We have previously described a reagentless, electrochemical DNA sensor (termed E-DNA) that is comprised of an electrode-immobilized stem-loop oligonucleotide [1,2]. The DNA probe, immobilized on a gold surface via thiol self-assembly, is tagged with a redox reporter at its 3' terminus which readily transfers electrons to the gold electrode in the absence of complementary target oligonucleotides. In the presence of target, the oligonucleotide target hybridizes to the loop, lifting the reporter tag away from the surface and inhibiting electron transfer.

In an effort to elucidate the sensing mechanism of the E-DNA sensor and to determine which sensor construct is optimal for such a surface-immobilized oligonucleotide sensor, we have characterized E-DNA sensors built using linear and stem-loop probe geometries of various lengths. We have also varied the location of our reporter element (Methylene Blue) on the probe to determine how the location of the redox tag affects signaling.

The results from these six E-DNA constructs indicate a distinction in sensor behavior not only between the linear and stem-loop designs, but also in the location of the reporter tag. Our results indicate that for example, a shorter linear E-DNA probe sequence gives greater signal gain upon target detection versus a probe that is twice as long, and that moving the Methylene Blue redox label from the 3'-terminal to the center of the E-DNA sequence, in both the linear and stem-loop constructs, greatly improves the sensor's behavior with the internally-labeled linear probe performing the best (ie: increased signal gain and faster equilibration times).

The sensors are all easily regenerated (with a water rinse), respond to nanomolar target concentrations and can be employed in contaminant-rich samples such as serum. The results of this comparative study will help guide future designs and applications of the E-DNA sensor.

References:

1. Fan, C.; Plaxco, K. W.; Heeger, A. J. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA. Proc. Natl.Acad. Sci 2003, 100, 9134-9137.

2. Lubin, A.A., Lai, R.Y., Baker, B.R., Heeger, A.J. and Plaxco, K.W. The sequence-specific, electronic detection of oligonucleotides in blood, soil and foodstuffs with the reagentless, reusable E-DNA sensor. Anal. Chem., 2006, 78, 5671-5677.

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