Two label-free electrochemical methods for the detection of human alpha-thrombin using a water-soluble, ferrocene-functionalized polythiophene transducer and a single-stranded oligonucleotide aptamer probe are described. The first approach is a direct method in which the recorded current decreases upon addition of the targeted protein. The second one requires more steps and the additional utilization of PNA probes and nuclease enzyme. This indirect method leads to an increase of the electrical signal as a function of the concentration of human alpha-thrombin with a detection limit of 75 fmol.

Many efforts have been made worldwide to develop and improve electrochemical methods for DNA detection with the aim of making portable and affordable devices.[1±4] In spite of many advances in this field, it is still a challenge to find new approaches that could improve the simplicity, selectivity, and sensitivity of DNA detection, in order to respond to the demands and needs of modern medical diagnostics and biomedical research applications. In this regard, some oligonucleotide-functionalized conjugated polymers have enabled the transduction of hybridization events into an electrical signal without labeling of the DNA target.[5±7] However, with this strategy, waterinsoluble electroactive polymers are always present on the electrodes, leading to a strong electrical background. Moreover, in those systems, hybridization often leads to a decrease in the electrical signal. To solve these problems, we propose a new solid-state electrostatic approach based on neutral peptide nucleic acid (PNA) capture probes [8] and an electroactive, cationic, water-soluble polythiophene transducer. [9,10] The solidstate electrochemical detection occurs when the neutral probes are hybridized with a complementary DNA target, leading to an attractive electrostatic interaction with a cationic polythiophene transducer (Scheme 1). A similar electrostatic strategy using fluorescent, cationic, conjugated polymers was recently utilized for the specific optical detection of oligonucleotides on PNA microarrays [10] and in aqueous solution.[11]To implement this new solid-state electrochemical approach, we first prepared gold electrodes with a monolayer of PNA capture probes (see Experimental), and then performed square-wave voltammetry (SWV). The cationic, water-soluble, and electroactive polymer 1 (see Scheme 2) does not bind to neutral PNA capture probes alone (Scheme 1a; Fig. 1, trace a) but interacts strongly with the negatively charged backbone of the complementary oligonucleotide bound to the PNA probes, [10] allowing transduction of room-temperature hybridization into an electrical signal (Scheme 1b; Fig. 1, COMMUNICATIONS

Electroactive polyfluorene films were obtained by anodic coupling of fluorene and a series of 9,9-disubstituted fluorenes and their related 2,7‘-dimers and trimer. The polymerization mechanism is discussed in light of cyclic voltammetry investigations in organic media using the classical millimetric electrode and ultramicroelectrodes, DFT theoretical calculations, and laser flash photolysis experiments. The first step of electropolymerization involves the formation of the carbon−carbon bond through the coupling between two fluorene radical cations. However, the radical cation of the produced dimer is not reactive enough to repeat the coupling reaction as in the classical electropolymerization mechanism. To continue the polymerization, formation of a higher oxidation state is required. This behavior is supported by theoretical expectations.

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