ABSTRACT
The probe oligonucleotide (38) was assembled on the Au-electrode, or the Au-quartz crystal, to yield a surface coverage of ca. 2.3×10−11 mole·cm−2 of the sensing probe. Figure 31(A) shows the faradaic impedance spectra (displayed as Nyquist plots) of the sensing interface, curve (a); the sensing surface after interaction with the mutant, (39), 3×10−9 mole·mL−1, curve (b); the sensing interface that includes the double-stranded probe-oligonucleotide/mutant (39) assembly after treatment with polymerase (Klenow fragment) and b-dCTP, curve (c); the resulting surface after treatment with the alkaline phosphatase-avidin conjugate, (41), curve (d); and upon the biocatalyzed precipitation of the insoluble product (12) for 10 minutes and 40 minutes, curves (e) and (f), respectively. The semicircle diameters of the different impedance spectra correspond to the electron transfer resistances at the electrode interface at the different phases of analysis. Using [Fe(CN)6]3−4− as a redox probe, the electron transfer resistance increases from 1.6 kΩ to ca. 4.1 kΩ upon the formation of the double-stranded assembly between the (38)-probe oligonucleotide and (39). This is consistent with the fact that the formation of the double-stranded assembly electrostatically repels the redox label from the electrode
interface, thereby introducing a barrier for interfacial electron transfer. While the treatment of the electrode with polymerase and b-dCTP does not affect the interfacial electron transfer resistance, the association of the hydrophobic alkaline phosphataseavidin conjugate introduces a barrier for electron transfer, Ret≈6.2 kΩ. Biocatalyzed precipitation of (12) onto the electrode insulates the electrode, a process that increases the electron transfer resistance at the electrode. The electron transfer resistance increases to 8.4 kΩ and 16 kΩ upon the precipitation of (12) for 10 and 40 minutes, respectively. Figure 31(B) shows the faradaic impedance spectra corresponding to similar experiments executed with the normal gene (40). It is evident that after the formation of the double-
stranded assembly, Ret=3.9 kΩ, no increase in the electron transfer resistance is observed upon treatment of the surface with alkaline phosphatase-avidin or an attempt to precipitate (12). Thus, the successful analysis of (39) is attributed to the specific polymerase-mediated coupling of b-dCTP to the mutant assembly, resulting in the biocatalytic precipitation of (12). Using this method, the mutant (39) was detected with a sensitivity limit that corresponded to 1.0×10−14 mole·mL−1.
