Biosensors and Bioelectronics 22 (2007) 1994–2002 A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips Panagiota S. Petrou a,∗ , Margarita Chatzichristidi b , Antonios M. Douvas b , Panagiotis Argitis b , Konstantinos Misiakos b , Sotirios E. Kakabakos a a Immunoassay/Immunosensors Laboratory, I/R-RP, NCSR “Demokritos”, 15310 Aghia Paraskevi, Greece b Microelectronics Institute, NCSR “Demokritos”, 15310 Aghia Paraskevi, Greece Received 28 April 2006; received in revised form 2 August 2006; accepted 23 August 2006 Available online 5 October 2006 Abstract The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures ≤50 ◦ C, development with dilute aqueous basic developer solutions and exposure at wavelengths above 300 nm. Following this photolithographic procedure onto the polymeric layer coated silicon surface, protein spots with diameters ranging from 2 to 50 m were created. The proposed methodology provided good intra- spot homogeneity (CV ≤5%) and inter-spot repeatability (CV ≤5%), as it was determined through epifluorescence microscopy after reaction of the immobilized proteins with their respective fluorescently labeled binding counterparts. Moreover, the polymeric film selected for immobilization of biomolecules presented high protein binding capacity, which was at least three folds higher than that obtained using aminosilanized surfaces. The proposed methodology is expected to facilitate considerably the fabrication of dense protein microarrays for bioanalytical applications. © 2006 Elsevier B.V. All rights reserved. Keywords: Protein arrays; Polymer coating; Photolithography; Protein friendly process; Photolithographic protein patterning 1. Introduction The driving force for the development of methods for biomolecule array fabrication is their prominent application in a variety of fields, including biomedical diagnostics, DNA anal- ysis, drug discovery and environmental monitoring (Howbrook et al., 2003; Stoll et al., 2004). There is a great number of available patterning methods that extend from delivery methods based on automated arrayers (Newman and Turner, 1992; Leuking et al., 1999; ∗ Corresponding author. Tel.: +30 210 6503820/6515573; fax: +30 210 6515573. E-mail address: ypetrou@rrp.demokritos.gr (P.S. Petrou). Delehanty, 2004) to more exploratory methods (Falconnet et al., 2006) which include microcontact printing using poly(dimethylsiloxane) stamps (Bernard et al., 1998; Lahiri et al., 1999; Kane et al., 1999; Martin et al., 2000), patterning along microfluidic channels (Delamarche et al., 1998), charged particle litghography (Bergman et al., 1998; Glezos et al., 2002), dip-pen lithography (MacBeath and Schreiber, 2000; Lee et al., 2003; Agarwal et al., 2003), and light-guided methods including in situ synthesis of biomolecules on the substrate (Pease et al., 1994; Pritchard et al., 1995; Hengsakul and Cass, 1996; Bock et al., 2004). Each of these methods has advantages and limitations concerning the resolution of the patterning or the number of spots that can be created simultaneously. Photoresist based lithography is in principle capable of pro- viding dense arrays of high-resolution structures on substrate 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.08.036