Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination

Abstract

Nanopores are increasingly utilized as tools for single-molecule detection in biotechnology. Many nanopores are fabricated through procedures that require special materials, expensive facilities and experienced operators, which limit their usefulness on a wider scale. We have developed a simple method of fabricating a robust, low-noise nanopore by externally penetrating a nanocavity enclosed in the terminal of a capillary pipet. The nanocavity was shown to have a pore size on the scale of a single molecule, verified by translocation of molecules of known sizes, including double-stranded DNA (2 nm), gold nanoparticles (10 nm), and ring-shaped cyclodextrin (1.5 nm). The small pore size allows entrapment of a single cyclodextrin molecule. The glass nanopore with a trapped cyclodextrin proves useful in single-molecule discrimination of chiral enantiomers.

Figure 1

Nanopore fabrication and electrical properties. a. Sealed glass ball (100 μm in radius) enclosing a nanocavity on the pipette terminal. b. External etching from bottom-up with electrical monitoring. c. Perforation on nanocavity terminal and nanopore formation. d. 20 min recording of nanopore current (+200 mV, 1 M NaCl).

Figure 2

Detection of nanocavity profile. a. SEM image of a nanopore-templated polydimethylsiloxane (PDMS) nanowire coated with a 3-nm platinum layer. The liquid PDMS, which has been vacuumed to remove the bubbles, was injected into the micropipette, followed by spinning in a microcentrifuge at 10,000 rpm for 2 minutes to deliver the polymer to the cavity end. After curing at 90 °C for 4 hours, the terminal glass was carefully removed manually to form a cavity-templated PDMS. b. Etching curve (g—t curve). The pipette was filled with 1 M NaCl (pH7.0) and etched in a bath containing 40% NH4F: 49% HF (30:1 v/v). When perforated, each pipette was transferred to a 1 M NaCl (pH7.0) solution every 5 to 10 s to determine the conductance at +20 mV. The unfilled symbols represent conductance of pores made from six independent nanocavities under standard fabrication processes (see text). Filled circles were mean conductance. Error bars represent standard deviation. Polynomial fitting of mean conductance yielded a continuous mean etching curve (black curve). c. The profile of the nanocavity (D—h curve) calculated from the etching curve (panel b), using Eq.1 in the text. The conductivity (K) of 1 M NaCl at 22 °C was 7.8 Sm^-1. d. Magnified profile of the narrow end of the nanocavity. This section resembled a conical shape with an aperture of 20°, as marked by the dashed line.

Figure 3

Translocation of dsDNA in nanopores. a. Current blocks by 1 kb DNA (10 nM) in the external solution (1 M NaCl) through a 3.8 nS nanopore (+100 mV). b. Current blocks with DNA through a 39 nS nanopore (+100 mV). c. Reveals DNA translocation by PCR. Lane-1, marker; Lane-2, 10 μl bath solution (containing DNA); Lane-3, 10 μl dd-H₂O; Lane-4, 10 μl internal solution without DNA (control); Lane-5, 10 μl internal solution near nanopore after the DNA translocation. d. Voltage-dependent duration of DNA translocation. e. Voltage-dependent occurrence of DNA translocation.

Figure 4

Translocation of Au-NP through nanopore. Recording solutions inside and outside nanopore contained 15 mM NaCl. Au-NP of 10 nm in diameter (4.75 nM) was presented in external solution. Current was monitored at +150 mV. a, Current blocks in a 5.9 nS nanopore and b, a 13 nS nanopore.

Figure 5

Translocation of βCDs in nanopore. a. Voltage-dependent current profile in the presence of 100 μM βCD in a 2.4 nS nanopore. b. Expanded current trace from a +1 V domain in a, showing stochastic translocation of individual βCD molecules. c. Expanded current trace from a -1 V domain in a, showing fewer translocation events compared with +1 V in b.

Figure 6

Chiral-discrimination by cyclodextrin trapped in nanopore. a. Current trace showing trapping and release of single βCD molecules (100 μM) in a 1 nS nanopore. b. Diagram showing single-molecule discrimination of chiral enantiomers with the cyclodextrin trapped in nanopore. c. Current profiles showing the binding of individual enantiomers of catechin to the βCD. The three traces were recorded for 50 μM 2S,3R-(-)-catechin (top), 50 μM 2R,3S-(+)-catechin (middle) and a mixture of 50 μM (-)-catechin and 50 μM (+)-catechin (bottom). D. Current profiles showing the binding of individual enantiomers of ibuprofen to the trapped βCD. The three traces were for 100 μM R-(-)-ibuprofen (top), 100 μM S-(+)-ibuprofen (middle) and a mixture 100 μM R-(-)-ibuprofen and 100 μM S-(+)-ibuprofen (bottom). The current block levels by R- and S-ibuprofen were marked with dash lines.