Electrochemical Quantitation of DNA Immobilized on Gold

We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensating redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1−10) × 1012 molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a function of the immobilized DNA surface density and found that it exhibits a maximum with increasing surface density.