QNP arrays (2.5 × 2.5 cm2
) were fabricated by a series of processes including colloidal-polystyrene (PS)-nanoparticle (NP) coating and templating, metallization for pattern transfer, and reactive ion etching (RIE) for constructing nanopillar structures
]. The PS NP monolayer was first coated onto a quartz substrate using a modified self-assembly technique we recently developed
]. Oxygen plasma etching was then performed for 5 s with a mixed gas of O2
/Ar and a RF power of 100 W for securing space between the PS NPs on the quartz substrate. Evaporated Cr metal of approximately 25-nm thickness was then lifted off with the solution of N
-methyl-2-pyrrolidone in ultrasonic bath. In producing the QNP arrays, RIE process was performed for 4 min with a mixture gas of SF6
/Ar, a pressure of 20 mTorr, RF power of 300 W, and bias power of 100 W. For the QNP RIE process, Ni metal of approximately 30 nm served as an etching mask. The remaining Ni metal was completely removed with nickel etchant (LCE-12 K, Cyantek Corporation, CA, USA). Figure
a shows the tilted scanning electron microscope (SEM) image of as-prepared QNP arrays.
Figure 1 Tilted SEM images. (a) QNP arrays and (b) wet chemically etched SiNW arrays. (c) Photographic images of artificial CTCs capturing/isolating assembly including nine cell-separation chamber (PDMS) and wet-chemically etched SiNW arrays (2.5 × 2.5 (more ...)
Next, SiNW arrays (2.5 × 2.5 cm2
) were prepared by Ag-assisted chemical wet-etching process of p-type Si wafer immersed into 10 wt% hydrofluoric (HF) acid for 5 min to remove the native oxide layer and sequentially treated in a boiled RCA solution (H2
O = 1:1:5) for 1 h to create a hydrophilic surface. An Ag film with a thickness of 30 nm was coated onto (100) Si substrates with the resistivity of 1 to 10 Ω·cm, which were cleaned by electroless deposition in an aqueous solution
]. The cleaned Si samples were placed in 10% HF and 5 × 10−3
solution at room temperature for 5 min. The Ag-coated Si samples were then immersed in an aqueous solution containing 10% HF and 0.3% H2
at room temperature for 30 min. The Ag metals remaining on the Si substrates were removed in boiling aqua regia (HCl/HNO3
= 3:1) for 1 h and by additional amorphous Si etching for 30 s in buffered oxide etchant (NH4
F/HF = 6:1). Figure
b shows the SEM images of wet chemically etched SiNW arrays with tilted view.
Prior to the surface functionalization, three as-prepared nanopatterned substrates (QNP, SiNW, and planar glass substrates for control samples) were carefully cleaned with H2
(1:1) for 10 min to remove all of the organic materials and impurities on the surface. Then, the substrates were washed using a conventional three-step cleaning process (acetone, isopropyl alcohol, and DI water) and dried with air. The surface was treated with O2
plasma for 20 s to confer the hydroxyl groups on the substrate surfaces. The surface of the two-nanopatterned substrates with planar glass substrates was functionalized by STR-immobilization method we developed previously (Figure
]. During this procedure, we first applied (3-aminopropyl)-triethoxysilane to aminate the nanowire surface, which can be further functionalized with STR via a two-step aldehyde/amine reaction using glutaraldehyde as the linker. Finally, biotinylated anti-human monoclonal anti-human epithelial cell adhesion molecule (EpCAM), where the targeted cells were pre-mixed, was introduced to the STR-functionalized nanowires through the high-affinity biotin-STR binding. Cell-capture chambers with nine circular wells (5 mm in diameter, Figure
c) were made by molding a polydimethylsiloxane (PDMS) elastomer. The solidified PDMS mold was cut to the size of 2.5 × 2.5 cm2
, which is the same size of the surface-functionalized nanopatterned substrates. A solution of the A549 cells (human lung carcinoma cell line, CCL-185) purchased from American Type Culture Collection (ATCC, VA, USA), which are conjugated with biotin-EpCAM-Abs in F12K:DMEM (500 mL, Invitrogen Corporation, NY, USA), with a final volume of approximately 50 μL was then pipetted into each of the nine cell-capture chambers with cell populations of approximately 103
cells/chamber. Three samples with nine cell-capture chambers were prepared in each group (0.5, 21, and 45 h), and the average spreading areas and sizes of each fixed cell on three different substrates were consequently calculated with standard deviation (n
For straightforward detection of the captured A549 cells, a standard immunofluorescent staining procedure was\ performed on cells fixed on STR-functionalized SiNW, QNP, and planar glass substrates. The PDMS cell-capture chambers were first washed out with 1 × PBS and Tween-20 (PBST, KPL Inc., USA) at least three times to remove unbound tumor cells on the SiNW arrays, and then fixed with 4% paraformaldehyde for 15 min. The cells were immersed in blocking buffer (5% bovine serum albumin and 0.3% Triton X-100 in 1 × PBS) for 60 min. After rinsing with PBS three times for at least 5 min each, the actin filaments and nuclei were stained with Alexa Fluor 594 conjugated phalloidin (Invitrogen, green 532 nm) for 20 min and with DRAQ5 (Cell Signaling Technology, Inc., MA, USA, red 632 nm) for 5 min (Figure
). The samples with cultured cells were then given a three-step cleaning process, using PBS, PBS in DI water (1:1), and DI water after peel-off of PDMS cell-capture chambers. The samples were finally transferred to a microarray scanner for further LSC analysis.
Figure 2 Optical and fluorescence images of A549 cells. (a) Flourescence images of A549 cells cultured on three different substrates (planar glass, QNP, and SiNW arrays) at 37°C for 0.5, 21, and 45 h. (b) Optical and fluorescence images of the immobilized (more ...)
For the image of surface-bound cells on the nanopatterned substrates, an Axon Genepix microarray scanner 4000B (Molecular Devices, LLC, CA, USA) was used. Green and red YAG lasers (532 and 635 nm) were used to visualize the captured cells (actin, green 532 nm) and nuclei (red 635 nm) on the three different STR-functionalized substrates with approximately 5-μm resolution. The cell-capture platform was automatically scanned, and the scanned images of PDMS cell-capture chambers that contained the captured cells were transported into CellProfilerTM
) cell image software for rapidly quantitation of the captured cells on STR-functionalized dual-nanopatterned substrates with planar glass substrates.