DNA sensing by electrocatalysis with hemoglobin
Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required high-density DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.
© 2012 by the National Academy of Sciences. Edited by Royce W. Murray, University of North Carolina at Chapel Hill, Chapel Hill, NC, and approved May 22, 2012 (received for review March 23, 2012). Published online before print June 25, 2012. This research was supported by the National Institute of Health (GM61077) and ONR (N00014-09-1-1117). We would like to thank Donald S. Clark for an undergraduate fellowship to L.F.G. The authors thank N. Muren for discussions and contributions in fabricating the multiplexed chips. This work was completed in part in the Caltech Micro/Nano Fabrication Laboratory. Author contributions: C.G.P. and J.K.B. designed research; C.G.P. and L.F.G. performed research; C.G.P., L.F.G., and J.K.B. analyzed data; and C.G.P. and J.K.B. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201551109/-/DCSupplemental.
Published - Pheeney2012p19089P_Natl_Acad_Sci_Usa.pdf
Supplemental Material - pnas.1201551109_SI.pdf