title: Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy.
abstract: The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) x10(12)/cm2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 x 10(12)/cm2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.
authors.affiliation: Electrical Engineering Division, Department of Engineering, University of Cambridge, 9 JJ Thomson Avenue, Cambridge CB3 0FA, UK. sdk25@cam.ac.uk
authors.forename: Simon D
authors.initials: SD
authors.lastname: Keighley
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Pedro
authors.initials: P
authors.lastname: Estrela
authors.forename: Piero
authors.initials: P
authors.lastname: Migliorato
mesh_terms: D000327:Adsorption; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D017097:Electric Impedance; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D008839:Microelectrodes; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D013057:Spectrum Analysis / Q000295:instrumentation* / Q000379:methods; D013194:Staining and Labeling
chemical_list: D015342:DNA Probes; D006046:Gold