pubs.acs.org/NanoLett
Novel Streptavidin-Functionalized Silicon Nanowire Arrays for CD4+ T Lymphocyte Separation Sung Tae Kim,†,§ Dong-Joo Kim,‡,§ Tae-Jin Kim,† Deok-Won Seo,‡ Tae-Hong Kim,‡ Seung-Yong Lee,‡ Kwanghee Kim,† Kyung-Mi Lee,*,† and Sang-Kwon Lee*,‡ †
Global Research Laboratory, Department of Biochemistry, Division of Brain Korea, College of Medicine, Korea University, Seoul 136-701, Korea, and ‡ Department of Semiconductor Science and Technology, Chonbuk National University, Jeonju 561-756, Korea ABSTRACT Silicon nanowires (SiNWs) offer promising inorganic nanostructures for biomedical application. Here, we report the development of a novel SiNW array designed for isolating primary CD4+ T lymphocytes from the heterogeneous mixture of cell populations. Our system employed the specific high-affinity binding features of streptavidin (STR)-functionalized SiNW with biotinlabeled CD4+ T lymphocytes. Fabricated SiNW arrays easily separated the CD4+ T lymphocytes from the mouse whole splenocytes with over ∼88% purity and demonstrated tight attachment to CD4+ T lymphocytes by scanning electron microscopy. Thus, our STR-SiNW arrays provide a potential tool for specific cell separation and further present a possibility to be applied to the other area of biomedical applications. KEYWORDS Silicon nanowire, streptavidin, CD4+ T lymphocyte, cell separation
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ilicon nanowire (SiNW) provides a good inorganic material for the fabrication of various nanoscale arrays including the field-effect transistors (FETs), lightemitting diodes (LEDs), solar cells, and gas-sensors.1-4 Because of its biocompatibility, controllable electrical conductivity, and high surface-to-volume ratio, SiNW also presents broad applicability in the biomedical field.5,6 For example, SiNW biosensors were developed to detect biological species such as DNAs, proteins, or viruses.7-10 Highdensity SiNW transistor arrays have been generated to monitor electrophysiological signals of living cells.11-13 These studies corroborate superior potential of SiNW as a highly sensitive biological sensor for the diagnosis of human diseases. Specific cell separation process is an obligatory step in a variety of biological and biomedical applications including cell transplantation and antitumor cell therapy.14,15 With the introduction of magnetic nanomaterials, cells of interest can now be specifically labeled and separated.16 The most common magnetic particles used for cell separation are made of nickel or iron oxide17 and chemically synthesized to be within the range of micrometer to nanometer sizes.18 Nanometer-sized magnetic particles have shown advantages due to their sensitivity and reduced toxicity,19 yet the synthesis of these nanoparticles uniform in size with good
dispersion properties has become a challenging task. Furthermore, isolation process required exposure of constant magnetic field, which may affect the viability and functionality of cells isolated. Therefore, the development of a novel cell separation system presenting consistent yield, and not requiring magnetic field, is immediately warranted. Herein, we construct a novel SiNW array, via functionalization with streptavidin (STR), specifically designed for isolating primary CD4+ T lymphocyte from the mixture of cell suspensions. SiNWs were fabricated by a vapor-liquid-solid method (VLS) in a hot wall chemical vapor deposition (HW-CVD) quartz-tube furnace (2 inch) with SiH4 as a silicon (Si) precursor and gold (Au) as a catalyst.20 Figure 1a shows the Si wafer (0.7 cm by 0.7 cm) and Figure 1b shows the densely grown SiNWs on silicon (111) substrate using the VLS method. Synthesized SiNWs demonstrated 100-200 nm of diameters with approximately 10 µm in length and the density measured to be ∼3.5 × 108/cm2. To characterize the physicochemical properties of generated SiNWs, we investigated the selective area electron diffraction (SAED) pattern and performed energy dispersive X-ray spectroscopy (EDX) analysis. As shown in Figure 1c, the SiNW was highly crystalline in nature with a preferred orientation in the [111] direction. EDX data showed that the SiNW consisted entirely of Si atoms when fabricated, as shown in Figure 1d. These observations demonstrate the synthesis of SiNW with superior quality and high density by the VLS method. To develop a specific cell separation system, the surface of SiNW arrays was conjugated with STR for allowing high affinity binding to biotin-conjugated monoclonal antibodies (Kd ) 10-15 M) directed against specific cell surface markers,
* To whom correspondence should be addressed. (S.-K.L.) E-mail: sk_lee@ chonbuk.ac.kr. Tel: +82-63-270-3973. Fax: +82-63-270-3585. (K.-M.L.) kyunglee@ korea.ac.kr. Tel: +82-2-920-6251. Fax: +82-2-920-6252. § These authors contributed equally to this study. Received for review: 3/17/2010 Published on Web: 07/13/2010
© 2010 American Chemical Society
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FIGURE 1. Generation of SiNW by a vapor-liquid-solid (VLS) method. (a) Photograph of SiNW grown on the Si wafer (0.7 by 0.7 cm2), (b) SEM image of SiNW arrays, (c) TEM image of SiNW arrays, and (d) EDX analysis of SiNW arrays.
as shown in Figure 2a. In brief, the silicon surface of the SiNW was first treated with O2 plasma to confer hydroxyl groups, which could allow conjugation with 3-aminoprophyltiethoxysilane (APTES, Sigma-Aldrich, U.S.A.). Then the amine groups of APTES were reacted with glutaraldehyde (GA, Sigma-Aldrich, U.S.A.) to functionalize the Si surface with an aldehyde group that sequentially reacted with STR (50 ng/mL, Calbiochem, Germany). APTES conjugation rendered the stability of STR binding on the SiNW surface. To ensure STR binding on the SiNW array, biotinylated gold nanoparticles ranging from 30 to 50 nm (50 µL, NANOC Inc., NY) were treated for 20 min and subjected to the scanning electron microscope (SEM) analysis (Figure 2b). Biotinylated gold nanoparticles, highlighted as yellow, were found to be tightly bound on STR-SiNW. These data demonstrate that STR was successfully functionalized on the surface of the SiNW, without the signs of denaturation or degradation. STR-conjugated SiNW arrays were then tested for their ability to isolate CD4+ T lymphocytes from the mixture of mouse splenocytes population (Figure 2c). Isolated and activated CD4+ T lymphocytes had been shown to possess strong antitumor responses in the cancer patients,21 thus CD4+ T lymphocytes were chosen as a primary candidate for our SiNW cell separation system. Splenocytes, prepared from the spleen of C57BL6/mice (6-8 weeks, Nara Biotech, Korea), were first passed through a nylon wool column to remove B lymphocytes and to enrich T lymphocytes. Resulting cell population (105 cells/plate), containing CD4+ T, CD8+ T, NK, and NKT cells, was then reacted with biotin-conju© 2010 American Chemical Society
gated anti-CD4 mAb (clone GK 1.5, eBioscience Inc., CA) and incubated with STR-SiNW for 20 min (Figure 2c). The remnants of splenocytes unbound were evaluated for the presence of CD4+ T lymphocytes by flow cytometer (Becton Dickinson, NJ) and analyzed using CellQuestPro software (BD Bioscience, U.S.A.).22 As shown in Figure 3a, the whole splenocytes mixture contained approximately 40.9% of CD4+ T lymphocytes and 59.1% of non-CD4+ T lymphocytes. After binding with STR-SiNW, the percentage of CD4+ T cells in the solution has decreased to 5.3%, indicating that the majority of CD4+ T cells were bound to STR-SiNW. In comparison, STR coated on Si wafer without NW (middle panel, Figure 3a) or glass wafer (bottom panel, Figure 3a) showed 13.0 and 14.3% of T lymphocytes remaining in the solution, respectively. When the average percentage of separation efficiency was calculated (n ) 3), STR-SiNW arrays show 87.6 ( 5.4% fidelity while that of STR-Si or glass wafer shows 68.0 ( 6.5 and 65.2 ( 2.3%, respectively (Figure 3b, bar graph). These data demonstrate that the SiNW displays greater cell separation efficiency over Si wafer (p ) 0.017) or glass wafer (p ) 0.003), presumably due to a large surface-to-volume ratio. During the SiNW surface modification process, O2 plasma treatment was found to be crucial in the first stage of STR immobilization. Without O2 plasma treatment, the percentage of unbound CD4+ T lymphocytes in cell suspensions has increased from 5.3 to 17.1%, approximately 3 folds higher than that of the O2 plasma-treated (Figure 4a). Furthermore, if the subsequent modification with APTES was delayed after 2878
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FIGURE 2. Surface functionalization on SiNW arrays. (a) Schematic diagram of the STR conjugation process on SiNW, (b) SEM image of STRSiNW bound to biotin-gold nanoparticles, where gold nanoparticles were highlighted as yellow for easy detection, and (c) schematic representation of the separation process for CD4+ T lymphocyte among mouse primary whole splenocytes.
followed with APTES conjugation for the effective functionalization with STR. To directly ensure the binding of CD4+ T lymphocytes on STR-SiNW, a field-emission scanning electron microscope (FE-SEM, S-4300, Hitachi, Japan) was performed. As can be seen in Figure 5a (tilted images) and Figure 5b (top images), NWs extruded from Si Wafer surface bound to multiple
O2 plasma treatment, a time-dependent decrease in the cell separation efficiency was observed (Figure 4b). When APTES conjugation was performed at 3 or 7 days after O2 plasma treatment, the percentage of unbound CD4+ T lymphocytes increased to 7.1 and 13.0%, respectively, compared with that conjugated at 1 day (5.3%). These results suggest that O2 plasma treatment should be adequately performed to be © 2010 American Chemical Society
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FIGURE 3. Flow cytometeric analysis of CD4+ T lymphocytes after cell separation using STR-functionalized SiNW arrays. (a) Flow cytometric analysis of CD4+ T lymphocytes using cell suspensions after binding to STR-SiNW, STR-Si wafer, or STR-glass wafer and (b) average cell separation efficiency of CD4+ T lymphocytes shown as bar graphs.
CD4+ T lymphocyte, highlighted as red, through specific STR-biotin binding. Bound CD4+ T lymphocytes were within 3-5 µm in diameter and found to be intact showing normal membrane protrusions on their surface (right panels, Figure 5). Upon higher magnification (top right panel, Figure 5), we found that multiple SiNWs were bound to single CD4+ T lymphocytes, providing strong docking sites for CD4+ T lymphocytes. To ensure that the SiNW-bound cells were indeed CD4+ T lymphocytes, we stained cells with phycoerythrin (PE)-conjugated antiCD4 mAb and attempted to detect the resulting fluorescence under fluorescence microscopy (Eclipse Ti-U, Nikon, Japan). However, visualization of SiNW-bound CD4+ T cells on Si wafer was difficult as the Si wafer was not able to transmit lights. Since cells on the glass wafer could be detected under optical light (Figure 6a) or UV light (Figure 6b), we confirmed the attachment of CD4+ T lymphocytes on STR-conjugated glass wafers instead. The majority of cells attached on STR-glass wafer (Figure 6a) were stained positive with PE-anti-CD4 mAb (Figure 6b), demonstrating overlapping fluorescent staining with cells of optical images (blue arrows, nos. 1-3, Figure 6). Though a few percentage of cells showed lack of fluorescence (red circles, no. 2, Figure 6a), most cells were found to be CD4+ T lymphocytes. Furthermore, cells attached on the SiNWs were found to maintain vitality, as shown by DAPI (4′-6diamidino-2-phenylindole, Sigma, MO) positive and PI (propidium iodide, BD Pharmigen, BD Bioscience, CA) negative staining (Figure 6c). Therefore, we confirm the fidelity and safety of our STR-SiNW array cell separation platform. Recent technologies capable of formulating uniform and high-density SiNWs with surface modification properties have enabled the development of diverse biomedical arrays detecting DNAs, RNAs, peptides, proteins, or viruses.7-10 In © 2010 American Chemical Society
this study, we developed a novel SiNW cell separation system capable of selectively isolating single cell population among the heterogeneous mixture of cells. Previously, cell separation using magnetic NWs has been demonstrated;23-25 however, their system did not present the ability to identify single cell population among the mixed populations since it utilized the NW’s properties to adhere common integrin proteins expressed in all mammalian cells. To confer sensitivity and selectivity, we have employed STR fabrication on the surface of SiNW. Surface STR allows specific binding of biotin-conjugated antibodies attached to cells of interest with high specificity and selectivity. Although we presented CD4+ T lymphocytes as an example, our STR-SiNW can be applied to the separation of a wide variety of cells, including various leukocytes, tumor, or stem cells, simply by changing the identity of biotin-conjugated antibodies. For example, antibodies against CD8 and NK1.1 can be applied to isolate CD8+ T cells and NK cells,26,27 while those against CD90, CD44, or CD105 can be applied to isolate mesenchymal stem cells.28,29 Tumor cells can also be specifically isolated by our SiNW system via using antibodies against tumorspecific antigens; for example, HER-2/Neu for breast and ovarian cancer, MUC-1 for breast and pancreatic cancer, and tyrosinase for melanoma. Therefore, our SiNW system provides a universal one step cell identification and separation system, regardless of the cell types and species used. With the capacity to be internalized and phagocytosed, magnetic nanowires have been shown to be utilized for the movement of cells30 and deliver specific genes inside the cell.31,32 Unlike their systems, our SiNW arrays did not appear to be internalized, as seen in the SEM images. Rather, cells were shown to be tightly attached on the surface of individual SiNWs via specific STR-biotin forces. Furthermore, 2880
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FIGURE 4. Effect of O2 plasma treatment in the immobilization of STR on SiNW. (a) Flow cytometric analysis of the CD4+ T lymphocytes using cell suspensions after binding to O2 plasma-treated or -untreated STR-SiNW (left panel). Average cell separation efficiency was shown as bar graphs (right panel). (b) Flow cytometric analysis of the CD4+ T lymphocytes separated on STR-SiNW arrays functionalized on 1, 3, and 7 days after O2 plasma treatment (left panel). Average cell separation efficiency was shown as bar graphs (right panel).
multiple SiNWs were bound to a single cell in various orientations, hence providing a tight multilayer matrix for the cells. Since biotin-conjugated anti-CD4 antibodies were present on the surface of cells, our arrays placed STR-SiNW on the surface, but not inside or cytoplasm, of the cell. Therefore, our SiNW arrays can be voluntarily targeted either on the cell surface or inside the cells via modification of the surface fabrication. Magnetic NWs have been shown to be advantageous over magnetic nanoparticles in cell separation, since elongated structures have a stronger magnetic moment and exert a larger force on the cell.33 Since magnetic field may affect the normal cellular behavior and physiology, we devised © 2010 American Chemical Society
SiNW cell separation system devoid of the use of magnetic field. Our SiNW was highly specific and efficient in isolating primary CD4+ T lymphocytes. When compared with flatsurfaced Si wafers or glass wafers, NW form of Si wafer was found much more efficient in isolating CD4+ T lymphocytes. This was likely due to their large surface to volume aspect ratios. Though our SiNWs were within the range of 100-200 nm in diameters, 10 µm in length, and the density of ∼ 3.5 × 108/cm2, their length and density could be easily manipulated within the desired window by adjusting the reaction temperature or the time and concentration of Au catalyst, which is dependent on the thickness of Au thin film on Si substrate, during the synthesis procedure. Experiments are 2881
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FIGURE 5. SEM images of CD4+ T lymphocytes bound on the surface of STR-SiNW. (a) Tilted SEM images of CD4+ T lymphocytes on STR-SiNW and (b) top SEM images of CD4+ T lymphocytes on STR-SiNW [left panel, low magnification (×800); right panel, high magnification (×6000)]. CD4+ T lymphocytes were highlighted as red for easy detection.
FIGURE 6. Optical images of CD4+ T lymphocytes bound on the surface of STR-glass wafer or SiNWs. (a) Light microscopic images (×40) and (b) fluorescence images of CD4+ T lymphocytes bound on the STR-glass wafer. Arrows indicate CD4+ T lymphocytes while circled cells show non-CD4+ T lymphocytes. (c) Fluorescence images of CD4+ T lymphocytes bound on the SiNWs stained with DAPI and PI, capable of detecting dead cells. DAPI, shown in blue, binds to all nuclei of the cells while PI, shown in red, binds to only dead cells. No dead (red) cells were present in CD4+ T lymphocytes bound to SiNWs.
currently in progress to identify the optimal length, diameter, and density of SiNWs to obtain maximum cell separation yield. © 2010 American Chemical Society
Finally, the property of STR-SiNW to bind specific cell populations may allow the development of “high-throughput cells on a chip” which can probe and analyze the complex 2882
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cellular processes. Via electrophysical recording, we can monitor the individual cell’s behavior as well as the secretion of various biogenic molecules including cytokines and chemokines in a timely controlled manner. In addition, separated cells on a chip can be used to screen for the development of a new drug, or concurrently stimulated via encapsulating and delivering biogenic molecules, for example, growth factors, anticancer drugs, and so forth. Furthermore, direct lysis of cells on a chip will permit binding of individual cell’s DNA, RNA, or proteins on the SiNW, which may allow simultaneous and multiple assessments of cellular physical responses in the normal or diseased states. Thus, our highly efficient STR-SiNW system offers a specific single cell isolation platform and launches a new avenue for the development of advanced biochips allowing simultaneous isolation and activation of cells with concomitant detection of biogenic molecules. Acknowledgment. This study was supported by Priority Research Centers Program and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0094032 and 2009-0075729) and also partially supported by a grant from the KRIBB Research Initiative Program. K.-M.L. was supported by a grant from KICOS (K20704000007-09A0500-00710, K2060100000209E0100-00210). S.T.K. and T.J.K. were supported by a grant from the Innovative Research Institute for Cell Therapy (A062260) and a grant from the National Nuclear R&D program (Grant BAERI), respectively. REFERENCES AND NOTES (1) (2) (3) (4) (5) (6) (7) (8) (9)
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