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Antibody-Modified Reduced Graphene Oxide Films with Extreme Sensitivity to Circulating Tumor Cells Yingying Li, Qihang Lu, Hongliang Liu, Jianfeng Wang, Pengchao Zhang, Huageng Liang, Lei Jiang, and Shutao Wang* Most cell–material interactions are mediated by bioactive interfaces, therefore it is important to understand the materials that comprise the interface and how they perform their functions. Many biointerfaces based on single chemical or physical factors have been successfully developed to direct cell behaviors, e.g., R: arginine; G: glycine; D: aspartic acid (RGD)confined cell adhesion,[1] geometry-controlled cell fate,[2] and topography-guided stem cell differentiation.[3] Nonetheless, the functions performed by these artificial biointerfaces are far simpler than those performed in the natural cell microenvironment, the complexity of which is multifactorial, and includes surface topography and chemistry, matrix stiffness and mechanical stress, molecular liquid composition, and other physiochemical parameters. This complexity presents a major challenge for designing biointerfaces that can complement several biophysical cell properties to achieve cell-specific recognition. For example, recent studies have attempted to improve the efficiency of circulating tumor cell (CTC) capture, several nanomaterial-based biointerfaces, such as silicon nanopillars[4] and polystyrene nanotubes,[5] have been developed to partially mimic the topographic features of cancer cells or their microenvironment. However, the sensitivity and selectivity of CTCspecific recognition in these nanomaterials is limited because they fail to complement multiple, complex cell properties. Y. Y. Li, Q. H. Lu, Dr. P. C. Zhang, Prof. L. Jiang, Prof. S. T. Wang Beijing National Laboratory for Molecular Sciences (BNLMS) Key Laboratory of Organic Solids Institute of Chemistry Chinese Academy of Sciences Beijing 100190, P. R. China E-mail: [email protected] Y. Y. Li, P. C. Zhang University of Chinese Academy of Sciences Beijing 100049, P. R. China Dr. H. L. Liu, Prof. S. T. Wang Laboratory of Bio-Inspired Smart Interface Science Technical Institute of Physics and Chemistry Chinese Academy of Sciences Beijing 100190, P. R. China Dr. J. F. Wang, Prof. L. Jiang School of Chemistry and Environment Beihang University Beijing 100191, P. R. China Dr. H. G. Liang Department of Urology Union Hospital of Tongji Medical College Huazhong University of Science and Technology Wuhan 430022, Hubei Province, P. R. China

DOI: 10.1002/adma.201502615

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Graphene based materials are considered potential auxiliary materials in biointerfaces for performing particular functions, e.g., photothermal for cancer therapy,[6] loading water insoluble drugs for drug delivery,[7] and providing functional groups for the introduction of antibodies.[8] However, the use of graphene based materials as a primary matrix nanomaterial has not been explored, nor is it known whether these materials can mimic several cellular microenvironment properties for the efficient capture of CTCs from whole blood. In this study, we describe the extreme sensitivity of antibodymodified reduced graphene oxide (rGO) films to CTCs that result from their ability to complement several physiochemical cell properties. The rough texture of rGO might enhance topographic interactions between CTCs and biointerfaces. Moreover, the low stiffness nature of rGO films might facilitate cell–matrix interactions, thereby contributing to highly efficient CTC capture. Additionally, the negative charge and superhydrophilicity of modified rGO films render the surface inert to nonspecific cell adhesion, which is helpful for decreasing the white blood cells (WBCs) background. We have clearly demonstrated that the antibody-modified rGO films prepared in this study can efficiently capture CTCs from fresh whole blood (which contains less than 10 CTCs mL−1) with high specificity and low background, without the need for complex microfluidic operations. The rGO films were prepared via a two-step process involving vacuum filtration and thermal reduction.[9,10] A representative image of a prepared sample is shown in Figure 1a. The rGO films exhibited a petal-like wrinkled architecture, and a more detailed image was demonstrated in Figure 1b,c. To achieve extremely sensitive CTCs recognition, the rGO films were designed and functionalized as follows (Figure 1d). First, pyrene carboxylic acid (PCA) was immobilized onto rGO film by π–π stacking,[7] thus introducing more carboxyl groups, compared to original rGO film, for conjugating streptavidin (SA). The immobilization of PCA onto rGO films was confirmed using surface enhanced Raman scattering (SERS)[11] (Figure S1, Supporting Information). Next, SA was conjugated onto the PCA-rGO films via an immobilization procedure assisted by N-hydroxysulfosuccinimide (Sulfo-NHS) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC).[12] Finally, biotinylated epithelial cell adhesion molecule antibody (anti-EpCAM), an agent that captures specific types of cancer cells, was immobilized onto the SA-rGO films. The resulting anti-EpCAM-rGO films can successfully act as sensitive biointerfaces to recognize specific CTCs. A series of rGO films was fabricated from GO solutions with various weight concentrations–rGO1 [0.5 mg mL−1], rGO2 [1.5 mg mL−1], rGO3 [2.5 mg mL−1], and rGO4 [3.5 mg mL−1] followed by thermal reduction. Figure S2 Supporting Information) shows

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COMMUNICATION Figure 1. Functionalization and characterization of rGO films. a) A photograph of the rGO4 film, which was used to capture CTCs directly from clinical blood samples. b) An ESEM image of the rGO4 film clearly shows the existence of a petal-like wrinkled architecture. c) An ESEM image of the rGO4 film at high magnification. d) Functionalization of the rGO film. PCA was immobilized onto the rGO film by π–π stacking. Next, SA was introduced after EDC/Sulfo-NHS activation. Finally, biotinylated-anti-EpCAM was added, creating the anti-EpCAM-rGO films that were used for cell capture. e–h) ESEM images of rGO1–rGO4 films. As the concentration of the GO solution used in preparing the film increases, the density of the bulging sheets coating the rGO surface also increases, thereby enhancing roughness.

that the deposited mass of GO films increased as concentrations of the GO solutions was increased. After thermal reduction of the GO films to rGO, the weight loss occurred due to the release of gaseous H2O, CO2, and CO.[13] Raman spectra confirmed the formation of rGO films, typical D, G, and 2D bands (Figure S1, Supporting Information) were observed at 1340, 1580, and 2650 cm−1, respectively, consistent with other graphene spectra.[14,15] Environmental scanning electron microscopy (ESEM) images of the rGO1–rGO4 films are shown in Figure 1e–h. These ESEM images clearly show the formation of a petal-like, wrinkled architecture, which was exfoliated during acid treatment.[15] As the concentration of the GO solutions increased, the density of the bulging sheets that coated the rGO film surface increased, thereby enhancing the roughness and three dimensional (3D) morphology of the surface (Figures S3 and S4, Supporting Information). Our previous studies[16,17] demonstrated that silicon nanowire arrays with morphologies that complement cell filopodia can efficiently enhance CTC capture yields. Here, structural control of the rGO films provided greater opportunity for optimizing CTCs capture. To test the performance of antibody-modified rGO films as sensitive biointerfaces for CTC recognition, we chose the EpCAM-positive human breast cancer (MCF-7) and human prostate cancer (PC3) cell lines, and the EpCAM-negative human T (Jurkat), Burkitt’s lymphoma (Daudi), and cervical cancer (HeLa) cell lines. The capture yields of MCF-7 and PC-3 cells

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on the anti-EpCAM-rGO4 film greatly increased after a 45 min incubation, reaching 92% ± 4% and 83% ± 6%, respectively (Figure 2a). In contrast, the capture yields of EpCAM-negative cell lines Jurkat, Daudi, and HeLa were less than 5%. The rGO4 film prepared in this study exhibited extremely high sensitivity and selectivity as a biointerface for the recognition of targeted cell lines. Next, four anti-EpCAM-rGO films with different morphologies and structures (anti-EpCAM-rGO1 through anti-EpCAM-rGO4) were tested under the optimal condition (45 min incubation), and a smooth anti-EpCAM highly ordered pyrolytic graphite (HOPG) film was chosen as a control. On the smooth anti-EpCAM-HOPG film, the capture yields of MCF-7 and PC-3 cells were only 22% ± 5% and 23% ± 1%, respectively. However, the efficiency of capturing these two cell lines was much higher on anti-EpCAM-rGO4 (Figure 2b). ESEM images of captured cells on the anti-EpCAM-rGO and antiEpCAM-HOPG films are shown in Figure 2c–h. We observed that morphology and 3D structures did affect the capture efficiency; the anti-EpCAM-HOPG film exhibited the lowest capture yield, whereas the anti-EpCAM-rGO4 film exhibited the highest capture yield. As the concentration of the GO solution used in film preparation increased, the rGO flakes were increasingly folded, resulting in a gradient in the morphology and roughness. An analysis of film roughness confirmed the rough surfaces of the rGO films (Figure S3, Supporting Information). On the smooth substrate of the control film, the tumor cells possessed few, short filopodia; as the roughness of the rGO



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Figure 2. Quantitative evaluations of cell-capture yields. a) The cell-capture yield of the anti-EpCAM-rGO4 film at various incubation times. Anti-EpCAMpositive cells (MCF-7, PC3) were captured with yields of greater than 90% after incubation for 45 min, whereas the capture yields for anti-EpCAMnegative cell lines (Jurkat, Daudi, and HeLa) were less than 5%. b) Cell-capture yields of various anti-EpCAM films. Anti-EpCAM-HOPG exhibited the lowest capture yield whereas anti-EpCAM-rGO4 exhibited the highest capture yield. c) An ESEM image of an anti-EpCAM-HOPG film after cell capture. d) An ESEM image of an anti-EpCAM-rGO2 film after cell capture. e) An ESEM image of an anti-EpCAM-rGO4 film after cell capture. f) An ESEM image of an MCF-7 cancer cell captured on an anti-EpCAM-HOPG film. g) An ESEM image of an MCF-7 cancer cell captured on an anti-EpCAM-rGO2 film. Films with rougher surfaces exhibited greater cell spreading and filopodia that were more elongated. h) An ESEM image of an MCF-7 cell captured on an anti-EpCAM-rGO4 film. This cancer cell exhibited extremely long protrusions along the wrinkles. It can be inferred that cancer cells are more likely to grasp the rough anti-EpCAM-rGO4 surface rather than smoother substrates due to the synergistic topographic interactions that occur between the nanostructure-induced matching effect and the microstructure-induced 3D trap effect.

films increased, the filopodia tended toward greater elongation. On the roughest rGO4 film, the substrate provided not only nanoscale interactions but also a 3D trap effect.[18] This synergy between the nanostructure-induced matching effect and the microstructure-induced 3D trap effect, complemented the sizes of the cell filopodia and whole cells, and this might have been crucial for the ultrahigh CTC capture yields that were achieved. The wettability of the rGO films was determined because it affects cell-capture yields (Figure 3 and Figure S4, Supporting Information). Wettability was determined by monitoring the dynamic change in contact angle (CA). As the concentration of the GO solution used in film preparation increased, with a concomitant increase in the density and roughness of the rGO flakes, the water droplet spread faster (Figure 3b,c). The CA of anti-EpCAM-rGO4 decreased to 0° after 4 s; in contrast, anti-EpCAM-HOPG exhibited no change in CA over time. The superhydrophilicity of the anti-EpCAM-rGO4 film increased water retention, thus preventing cell–interface interactions and most likely, decreasing nonspecific cell adhesion.

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Material stiffness is important in cellular processes, such as cell adhesion,[19] stem cell proliferation and differentiation,[20] and tumor generation.[21] Cells are fragile and live in a low-stiffness tissue environment,[5] films with stiffness values that are similar to that of the cellular microenvironment might facilitate cell adhesion and increase cell capture. To investigate the effect of stiffness on cell capture, the stiffness of various samples was tested using a triboindenter. The Young’s modulus of flat silicon (Figure S5, Supporting Information, E = 187.9 ± 4.2 GPa) is 20-fold greater than that of flat HOPG (Figure 3d, E = 9.0 ± 0.4 GPa). In contrast, the CTC yield of antiEpCAM-flat silicon is ten times lower than that of anti-EpCAMHOPG (Figure S6, Supporting Information, and Figure 2b). Thus, for these similarly smooth surfaces, the material with a lower stiffness generated a higher CTC yield, revealing that low stiffness substrates are probably more beneficial than rigid substrates for CTC capture. These data are consistent with our previous study on polystyrene (PS) nanotubes,[5] in which PS nanotubes of lower stiffness generated an 80% CTC yield


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COMMUNICATION Figure 3. Stiffness and wettability of the anti-EpCAM-HOPG and rGO films. a) Contact angles (CAs) of the anti-EpCAM-HOPG and anti-EpCAM-rGO1 to rGO4 films. Anti-EpCAM-HOPG remained stable with a CA of 63.5°, whereas the rGO films were superhydrophilic with a CA of 0°. b) Dynamic CAs of anti-EpCAM-HOPG and anti-EpCAM-rGO1 to rGO4 films. As the concentration of the GO solution used in preparing the film increased, the water droplet behaved to spread faster. c) Photographs of the CAs of anti-EpCAM-HOPG and anti-EpCAM-rGO4. The CA of anti-EpCAM-rGO4 decreased to 0° at 4 s, in contrast, anti-EpCAM-HOPG exhibited no change in CA over time. d) Stiffness of the HOPG, rGO1–rGO4 films. In the current study, the Young’s moduli of all the prepared rGO were comparable. It can be deduced that the small increase in stiffness from rGO1 to rGO4 did not directly enhance cell yield. Therefore, the low stiffness of the rGO films is more likely to mimic natural microenvironments, thereby providing suitable conditions for cell capture.

efficiency (unfortunately also resulted in comparably high nonspecific cell adhesion). In the current study, the Young’s moduli of all prepared rGO films were comparable. It can be deduced that small increase in stiffness from rGO1 to rGO4 does not directly enhance cell-capture yield. Therefore, the low stiffness of the rGO films is more likely to mimic natural microenvironments, thus offering suitable conditions for cell capture. The ability of cells to apply cytoskeletal force to the extracellular matrix (ECM) through integrin receptors is essential for embryogenesis, migration, and metastasis.[22] To further investigate the interactions between cells and substrates, immunochemistry experiments were performed to observe the actin cytoskeleton, filopodia, lamellipodia, and focal adhesions of CTCs. Figure 4 shows cells that were costained for the nuclei (4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), blue), actin filaments (FITC-conjugated phalloidin, red), and vinculin (universal FA marker, green). Cellular vinculin distribution differed between cells that were captured on the anti-EpCAMrGO films and cells that were captured on anti-EpCAM-HOPG films. Cells that were captured on anti-EpCAM-HOPG exhibited small circular plaques of vinculin, mainly at the peripheries of the cells, whereas larger vinculin-containing focal contacts were localized at the peripheral and ventral regions of cells that were captured on the anti-EpCAM-rGO films. Because vinculin is involved in the adhesion strengthening response,[22] it can be inferred that substrate–cell focal adhesion is lower in the anti-EpCAM-HOPG film than in the anti-EpCAM-rGO4 film. Cell area was also wider on the anti-EpCAM-rGO films than

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on the anti-EpCAM-HOPG film. The synergistic topographic interactions including the nanostructure-induced cell filopodia matching effect and the microstructure-induced 3D trap effect, and the low stiffness nature of the rGO films likely contributed to higher FA of the anti-EpCAM-rGO films. Optimal cell capture conditions were applied in static cellcapture studies on whole blood samples. The rGO4 films were modified with anti-EpCAM before testing, and a typical image is shown in Figure S7 (Supporting Information). To determine the capture efficiency of anti-EpCAM-rGO4 film in blood samples, various numbers of MCF-7 cells (5, 10, 25, 50, 100, and 200 cells) were spiked into 1 mL of whole blood and dropped onto the surfaces of anti-EpCAM-rGO4 films (Figure S8, Supporting Information). The average recovery rates when using 5, 10, 25, 50, 100, and 200 spiked cells mL−1 were 67% ± 12%, 83% ± 11%, 84% ± 9%, 83% ± 9%, 89% ± 4%, and 93% ± 5%, respectively. Fresh blood samples obtained from patients with metastatic breast cancer (n = 8) (Figure 5) were processed on anti-EpCAMrGO4 films. Clinical data regarding these patients are summarized in Table S1 (Supporting Information). All blood samples were drawn after obtaining informed signed consent from patients. DAPI-positive, nucleated cells that also stained positive for cytokeratin and negative for CD45[5] were deemed CTCs in these breast cancer patients samples (Figure 5a,d). WBCs were identified as DAPI-positive nucleated cells that stained positive for CD45 and negative for cytokeratin (Figure 5e). A scatter plot (Figure 5c) demonstrates the fluorescence intensity limitation for separating clinical cytokeratin-positive CTCs and



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Figure 4. Immunofluorescent staining of cells on anti-EpCAM-substrates. Actin cytoskeleton (red); filopodia, lamellipodia, and focal adhesions (vinculin, green); and nuclei (4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) blue). Cells captured on anti-EpCAM-HOPG exhibited small circular plaques of vinculin, mainly at the peripheries of the cells, whereas larger vinculin-containing focal contacts were localized at the peripheral and ventral regions of cells that were captured on the anti-EpCAM-rGO films. These data demonstrate that the cell–substrate adhesion is progressively higher from anti-EpCAM-HOPG film to the anti-EpCAM-rGO4 film.

CD45-positive WBCs on the anti-EpCAM-rGO4 film. The size of each dot reflects the footprint of each cell. The CTC number reached as high as 6 in 1 mL of fresh blood, demonstrating the extremely high sensitivity and selectivity of the capture. The WBC numbered less than 1 per 2 mm2 area of rGO chip, compared to their original large number of 4–11 × 106 mL−1 of blood, we greatly decreased the nonspecific adhesion. In conclusion, we prepared antibody-modified rGO films that achieved extremely sensitive CTC static capture without the need for complex microfluidic operations. The extremely high efficiency of CTC capture obtained is ascribed to the synergistic topographic interactions provided by low stiffness of the prepared

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rGO films. In addition to the topographic interactions provided by the film, the low stiffness of the rGO films compared to other rigid artificial substrates more closely mimics the cellular microenvironment of living organisms, thus facilitating cell–environment interactions. In contrast, the negative charge and the superhydrophilicity of the prepared rGO films might contributed to the observed ultralow levels of nonspecific cell adhesion (Figure S9, Supporting Information). The sharply decrease of nonspecific cell adhesion on negative charged or superhydrophilic rGO films, compared to that on bare hydrophobic rGO films, demonstrated that the negative charge and superhydrophilicity are most likely to hinder nonspecific cell adhesion.


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COMMUNICATION Figure 5. Quantification and fluorescence microscopy images of CTCs captured from clinical samples. a) Quantification of CTCs captured from breast cancer patients. Up to six CTCs mL−1 were captured from samples obtained from patients with breast cancer. The anti-EpCAM-rGO4 films exhibited excellent capture ability using little blood volume. b) Quantification of WBCs captured from patients with breast cancer. The number of WBCs is markedly low. c) A scatter plot demonstrating the fluorescence intensity limitation for separating cytokeratin-positive CTCs and CD45-positive WBCs on the anti-EpCAM-rGO4 film using clinical samples. d) Fluorescence demonstration of CTC captured from a patient with breast cancer. e) Fluorescence demonstration of WBC captured from a patient with breast cancer. The size of each dot reflects the footprint of each cell.

Utilizing the multiple cell complementing properties of the antibody-modified rGO films, sensitive CTC specific recognition was realized via a simple static method, without the need for complex microfluidic devices or operations. These results provide clues and guidance for the engineering of the biointerfaces that mimic natural microenvironments, by complementing multiple complex cell properties. Further extension of this multimatching principle to cell-specific recognition using other platforms is warranted by this work; the results obtained highlight the importance of engineering the surface chemistry and physical properties of a material and of understanding the mechanisms of interface–cell adhesion interactions.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This research was supported by the National Research Fund for Fundamental Key Project (2012CB933800, 2013CB933000, and 2012CB934100), National Natural Science Foundation (21121001,

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21127025, and 20974113), the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-M01), the Major Program of National Natural Science Foundation of China (21434009), and the National High Technology Research and Development Program of China (863 Program) (2013AA031903). Received: June 2, 2015 Revised: July 9, 2015 Published online: October 1, 2015

[1] S. Petersen, J. M. Alonso, A. Specht, P. Duodu, M. Goeldner, A. del Campo, Angew. Chem. Int. Ed. 2008, 47, 3192. [2] C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E. Ingber, Science 1997, 276, 1425. [3] C. J. Bettinger, R. Langer, J. T. Borenstein, Angew. Chem. Int. Ed. 2009, 48, 5406. [4] J. Sekine, S. C. Luo, S. Wang, B. Zhu, H. R. Tseng, H. H. Yu, Adv. Mater. 2011, 23, 4788. [5] X. Liu, L. Chen, H. Liu, G. Yang, P. Zhang, D. Han, S. Wang, L. Jiang, NPG Asia Mater. 2013, 5, e63. [6] K. Yang, S. Zhang, G. Zhang, X. Sun, S. T. Lee, Z. Liu, Nano Lett. 2010, 10, 3318. [7] Z. Liu, J. T. Robinson, X. Sun, H. Dai, J. Am. Chem. Soc. 2008, 130, 10876.



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[8] H. J. Yoon, J. M. Kim, Z. Zhang, E. Azizi, T. M. Pham, C. Paoletti, J. Lin, N. Ramnath, M. Wicha, D. Hayes, D. M. Simenoe, S. Nagrath, Nat. Nanotechnol. 2013, 8, 735. [9] M. J. McAllister, J. Li, D. H. Adamson, H. C. Schniepp, A. A. Abdala, J. Liu, M. Herrera-Alonso, D. L. Milius, R. Car, R. K. Prud’homme, I. A. Aksay, Chem. Mater. 2007, 19, 4396. [10] S. Pei, H. Cheng, Carbon 2012, 50, 3210. [11] S. Nie, S. Emory, Science 1997, 275, 1102. [12] G. T. Hermanson, Bioconjugate Techniques, Elsevier, Rockford, IL, USA 2008. [13] H. Guo, M. Peng, Z. Zhu, L. Sun, Nanoscale 2013, 5, 9040. [14] M. S. Mannoor, H. Tao, J. D. Clayton, A. Sengupta, D. L. Kaplan, R. R. Naik, N. Verma, F. G. Omenetto, M. C. McAlpine, Nat. Commun. 2012, 3, 763. [15] S. Stankovich, D. A. Dikin, R. D. Piner, K. A. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S. T. Nguyen R. S. Ruoff, Carbon 2007, 45, 1558.


[16] S. Wang, H. Wang, J. Jiao, K. J. Chen, G. E. Owens, K. Kamei, J. Sun, D. J. Sherman, C. P. Behrenbruch, H. Wu, H. R. Tseng, Angew. Chem. Int. Ed. 2009, 48, 8970. [17] X. Liu, S. Wang, Chem. Soc. Rev. 2014, 43, 2385. [18] L. Ma, G. Yang, N. Wang, P. Zhang, F. Guo, J. Meng, F. Zhang, Z. Hu, S. Wang, Y. Zhao, Adv Healthcare Mater. 2015, 4, 838. [19] R. Pelhan, Y. Wang, Proc. Natl. Acad. Sci. USA 1997, 94, 13661. [20] B. Trappmann, J. E. Gautrot, J. T. Connelly, D. G. Strange, Y. Li, M. L. Oyen, M. A. C. Stuart, H. B. Trappmann, B. Li, V. Vogel, J. P. Spatz, F. M. Watt, T. S. H. Wilhelm, Nat. Mater. 2012, 11, 642. [21] M. J. Paszek, N. Zahir, K. R. Johnson, J. N. Lakins, G. I. Rozenberg, A. Gefen, C. A. Reinhart-King, S. S. Margulies, M. Dembo, D. Boettiger, D. A. Hammer, V. M. Weaver, Cancer Cell 2005, 8, 241. [22] C. G. Galbraith, K. M. Yamada, M. P. Sheetz, J. Cell Biol. 2002, 159, 695.


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