Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore

Abstract

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

Figure 1

(a) Photograph of the microdevice used for lipid bilayer preparation. Two chambers are separated by a separator with a parylene film. (b) Illustration of the droplet contact method and collection of single molecules of DNA. Immediately after a single molecule of DNA passes through the αHL pore in the lipid bilayer, the separator is slid across to intercept the bilayer.

Figure 2

(a) Schematic illustration of the nanopore filter experiments. After observing 30 blockage signals, the solution in the chamber connected to the recording terminal of a patch-clamp amplifier was collected. Then, the DNA was quantified by qPCR assay. (b) Amplification curves obtained from the filtered target DNA and 10⁶ molecules of the target DNA by real-time PCR assay. Similar C_t values were obtained from these samples. (c) Amplification curves obtained from 10² molecules of the target DNA in different salt solutions. DNA amplification was observed only when using NH₄Cl and MilliQ with the PCR solution. (d) Calibration curves of the C_t value versus the molecular number with 94 mM NH₄Cl and MilliQ with the PCR solution. The C_t value can fit linearly with the natural logarithmic value of the DNA number. The literature value was referenced from N. Jothikumar et al., J. Virol. Methods, 2006.

Figure 3

(a) Amplification curves of the negative control before and after improvements. In the improved situation, there was no DNA amplification after 45 cycles. (b) DNA numbers obtained from controlled experiments. The DNA numbers increased with increasing concentration of DNA added to the droplet. (c) Amplification curves with and without an applied negative transmembrane potential (−180 mV) and negatively charged lipid in the oil. The C_t value were 14.0, 17.9, and 15.6 for the normal, −180 mV, and the negatively charged lipid conditions, respectively.

Figure 4

(a) Schematic illustration of the CBB system. A lipid bilayer is formed by the contact of two water droplets bubbled from glass pipettes on the microscope. (b) Microscopic image during pBLM formation by the CBB method. The diameters of the emulsion and the lipid bilayer were around 50 and 5 μm, respectively. (c) Control data of the nanopore filter experiments using the CBB method. One droplet contained 100 nM (10¹¹ molecules) or 0 M target DNA. 10 min after forming pBLMs, another droplet was collected, and the number of the target DNA was quantified by qPCR assay.

Figure 5

(a) Schematic illustration of the nanopore filter using PNA-DNA duplexes. Because the contaminated DNA is hydrolyzed with PNA, PCR amplification is inhibited. (b) Typical recorded channel current obtained by translocating PNA-DNA duplexes through the αHL nanopore. (c) Number of DNA molecules obtained from controlled experiments with and without 3 nM PNA. (d) Amplification curves and the quantitated DNA numbers for the nanopore-filtered DNA solution and the control experiment with 10 pM DNA and 30 pM PNA. The R² value is 0.9.