ABSTRACT
The thiophosphate-thymine-tagged oligonucleotide (13) was assembled on an Auelectrode. The surface-coverage of the sensing oligonucleotide monolayer was determined by microgravimetric measurements as well as by chronocoulometry [33] using Ru(NH3)63+ as a redox-label to be 1.5×10−11 mole·cm−2. Figure 10(A) shows the impedance features of the electrode interface upon the buildup of the double-stranded assembly on the conductive support, using Fe(CN)63−/ Fe(CN)64− as a redox-probe. While the bare electrode shows the impedance spectrum depicted in curve (a), the assembly of the sensing interface modified with (13), and then the formation of the ds-complex with the analyte (14) and the biotinylated oligonucleotide (15), yield the impedance spectra shown in curves (b) and (c), respectively. The respective semicircle diameters correspond to the interfacial electron-transfer resistances Ret. It can be seen that the electron-transfer resistance increases upon the buildup of the biotinylated oligonucleotide-DNA assembly. For example, for the (13)-functionalized electrode Ret=1.1 kΩ, Ret increases to approximately 2 kΩ upon the association of the complex between (14) and the biotinylated oligonucleotide (15). These results are consistent with the fact that the negative charge associated with the phosphate groups of the different oligonucleotides increases upon the two-step organization of the assembly. This results in the enhanced electrostatic repulsion of the redox probe and introduces higher interfacial electrontransfer resistances. Figure 10B shows the impedance spectra of the bifunctional doublestranded assembly consisting of the target DNA, (14), linked to the sensing interface and the biotinylated oligonucleotide, (15), before (curve c) and after (curve d) interaction with the avidin-HRP conjugate, (16). Upon the association of the avidin-HRP biocatalytic conjugate to the layer, a considerable increase in the electron-transfer resistance is observed due to the partial insulation of the electrode by the proteins. In the presence of H2O2 and the substrate (7), biocatalytic precipitation of the product (8) onto the electrode occurs. This insulates the conductive support, resulting in a very high increase in the electron-transfer resistance (curve e; Ret=17 kΩ). Note the difference in the scales of the Zre and Zim axes of parts A and B of Figure 10. The association of the avidin-HRP conjugate to the oligonucleotide-DNA assembly, and the precipitation of the product, induce an approximately 10-fold increase in the interfacial electron-transfer resistance as compared to that for the changes that occurred upon the formation of the ds-assembly between the sensing interface and the complex between the DNA analyte (14) and the biotin-labeled oligonucleotide (15). It should be noted that the two parameters controlling the sensitivity of the DNA-sensing devices are the time of incubation of the (13)- functionalized monolayer electrode with the complex between the analyte DNA (14) and the biotinylated oligonucleotide (15), and, more important, the time interval used to precipitate the product (8) by the avidin-HRP biocatalytic conjugate. Figure 10(B) (inset) shows the electron-transfer resistance at the sensing interface upon precipitation of the insoluble product at different concentrations of the analyte DNA (14). It is evident that as the bulk concentration of the DNA is lowered, the observed electron-transfer resistance decreases as a result of the precipitation of the insoluble product. This is consistent with the fact that lower bulk concentrations of (14) yield a lower coverage of the (14) and biotin-labeled oligonucleotide complex on the sensing interface. This results in lower coverage of the interface with avidin-HRP (16), and consequently, a decreased efficiency in the deposition of the insoluble product (8) is observed. Using this configuration, and
upon precipitation of (8) for 40 min, we were able to sense (14) at a concentration of 2×10−8 mg·mL−1, Ret=7.9 kΩ. It should be noted
