The development of a biosensor with adequate sensitivity generally requires a biospecific interaction with high binding affinity. The affinity constants for most antibody- antigen interactions are determined largely by the dissociation constants, kd, with little variation observed in rates of associated. additionally, surface immobilization typically results in a reduced kd. In this case, the sensor binds analyte kinetically irreversibly preventing response to changes in analytic concentration or reuse. Regeneration of the sensor surface is difficult, at best. On the other hand, a higher dissociation rate which would lend itself to a linear and reusable sensor, results in lower affinity and poor sensitivity. Consequently, most biosensors are disposable devices and quantitation is obtained using multiple single-use sensors. In this work, a new reusable biosensor platform which provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed. Biotin was covalently attached to a transparent indium tin oxide electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions while electrochemical polarization was employed to control the interactions. Two possible mechanisms of the electric field effect involving interactions with induced and permanent dipoles of proteins will be discussed.
A total internal reflection fluorescence (TIRF) flow system was used to detect DNA adsorption and hybridization of a single stranded target DNA with a surface immobilized DNA probe. A transparent SnO2 or indium tin oxide (ITO) film served as a spectroscopic surface in the TIRF flow system and simultaneously as a working electrode for electrochemical (EC) control. The SnO2 and ITO electrodes were chemically modified to provide hydrophilic or hydrophobic surfaces with different functional groups. Charge of the sensor surface was modulated by external electrochemical polarization. Results indicate that DNA oligonucleotides exhibit higher adsorption affinity to positively charged aminated sensor surfaces, while negative polarization stimulates desorption of DNA molecules. To investigate DNA hybridization at the TIRF EC sensor surface, a thiolated probe oligonucleotide, with the complementary sequence to that of the target DNA, was covalently attached to the aminated sensor surface using a heterobifunctional crosslinker. A solution phase 27-base target DNA labeled by fluorescein was exposed to the sensor surface. The kinetics of heterogeneous hybridization between target DNA and surface immobilized probe was studied at different electrode potentials. The results show that cathodic polarization can accelerate hybridization and, at the same time, suppress nonspecific DNA adsorption.
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