Capturing single molecules of immunoglobulin and ricin with an aptamer-encoded glass nanopore

Abstract

Nanopore-based single-molecule biosensors have been extensively studied. Protein pores that have receptors attached to them are target-selective, but their real-time applications are limited by the fragility of the lipid membrane into which the protein pores are embedded. Synthetic nanopores are more stable and provide flexible pore sizes, but the selectivity is low when detecting in the translocation mode. In spite of modifications with probing molecules, such as antibodies, to potentiate specific targeting, these nanopores fail to bind individual target molecules. Distinguishing between binding and translocation blocks remains unsolved. Here, we propose an aptamer-encoded nanopore that overcomes these challenges. Aptamers are well-known probing oligonucleotides that have high sensitivity and selectivity. In contrast to antibodies, aptamers are much smaller than their targets, rendering target blockades in the nanopore much more distinguishable. We used aptamer-encoded nanopores to detect single molecules of immunoglobulin E and the bioterrorist agent ricin, sequentially captured by the immobilized aptamer in the sensing zone of the pore. The functional nanopore also probed sequence-dependent aptamer-protein interactions. These findings will facilitate the development of a universal nanopore for multitarget detection.

Figure 1

Fabrication of the nanopore. a. Microscopic image of a sealed micropipette tip enclosing the nanocavity. b. Cartoon depicting the external etching of the pipette tip. c. Perforation of nanocavity to form a nanopore.

Figure 2

Capture of single IgE molecules by immobilized aptamers in the nanopore. The voltage was given from the electrode in the pipette and the external bath was grounded. a. Current in a 63 nm A_IgE-encoded nanopore at +100 mV without IgE in the pipette. b. Current through the same pore as in a, with 5 nM IgE in the pipette. c. Sensing zone at the narrow opening of the nanopore. d. The detection time of each sequentially-occurring stepwise block in b. e. Amplitudes of conductance change for stepwise blocks.

Figure 3

Selectivity of aptamer-encoded nanopores. a. Current for 5 nM IgG in an 53 nm A_IgE-modified nanopore at +100 mV. b. Current for 5 nM IgE in a 57 nm ΔC20 (mutant of A_IgE)-modified nanopore at +100 mV.

Figure 4

Detection of IgE in the external solution with an A_IgE-modified nanopore. a. A model showing the detection of protein presented in the exterior solution (left) and the expected current trace (right). b. Current in an 55 mm A_IgE-encoded nanopore at −100 mV with 5 nM IgE in the external solution. c. Nanopore current for addition of 5 nM IgE in the pipette used in panel a. d. The detection time interval between the first and the second IgE block (t₁− t₂) in various IgE concentrations ([IgE]).

Figure 5

Single-molecule detection of ricin A-chain protein in the external solution using an A_ricin-encoded nanopore. a. Current in a 56 nm A_Ricin-encoded nanopore at −100 mV, with 100 nM ricin A-chain protein in the external solution. b. The detection time interval between the first and the second ricin block (t₁− t₂) in various concentrations of ricin A-chain protein.