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Biological and Medical Applications of Materials and Interfaces

Frosted Slides Decorated with Silica Nanowires for Detecting Circulating Tumor Cells from Prostate Cancer Patients Haijun Cui, Binshuai Wang, Wenshuo Wang, Yuwei Hao, Chuanyong Liu, Kai Song, Shudong Zhang, and Shutao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06072 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Applied Materials & Interfaces

Frosted Slides Decorated with Silica Nanowires for Detecting Circulating Tumor Cells from Prostate Cancer Patients Haijun Cui,†,‡ Binshuai Wang,ǁ Wenshuo Wang,†,‡ Yuwei Hao,‡,§ Chuanyong Liu,‡ Kai Song,† Shudong Zhang*,ǁ and Shutao Wang*,†,‡ †

CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, CAS Center for

Excellence in Nanoscience, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡

§

University of Chinese Academy of Sciences, Beijing, 100049, China

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Green Printing,

Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ǁ

Department of Urology, Peking University Third Hospital, Beijing, 100191, China

KEYWORDS: nano-biochip, circulating tumor cells, silica nanowires, interfacial growth, topographic interaction.

ACS Paragon Plus Environment

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ABSTRACT: Developing low-cost and highly efficient nano-biochips are important for liquid biopsies, real-time monitoring, and precision medicine. By in situ growth of silica nanowires on a commercial frosted slide, we develop a biochip for effective circulating tumor cells (CTCs) detection after modifying epithelial cell adhesion molecule antibody (anti-EpCAM). The biochip shows the specificity and high capture efficiency of 85.4 ± 8.3% for prostate cancer cell line (PC-3). The micro-sized frosted slides and silica nanowires allow enhanced efficiency in capture EpCAM positive cells by synergistic topographic interactions. And the capture efficiency of biochip increased with the increase of silica nanowires length on frosted slide. The biochip shows that micro/nano composite structures improve the capture efficiency of PC-3 over 70% towards plain slide. Furthermore, the nano-biochip has been successfully applied to identify CTCs from whole blood specimens of prostate cancer patients. Thus, this frosted slide based biochip may provide a cheap and effective way of clinical monitoring of CTCs.

1. INTRODUCTION

Circulating tumor cells (CTCs) in peripheral blood1-2 provide clinical significance for cancer diagnosis or prognosis as liquid biopsies.3-6 In the past decade, scientists and medical doctors have developed sorts of devices or biochips detecting CTCs from patient blood,7-16 such as immunomagnetic separation (the gold standard CellSearch™17) ,or utilizing size,18-21 density,22 electrical properties23 etc. Unfortunately, expensive equipment or complicated fabrication processes (in especial microfluidic24-28) of these chips become enormous obstacles for feasible and cheap blood detection of CTCs. Recently, our nano-biochips have showed the highly efficient capture of CTCs with molecular and topographic interactions synergistically.29-35 Further results demonstrate that the hierarchical structure enhance the capture efficiency of CTCs.


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The microscale topography provides suitable sites for cancer cells trapping and the nanoscale structure matches the size of cancer cell filopodia.35-36 However, it still remains challenge to develop glass-based biochips which are easily prepared and low-cost. Frosted slides are commonly used for microscope in high schools, university laboratories and hospitals. The frosted side (for easy labeling purpose) can improve adhesion of cytological preparations, and diffuse the light to protect eyes when the light source of microscope is glaring.37-39 Frosted slides (in this paper) are sandblasted with the micro-size in 0.26-24.6 µm (average size is 4.5 ± 3.7 µm, Figure S1). The inherent microstructures make the commercial frosted slide potential glass-based chip to snag CTCs. Besides, nano-sized silica is biocompatible and synthesized silica nanostructures (spherical, fibrous, tubular, and rod-like) have been reported.40-41 Typically, in a pentanol/water system, silica nanowires (SNW) or SNW hybrid microstructures are well controlled in a solution-phase process which is suitable for large scale preparation.42-45 Thus, we consider a facile method fabricating the hierarchical structure by in situ growth of silica nanowires on frosted slide as a cheap and easily prepared CTC biochip. Herein, we exhibited the interfacial growth of silica nanowires on frosted slide (Fr-S-SNW) and Fr-S-SNW was modified with epithelial-cell adhesion molecule antibody (anti-EpCAM) to execute specifically capture of cancer cells in culture medium or blood. In a pentanol/water system,42-45 water nano-droplets were stabilized by sodium citrate in pentanol solution of polyvinyl pyrrolidone (PVP, 0.1 g/mL). With the anchor of water nano-droplets on the frosted slide, silica nanowires can grow in situ on the surface after the hydrolysis of tetraethyl orthosilicate (TEOS). To specifically capture of cancer cells in culture medium or whole blood, we introduced the anti-EpCAM on the surfaces. After modification of anti-EpCAM, Fr-S-SNW with higher capture efficiency towards frosted slides (Fr-S) or plain glass slides (Pl-G) revealed


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the micro- and nanotopographic interaction with cancer cells. Notably, as an available biochip for capture of cancer cells, we further demonstrated blood tests of prostate cancer patients to identify CTCs. 2. EXPERIMENTAL SECTION 2.1 Materials. 1-Pentanol (ACS reagent, ≥99%, Sigma-Aldrich); Polyvinylpyrrolidone (PVP, average mol wt 40,000, Sigma-Aldrich); Ethanol, acetone (AR, Beijing Chemical Works); Ammonium hydroxide (ACS, 28.0-30.0% NH3, Alfa Aesar); Tetraethyl orthosilicate (TEOS, reagent grade, 98%, Sigma-Aldrich); Purified water (Milli-Q reference, Millipore); Sodium citrate tribasic dehydrate (ACS reagent, ≥99.0%, Sigma-Aldrich); Frosted glass was cut from glass slide (SAIL BRAND, CAT. NO.7105); Glutaraldehyde (50 wt. % solution in water, J&K Scientific Ltd.); DAPI solution (ready-to-use), paraformaldehyde (4% in PBS) (Solarbio®); Phalloidin-Tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC, phalloidin from Amanita phalloides,

Sigma-Aldrich);

Triton™

X-100

(BioXtra,

Sigma-Aldrich);

(3-

Mercaptopropyl)trimethoxysilane (MPTS, 95%, Sigma-Aldrich); 4-Maleimidobutyric acid Nhydroxysuccinimide ester (GMBS, ≥98.0%, HPLC, Sigma-Aldrich); Streptavidin, biotinylated anti-human EpCAM (biotin-anti-EpCAM) (R&D systems); Sodium azide, Bovine serum albumin (BSA, lyophilized powder, Sigma-Aldrich); Dulbecco's modified eagle medium (DMEM), phosphate buffer solution (PBS) (HyClone®); Ham's F-12K (Kaighn's) Medium (Gibco™); Hexamethyldisilazane (HMDS, reagent grade, ≥99%, Sigma-Aldrich); Acridine Orange (AO, bioreagent, for molecular biology, Sigma-Aldrich); Propidium Iodide (PI, ≥94.0%, HPLC, Sigma-Aldrich).


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2.2 Growth of silica nanowires on frosted slide (Fr-S-SNW). The synthesis of silica nanorods or nanowires in solution has been published before.42-45 We used the system to grow silica nanowires on frosted slides in-situ. Briefly, PVP in 1-pentanol (0.1 g/mL) and sodium citrate in water (0.18 M) were prepared under stirring overnight. Typically, pentanol (PVP, 5 mL), ethanol (0.5 mL), water (140 µL) and sodium citrate solution (50 µL) were added into a centrifuge tube (10 mL). Then the sealed tube was shaken vigorously (vortex shaker, 2400 rpm, WH-866) after placing the clean frosted slide (1×2 cm) for 5 minutes. The tube was shaken again after adding ammonia (100 µL). Then TEOS (50 µL for Fr-S-SNW0.5, 100 µL for Fr-S-SNW1, 150 µL for Fr-S-SNW1.5, 200 µL for Fr-S-SNW2, 250 µL for Fr-S-SNW2.5) was dropwise added into the mixture and the tube was shaken again for another minute. The sealed tube was placed undisturbed in the oven for 12 hour at 50 °C. The substrates were washed totally by ethanol and water after reaction. 2.3 Modification of anti-EpCAM on biochips. The antibody was modified according our previous work. Firstly, the substrates were treated with oxygen plasma and immersed in ethanol solution of MPTS (4% v/v) overnight. The substrates were washed with ethanol and DMSO for three times. Then the substrates were reacted with GMBS (0.5 mM in DMSO) for one hour. After washing with PBS for three times, the substrates were immersed in PBS of SA (10 µg/mL) for one hour. The excess SA was flushed away with PBS, and the biotin-anti-EpCAM (10 µg/mL with 1% (w/v) BSA and 0.09% (w/v) sodium azide, 50 µL was used for each 1×1 cm substrate) was freshly introduced (reaction time was 1 h) before each cell-capture experiment or blood assay. 2.4 Cancer cell capture assays. All cell lines were cultured according to the general process from the American Type Culture Collection (ATCC). For PC-3, substrates (Pl-G, Fr-S, Fr-S-


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SNW1, Fr-S-SNW2, and each substrate was 1×1 cm) were treated for anti-EpCAM coating and substrates with suspended cells (105 /mL, 3 mL each well in F-12K) in 6-well plate were cultured for different times at 37 °C (5% CO2). After culture for intervals (15 min), the substrates were washed with warm PBS (37 °C) for three times and placed in paraformaldehyde solution (4%) for 10 min. After washing three times with PBS, the substrates were treated with Triton™ X-100 (0.2%) for another 10 min. To distinguish the captured cells, the substrates were incubated with DAPI solution (10 µg/mL) for 10 min. Then the cells on substrates were counted and imaged using a fluorescence microscope (Nikon, Ti-E). 2.5 Calculation of cell-capture efficiency. Cell capture efficiency was calculated with the following formula in our previous work.46

=

/ /

× 100%

(1)

The number of capture cells (per unit area, 1 cm2 in this experiment) was /. And x was the number of cells on a fluorescent image (10X). was the area of the fluorescent image (10X, and was 0.0114 cm2 in this experiment). The theoretical cells number (per unit area, 1 cm2 in this experiment) was /. N was the total cells number (3×105 in this experiment) and A was the area of 6-well plate (9.5 cm2 in this experiment). Ten fluorescent images were randomly taken from the substrate (1×1 cm) to give an average number of x. Three parallel experiments (substrates were different batches) were done to give an error bar. 2.6 Immunofluorescent staining for cells. After 45 min cell-capture experiment, the substrates were washed and fixed in paraformaldehyde solution (4%) for 10 min. After washing three times with PBS, the substrates were treated with Triton™ X-100 (0.2%) for 10 min. Then


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the substrates were washed with PBS and immersed in a blocking solution (2% BSA in PBS) for 2 h. The blocking solution was removed without wash and the substrates were incubated with 100 µL TRITC-conjugated phalloidin (15 µg in 250 µL methanol and used 1/40 in PBS) for 30 min. After washing three times with PBS, the substrates were incubated with 100 µL DAPI solution (10 µg/mL) for 10 min. Finally, high resolution fluorescence images (100X) were recorded by a laser scanning confocal microscopy (Nikon, N-C2-SIM). 2.7 SEM images for the captured cells on the substrates. After 45 min cell capture, the substrates were washed and fixed in glutaraldehyde solution (2.5 wt. %) for 60 min. After that, glutaraldehyde was totally washed away and dehydration was used with different concentration of ethanol and water (30%, 50%, 70%, 85%, 95%, and 100%). And HMDS (50% in ethanol and 100%, each for 30 min) was used to remove the water in cells totally. The substrates were adhered on a slant sample stage (45°) and imaged with a scanning electron microscope (QUANTA FEG 250, high voltage on 10 kV, gold sputtering for 60 s). 2.8 Cell viability of captured cells. Firstly, the mixed solution was prepared by mixing 0.5 µg/mL AO solutions and 0.5 µg/mL PI solutions in 1:1 volume ratio. 0.5 mL AO/PI solution was added into the culture medium during the cell-capture experiment (45 min). The cells were stained with AO/PI for 5 min and the fluorescent images were taken in real time. Ten images were used to figure out the cell viability for each substrate. 2.9 Cell capture from artificial whole blood samples. Artificial blood samples were prepared using healthy mouse blood with loaded cells (10, 20, 50, 100 cells/mL). The samples were added into a 4-well Lab-Tek™ chamber slide with the anti-EpCAM modified Fr-S-SNW2 (1×2 cm). After 45 min cell-capture, the substrates were washed with warm PBS (37 °C) for five


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times. Then, the captured cells were fixed with paraformaldehyde solution (4%) for 10 min. After washing with PBS, Triton™ X-100 (0.2%) were added for another 10 min. To distinguish the captured cells, the substrates were stained with DAPI solution (10 µg/mL) for 10 min. Finally, the cells on substrates were counted using a fluorescence microscope (Nikon, Ti-E). 2.10 Capture CTCs from the whole blood specimens. We finished the assay with the fresh blood from prostate cancer patients. Firstly, the blood (1 mL) was added into a 4-well Lab-Tek™ chamber slide with the anti-EpCAM modified Fr-S-SNW2 (1×2 cm). After 45 min incubation (37 °C, 5% CO2), the substrates were washed totally with warm PBS (37 °C) for five times. Then, the captured cells on substrates were fixed with paraformaldehyde solution (4%) for 10 min. After washing three times with PBS, the substrates were treated with Triton™ X-100 (0.2%) for 10 min. Then the substrates were washed with PBS and immersed in a blocking solution (2% BSA in PBS) for 2 h. The blocking solution was removed without wash and the substrates were incubated with antibody solution (50 µL for each, CD45 Monoclonal Antibody, FITC, ThermoFisher Scientific; PE-CF594 Mouse Anti-Human Cytokeratin, BD Biosciences) in the dark overnight. After washing with PBS, the substrates were incubated with DAPI solution (10 µg/mL) for 10 min. Then a fluorescence microscope was used for imaging and counting the captured CTCs (Nikon, Ti-E). 3. RESULTS AND DISCUSSION Firstly, we have prepared glass-based biochips by synthesis of silica nanowires on frosted slides. The silica nanowires were fabricated by an emulsion system42-45 as shown in Scheme 1. Briefly, PVP in pentanol (0.1 g/mL, 5 mL), ethanol (0.5 mL), water (140 µL), and sodium citrate solution (0.18 M, 50 µL) were mixed (vortex shaker, 2400 rpm) in a sealed centrifuge tube. The


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water nano-droplets were formatted in the mixture after vigorous shake (vortex shaker, 2400 rpm, Scheme 1a). The frosted slide (Fr-S) was vertically placed in the tube and the tube was vigorously shaken again after ammonia was added. Then following by the addition of TEOS, silica nanowires started to grow on Fr-S surface after the hydrolysis of TEOS. After 12 hour reaction, we obtained the silica nanowires-decorated frosted slides (Fr-S-SNW). The substrates (with SNW or not) were then reacted with (3-mercaptopropyl)trimethoxysilane (MPTS) after oxygen-plasma treatment, and a heterobifunctional cross-linking reagent (4-maleimidobutyric acid N-hydroxysuccinimide ester, GMBS) was employed to link with sulfhydryl and streptavidin (SA). After the terminated SA reacting with biotinylated anti-EpCAM, the substrates have realized the molecular modification for EpCAM-positive cancer cells capture (Scheme 1b). Thus, we have obtained the silica nanowires decorated frosted slide as glass-based biochips. In our experiments, we firstly chose four kinds of substrates (Pl-G were short for plain glass slides, Fr-S were short for frosted slides, silica nanowires on frosted slides were named as Fr-SSNW1 (100 µL of TEOS) and Fr-S-SNW2 (200 µL of TEOS)) to investigate the capture efficiencies of specific cancer cells and the topographical interactions of cells and surfaces (Figure 1). Scanning electron microscope (SEM) images of Pl-G (Figure 1a and insert) showed a flat glass without any micro- or nano-structure. Fr-S (Figure 1b and insert) showed the special convex or concave at the micro scale (Figure S1). With two different feed ratios of ammonia water and TEOS, we gained two lengths of silica nanowires on the frosted slides. Fr-S-SNW1 (NH3·H2O:TEOS = 1:1, 100 µL of TEOS, Figure 1c and insert) had diameter of 186.2 ± 43.5 nm and length of 1.2 ± 0.3 µm, and Fr-S-SNW2 (NH3·H2O:TEOS = 1:2, 200 µL of TEOS, Figure 1d and insert) had diameter of 195.8 ± 52.4 nm and length of 2.6 ± 0.9 µm (Figure S2). From the side views of Fr-S-SNW1 and Fr-S-SNW2, we found the silica nanowires were vertically


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anchored on the frosted slide (Figure S3). Thus, we exhibited four kinds of substrates (structureless, with micro-structure and micro/nano composite structure) to further study the topographical interaction of cells and surfaces. We then investigated the capture efficiency of the four kinds of substrates for targeted cancer cells. Five types of cell lines were examined. PC-3 (human prostatic cancer cell) and MCF-7 (human breast cancer cell) are the EpCAM positive cell lines; HeLa (human cervical carcinoma cell), Jurkat T (suspension cell) and Daudi (human lymphoma cell lines) are the EpCAM negative cell lines. After the modification of anti-EpCAM, Pl-G, Fr-S, Fr-S-SNW1 and Fr-SSNW2 all showed abilities to capture PC-3 with the increase of time in culture medium (Figure 2a). The capture efficiencies revealed smaller change after 45 min. For Fr-S-SNW2, the efficiencies were 85.4 ± 8.3% in 45 min, 80.9 ± 9.0% in one hour and 84.9 ± 5.9% in 75 min. Then we chose 45 min as an optimal incubation time for other control cell lines. The antiEpCAM coated substrates showed the specific capture for the EpCAM positive cell lines (PC-3 and MCF-7) and much lower capture efficiency for the EpCAM negative cell lines (Figure 2b, Figure S4, S5). From Figure 2b, the efficiencies were all below 20%, and the highest efficiency for HeLa on Fr-S-SNW2 was 14.9 ± 7.6% (Typical fluorescent images were provided in Figure S4, S5). Also, the cell capture assays indicated that Fr-S had more adhered cells than the Pl-G and both Fr-S-SNW1 and Fr-S-SNW2 had enhanced capture efficiency than the frosted slides after the modification of anti-EpCAM. The enhanced capture efficiency could be attributed to the integration of micro-structure of frosted slides and nano-structure of silica nanowires. The typical fluorescent images on the boundary of Fr-S-SNW2 and Pl-G showed that more cells were captured on the micro/nano-structured part (Figure S6). In contrast, the substrates without the modification of anti-EpCAM were also tested in the same conditions. The substrates showed low


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capture efficiency for PC-3 cells without modification of specific antibody (Figure S7). The efficiencies were 1.0 ± 0.2% for Pl-G, 6.8 ± 2.1% for Fr-S, 14.1 ± 3.0% for Fr-S-SNW1 and 10.1 ± 6.4% for Fr-S-SNW2. As the silica was biocompatible, the cells viabilities were also test with the substrates during cell-capture experiments. The captured cells (PC-3) on substrates (Pl-G, FrS, Fr-S-SNW1 and Fr-S-SNW2) had good viability with Live/Dead staining (AO/PI). The substrates were non-invasive for captured cells and over 97% of captured cells showed good viability (Figure S8). Thus, these glass-based biochips were potential options for clinical detection of CTCs. Besides the molecular interaction of antibody and antigen, the topographical interaction and molecular interaction enhanced capture efficiency for targeted cancer cells synergistically. Because the silica nanowires on frosted slides affected the capture efficiency, we then investigated the effect of silica nanowires length on capture efficiency. As we had showed in the experiment sections, the amount of TEOS was one of the influencing factors on silica nanowires lengths. Keeping same conditions, we changed the dosage of TEOS to obtain varying length of silica nanowires (0 µL for Fr-S, 50 µL for Fr-S-SNW0.5, 100 µL for Fr-S-SNW1, 150 µL for FrS-SNW1.5, 200 µL for Fr-S-SNW2, 250 µL for Fr-S-SNW2.5). As showed in Figure 3a, the capture efficiency (PC-3, and capture time was 45 min) increased with the increased silica nanowires length. The efficiencies were 38.8 ± 3.4% for Fr-S (length of silica nanowires was 0 µm, Figure 3b), 41.7 ± 4.2% for Fr-S-SNW0.5 (length of silica nanowires was 0.5 ± 0.1 µm, Figure 3c), 62.4 ± 9.6% for Fr-S-SNW1 (length of silica nanowires was 1.2 ± 0.3 µm, Figure 3d), 66.9 ± 7.3% for Fr-S-SNW1.5 (length of silica nanowires was 1.8 ± 0.6 µm, Figure 3e), 85.4 ± 8.3% for Fr-S-SNW2 (length of silica nanowires was 2.6 ± 0.9 µm, Figure 3f), and 80.5 ± 7.6% for Fr-S-SNW2.5 (length of silica nanowires was 2.8 ± 0.4 µm, Figure 3g). With the growth of


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silica nanowires, the topographic interactions between silica nanowires and cells helped to improve the cell capture performance. However, the morphology of silica nanowires was failed to control with excessive amounts of TEOS (Figure 3g). Micro-sized silica particles were formed during the growth of silica nanowires. Thus, we chose Pl-G, Fr-S, Fr-S-SNW1 and Fr-S-SNW2 as the typical substrates to study the topographic interactions between cells and surfaces. And FrS-SNW2 was option for clinical detection of CTCs which had better capture performance than other biochips with shorter silica nanowire lengths. To further demonstrate the interactions of cells and substrates, we carried out the immunofluorescent staining for the captured cancer cells on anti-EpCAM coated surfaces after 45 min cell (PC-3) capture assays. Tetramethylrhodamine B isothiocyanate (TRITC, red fluorescence) conjugated phalloidin bound to actins to show the cytoskeletal structure and 4’,6Diamidino-2-phenylindole dihydrochloride (DAPI, blue fluorescence) was used as a nuclear fluorescent indicator (Figure 4a, 4c, 4e, 4g). In addition, to directly observe the morphologies of cells on the surfaces, the cells were fixed with glutaraldehyde and gradient dewatered after cell capture experiment (45 min). SEM images were recorded and listed to proof the cell-substrate interactions (Figure 4b, 4d, 4f, 4h). For the Pl-G, both fluorescent images and SEM image showed that the cells tend to be spherical with little filopodia on the surface. While, the images of Fr-S showed that the micro-sized convex and concave of frosted slide were helpful to trap cells. What’s more, the fluorescent and SEM images showed the cells on Fr-S-SNW1 and Fr-SSNW2 protruded large amount of filopodia. Besides the micro-sized trap effect of frosted slide, the vertical silica nanowires would provide a three dimensional environment to match filopodia of cells. And these improved interactions of cell-surface demonstrated over 40% enhancement of capture efficiency for specific cancer cells (i.e. Fr-S-SNW1 and Fr-S-SNW2 versus Fr-S). Thus,


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these anti-EpCAM coated biochips as micro/nano-composites revealed the topographic interaction of cells and substrates which was helpful to fabricate high-performance glass-based biochips. Besides this work, various nanowires arrays had been fabricated to fulfill detection of circulating tumor cells.28, 46-56 The nanowires arrays were three-dimensional (3D) nanostructured substrates which had wide application in energy conversion, biological and biomedical research.57-59 Numerous materials (Such as, silicon, quartz, indium tin oxides (ITO), polymer, and gold) had been used to make these arrays. As listing in Table 1, chemical etching, reactive ion etching (RIE), electron-beam lithography (EBL), chemical vapor deposition (CVD), and template method were commonly used methods. In this work, we demonstrated a template-free solution method to grow silica nanowires on frosted slides as the low-cost glass-based biochip. We believe that this facile process will expand varieties of nanowires arrays for cell-capture application. We further studied the preoperative CTCs detection from whole blood specimens with antiEpCAM coated Fr-S-SNW2 at the optimal condition (45 min) (Figure 5a). Firstly, we tested the ability of the anti-EpCAM coated Fr-S-SNW2 to capture spiked cells in artificial whole blood. Figure S9 showed the cell captured efficiency of PC-3 with spiked cell number of 10, 20, 50 and 100 (1 mL mouse blood). The capture efficiencies were 40 ± 10% for 10 cells, 48.3 ± 10.4% for 20 cells, 62.7 ± 6.4% for 50 cells and 63.3 ± 5.0% for 100 cells, respectively. We then used the anti-EpCAM coated Fr-S-SNW2 to detect CTCs in patients’ blood. Fresh blood from patients with prostate cancer (numbers=18, Table S1) placed onto anti-EpCAM coated Fr-S-SNW2 to detect the CTCs (with 1 mL blood, 1×2 cm substrate). After capture, the immunofluorescent staining was used to distinguish the cytokeratin (CK)-positive CTCs (DAPI+/CD45-/CK+) and


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CD45-positive white blood cells (WBCs, DAPI+/CD45+/CK-). Clinical prostate cancer cell are usually EpCAM positive with high cytoplasmic epithelial cytokeratins.60-62 The phycoerythrin (PE-CF594, red fluorescent) conjugated anti-cytokeratin would bind to the CTCs (Figure 5b) and the prostatic CTCs were identified with the absence of CD45. The biochips were sensitive and selected to capture even 10 CTCs in 1 mL of blood (Table S1). The hematopoietic CD45 marker staining with FITC (fluorescein isothiocyanate, green fluorescence, and Figure 5c, Figure S10) helped to mark out the WBCs. And the number of WBC was less than 3 /mm2 (Figure S10), while the regular number of WBCs was 106 /mL. Thus, we realized the capture/identify of CTCs from true blood of prostate cancer patients with specificity and low nonspecific adhesion. 4. CONCLUSIONS In conclusion, we realized the in situ growth of silica nanowires on frosted slide as a cheap nano-biochip for detecting circulating tumor cells form prostate cancer patients’ blood. The specific cell capture assays revealed the micro/nano-structures were helpful to enhance the capture efficiency with topographic interactions between cells and silica nanowires. These nanobiochips may provide guides for investigation of cell-matrix interaction or development of novel functional devices with synergetic effect. Furthermore, the anti-EpCAM coated Fr-SSNW2 was successfully used to capture CTCs from whole blood specimens (prostate cancer patient) to show the ability to clinical preoperative test with low nonspecific adhesion of white blood cells. Thus, we foresee this frosted slide-based biochip can provide more candidates for low-cost detection of CTCs. ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: www.acs.org. Statistical analyses of the sizes of frosted slides (Figure S1) and silica nanowires on the frosted glass (Figure S2). Side views of silica nanowires growth on the frosted slide (Figure S3). Typical fluorescent images of captured PC-3 cells on the different substrates (Figure S4). Typical fluorescent images of different cells on the Fr-S-SNW2 (Figure S5). Cell capture on the edge of Fr-S-SNW2 and plain glass (Figure S6). The substrates showed low capture efficiency for PC-3 cells without modification of specific antibody (Figure S7). Cell viability (Figure S8). Cell captured efficiency of PC-3 on anti-EpCAM modified Fr-S-SNW2 with spiked cell number of 10, 20, 50 and 100 (Figure S9). Numbers of white blood cells (WBCs) on the anti-EpCAM modified Fr-S-SNW2 with 1 mL blood of the patient blood (Figure S10). The information of prostate cancer patients and the detected CTC numbers (Table S1). (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Shudong Zhang) * E-mail: [email protected] (Shutao Wang) Author Contributions S. T. Wang designed the project. H. J. Cui and B. S. Wang conducted the experiments. C. Y. Liu and K. Song helped to synthesis the silica nanowires. B. S. Wang and S. D. Zhang provided the clinical samples. The manuscript was written by H. J. Cui. All authors have given approval to the final version of the manuscript. H. J. Cui and B. S. Wang contributed equally to this work.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research is supported by the National Natural Science Foundation (21425314, 21501184, and 20141061), Beijing Municipal Science & Technology Commission (Z161100000116037), the Top-Notch Young Talents Program of China, and Youth Innovation Promotion Association, CAS (2017036). REFERENCES (1) Alix-Panabieres, C.; Pantel, K. Challenges in Circulating Tumour Cell Research. Nat. Rev. Cancer 2014, 14, 623-631. (2) Plaks, V.; Koopman, C. D.; Werb, Z. Circulating Tumor Cells. Science 2013, 341, 11861188. (3) Yu, M.; Stott, S.; Toner, M.; Maheswaran, S.; Haber, D. A. Circulating Tumor Cells: Approaches to Isolation and Characterization. J. Cell Biol. 2011, 192, 373-382. (4) Kaiser, J. Cancer's Circulation Problem. Science 2010, 327, 1072-1074. (5) Gupta, G. P.; Massagué, J. Cancer Metastasis: Building a Framework. Cell 2006, 127, 679695. (6) Cristofanilli, M.; Mendelsohn, J. Circulating Tumor Cells in Breast Cancer: Advanced Tools for “Tailored” Therapy? Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 17073-17074. (7) Talasaz, A. H.; Powell, A. A.; Huber, D. E.; Berbee, J. G.; Roh, K.-H.; Yu, W.; Xiao, W.; Davis, M. M.; Pease, R. F.; Mindrinos, M. N.; Jeffrey, S. S.; Davis, R. W. Isolating Highly


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Scheme 1. Interfacial growth of silica nanowires and surface modification of antibody on silica nanowires. a) Water emulsions took shape after vigorous shake of water and 1-pentanol (7:250, v/v, vortex shaker, and 2400 rpm). Then, water emulsion anchored onto the frosted slides to array the silica growth sites. After addition of ammonia water and TEOS, silica nanowires sprouted out with time. b) The substrates were modified with epithelial cellular adhesion molecule antibody (anti-EpCAM). The silica nanowires were firstly treated with O2-plasma. The thiol-functionalized substrates were reacted with 4-Maleimidobutyric acid Nhydroxysuccinimide ester (GMBS). After GMBS connecting streptavidin (SA), biotinylated antiEpCAM was introduced via biotin−streptavidin interactions.


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Figure 1. Surface morphologies of plain glass slides (Pl-G), frosted slides (Fr-S) and silica nanowires on frosted slides (Fr-S-SNW1, Fr-S-SNW2). a) The Pl-G showed no distinguished structure and the insert was partial enlarged image. b) The Fr-S had disordered micro-structure and the insert was a raised border. c) Fr-S-SNW1 (NH3·H2O: TEOS=1:1, 100 µL of TEOS) and d) Fr-S-SNW2 (NH3·H2O: TEOS=1:2, 200 µL of TEOS) showed the structured silica nanowires on surface of frosted slides and the inserts were terminated parts of silica nanowires.


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Figure 2. Enhanced capture efficiency for EpCAM positive cancer cells. a) Histograms of capture yields (PC-3) were plotted to describe that capture efficiencies increased with time for Pl-G, Fr-S, Fr-S-SNW1 and Fr-S-SNW2. b) The Fr-S-SNW1 and Fr-S-SNW2 showed the specificity between EpCAM positive cell lines (PC-3, MCF-7) and EpCAM negative cell lines (HeLa, Jurkat and Daudi).


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Figure 3. Topographic interactions between silica nanowires and targeted cancer cells significantly improve the cell-capture (PC-3) performance. The capture yields increased with the increased lengths of silica nanowires. a) was plot of the capture efficiencies on substrates with varying lengths. From b) to g), the amounts of TEOS were 0 µL, 50 µL, 100 µL, 150 µL, 200 µL, 250 µL, respectively. Increase of TEOS resulted in longer silica nanowires and better performance. However, the morphology of silica nanowires was failed to control with excessive amounts of TEOS. (The time of cell-capture was 45 min in 37 °C incubator.)


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Figure 4. Interactions of cancer cells and interfacial structures. Immunofluorescent staining provided fluorescent profiles of the captured cells (PC-3) on substrates (Pl-G (a), Fr-S (c), Fr-SSNW1 (e) and Fr-S-SNW2 (g)). Actin, TRITC-phalloidine, red; Nucleus, DAPI, blue. Typical SEM images of PC-3 cells on Pl-G (b), Fr-S (d), Fr-S-SNW1 (f) and Fr-S-SNW2 (h) revealed the interaction of cells and substrates. There was no obvious interaction on Pl-G, while cells protruded more filopodia to match the nanowires structure of Fr-S-SNW1 and Fr-S-SNW2. SEM images were taken with a slant sample stage (45º and high voltage on 10 kV).


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Figure 5. Counting and fluorescent images of captured CTCs from whole blood of prostate cancer patients. a) Numbers of captured CTCs in 1 mL blood with the anti-EpCAM modified FrS-SNW2. b) Typical fluorescent images of CTCs showed the cytokeratin-positive CTCs with red fluorescence for counting (DAPI+/CD45-/CK+). c) Typical fluorescent images of WBCs showed the CD45-positive WBCs with green fluorescence for counting (DAPI+/CD45+/CK-).


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Table 1. Nanowires arrays for circulating tumor cells detection. Substrates a

Preparation method

Cell lines c

b

Silicon nanopillar

Capture

Feature

Reference

3D nanostructured substrates

47

Integration of nanostructured

28

efficiency

Chemical etching

MCF-7

Chemical etching

MCF-7

70%

(SiNP) array

Silicon nanopillar

>95%

(SiNP) array +

substrate with microfluidic

microfluidic

Quartz nanowire

Reactive ion etching

A549

(QNW) arrays

Silicon nanowires +

Rapid thermal

gold nanoclusters

chemical vapor

MCF-7

Average

Laser scanning imaging

89.2%

cytometry

∼88%

Photothermal destruction of

48

49

cancer cells

deposition

Silicon nanopillars +

Chemical etching

MCF-7

N/A

thermoresponsive

Thermoresponsive capture and

50-51

release

polymer

Polystyrene nanotube

Replication with

substrate

AAO template

PEDOT-based

Chemical oxidative

micro/nanorods array

polymerization with

MCF-7

~80%

Rapid capture with low non-

52

specific cell adhesion

A549

over 80%

Tunable structures and tunable

53

from non-specific to specific

a PDMS replicate

Silicon nanopillars

Metal assisted

(NPs)

chemical etching

PC-3

over 80%

NPs with smaller diameter are

54

preferred to achieve higher capture yield due to their large


32

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effective contact area.

ITO nanowire array

Chemical vapor

MCF-7

89%

Hierarchical structure

46

MCF-7

86.8%

Lipid coating to prevent

55

deposition

Quartz nanopillar

Electron-beam

arrays.

lithography

Gold Nanowire

Electrochemical

human

Arrays

deposition

leukemic

nonspecific cell adhesion

83%

Electrochemical release of

56

captured cells

lymphoblasts

Frosted slide decorated with silica

Solution growth

PC-3

85.4%

Easily prepared glass-based

This work

biochip

nanowires

a

PEDOT is short for Poly(3,4-ethylenedioxythiophene); ITO is short for indium tin oxide.

b

AAO is short for anodic aluminum oxide; PDMS is short for polydimethylsiloxane.

c

MCF-7 is a breast cancer cell line; A549 is a human lung tumor cell line; PC-3 is a human prostate cancer cell line.


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Table of Contents：


34

Frosted Slides Decorated with Silica Nanowires for ... - ACS Publications

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