Selective Capture and Collection of Live Target Cells Using a

Aug 13, 2012 - A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an ant...
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Selective Capture and Collection of Live Target Cells Using a Photoreactive Silicon Wafer Device Modified with Antibodies via a Photocleavable Linker Shinya Ariyasu,† Kengo Hanaya,‡ Eita Watanabe,§ Toshihiro Suzuki,†,∥ Kazutaka Horie,† Masanori Hayase,†,§ Ryo Abe,†,‡,∥ and Shin Aoki*,†,‡ †

Center for Technologies against Cancer, ‡Faculty of Pharmaceutical Sciences, §Faculty of Science and Technology, and ∥Research Institute for Biomedical Science, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510 Japan S Supporting Information *

ABSTRACT: A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy−attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FSAFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody− alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.

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INTRODUCTION

is capable of isolating and retrieving rare viable CTCs from a cancer patient’s blood without the need to use biomarkers.23−26 The second type is IgG-modified devices, which are effective for trapping and/or counting immunologically specific cells such as CTCs.30,31 In these devices, the detachment of cells from the captured solid phase is also an important factor. Trapped cells could be detached by sweeping with a strong buffer stream. However, the required flow rate is dependent on the characteristics of the specific type of target cells10 and the mechanical stress associated with flow acceleration, may cause severe damage to cells.32 In the case that cells are fixed by ligand−receptor interactions such as strong antibody−antigen complexation, detaching viable target cells from such devices under mild conditions without a detergent, high or low pH, or high salt concentrations can be a difficult task.33 This background information prompted us to develop a Si wafer device modified with an antibody via a chemically cleavable linker for capturing and collecting target cells. The chemical linkers have been used in various areas such as proteome analysis34,35 and the isolation of biomolecules.36,37 The use of photocleavable moieties has been reported for

The isolation, enrichment, and identification of specific target proteins and cells are very important manipulations in the biological and medicinal sciences and in the diagnosis and treatment of a variety of diseases.1,2 Currently, flow cytometory,3,4 immunoglobulin G (IgG)-coated magnetic beads,5,6 and membrane filtration7,8 are routinely used for the separation and analysis of cells. Recent cell-enrichment and/or cell-trapping systems have been developed using silicon (Si)based wafer platforms9−13 because Si wafers can be easily processed to the desired shape via microfabrication techniques and the surfaces of Si-based materials can readily be modified with chemical reagents such as silane coupling reagents14−16 and then with specific biomolecules such as IgG17 and celladhesion proteins/peptides.9,18−20 Si-based biodevices for cell enrichment can be classified into two types. One is based on size selection and includes microfluidic devices10−12 and consists of uniformly sized holes21,22 and an appropriate flow channel to control solvent flow precisely.23−26 For example, Matsunaga et al. reported the enrichment of model cells such as MCF-7, MDA-MB-231, and circulating tumor cells (CTC) in peripheral whole blood without the need for any pretreatment procedures.21,22,27−29 Lim et al. reported a mechanics-based microfluidic biochip that © 2012 American Chemical Society

Received: June 13, 2012 Revised: August 10, 2012 Published: August 13, 2012 13118

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Scheme 1. Capture and Recollection of Target Cells on a Si Wafer Modified with an Antibody through a Photocleavable ANP Linker

Scheme 2. Modification of Si Wafers with ANP Linker 1a

a

For details, see Schemes S1 and S2 in the Supporting Information.

analyses of protein function,38−40 the isolation of target protein−ligand complexes,41−47 and the control of celladhesion properties of a solid surface.18,20 In this study, we used a 3-amino-3-(2-nitrophenyl)propionic acid (ANP) unit in the linker part and anti-HEL-IgG (HEL, hen egg lysozyme) as a model antibody to trap model cells that express HEL as an antigen on the cell membrane (SP2/O-HEL cell, Scheme 1). The ANP unit contains a 2-nitrobenzyl amino group that is often used for the “photocages” and other applications.38−40,48,49 Besides, it is commercially available and includes amino and carboxylate groups to which different functionalities can be selectively and readily attached. The modification of the Si wafer with the ANP linker and then with anti-HEL-IgG was monitored using direct methods (Fourier transform infrared spectroscopy by attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FSAFM)) and an indirect method (enzyme-linked immunosorbent assay (ELISA)). In addition, we were able to observe the photorelease of IgG directly from the Si surface by means of FS-AFM. The selective capture and detachment of SP2/O-HEL

cells and the reculturing of the recovered SP2/O-HEL cells are presented.50

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RESULTS AND DISCUSSION

Synthesis of the ANP Linker and Modification of the Si Wafer with IgG through an ANP Linker. The ANP linker containing Si(OEt)3 (1) was prepared from Fmoc-ANP (5) and 6 via intermediates 7 and 8, as shown in Scheme S1 in the Supporting Information. The modification of the Si wafer with 1 was carried out as shown in Schemes 2 and S2 in the Supporting Information. Si wafer 2a that had been cleaned with a piranha solution was reacted with 1 in toluene at 80 °C for 3 h to give 3a. The tBu protecting group was removed by treatment with trifluoroacetic acid (TFA) to afford 10a (Scheme S2 in the Supporting Information), which was reacted with N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in H2O to give 11a (Scheme S2 in the Supporting Information). Finally, 11a was reacted with anti-HEL-IgG (2 μg/mL), and the subsequent blocking reaction of unreacted NHS ester groups with ethanolamine 13119

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afforded photoreactive Si wafer 4a. In this experiment, the concentration of anti-HEL-IgG in solution was changed from 0.2 to 20 μg/mL. The numbers of trapped SP2/O-HEL cells, which are model target cells used in this work (see below), were almost the same in the presence of 2 and 20 μg/mL antiHEL-IgG, a fact that allowed us to conclude that 2 μg/mL is enough to trap SP2/O-HEL cells. Similarly, porous Si 2b was reacted with 1 and then converted to 4b for the analysis of its surface modification processes by FTIR-ATR. Characterization of the Si Wafer Modified with IgG through the ANP Linker by FTIR-ATR and FS-AFM before and after Photoirradiation. The chemical modification of the Si surface with the ANP linker and IgG was followed by FTIR-ATR spectroscopy of porous Si (2b → 4b). FTIR-ATR spectra between 1900 and 1300 cm−1 of porous Si at each modification step (2b → 3b → 10b → 11b → 4b) are shown in Figure S1 in the Supporting Information. The IR spectrum of 3b exhibited absorption peaks at 1650 and 1540 cm−1 that correspond to the vibrational mode of amide and nitro groups, respectively. The activation of 10b by NHS/EDC was confirmed by the IR peaks at 1830, 1790, and 1750 cm−1, which are characteristic peaks of a succinimide ester. The IR spectrum of 4b maintained the absorption peak at 1540 cm−1 because of the nitro group in the ANP linker and exhibited rather strong absorption around the 1300−1700 cm−1 region (Figure S1a in the Supporting Information), which was assigned to various carbonyl groups in IgG. The broad signal possibly corresponding to H2O was strongly observed in the IR spectra of IgG-coated Si wafer 4b in the CH2 region (Figure S1c in the Supporting Information). In addition, FT-IR signals of modified Si surface 4b are not as strong. Therefore, the anti-HEL-IgG on Si wafer 4a and its release by the photochemical cleavage of the ANP linker was directly observed by fast-scanning atomic force microscopy (FS-AFM) (Figure 1). The FS-AFM images of anti-HEL-IgG adsorbed on a mica surface without a covalent linker (Figure 1a) showed Yshaped objects that were ca. 20 nm in size that possibly correspond to the typical shape of IgG (the size of its monomer is 15 × 9 × 4 nm3).51 In Figure 1c, the spherically shaped objects of 20−30 nm were observed on the surface of 4a. In Figure 1a−d, scanning was performed several times over the same area on FS-AFM, and almost identical images were observed. In addition, similar AFM images were observed at several points on the same Si wafer sample. Therefore, these spherelike objects were assigned to anti-HEL-IgG molecules (Supporting Information Movie S1 described below). We consider that different shapes of IgG in Figure 1c are from that in Figure 1a as a result of the random orientation of IgG caused by the rather rough surface of the Si wafer as observed in Figure 1b.52 The approximate number of IgG's on the Si wafer over the μm2 area was determined to be ca. 5.2 × 103/μm2. The photorelease of IgG from 4a was directly observed using FS-AFM. The AFM image of 4a after photoirradiation at 330 nm for 1 h strongly suggests that IgG was detached from 4a by the photochemical cleavage of the ANP linkers (Figure 1d). Besides, the photorelease of IgG from 4a was monitored by FSAFM, as displayed in Supporting Information Movie S1. Photorelease of IgG from the Surface of 4a Followed by ELISA. The photorelease of IgG from the surface of 4a upon photoirradiation was also confirmed by ELISA using antimouse IgG-IgG (secondary antibody) conjugated with alkaline phosphatase (AP, 2nd IgG-AP, Scheme 3). The second IgG-AP was first complexed with anti-HEL-IgG in 4a (A in Scheme 3),

Figure 1. Topographic AFM images of anti-HEL-IgG on (a) mica or (b−d) the Si surface. (a) AFM images of anti-HEL-IgG deposited on mica. AFM images of modified Si wafers (b) 3a, (c) 4a, and (d) 4a after photoirradiation at 330 nm for 1 h.

and its AP activity was then measured by monitoring the release of 4-nitrophenol from mono(4-nitrophenyl) phosphate (MNP), which is a typical substrate of AP.53 The activity of AP on the Si wafer and that in the supernatant (Scheme 3B,C after photoirradiation for 15 min, Scheme 3D,E after 30 min, Scheme 3F,G after 45 min, and Scheme 3H,I after 60 min) were measured. As summarized in Figure 2, the sum of AP activity for the Si wafer (B in Scheme 3 and Figure 2) and the supernatant after a 15 min photoreaction (C in Scheme 3 and Figure 2) was nearly equal to the activity of A. Similar results were obtained after photoirradiation for 30 (D + E = B), 45 (F + G = D), and 60 min (H + I = F), indicating that the total AP activity after photoirradiation is largely retained. On the basis of these results, we conclude that IgG and enzymes (AP in this experiment) can be released from 4a by photoirradiation without critical damage. Capture and Recollection of SP2/O-HEL Cells on a Photoreactive IgG-Coated Si Wafer. The capture and photorelease of SP2/O cells expressing HEL on their cell 13120

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Scheme 3. ELISA Based on the AP Enzymatic Reaction of the Photorelease of IgG from 4a

Figure 3. Microfluidic device for the capture and release of target cells.

device is ca. 0.001 mL, implying that ca. 103 target cells can be trapped by the injection of a 106 cells/mL target cell suspension. The specific capture of SP2/O-HEL cells on 4a was examined using an approximately a 1:1 mixture (in number) of SP2/O-HEL cells that are stained with carboxyfluorescein succinimidyl ester (CFSE) (SP2/O-HEL-CFSE cells) and SP2/ O cells that are not. The device was first filled with PBS buffer from port 1 to remove bubbles, and the cell suspension was then introduced into the device from port 2 (Scheme S3b in the Supporting Information). After a 5 min incubation to permit cell adhesion to occur (Scheme S3c in the Supporting Information), the PBS buffer was allowed to flow from port 1 to port 2 at a rate of 5 μL/min for 5 min in order to wash out nonadhesive cells (SP2/O cells in this case, Scheme S3d in the Supporting Information). The cells on the Si surface at each step were observed as bright-field images and CFSE fluorescent images (NIBA filter λex = 470−490 nm, λem = 510−550 nm) by an upright fluorescent microscope (Figure S4a−d in the Supporting Information). After these manipulations, the ratio of the numbers of SP2/O-HEL-CFSE cells and SP2/O cells was almost 1:1 (Figures 4 and S4a,b). However, the

Figure 2. Relative AP activities of 4a complexed with secondary IgGAP at each photoreaction step (A−I corresponds to those in Scheme 3). (A) Relative AP activity of the complex of 4a with secondary IgGAP before photoirradiation. B, D, F, and H are the relative AP activities of the Si wafer after photoirradiation for 15, 30, 45, and 60 min, respectively. C, E, G, and I are the relative AP activities of the supernatant after photoirradiation for 15, 30, 45, and 60 min, respectively. AP activity was calculated on the basis of the absorbance at 405 nm of p-nitrophenol, the hydrolytic product of MNP, in Trisbuffered saline (pH 7.4) containing 0.05% (w/v) Tween 20 (TBST).

Figure 4. Relative cell numbers of SP2/O-HEL-CFSE and SP2/O for each process are shown as gray and white bars, respectively.

membrane (SP2/O-HEL cells) and SP2/O cells not expressing HEL (SP2/O cells) on 3a, 4a, and 4b were examined.54 For this purpose, we developed a microfluidic device for the flow of PBS buffer and used the device to count the numbers of cells on the Si surface. As shown in Figure 3 and Scheme S3a in the Supporting Information, this device is composed of an IgGcoated Si wafer, an aluminum plate, a brass plate, a glass window for microscopically counting cells, and three ports (1− 3) for access to the PBS buffer in the vessel, the injection of the cell suspension, and collecting the recovered cells, respectively. (A picture showing the whole system is presented in Figure S3 in the Supporting Information.) The IgG-coated Si wafer and glass window were placed between an aluminum plate (bottom side) and a brass plate (top side) to give a microvessel (30 μm (height) × 12 mm (length) × 3 mm (width)) on the Si wafer at the center of the device. The volume inside our IgG-coated Si

nonfluorescent SP2/O cells observed in Figure S3a were removed from 4a after a slow washing (5 μL/min), and nearly all of the cells remaining were SP2/O-HEL-CFSE cells (Figure 4 and S4c,d), indicating that SP2/O-HEL-CFSE cells are selectively trapped on 4a. As far as we use a combination of anti-HEL-IgG and HEL, nonspecific binding has been scarcely observed. One of the main purposes of this device is to trap a small number of specific cells selectively in the presence of the large number of other cells. We examined the selective trapping of SP2/O-HEL cells from a 1:100 mixture (in number) of SP2/OHEL cells and healthy mouse blood cells (Figure 5). A 1:100 mixture (in number) of medium contained SP2/O-HEL cells (ca. 1 × 106 cells/mL), and mouse blood was introduced into the device (corresponding to Scheme S3b in the Supporting Information). Figure 5a is a microscope image of the Si surface 13121

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that the adhesion of SP2/O-HEL cells on 4a occurs through specific antigen−antibody complexation. The photorelease of SP2/O-HEL trapped on 4a and 4b was achieved, as described above (Scheme S3 and Figures 6b, 7, S5,

Figure 5. Bright-field images of a mixture of SP2/O-HEL cells and red blood cells on 4a (1:100 mixture (in number)) (a) before and (b) after being washed. Trapped SP2/O-HEL cells are indicated by the arrows in b.

after a 5 min incubation to permit cell adhesion to occur (corresponding to Scheme S3c) in which many red blood cells were observed. After the flow of PBS buffer at a rate of 5 μL/ min, almost all of the red blood cells were washed out and only SP2/O-HEL cells remained on 4a, as shown in Figure 5b (as indicated by arrows). We also tested that the reactivity of the anti-HEL-IgG-coated Si wafer remained >90%, even after it was kept in mouse blood containing 10 mM EDTA for 10 min, suggesting that this system is stable under physiological and experimental conditions. We measured the adhesion of SP2/O-HEL and SP2/O cells with modified Si wafers 3a, 4a, and 4b. After a 5 min incubation to permit cell adhesion to occur, PBS buffer was allowed to flow from port 1 to port 3 (Figure 3 and Scheme S3 in the Supporting Information) at an increasing flow rate, and the numbers of cells on the modified Si wafer were visually counted at each flow rate. As summarized in Figure 6a, a flow of 100− 150 μL/min was required to detach nearly all of the SP2/OHEL cells from 4a (closed circles). In control experiments in the absence of either IgG (SP2/O-HEL + 3a) (solid triangles) or HEL (SP2/O + 4a) (solid squares), almost all of the cells were washed out at flow rates of below 10 μL/min, indicating

Figure 7. Relative numbers of SP2/O-HEL on 4a with and without photoirradiation are shown as solid circles and squares, respectively.

and S6). After photoirradiation at 365 nm for 30 min (Scheme S3e in the Supporting Information), the captured SP2/O-HEL cells on 4a were detached when the flow rate of the PBS buffer was increased (Scheme S3f in the Supporting Information). As shown in Figure 6b (solid squares), almost all of the cells were released from the Si surface at a rate below 50 μL/min. Figure S5a−d in the Supporting Information shows photographs of SP2/O-HEL cells on 4a. After photoirradiation at 365 nm for 30 min, ca. 20% of the SP2/O-HEL cells were removed from 4a (Figure 7) and the remaining cells were detached by the flow of PBS. A comparison of these curves with and without photoirradiation suggests that the SP2/O-HEL cells can be detached at flow rate of 50 μL/min after photoreaction whereas the release of SP2/O-HEL cells occurs to a small extent at a 50 μL/min flow rate without photoreaction and 200 μL/min is required to detach almost all of the SP2/O-HEL cells. Moreover, the SP2/O-HEL cells trapped on 4a were negligibly detached in the PBS buffer containing anti-HEL-IgG (0.2−2 μg/mL) or HEL (0.2 μg/mL−2 mg/mL) (after treatment for 30 min). In this study, we tried to estimate the adhesion force between SP2/O-HEL cells and a Si wafer under practical conditions in which the cells are detached by fluid flow. The fluid flow was changed from 0 to 300 μL/min over 5 min, and the adhesion force was calculated from the fluid velocity when the cells were detached from the Si surface. It is assumed that the flow velocity in this flow system obeys 2D Poiseille flow (eq 2), and then the flow velocity at the center of cells assuming Poiseille flow was used in the Stokes’ formula (eq 1). The radius of the cells was estimated to be 10−15 μm from microscope images, and their shape was almost symmetric, as depicted in Figure 8. An analysis of the typical release curve for 4a-SP2/O-HEL in Figure 6b gave a cell-adhesion force of ca. 1.47 ± 0.26 nN (Figure 8). Considering that the reported attractive force for the interaction between a single molecule of IgG and an antigen is several hundred piconewtons,55−57 the adhesion of SP2/OHEL cells with anti-HEL-IgG on 4a likely occurs via multiple interactions. The cell-adhesion force after photoirradiation was estimated to be 0.49 ± 0.09 nN (Figure 8).59 This value is not very small, possibly because the photochemical cleavage of the linker does not proceed completely and hence there must be some unreacted chemical linkers that still connect SP2/O-HEL cells with the Si surface.

Figure 6. Relative numbers of cells observed on the Si wafer with increasing PBS buffer flow rate. (a) The results of SP2/O-HEL + 4a, SP2/O-HEL + 3a, and SP2/O + 4a are shown with solid circles, triangles, and squares, respectively. (b) Relative numbers of SP2/OHEL on 4a before and after photoirradiation are shown as solid circles and squares, respectively. 13122

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photoirradiation and that the malignancy of SP2/O cells, which are a cancer cell line, is not as advanced upon photoirradiation.

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CONCLUSIONS In this article, we report the synthesis of a photocleavable linker containing an ANP moiety and the preparation of a photoreactive IgG-coated Si wafer for the capture and release of specific types of cells. The modification of the Si wafer surface by the ANP linker and then IgG was confirmed by FTIR-ATR analyses and FS-AFM observations. Comparisons of AFM images of 4a before and after photoirradiation indicate that the photocleavage of the ANP linker promotes the effective removal of IgG. In addition, the results of ELISA using an enzymatic reaction of AP suggest that the complex of IgG fixed on the Si wafer with second IgG-AP can be released from the Si surface with minimum damage by photoirradiation. These results imply that a photoreactive IgG-coated Si wafer such as 4a can be used for the isolation and identification of intact protein−ligand and protein−protein complexes. Moreover, it was possible to capture SP2/O-HEL cells on 4a selectively from a mixture of SP2/O-HEL and SP2/O cells and a mixture of SP2/O-HEL and blood cells. The measurement of the celladhesion force between 4a and SP2/O-HEL cells indicates that the capture of SP2/O-HEL with 4a is based on multiple antigen−antibody complexation. Finally, the photocleavage of the ANP linker facilitated the release of the trapped cells from the Si surface, and it was possible to reculture the cells. This methodology might be useful for the separation of specific cells that have similar sizes or morphological properties but express different antigens or receptors on their membranes. This information will contribute to further progress in the biological and medicinal sciences as well as in related scientific areas.

Figure 8. Estimation of the cell-adhesion force (F (nN)) of modified Si wafers on the assumption that the flow in the device causes an approximate 2D Poiseille flow.

F = 6πrμu(r )

(1)

⎛ r ⎞r u(r ) = 6u ̅ ⎜1 − ⎟ ⎝ b⎠b

(2)

Culturing the SP2/O-HEL Cells Recovered from the Silicon Device by Photochemical Cleavage. The viabilities of SP2/O-HEL cells recovered by flow only (0−200 μL/min) and photoirradiation and flow (0−50 μL/min) were determined to be >80% by tripan blue staining (data not shown). Moreover, the collected SP2/O-HEL cells recovered by photoirradiation and flow (50 μL/min) were incubated in a medium and 5% CO2 at 37 °C for 5 days, and the number of cells was found to be increased by a factor of ca. 5, as displayed in Figure 9. This growth rate was almost equivalent to that of

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EXPERIMENTAL SECTION

General Information. Reagents and solvents were purchased at the highest commercial quality and used without further purification. Goat polyclonal secondary antibody to mouse IgG-HL conjugated with AP (2nd IgG-AP) was purchased from Abcam. All aqueous solutions were prepared using deionized and distilled water. IR spectra were recorded on a Perkin-Elmer FT-IR spectrophotometer (Spectrum100) at room temperature. 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a JEOL Always 300 spectrometer. Tetramethylsilane was used as an internal reference for 1H and 13C NMR measurements in CDCl3. MS measurements were performed on a JEOL JMS-SX-102A and Agilent (Varian) 910MS. Thin-layer (TLC) and silica gel column chromatography were performed using a Merck Silica gel 60 F254 TLC plate and Fuji Silysia Chemical FL-100D, respectively. FTIR-ATR Analysis of Modified Porous Si 4b. Porous Si wafers at modification steps 2b, 3b, 10b, 11b, and 4b were dried in a stream of Ar gas before the IR measurements. FTIR-ATR spectra of the porous Si wafers in the range of 4000−380 cm−1 were obtained using a Perkin-Elmer FT-IR spectrophotometer (Spectrum100) with an ATR accessory unit with a diamond crystal. Each IR spectrum represents an average of 20 scans at 4 cm−1. FS-AFM Observation of Anti-HEL-IgG and Modified Si Wafers. Stock solutions of anti-HEL-IgG were deposited and adsorbed on a freshly cleaved mica surface, and the mica surface was then washed with distilled water. Modified Si wafers 3a and 4a (7.5 mm × 7.5 mm) were very carefully cut in PBS buffer to a small size (about 2 mm × 2 mm). These samples were set on an FS-AFM (Research Institute of Biomolecule Metrology, Co. Ltd., Nano Live Vision). Si cantilevers (Olympus, BL-AC-10EGS-A2) were used for imaging in distilled water. A scan of each sample was taken at a 500 × 375 nm2 size at a scan rate of 0.2 frames/s. The photorelease of IgG

Figure 9. Results of culturing SP2/O-HEL cells that were recovered via flow (50 μL/min) after photoirradiation and via flow (200 μL/ min) alone (without photoirradiation) in an RPMI 1640 medium.

SP2/O-HEL cells recovered by only a flow rate of 200 μL/min, as indicated by the solid squares in Figure 9. Note that the numbers of SP2/O-HEL cells at the starting point in Figure 9 are almost identical because the same number of cells (1.0 × 103 cells) was used in the two procedures for the cell detachment experiment (photochemical and nonphotochemical release (by a 200 μL/min)) in Figure 7 and cells were recovered almost completely in both experiments. These results imply that the photoirradiation of 4a permits SP2/O-HEL cells to be detached from 4a with minimum damage upon 13123

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from 4a was carried out upon photoirradiation at 330 nm for 1 h by a high-pressure mercury lamp (USHIO, USH-500SC (500 W)). For direct observation of the photorelease of IgG from 4a, photoirradiation was carried out using a high-pressure mercury lamp (USHIO, USH103OL (100 W)) equipped with FS-AFM. ELISA of 4a with Photoirradiation. ELISA of 4a with photoirradiation was carried out as shown in Scheme 3. First, 4a in PBS was washed and replaced with TBST, and a second IgG-AP (1:1000 dilution) in TBST solution was reacted with 4a to form a complex between anti-HEL-IgG on the Si surface and second IgG-AP. After unreacted second IgG-AP was removed by washing with TBST, the Si wafer was placed in 100 μM MNP/TBST (3 mL) in another glass bottle, and this bottle was incubated at 37 °C for 30 min (A in Scheme 3). The absorbance of this reaction solution was measured by using a UV−vis spectrophotometer (Jasco, V-630BIO), and the AP activity was calculated by the 405 nm absorbance of 4-nitrophenol, which is the MNP hydrolysis product. Next, the Si wafer having an IgG-AP complex in 3 mL of TBST was irradiated at 365 nm with a high-pressure mercury lamp (USHIO, USH-500SC (500 W)) equipped with an optical filter (Asahi Spectra, Co. Ltd., HQBP365UV). After photoirradiation for 15 min, the wafer was added to 100 μM MNP/TBST (3 mL) in another glass bottle and MNP was added to the residual TBST supernatant containing the photoreleased IgGAP complex MNP. Both samples were incubated at 37 °C for 30 min (B and C in Scheme 3), and the AP activities were measured. As for the light source, other light sources such as the Xe lamp of the spectrofluorometer were also found to work for the cleavage of the ANP linker. Preparation of Stable HEL Transfectant Cell Lines (SP2/OHEL). Parent SP2/O cells were a generous gift from the Takachika Azuma laboratory (Tokyo University of Science). Stable cell lines expressing the hCD20-HEL fusion protein were generated by electroporation with an expression vector. One day after transfection, the cells were seeded on 24-well plates and selected in culture medium (IMDM supplemented with 10% FCS) containing 0.4 mg/mL Zeocine. To test the expression of the hCD20-HEL fusion protein on the cell surface, selected clones were prepared in FACS medium (PBS containing 0.5% Calf Serum and 0.1% sodium azide) and incubated with unlabeled anti-FcR (2.4G2) to block nonspecific binding and then stained with biotin-conjugated anti-HEL-IgG. After being washing with FACS medium, the cells were stained with PE conjugated streptavidin and analyzed with a FACS Sort with CellQuest software (BD Biosciences, San Jose, CA) for three-color flow cytometric analysis. For analysis by fluorescence microscopy, SP2/O-HEL was labeled with CFSE prior to each experiment. Typically, SP2/O-HEL was prepared in PBS (2 × 106 cells/mL) and incubated with 1.7 μM CFSE in PBS at 37 °C for 3 min. After FCS was added to stop the reaction, the cells were washed twice with PBS and were then used in the following experiments. Cell-Adhesion Experiment. Cell-capture and recovery by the photoirradiation of 4a and 4b were conducted in a microfluidic device (Figure 3) to count the number of cells on the Si surface (Scheme S3). PBS buffer was first injected into this device in order to remove bubbles, and a solution containing SP2/O-HEL (about 106 cells/mL) was then injected. After a period of 5 min to permit cell adhesion, PBS buffer was allowed to flow at increasing flow rates using a syringe pump (Harvard Apparatus, Harvard Pump 11 Plus). The number of cells on the Si surface were visually counted using microscopy (Olympus, BX51). The photorelease of cells from the Si wafer was achieved by photoirradiation at 365 nm for 30 min using a highpressure mercury lamp (USHIO, USH-500SC (500 W)) with an optical filter (Asahi Spectra, Co. Ltd., HQBP365-UV). The collected SP2O-HEL cells recovered by photoirradiation and flow (50 μL/min) were incubated in Roswell Park Memorial Institute (RPMI) 1640 medium in a 96-well plate under 5% CO2 at 37 °C.

Article

ASSOCIATED CONTENT

S Supporting Information *

Synthesis of 1 and preparation of a Si wafer with 1. Experimental procedure of the selective cell-adhesion experiment. FTIR-ATR spectra of IgG-modified porous Si 4b. QCM analysis of complexation between anti-HEL-IgG and HEL. Picture showing the whole system of the Si device. Microscopy images for the selective capture of CFSE-stained SP2/O-HEL cells and the trapping of SP2/O-HEL. Results of cell-adhesion experiment SP2/O-HEL on 4b. Movie showing the photorelease of IgG from 4a as observed by FS-AFM (scale = 500 nm × 375 nm). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ms. Ai Shibuya (Faculty of Pharmaceutical Sciences, Tokyo University of Science) for the 27 MHz QCM experiments to determine the Kd value of HEL and antiHEL-IgG and Dr. Tetsuya Nakatsura (National Cancer Center Hospital East) for helpful discussion. This study was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (no. 23790023 for S. Ariyasu and nos. 18390009, 19659026, 22390005, 22659005, and 24659058 for S. Aoki) and the Academic Frontier project for private universities, including a matching fund subsidy from MEXT, 2009−2013.

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REFERENCES

(1) Steeg, P. S. Tumor Metastasis: Mechanistic Insights and Clinical Challenges. Nat. Med. 2006, 12, 895−904. (2) Gupta, G. P.; Massague, J. Cancer Metastasis: Building a Framework. Cell 2006, 127, 679−695. (3) Cho, S. H.; Chen, C. H.; Tsai, F. S.; Godin, J. M.; Lo, Y.-H. Human Mammalian Cell Sorting Using a Highly Integrated MicroFabricated Fluorescence-Activated Cell Sorter (μFACS). Lab Chip 2010, 10, 1567−1573. (4) Krivacic, R. T.; Ladanyi, A.; Curry, D. N.; Hsieh, H. B.; Kuhn, P.; Bergsrud, D. E.; KepJ., F.; Barbera, T.; Ho, M. Y.; Chen, L. B.; Lerner, R. A.; Bruce, R. H. A Rare-Cell Detector for Cancer. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10501−10504. (5) Miltenyi, S.; Müller, W.; Weichel, W.; Radbruch, A. High Gradient Magnetic Cell Separation with MACS. Cytometry 1990, 11, 231−238. (6) Kuhara, M.; Takeyama, H.; Tanaka, T.; Matsunaga, T. Magnetic Cell Separation Using Antibody Binding with Protein A Expressed on Bacterial Magnetic Particles. Anal. Chem. 2004, 76, 6207−6213. (7) Zabaglo, L.; Ormerod, M., G.; Parton, M.; Ring, A.; Smith, I., E.; Dowsett, M. Cell Filtration-Laser Scanning Cytometry for the Characterisation of Circulating Breast Cancer Cells. Cytometry A 2003, 55A, 102−108. (8) Kahn, H. J.; Presta, A.; Yang, L.-Y.; Blondal, J.; Trudeau, M.; Lickley, L.; Holloway, C.; McCready, D. R.; Maclean, D.; Marks, A. Enumeration of Circulating Tumor Cells in the Blood of Breast Cancer Patients after Filtration Enrichment: Correlation with Disease Stage. Breast Can. Res. Treat. 2004, 86, 237−247. (9) Robertus, J.; Browne, W. R.; Feringa, B. L. Dynamic Control over Cell Adhesive Properties Using Molecular-Based Surface Engineering Strategies. Chem. Soc. Rev. 2010, 39, 354−378.

13124

dx.doi.org/10.1021/la302393p | Langmuir 2012, 28, 13118−13126

Langmuir

Article

(29) Reighdorf, S.; Muller, V.; Zhang, L.; Rau, T.; Loibl, S.; Komor, M.; Roller, M.; Huober, J.; Ferhm, T.; Schrader, I.; Hifrich, J.; Holms, F.; Tesch, H.; Eidtman, H.; Untch, M.; von Minckwitz, G.; Pantel, K. Detection and HER2 Expression of Circulating Tumor Cells: Prospective Monitoring in Breast Cancer Patients Treated in the Neoadjuvant GeparQuattro Trial. Clin. Cancer Res. 2010, 16, 2634− 2645. (30) Nagrath, S.; Sequeist, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. J.; Digumarthy, S.; Muzikansky, A.; Ryan, P.; Balis, U. J.; Tompkins, R. G.; Haber, D. A.; Toner, M. Isolation of Rare Circulating Tumor Cells in Cancer Patients by Microchip Technology. Nature 2007, 450, 1235−1239. (31) Wang, S.; Wang, H.; Jiao, J.; Chen, K.-J.; Owens, G. E.; Kamei, K.; Sun, J.; Sherman, D. J.; Behrenbruch, C. P.; Wu, H.; Tseng, H.-R. Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells. Angew. Chem., Int. Ed. 2009, 48, 8970−8973. (32) Fukuda, S.; Schmid-Schönbein, G. W. Centrifugation Attenuates the Fluid Shear Response of Circulating Leukocytes. J. Leuk. Biol. 2002, 72, 133−139. (33) Finn, F. M.; Titus, G.; Montibeller, J. A.; Hofmann, K. Hormone-Receptor Studies with Avidin and Biotinylinsulin-Avidin Complexes. J. Biol. Chem. 1980, 255, 5742−5746. (34) Speers, A. E.; Cravatt, B. F. Chemical Strategies for ActivityBased Proteomics. ChemBioChem 2004, 5, 41−47. (35) Jessani, N; Cravatt, B. F. The Development and Application of Methods for Activity-Based Protein Profiling. Curr. Opin. Chem. Biol. 2004, 8, 54−59. (36) Sato, S.; Murata, A.; Shirakawa, T.; Uesugi, M. Biochemical Target Isolation for Novices: Affinity-Based Strategies. Chem. Biol. 2010, 17, 617−623. (37) Sato, S.; Kwon, Y.; Kamisuki, S.; Srivastava, N.; Mao, Q.; Kawazoe, Y.; Uesugi, M. Polyproline-Rod Approach to Isolating Protein Targets of Bioactive Small Molecules: Isolation of a New Target of Indomethacin. J. Am. Chem. Soc. 2007, 129, 873−880. (38) Breitinger, H.-G. A.; Wieboldt, R.; Ramesh, D.; Carpenter, B. K.; Hess, G. P. Synthesis and Characterization of Photolabile Derivatives of Serotonin for Chemical Kinetic Investigations of the Serotonin 5HT3 Receptor. Biochemistry 2000, 39, 5500−5508. (39) Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Coumarin-Caged Glycine That Can Be Photolyzed within 3 μs by Visible Light. Biochemistry 2007, 46, 5479−5484. (40) Nomura, W.; Narumi, T.; Ohashi, N.; Serizawa, Y.; Lewin, N. E.; Blumberg, P. M.; Furuta, T.; Tamamura, H. Synthetic Caged DAGLactones for Photochemically Controlled Activation of Protein Kinase C. ChemBioChem 2011, 12, 535−539 and referenced cited therein. (41) Bochet, C. G. Photolabile Protecting Groups and Linkers. J. Chem. Soc., Perkin Trans. 1 2002, 125−142. (42) Thiele, C.; Fahrenholz, F. Photocleavable Biotinylated Ligands for Affinity Chromatography. Anal. Biochem. 1994, 218, 330−337. (43) Olejnik, J.; Sonar, A.; Krzymañska-Olenjnik, E.; Rothschild, K. J. Photocleavable Biotin Derivatives: A Versatile Approach for the Isolation of Biomolecules. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7590− 7594. (44) Aoki, S.; Matsuo, N.; Hanaya, K.; Yamada, Y.; Kageyama, Y. Design and Synthesis of a Photocleavable Biotin-Linker for the Photoisolation of Ligand-Receptor Complexes Based on the Photolysis of 8-Quinolinyl Sulfonates in Aqueous Solution. Bioorg. Med. Chem. 2009, 17, 3405−3413. (45) Hanaya, K.; Kageyama, Y.; Kitamura, M.; Aoki, S. Design and Synthesis of Photocleavable Biotinylated-Dopamine with Polyethyleneoxy Photocleavable Linkers. Heterocycles 2011, 82, 1601−1615. (46) Kageyama, Y.; Ohshima, R.; Sakurama, K.; Fujiwara, Y.; Tanimoto, Y.; Yamada, Y.; Aoki, S. Photochemical Cleavage Reactions of 8-Quinolinyl Sulfonates in Aqueous Solution. Chem. Pharm. Bull. 2009, 57, 1257−1266. (47) Ariyasu, S.; Hanaya, K.; Tsunoda, M.; Kitamura, M.; Hayase, M.; Abe, R.; Aoki, S. Photochemical Cleavage Reaction of 8-Quinolinyl

(10) Didar, T. F.; Tabrizian, M. Adhesion Based Detection, Sorting and Enrichment of Cells in Microfluidic Lab-on-Chip Devices. Lab Chip 2010, 10, 3043−3053. (11) Pamme, N. Continuous Flow Separations in Microfluidic Devices. Lab Chip 2007, 7, 1644−1659. (12) Kim, S. M.; Lee, S. H.; Suh, K. Y. Cell Research with Physically Modified Microfluidic Channels: A Review. Lab Chip 2008, 8, 1015− 1023. (13) Kim, W.; Ng, J. K.; Kunitake, M. E.; Conklin, B. R.; Yang, P. Interfacing Silicon Nanowires with Mammalian Cells. J. Am. Chem. Soc. 2007, 129, 7228−7229. (14) Herzer, N.; Hoeppener, S.; Schubert, U. S. Fabrication of Patterned Silane Based Self-Assembled Monolayers by Photolithography and Surface reactions on Silicon-Oxide Substrates. Chem. Commun. 2010, 46, 5634−5652. (15) Haensch, C.; Hoeppener, S.; Schubert, U. S. Chemical Modification of Self-Assembled Silane Based Monolayers by Surface Reactions. Chem. Soc. Rev. 2010, 39, 2323−2334. (16) Samanta, D.; Sarkar, A. Immobilization of Bio-Macromolecules on Self-Assembled Monolayers: Methods and Sensor Applications. Chem. Soc. Rev. 2011, 40, 2567−2592. (17) Kim, J.; Cho, J.; Seidler, P. M.; Kurland, N. E.; Yadavalli, V. K. Investigation of Chemical Modifications of Amino-Terminated Organic Films on Silicon Substrates and Controlled Protein Immobilization. Langmuir 2010, 26, 2599−2608. (18) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. Photoactivation of a Substrate for Cell Adhesion under Standard Fluorescence Microscopes. J. Am. Chem. Soc. 2004, 126, 16314−16315. (19) Sivagnanam, V.; Song, B.; Vandevyver, C.; Bünzli, J.-C. G.; Gijis, M. A. M. Selective Breast Cancer Cell Capture, Culture, and Immunocytochemical Analysis Using Self-Assembled Magnetic Bead Patterns in a Microfluidic Chip. Langmuir 2010, 26, 6091−6096. (20) Gong, Y.-H.; Li, C.; Yang, J.; Wang, H.-Y.; Zhuo, R.-X.; Zhang, X.-Z. Photoresponsive “Smart Template” via Host-Guest Interaction for Reversible Cell Adhesion. Macromolecules 2011, 44, 7499−7502. (21) Hosokawa, M.; Hayata, T.; Fukuda, Y.; Arakaki, A.; Yoshino, T.; Tanaka, T.; Matsunaga, T. Size-Selective Microcavity Array for Rapid and Efficient Detection of Circulating Tumor Cells. Anal. Chem. 2010, 82, 6629−6635. (22) Matsunaga, T.; Hosokawa, M.; Arakaki, A.; Taguchi, T.; Mori, T.; Tanaka, T.; Takeyama, H. High-Efficiency Single-Cell Entrapment and Fluorescence in Situ Hybridization Analysis Using a Poly(dimethylsiloxane) Microfluidic Device Integrated with a Black Poly(ethyleneterephthalate) Micromesh. Anal. Chem. 2008, 80, 5139−5145. (23) Bhagat, A. A. S.; Hou, H. W.; Li, L. D.; Lim, C. T.; Han, J. Pinched Flow Coupled Shear-Modulated Inertial Microfluidics for High-Throughput Rare Blood Cell Separation. Lab Chip 2011, 11, 1870−1878. (24) Lee, W. C.; Bhagat, A. A. S.; Hung, S.; Vilet, K. J. V.; Han, J.; Lim, C. T. High-Throughput Cell Cycle Synchronization Using Inertial Forces in Spiral Microchannels. Lab Chip 2011, 11, 1359− 1367. (25) Tan, S. J.; Lakshmi, R. L.; Chen, P.; Lim, W.; Yobas, L.; Lim, C. T. Versatile Label Free Biochip for the Detection of Circulating Tumor Cells from Peripheral Blood in Cancer Patients. Biosens. Bioelectron. 2010, 26, 1701−1705. (26) Tan, S. J.; Yobas, L.; Lee, G. Y. H.; Ong, C. N.; Lim, C. T. Microdevice for the Isolation and Enumeration of Cancer Cells from Blood. Biomed. Microdev. 2009, 11, 883−892. (27) Cristofanilli, M.; Thomas, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Tersptappen, L. W. M. M.; Hayes, D. F. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. New Engl. J. Med. 2004, 351, 781−791. (28) Kim, M.-Y.; Oskarsson, T.; Acharyya, S.; Nguyen, D. X.; Zhang, X. H.-F.; Norton, L.; Massague, J. Tumor Self-Seeding by Circulating Cancer Cells. Cell 2009, 139, 1315−1326. 13125

dx.doi.org/10.1021/la302393p | Langmuir 2012, 28, 13118−13126

Langmuir

Article

Sulfonates That Are Halogenated and Nitrated at the 7-Position. Chem. Pharm. Bull. 2011, 59, 1355−1362. (48) Yu, H.; Li, J.; Wu, D.; Qiu, Z.; Zhang, Y. Chemistry and Biological Applications of Photo-Labile Organic Molecules. Chem. Soc. Rev. 2010, 39, 464−473. (49) Celie, P. H. N.; Toebes, M.; Rodenko, B.; Ovaa, H.; Perrakis, A.; Schumacher, T. N. M. UV-Induced Ligand Exchange in MHS Class I Protein Crystals. J. Am. Chem. Soc. 2009, 131, 12298−12304. (50) For a review and foresight of photochemical devices and their applications, see Garcia-Garibay, M. A. Advances at the Frontiers of Photochemical Sciences. J. Am. Chem. Soc. 2012, 134, 8289−8292 and references cited therein. (51) Silverton, E. W.; Navia, M. A.; Davies, D. R. Three-Dimensional Structure of an Intact Human Immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140−5144. (52) Chen, S.; Liu, L.; Zhou, J.; Jiang, S. Controlling Antibody Orientation on Charged Self-Assembled Monolayers. Langmuir 2003, 19, 2859−2864. (53) Zulkefeli, M.; Suzuki, A; Shiro, M.; Hisamatsu, Y.; Kimura, E.; Aoki, S. Selective Hydrolysis of Phosphate Monoester by a Supramolecular Phosphatase Fromed by the Self-Assembly of a Bis(Zn2+-cyclen) Complex, Cyanuric Acid, and Copper in an Aqueous Solution (Cyclen = 1,4,7,10-Tetraazacyclododecane). Inorg. Chem. 2011, 50, 10113−10123 and references cited therein. (54) The association constant Ka between anti-HEL-IgG and HEL was estimated to be 1.35 × 108 [M−1] (dissociation constant Kd = 7.4 nM) by a 27 MHz quartz crystal microbalance (27 MHz QCM). Typical QCM titration curves are shown in Figure S2 in the Supporting Information. (55) Helenius, J.; Heisenberg, C.-P.; Gaub, H. E.; Muller, D. J. SingleCell Force Spectroscopy. J. Cell Sci. 2008, 121, 1785−1791. (56) Chen, Y.; Zeng, G.; Chen, S. S.; Feng, Q.; Chen, Z. W. AFM Force Measurements of the gp120-sCD4 and gp120 or CD4 AntigenAntibody Interactions. Biochem. Biophys. Res. Commun. 2011, 407, 301−306. (57) Quite recently, the adhesion force of human mesenchymal stem cells (MSCs) on TiO2 flat cells was reported to be 1000−2500 pN. See Bertoncini, P.; Le Chevalier, S.; Lavenus, S.; Layrolle, P.; Louarn, G. Early Adhesion of Human Mesenchymal Stem Cells on TiO2 Surfaces Studied by Single-Cell Force Spectroscopy Measurements. J. Mol. Recognit. 2012, 25, 262−269. (58) SP2/O-HEL cells on modified porous Si 4b after photoirradiation were also detached (open squares in Figure S5 in the Supporting Information) more easily than without photoirradiation (open circles in Figure S5 in the Supporting Information). Although it was reported that the microstructure of a Si surface can effectively trap cells (ref 31), the adhesion force of 4b was smaller than that of 4a, possibly because of the smaller surface contact area between the porous Si surface 4b and SP2/O-HEL cells than that between 4a and SP2/O-HEL cells.

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