在分離細胞及分析細胞表現特徵之研究上，抗體抗原反應(antibody-antigen interaction)為一常用的工具，其反應結合具有相當高的專一性，目前已廣泛應用在癌症細胞的篩選及幹細胞研究上，然目前抗體陣列晶片僅具有分析，而不具有收集標的細胞的功能，其表面材料的不可變換性，限制了晶片的應用性。釋放、收集細胞之關鍵在於可變換表面特性基材之效能，其高低影響篩選的能力。近年來發現很多具有可變換表面特性的智慧型表面(intelligent surface)，大多是具有生物相容性的高分子為材料，在不同的酸鹼值或溫度下，高分子構造改變而使表面具有可變換性，其中溫度敏感型智慧型表面，以聚異丙基丙烯醯胺材料為代表，能以較溫和的方式使細胞脫附，除了在組織工程上的應用，在細胞篩選研究上有越來越多的應用。本研究旨在利用溫度敏感型表面設計一具有捕捉和釋放機制之細胞篩選晶片，並期望能結合微流體系統及抗體晶片之優點，增加細胞篩選的專一性及收集性。
本研究採用溫度敏感型智慧水膠材料聚異丙基丙烯醯胺，將其修飾在生醫晶片常用的材料PDMS上，聚異丙基丙烯醯胺具有隨溫度改變表面親疏水性的特色，能使細胞釋放的過程較溫和，實驗上分別針對基材與抗體之特性，進行最佳化之實驗參數研究，在溫感表面的製備方面，利用聚異丙基丙烯醯胺單體製備的高分子表面，表面親水角數據顯示在不同的溫度下該表面具有親疏水性的差異。在抗體修飾聚苯乙烯球的實驗方面，已利用標準抗體實驗，得到各種反應物的濃度參數和實驗的環境參數，可得到分散度較高的聚苯乙烯球，再以實際能夠辨認細胞表面抗原的抗體修飾在聚苯乙烯球表面，進行細胞表面抗原抗體染色反應。應用在細胞上，抗體修飾的聚苯乙烯球在表面抗原上亦具有可行性，在標的細胞上可觀察到聚苯乙烯球上二級抗體的染色。本研究並結合溫感表面與抗體修飾的聚苯乙烯球，分析在不同溫度下，抗體聚苯乙烯球輔助細胞被表面捕捉和釋放的數量，以評估以疏水性作用力的差異來捕捉和釋放細胞的可行性。

Protein-based cell microarray is a powerful tool used in both clinical diagnostics and fundamental researches. Protein such as antibodies were immobilized on the surface through covalent bond and the composition of cell mixture and characteristic of surface proteins could be determined in single assay within several minutes using this technology. However, the cell of interest after screening could not be released from cell microarray chip due to the unchangeable property of the microarray chip substrate.
An intelligent surface with intrinsic thermoresponsive property could be used to improve surface property for cell release. Poly N-isopropylacrylamide (pNIPAAM) is a temperature sensitive hydrogel, which have been extensively studied in grafting on many kinds of surfaces due to its unique properties, such as its lower critical solution temperature (LCST) around 32-33℃ in aqueous solution, and the anti-fouling characteristic in room temperature. Poly N-isopropylacrylamide has been applied to the separation of biomolecules in the form of chromatographic matrices and cell substrates. Temperature dependent hydrophobic-hydrophilic changeable surface which is grafted by pNIPAAm (Poly N-isopropylacrylamide) have also been used for controllable capture and release of proteins in microfluidic devices.
In this paper, a surface was fabricated by grafting thermo-responsive polymer to PDMS (Polydimethylsiloxane) for cell capture and release, and the difference in surface hydrophobicity were demonstrated by contact angle change and the binding of hydrophobic polystyrene beads to the surface. Furthermore, polystyrene beads that were modified with antibodies for cell recognition also showed activity to bind to cell surface through antibody-antigen interaction. The cells stained with conjugates of the antibody and polystyrene beads were applied to the surface to test the capture and release at different temperature. Polystyrene beads show the capability of both selection of specific cells and assisting the binding of cells to pNIPAAm surface through hydrophobic interaction.
This research presents the idea of utilizing temperature-controlled hydrophobic interaction for capturing and releasing cells. It seeks to combine antibody-antigen interaction and cell sorting system to improve the specificity. This platform can be optimized and applied to provide further collection of cells of interest from protein-based cell microarray.

[1] M. Radisic, R. K. Iyer, and S. K. Murthy, "Micro- and nanotechnology in cell separation," International Journal of Nanomedicine, vol. 1, p. 10, 2006.
[2] J. M. Ramsey, "The burgeoning power of the shrinking laboratory," Nat Biotechnol, vol. 17, pp. 1061-2, Nov 1999.
[3] S. J. Lee and S. Y. Lee, "Micro total analysis system (micro-TAS) in biotechnology," Appl Microbiol Biotechnol, vol. 64, pp. 289-99, Apr 2004.
[4] K. W. Dimitri Pappas, "Cellular separations: A review of new chanllenges in analytical chemistry," Analytica Chimica Acta, vol. 601, pp. 26-35, 2007.
[5] L. R. Huang, E. C. Cox, R. H. Austin, and J. C. Sturm, "Continuous particle separation through deterministic lateral displacement," Science, vol. 304, pp. 987-990, May 14 2004.
[6] H. Mohamed, L. D. McCurdy, D. H. Szarowski, S. A. D. S. Duva, J. N. A. T. J. N. Turner, and M. A. C. M. Caggana, "Development of a rare cell fractionation device: application for cancer detection," Nanobioscience, IEEE Transactions on, vol. 3, pp. 251-256, 2004.
[7] J. Voldman, M. L. Gray, M. Toner, and M. A. Schmidt, "A microfabrication-based dynamic array cytometer," Analytical Chemistry, vol. 74, pp. 3984-3990, Aug 15 2002.
[8] Y. Huang, J. Yang, X. B. Wang, F. F. Becker, and P. R. Gascoyne, "The removal of human breast cancer cells from hematopoietic CD34+ stem cells by dielectrophoretic field-flow-fractionation," J Hematother Stem Cell Res, vol. 8, pp. 481-90, Oct 1999.
[9] K. H. Han and A. B. Frazier, "Lateral-driven continuous dielectrophoretic microseparators for blood cells suspended in a highly conductive medium," Lab Chip, vol. 8, pp. 1079-86, Jul 2008.
[10] A. Y. Fu, C. Spence, A. Scherer, F. H. Arnold, and S. R. Quake, "A microfabricated fluorescence-activated cell sorter," Nat Biotechnol, vol. 17, pp. 1109-11, Nov 1999.
[11] D. Huh, W. Gu, Y. Kamotani, J. B. Grotberg, and S. Takayama, "Microfluidics for flow cytometric analysis of cells and particles," Physiol Meas, vol. 26, pp. R73-98, Jun 2005.
[12] A. Y. Fu, H. P. Chou, C. Spence, F. H. Arnold, and S. R. Quake, "An integrated microfabricated cell sorter," Anal Chem, vol. 74, pp. 2451-7, Jun 1 2002.
[13] A. Wolff, I. R. Perch-Nielsen, U. D. Larsen, P. Friis, G. Goranovic, C. R. Poulsen, J. P. Kutter, and P. Telleman, "Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter," Lab Chip, vol. 3, pp. 22-7, Feb 2003.
[14] K. M. Partington, E. J. Jenkinson, and G. Anderson, "A novel method of cell separation based on dual parameter immunomagnetic cell selection," J Immunol Methods, vol. 223, pp. 195-205, Mar 4 1999.
[15] N. Xia, T. P. Hunt, B. T. Mayers, E. Alsberg, G. M. Whitesides, R. M. Westervelt, and D. E. Ingber, "Combined microfluidic-micromagnetic separation of living cells in continuous flow," Biomed Microdevices, vol. 8, pp. 299-308, Dec 2006.
[16] T. M. Said, A. Agarwal, M. Zborowski, S. Grunewald, H. J. Glander, and U. Paasch, "Utility of magnetic cell separation as a molecular sperm preparation technique," Journal of Andrology, vol. 29, pp. 134-142, Mar-Apr 2008.
[17] A. K. Gupta and M. Gupta, "Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications," Biomaterials, vol. 26, pp. 3995-4021, Jun 2005.
[18] I. K. Ko, K. Kato, and H. Iwata, "Antibody microarray for correlating cell phenotype with surface marker," Biomaterials, vol. 26, pp. 687-696, 2005.
[19] L. Belov, O. de la Vega, C. G. dos Remedios, S. P. Mulligan, and R. I. Christopherson, "Immunophenotyping of leukemias using a cluster of differentiation antibody microarray," Cancer Res, vol. 61, pp. 4483-9, Jun 1 2001.
[20] I. K. Ko, K. Kato, and H. Iwata, "Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays," Biomaterials, vol. 26, pp. 4882-4891, Aug 2005.
[21] M. Nakajima, T. Ishimuro, K. Kato, I. K. Ko, I. Hirata, Y. Arima, and H. Iwata, "Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation," Biomaterials, vol. 28, pp. 1048-1060, Feb 2007.
[22] M. A. Vijayalakshmi, Biochromatography : theory and practice: London ; New York : Taylor & Francis, 2002.
[23] K. A. Dill, "The meaning of hydrophobicity," Science, vol. 250, pp. 297-8, Oct 12 1990.
[24] G. Healthcare, "Affinity Chromatography: Principles and Methods," General Electric Company, 2007.
[25] Y. Shibusawa, "Separation and retention of human blood cells by surface-affinity chromatography," J Biochem Biophys Methods, vol. 49, pp. 683-703, Oct 30 2001.
[26] P. A. Albertson, Partition of Cell Particles and Macromolecules, 2nd ed.: John Wiley and Sons, 1986.
[27] A. Kumar, M. Kamihira, I. Y. Galaev, B. Mattiasson, and S. Iijima, "Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive polymers," Biotechnology and Bioengineering, vol. 75, pp. 570-580, Dec 5 2001.
[28] K. H. Nam, W. J. Chang, H. Hong, S. M. Lim, D. I. Kim, and Y. M. Koo, "Continuous-flow fractionation of animal cells in microfluidic device using aqueous two-phase extraction," Biomedical Microdevices, vol. 7, pp. 189-195, Sep 2005.
[29] M. Tsukamoto, S. Taira, S. Yamamura, Y. Morita, N. Nagatani, Y. Takamura, and E. Tamiya, "Cell separation by an aqueous two-phase system in a microfluidic device," Analyst, vol. 134, pp. 1994-8, Oct 2009.
[30] D. D. M. J.S. Scarpa, I.M. Klotz, "Slow hydrogen-deuterium exchange in a non-a-helical polyamide," Journal of the American Chemical Society, vol. 89, pp. 6024-6030, 1967.
[31] H. G. Schild, "Poly(N-isopropylacrylamide): experiment, theory and application," Progress in Polymer Science, vol. 17, pp. 163-249, 1992.
[32] T. Okano, N. Yamada, M. Okuhara, H. Sakai, and Y. Sakurai, "Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces," Biomaterials, vol. 16, pp. 297-303, 1995.
[33] K. Nagase, J. Kobayashi, and T. Okano, "Temperature-responsive intelligent interfaces for biomolecular separation and cell sheet engineering," J R Soc Interface, vol. 6 Suppl 3, pp. S293-309, Jun 6 2009.
[34] S.-Y. Lin, K.-S. Chen, and R.-C. Liang, "Thermal micro ATR/FT-IR spectroscopic system for quantitative study of the molecular structure of poly(N-isopropylacrylamide) in water," Polymer, vol. 40, pp. 2619-2624, 1999.
[35] A. Kikuchi and T. Okano, "Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs," J Control Release, vol. 101, pp. 69-84, Jan 3 2005.
[36] H. E. Canavan, X. Cheng, D. J. Graham, B. D. Ratner, and D. G. Castner, "Cell sheet detachment affects the extracellular matrix: a surface science study comparing thermal liftoff, enzymatic, and mechanical methods," J Biomed Mater Res A, vol. 75, pp. 1-13, Oct 1 2005.
[37] K. Nakajima, S. Honda, Y. Nakamura, F. Lopez-Redondo, S. Kohsaka, M. Yamato, A. Kikuchi, and T. Okano, "Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method," Biomaterials, vol. 22, pp. 1213-23, Jun 2001.
[38] A. Luchini, D. H. Geho, B. Bishop, D. Tran, C. Xia, R. L. Dufour, C. D. Jones, V. Espina, A. Patanarut, W. Zhou, M. M. Ross, A. Tessitore, E. F. Petricoin, 3rd, and L. A. Liotta, "Smart hydrogel particles: biomarker harvesting: one-step affinity purification, size exclusion, and protection against degradation," Nano Lett, vol. 8, pp. 350-61, Jan 2008.
[39] H. Kanazawa, T. Sunamoto, E. Ayano, Y. Matsushima, A. Kikuchi, and T. Okano, "Temperature-responsive chromatography using poly(N-isopropylacrylamide) hydrogel-modified silica," Anal Sci, vol. 18, pp. 45-8, Jan 2002.
[40] H. Kanazawa, Y. Kashiwase, K. Yamamoto, Y. Matsushima, A. Kikuchi, Y. Sakurai, and T. Okano, "Temperature-responsive liquid chromatography. 2. Effects of hydrophobic groups in N-isopropylacrylamide copolymer-modified silica," Anal Chem, vol. 69, pp. 823-30, Mar 1 1997.
[41] N. Malmstadt, P. Yager, A. S. Hoffman, and P. S. Stayton, "A smart microfluidic affinity chromatography matrix composed of poly(N-isopropylacrylamide)-coated beads," Anal Chem, vol. 75, pp. 2943-9, Jul 1 2003.
[42] W. L. KS. Chen, JC Tsai, SH. Chiou, "Surface modification of materials by plasma process and UV-induced grafted polymerization for biomedical applications," Journal of the Vacuum Society of Japan, vol. 50, pp. 19-24, 2007.
[43] V. P. Gilcreest, W. M. Carroll, Y. A. Rochev, I. Blute, K. A. Dawson, and A. V. Gorelov, "Thermoresponsive poly(N-isopropylacrylamide) copolymers: Contact angles and surface energies of polymer films," Langmuir, vol. 20, pp. 10138-10145, Nov 9 2004.
[44] P. Schmidt, J. Dybal, J. C. Rodriguez-Cabello, and V. Reboto, "Role of water in structural changes of poly(AVGVP) and poly(GVGVP) Studied by FTIR and Raman spectroscopy and ab initio calculations," Biomacromolecules, vol. 6, pp. 697-706, Mar-Apr 2005.
[45] S. Y. X. L. He, Y. Y. Dong, F. Y. Yan, and L. Chen, "Preparation and properties of a novel thermo-responsive poly(N-isopropylacrylamide) hydrogel containing glycyrrhetinic acid," Journal of Materials Science, vol. 44, pp. 4078-4086, 2009.
[46] L. Y. C. X. J. Ju, P. Mi, H. Song, and Y. M. Lee, "Synthesis and characterization of a novel thermo-sensitive copolymer of N-isopropylacrylamide and dibenzo-18-crown-6-diacrylamide," Macromolecular Rapid Communications, vol. 27, pp. 2072-2077, 2006.
[47] A. Khan, "Preparation and characterization of N-isopropylacrylamide/acrylic acid copolymer core-shell microgel particles," J Colloid Interface Sci, vol. 313, pp. 697-704, Sep 15 2007.
[48] Y. V. Pan, R. A. Wesley, R. Luginbuhl, D. D. Denton, and B. D. Ratner, "Plasma polymerized N-isopropylacrylamide: synthesis and characterization of a smart thermally responsive coating," Biomacromolecules, vol. 2, pp. 32-6, Spring 2001.