FIGURE 11.13 Schematic showing analyte capture on the trans side of the a-hemolysin pore. A +200 mV forward potential is used to capture the probe in the pore; probe capture is observable as a decrease in current to ~25% of the open channel current. The potential is then reduced to +10 mV, and probe exit is prevented by bound analyte. The potential is then reversed to —60 mV. The blockade duration (toff) lasts until the probe and analyte dissociate under the applied force is measured. Dissociation event durations provide the identifying parameter in this detection method. (From Nakane, J., Wiggin, M., and Marziali, A., Biophys. J., 87, 615, 2004. With permission.)

Nakane and colleagues23 used a 65-nucleotide probe DNA to detect oligomers on the trans side of the pore (Figure 11.13). The DNA probe extended through the channel so that the last 14 nucleotides extended out from the trans side. A 200 mV potential was used to capture the probe, and the potential was lowered to 10 mV to determine whether the analyte had associated with the probe. The potential was then reversed and ramped from —30 to —90 mVor until the analyte dissociated and the probe exited the pore. The durations of the current blockades were proportional to the stability of the hybridized region, such that a single mismatch in the strand could be distinguished.

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