Nanopore biosensor for label-free and real-time detection of anthrax lethal factor

Abstract

We report a label-free real-time nanopore sensing method for the detection of anthrax lethal factor, a component of the anthrax toxin, by using a complementary single-stranded DNA as a molecular probe. The method is rapid and sensitive: sub-nanomolar concentrations of the target anthrax lethal factor DNA could be detected in ∼1 min. Further, our method is selective, which can differentiate the target DNA from other single-stranded DNA molecules at the single-base resolution. This sequence-specific detection approach should find useful application in the development of nanopore sensors for the detection of other pathogens.

Scheme 1. Detection of Pathogens in a Nanopore

The hybridization of the target pathogen gene segment by an unlabeled complementary DNA probe produces current modulation events in the nanopore having significantly different signatures from those of the cDNA probe.

Figure 1

Monitoring the hybridization of the target aLF strand by the cDNA probe. (a) Representative single-channel current recording trace segments, and (b) the corresponding scatter plots of event residence time vs. amplitude. The experiments were performed at +120 mV with the (M113F)₇ αHL pore in a 1 M NaCl solution buffered with 10 mM Tris·HCl (pH 7.5).

Figure 2

Selectivity of the aLF nanopore sensor. The experiments were performed with the (M113F)₇ αHL pore in a buffer solution comprising 1.0 M NaCl and 10 mM Tris·HCl (pH 7.5) at +120 mV in the presence of a 20-base ssDNA (sequence 5′-TCAATATTTAACAATAATCC-3′) as the molecular probe.

Figure 3

Dose–response curve for aLF detection. The experiments were performed with the (M113F)₇ αHL pore at +180 mV in the presence of a 20-base ssDNA (sequence 5′-TCAATATTTAACAATAATCC-3′) as the molecular probe. An asymmetric buffer condition (with 3 M NaCl and 10 mM Tris·HCl (pH 7.5) in the trans compartment, while 0.15 M NaCl and 10 mM Tris·HCl (pH 7.5) in the cis compartment) was used. The event frequency was calculated by dividing the number of long-lived DNA duplex events by the recording time. The concentration of the cDNA probe used was 10 nM.

Figure 4

(a) Effect of incubation on the frequency of the long-lived DNA duplex events and effect of matrix components on the (b) mean residence time and (c) frequency of the aLF-cDNA duplex events. The experiments were performed at +180 mV using the (M113F)₇ αHL pore in a solution comprising 1.0 M NaCl and 10 mM Tris·HCl (pH 7.5). In the experiment with the unincubated aLF sample, the electrolyte solution contained an additional 20-base ssDNA (sequence 5′-TCAATATTTAACAATAATCC-3′) as the molecular probe. The concentrations of HSA, aLF, T₂₀, and cDNA probe used in experiments shown in panels b and c were 10 μg/mL, 100 nM, 100 nM, and 1 μM, respectively.