Quantification of protein immobilization on substrates for cellular microarray applications

• Published in: Journal of biomedical materials research. Part A Vol. 98A; no. 2; pp. 245 - 256
• Main Authors: Rodríguez-Seguí, Santiago A., Pons Ximénez, José Ignacio, Sevilla, Lídia, Ruiz, Ana, Colpo, Pascal, Rossi, François, Martínez, Elena, Samitier, Josep
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.08.2011; Wiley-Blackwell
• Subjects: Animals; Biological and medical sciences; Calibration; Cell Proliferation; Cell Survival; Fibronectins - analysis; Fluorescence; Humans; Immobilized Proteins - analysis; Medical sciences; Mice; microarray; Molecular Weight; Myoblasts - cytology; Myoblasts - metabolism; protein immobilization; quantification; Streptavidin - analysis; substrate; surface chemistry; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Technology. Biomaterials. Equipments; Tissue Array Analysis - methods; microarray; quantification; surface chemistry; Immobilization; Surface properties; protein immobilization; Biomaterial; Protein; substrate; Biomedical engineering
• ISSN: 1549-3296; 1552-4965; 1552-4965
• DOI: 10.1002/jbm.a.33089

Cellular microarray developments and its applications are the next step after DNA and protein microarrays. The choice of the surface chemistry of the substrates used for the implementation of this technique, that must favor proper protein immobilization while avoiding cell adhesion on the nonspotted areas, presents a complex challenge. This is a key issue since usually the best nonfouling surfaces are also the ones that retain immobilized the smallest amounts of printed protein. To quantitatively assess the amount of protein immobilization, in this study several combinations of fluorescently labeled fibronectin (Fn*) and streptavidin (SA*) were microspotted, with and without glycerol addition in the printing buffer, on several substrates suitable for cellular microarrays. The substrates assayed included chemically activated surfaces as well as Poly ethylene oxide (PEO) films that are nonfouling in solution but accept adhesion of proteins in dry conditions. The results showed that the spotted Fn* was retained by all the surfaces, although the PEO surface did show smaller amounts of immobilization. The SA*, on the other hand, was only retained by the chemically activated surfaces. The inclusion of glycerol in the printing buffer significantly reduced the immobilization of both proteins. The results presented in this article provide quantitative evidence of the convenience of using a chemically activated surface to immobilize proteins relevant for cellular microarray applications, particularly when ECM proteins are cospotted with smaller factors which are more difficult to be retained by the surfaces. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 2011.