Steel A B, Herne T M, Tarlov M J
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001, USA.
Anal Chem. 1998 Nov 15;70(22):4670-7. doi: 10.1021/ac980037q.
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-compensation 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) x 10(12) 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.
我们开发了一种电化学方法来量化固定在金表面的DNA的表面密度。DNA的表面密度，更具体地说是核苷酸磷酸残基的数量，是根据在电极表面测量的阳离子氧化还原标记物的量来计算的。通过形成硫醇衍生的单链寡核苷酸和6-巯基-1-己醇的混合单层，将DNA固定在金上。使用计时库仑法测定的DNA单层中电荷补偿氧化还原标记物的饱和量与磷酸残基的数量直接成正比，从而与DNA的表面密度成正比。该方法允许在电极上定量测定单链和双链DNA。通过电化学方法，使用混合单层，确定单链DNA的表面密度在(1-10)×10(12)个分子/cm2范围内精确变化。我们测量了固定化单链DNA与互补链的杂交效率作为固定化DNA表面密度的函数，发现随着表面密度的增加，杂交效率呈现最大值。
