Published: April 14, 2011 r2011 American Chemical Society 6116 dx.doi.org/10.1021/la1050833 | Langmuir 2011, 27, 6116–6123 ARTICLE pubs.acs.org/Langmuir Simple One-Step Process for Immobilization of Biomolecules on Polymer Substrates Based on Surface-Attached Polymer Networks Martin Rendl, † Andreas B€ onisch, † Andreas Mader, †,‡ Kerstin Schuh, † Oswald Prucker, † Thomas Brandstetter,* ,† and J€ urgen R€ uhe † † Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-K€ ohler-Allee 103, D-79110 Freiburg, Germany ‡ Institute of Pathology, Freiburg University Medical Center, Breisacher Strasse 115a, D-79106 Freiburg, Germany b S Supporting Information 1. INTRODUCTION Microarrays are versatile tools in biomedical research, as they permit highly parallel analytics of various samples such as blood, urine, saliva, or tissues. 1 However, despite considerable effort, this technology still suffers from rather complex and cost-intensive manufacturing processes, tedious handling, and poor reproducibi- lity 2,3 which still prevents a wider acceptance in today’ s clinical routine diagnostics. The development of a suitable strategy, which allows the simple and efficient immobilization of probe molecules to the chip surfaces, plays a key role in the successful design and implementation of a microarray. 2 High surface concentration of probe molecules and retention of their biological activity, high sensitivity, and signal to background ratio as well as low unspecific binding are crucial parameters of such a process. To permit quantitative or semiquantitative bioassays and to allow for low intra- and interchip variance, control over the amount of probe molecules per area, their orientation, and reliable and robust immobilization are decisive requirements. To meet these requirements, a wide spectrum of different immobilization strategies has been developed and published over the years. Commonly, biomolecules such as DNA strands or proteins are immobilized on substrates through binding to a monolayer for the functionalization of surfaces. To achieve efficient coupling, several different chemistries have been developed. Most tech- nologies are based on surface modification of the substrate with active groups reacting with more or less specific parts of the biomolecule itself or conjugated linker groups such as biotiny- lated or amino-modified DNA or proteins. 3,4 The strategies for the immobilization of biomolecules can be divided into three categories, in which the surface attachment of the biomolecules is based on either covalent, physical, or affinity based binding interactions. 5 Strategies which use covalent binding are often based on amine chemistry, where, for example, aminosilanes or lysine moieties are used as anchoring groups for biomolecules. Probably the most commonly used binding mechanisms rely on succinimidyl esters (NHS), epoxy, aldehyde, or carbodiimide containing compounds, which covalently bind to amine groups of the biomolecule. 2 Covalent binding strategies yielding or- iented immobilization of proteins are based on maleimides and disulfide derivates binding to cysteines of disulfide bridges in the hinge region of antibodies. Amino groups or hydrazines binding to carbohydrate residues of the Fc portion of antibodies provide Received: December 23, 2010 Revised: March 16, 2011 ABSTRACT: For the miniaturization of biological assays, espe- cially for the fabrication of microarrays, immobilization of biomo- lecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such sub- strates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe mol- ecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.