Novel biosensors are always needed for improving modern biomedical research as well as for environmental and forensic sciences. Among the proposed techniques, the (micro) fabrication of addressable 2D arrays (biochips) has become a powerful tool. However, in most cases this method relies on fluorescent tagging of the analytes for detection. In addition to implying the use of careful synthetic procedures, it is worth noting that labeling may even alter the binding properties of the analytes. Assays that do not require any chemical manipulation of the biological targets or sophisticated experimental techniques (mass spectrometry, surface plasmon resonance, etc.) would be greatly advantageous. These limitations could be solved by a new generation of responsive supports with optical or electrical properties modified upon specific and efficient binding of a specific label-free target. In parallel, because of their central importance in many biological processes and as biomarkers related to human diseases, there is a high demand for convenient methodologies for detecting specific proteins. Along these lines, RNA and DNA aptamers have recently received considerable attention as new protein recognition elements. Aptamers are usually isolated from combinatorial libraries of synthetic nucleic acids by an iterative process of adsorption, recovery, and amplification. Therefore, by combining smart polymeric transducers and aptamer ligands, we report new responsive supports for the reagentless, sensitive, and specific optical detection of proteins. For instance, these simple integrated biochips allow the direct and specific detection of human thrombin in the attomole range in less than one hour at room temperature. The development of these novel responsive biochips is based on the utilization of a chromic, cationic, water-soluble polythiophene derivative (Scheme 1). This polymer exhibits different conformational structures and optical properties when it is in the presence of free single-stranded (ss) nucleic acids or bound nucleic acids. More precisely, stoichiometric complexes between this polythiophene derivative and ssDNA form neutral rigid-rod moieties that self-assemble into nanoaggregates and result in significant quenching of the fluorescence of the conjugated polymer. In solution, it has been shown that the polythiophene becomes fluorescent again through specific hybridization or DNA-aptamer– protein interactions. For instance, the negatively charged DNA aptamer bound to a specific protein undergoes a conformational transition from an unfolded to a folded (G-quadruplex) structure that can be detected by the cationic polythiophene derivative. Moreover, it has been recently reported that a significant fluorescence signal amplification (fluorescence chain reaction (FCR)) takes place within micelle-like structures formed from labeled ssDNA–polythiophene complexes in solution. This new detection method is based on the efficient and fast energy transfer (Forster resonance energy transfer (FRET)) between one resulting fluorescent polythiophene chain and many fluorophores attached to neighboring ssDNA probes. Stoichiometric complexes (duplexes) were therefore prepared by mixing the polythiophene optical transducer with a Cy3-3′-labeled ssDNA aptamer. This chromophore was chosen because its absorption spectrum overlaps well with the emission spectrum of the polythiophene, a necessary condition for efficient FRET. However, to allow the covalent binding of these aggregated duplexes onto surface-treated glass slides, an amine group was also inserted at the 5′-end of the ssDNA capture probes. Upon spotting, these aggregates made of hybrid polythiophene–ssDNA complexes were therefore bound onto glass slides (Scheme 1). Then, for the specific detection of human thrombin, the following strategy was designed: First, P1 (5′-NH2-C6-GGT TGG TGT GGT TGG-Cy3-3′), P2 (5′-NH2-C6-GGT TGG TGT GGT TGG3′), or P3 (5′-NH2-C6-GGT GGT GGT TGT GGT-Cy3-3′) were combined with cationic polythiophene in order to form stoichiometric duplexes. P1 and P2 are both sequences specific to thrombin; however, P1 is labeled with Cy3 fluorophore whereas P2 is not. P3 is a labeled sequence that does not bind to thrombin. The diameter of the spots is about 1.5–1.7 mm (see Fig. 1), each including about 1 × 10 probes. C O M M U N IC A IO N S
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Aug 1, 2008
E.Y Song + 1
Open Access