DNA表面密度的计算.docVIP

DNA表面密度的计算[1] 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. 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. We have quantified the surface density of DNA by taking advantage of the electrostatic attraction of specific redox cations with the nucleotide phosphate backbone. There are two types of interactions for small redox molecules with DNA: electrostatic attraction and intercalation. Redox cations exchange for native counterions associated with the nucleotide phosphate residues of the probe. The amount of redox marker “electrostatically trapped” at the DNA-modified electrode is then determined using chronocoulometry. At saturation coverage of the redox marker, the surface density of the probe is calculated assuming complete charge compensation of the DNA phosphate residues by redox cations. A strong appeal of this approach is that this measurement, unlike measurements using other noncovalent labels, is insensitive to both the base composition and the chain order (single-stranded vs duplex). The measured redox marker is directly proportional to the number of phosphate groups present at the surface. An attractive aspect of chronocoulometry(计时库伦分析法) is that the doublelayer charg