blood sample confirm the potential applicability of AuNWs in practical sample analysis.

Figure 5. SEM characterization of the captured CCRF-CEM cells. (a) CCRF-CEM cells on the aptamer/mPEG/BSA modified flat gold substrate. (b) CCRF-CEM cells on the aptamer/mPEG/BSA modified AuNWs. The scale bars represent 5μm. (c) Fluorescent microscope image of captured CCRF-CEM cells (stained green) in blood cell (stained red) sample. The scale bar represents 50 μm. (d) The capture yield of rare CCRF-CEM cells and CCRF-CEM cells in blood sample. In addition, for further medical research the capture progress should not influence


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the cell viability. An AO/PI staining assay was performed to examine the cell viability variation after captured. The results were shown in Figure S6 and Figure 6c reveals that that most of the captured cells remained viable after been captured on AuNWs. And the cell viability (ca. 90%) has little change compared to that of cells cultured in incubator (ca. 95%). Thus, we can conclude that the capture process cause little damage to cells and the captured cells can be used for further research.

Cell release and Viability Analysis. Efficient release of CTCs from substrate without damage is also full of significance for subsequent cell study. Here, we designed an electrochemical method to efficiently release the captured cells by cleaving sulfur-gold bonds between aptamers and AuNWs. Sulfur-gold bond is very stable under the mild condition, which can help many kinds of molecules immobilization on gold surface. However, Au-S bonds can be broken down through negative potential (electroreduction process).49 Thus, release of captured cells could be realized by applying a proper voltage on the AuNWs. After incubation for 60min, the CCRF-CEM cells were well captured on aptamer modified AuNWs (Figure 6a). It a potential of -1.2 V is applied to the AuNWs for 30 s, 96.2% of the captured CCRF-CEM are released from the AuNWs (Figure 6b). Moreover, we examine cell viability of the released cells using an AO/PI staining assay. The results indicate that the released cells maintain excellent cell activity (ca.90%, Figure S6c and Figure 6c). The breaking of Au-S bonds ended the linking of cell/aptamers to AuNWs. In addition, the generated hydrogen might accelerate the release of bounded cells and reduce the damage of interfacial environment change to captured cells.



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Figure 6. Potential triggered cell release from AuNWs. (a) Fluorescent microscope images of captured target cells on the aptamer modified AuNWs. (b) Fluorescent microscope images of residual cells on AuNWs after eletrochemical release. The scale bars represent 200 μm. (c) Change of release efficiency and cell viability during the releasing process.

CONCLUSIONS In summary, we propose an efficient and reversible CTCs capture and release strategy based on aptamers modified AuNWs array. The AuNWs are fabricated by electrochemical deposition using AAO as the template and their morphology can be


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controlled by adjusting the deposition current/time. The fabrication method of AuNWs is simple and economical. Combining with cell aptamers, the AuNWs can specifically capture target cells with a high capture yield (ca. 83% in 60 min) and achieve efficient release of the captured cells with negative potential stimulation. The captured cells still maintain high viability (90%) for further research. In addition, the AuNWs can be easily integrated into microfluidic and electrochemical device to better isolate target cells from peripheral blood and analyze the cell properties with electrochemical methods in the future. It is conceivable that our low-cost, rapid, reversible strategy is promising to provide a platform for CTCs isolation, early diagnosis, and therapy of cancer.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. SEM characterization of the AuNWs fabricated under different condition; top-view of SEM characterization of the AuNWs fabricated under the optimized condition; statistical analysis of gold nanowires’ diameter measured from SEM characterization results; statistical analysis of gold nanowires’ distance measured from SEM characterization results; SEM characterization of the AuNWs length; fluorescent microscope images of the captured CCRF-CEM cells on AuNWs with different deposition times; SEM characterization of the flat gold substrate before and after annealing in acetylene flame; PI/AO stained CCRF-CEM cells before captured; PI/AO



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stained CCRF-CEM cells after captured by AuNWs; PI/AO stained CCRF-CEM cells after electrochemical release. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (X.H. Xia), Fax: (+86)-25-83686106.

Author Contributions All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21635004, 21627806).


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Highly Efficient Capture and Electrochemical Release of Circulating

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