Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay Lotta Rö mhildt, †,‡ Claudia Pahlke, † Felix Zö rgiebel, †,§ Hans-Georg Braun, ⊥ Jö rg Opitz, †,‡ Larysa Baraban, †, * and Gianaurelio Cuniberti †,§ † Institute for Materials Science and Max Bergmann Center of Biomaterials and § Center for Advancing Electronics Dresden, TU Dresden, 01062 Dresden, Germany ‡ Fraunhofer Institute IZFP Dresden, 01109 Dresden, Germany ⊥ Max Bergmann Center of Biomaterials, Leibniz Institute of Polymer Research Dresden, 01069 Dresden, Germany * S Supporting Information ABSTRACT: Here we propose a platform for the detection of unlabeled human α-thrombin down to the picomolar range in a fluorescence-based aptamer assay. In this concept, thrombin is captured between two different thrombin binding aptamers, TBA1 (15mer) and TBA2 (29mer), each labeled with a specific fluorescent dye. One aptamer is attached to the surface, the second one is in solution and recognizes surface-captured thrombin. To improve the limit of detection and the comparability of measurements, we employed and compared two approaches to pattern the chip substrate-microcontact printing of organosilanes onto bare glass slides, and controlled printing of the capture aptamer TBA1 in arrays onto functionalized glass substrates using a nanoplotter device. The parallel presence of functionalized and control areas acts as an internal reference. We demonstrate that both techniques enable the detection of thrombin concentrations in a wide range from 0.02 to 200 nM with a detection limit at 20 pM. Finally, the developed method could be transferred to any substrate to probe different targets that have two distinct possible receptors without the need for direct target labeling. KEYWORDS: biosensor, selective functionalization, aptamer, thrombin, sandwich assay ■ INTRODUCTION The early detection of disease markers, pathogenic cells, molecules, or drugs is a key driving force for the development of simple and rapid diagnostic techniques to assure specific and timely treatment. 1,2 Numerous efforts are nowadays directed towards miniaturization and multiplexed sensing for portable and cost-effective devices aiming at personalized diagnostics. To date, standard technologies like polymerase chain reaction (PCR) or immunoassays (e.g., ELISA) using antibodies for sensing 3 are highly specific but complex and require highly qualified employees, laboratory equipment, relatively long assay times, or a large sample volume. Recently reported DNA aptamers (i.e., oligonucleotide chains) 4-6 represent an alternative to antibodies as highly specific receptors for cost- effective biosensors. These aptasensors rely mostly on either monitoring of electrical characteristics 7-10 or optical properties of devices 11-13 and open up possibilities for real-time or label- free biosensing. Among the most frequently employed techniques, microarray technologies enable integration of several experiments on one chip for parallel detection of different analytes. 14-17 In combination with lab-on-a-chip technologies, fast and sensitive sensors can be developed. One of the remaining challenges in the field of biological detection is related to improving the selectivity of the assay and reducing the background noise caused by unspecific adsorp- tion. 18,19 High specificity of the whole biochemical scheme determines the detection limit of the technique. In parallel to already well-studied antibodies, 18,20 the use of engineered receptor molecules, which are more stable and can be developed at low cost, i.e., aptamers, still need to be optimized for their wider implementation into biochemical assays. 21-23 Because of its distinct sequence, each aptamer forms a tertiary structure (e.g., hairpin or quadruplex 24,25 ), which interacts with a certain recognition site of the analyte such as proteins or organic molecules. 26 Easy labeling protocols or the attachment of functional end groups to the nucleotides, the higher stability under varying conditions such as temperature or ionic strength compared to antibodies as well as the wide variety of possible targets make aptamers a promising candidate for diverse biosensor applications in the future. 13,21,26,27 Received: September 5, 2013 Accepted: October 31, 2013 Research Article www.acsami.org © XXXX American Chemical Society A dx.doi.org/10.1021/am4038245 | ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX