Probing nanopore surface chemistry through real-time measurements of nanopore conductance response to pH changes

Abstract

We developed a flow cell apparatus and method for streamlined, real-time measurements of nanopore conductance (G) in response to pH changes. By time-resolving the measurements of interfacial kinetics, we were able to probe nanopore surface coating presence and properties more thoroughly than in our previous work. Nanopores have emerged as a prominent tool for single-molecule sensing, characterization, and sequencing of DNA, proteins, and carbohydrates. Nanopore surface chemistry affects analyte passage, signal characteristics, and sensor lifetime through a range of electrostatic, electrokinetic, and chemical phenomena, and optimizing nanopore surface chemistry has become increasingly important. Our work makes nanopore surface chemistry characterizations more accessible as a complement to routine single-pH conductance measurements used to infer nanopore size. We detail the design and operation of the apparatus and discuss the trends in G and capacitance. Characteristic G vs pH curves matching those obtained in previous work could be obtained with the addition of time-resolved interfacial kinetic information. We characterized native and chemically functionalized (carboxylated) silicon nitride (SiNx) nanopores, illustrating how the method can inform of thin film compositions, interfacial kinetics, and nanoscale chemical phenomena.

FIG. 1.

Plots of four different nanopore surfaces characterized by G vs pH. (a) Carboxylic acid-terminated SiN_x, (b) hydroxyl-terminated SiN_x, (c) amine-terminated SiN_x, and (d) native SiN_x surfaces. These <10 nm diameter nanopores were profiled in pH ≈ 7, 1.0M KCl solutions, with data taken from Ref. .

FIG. 2.

(a) 3D printed polymethacrylate (PMA) nanopore flow cell. (b) The transparent schematic shows the interior fluid channels and volumes. (1) Twist-fit connection ports, (2) threaded electrode ports, (3) silicon nanopore support wafer, (4) silicone gaskets, (5) alignment guides, (6) flow channels, (7) screw through-holes, (8) nanopore sealing surfaces, (9) nanopore sensing volume, (10) fluid inlet, (11) fluid outlet, and (12) resin drainage hole.

FIG. 3.

(a) Diagram of the fluid handling apparatus. The black lines indicate fluid-filled lines, the green lines indicate vacuum or suction lines, and the blue lines indicate pressurized air lines. Four syringes were used in a synchronized push–pull configuration to drive fluid flow. Fluid and air lines to each half of the apparatus were of equal length to ensure synchronized arrival times. (b) Image of apparatus in an open Faraday cage (top-down image). The air-filled drive lines are shown exiting the Faraday cage at the top of the image, while all fluid-filled components are housed within the Faraday cage.

SCHEME 1.

Flow chart of the streamlined nanopore characterization implementation. The flow is driven at 0.5 mL/min through both halves of the flow cell simultaneously (only one half is shown). The current is measured over the last ∼50 min of immersion without flow while the voltage is cycled. This was repeated across a range of pH values. The first second of data from each voltage state was omitted from the conductance analysis because of the capacitive spike. The conductance of the pore for each 26 s voltage cycle was calculated using one second of current data from each voltage state and performing a linear fit. This calculation was repeated for all iterations of the voltage cycle for each pH. These data, in series, then marked the time evolution of the pore conductance at each pH.

FIG. 4.

Conductance vs pH plots from measurements averaged over 10-minute windows (red: 10–20 min and black: 50–60 min) for (a) a native SiN_x nanopore (16.81 ± 0.04 nm diameter) and (b) a 4-pentenoic acid-functionalized nanopore (9.49 ± 0.02 nm diameter). The characterizations were performed in 0.1 M KCl with an acquisition rate of 10 kHz and a low-pass Bessel filter set to 1 kHz.

FIG. 5.

Conductance vs time plots at three representative pH values for (a) a native SiN_x nanopore (16.81 ± 0.04 nm diameter) and (b) a 4-pentenoic acid functionalized nanopore (9.49 ± 0.02 nm diameter). σ denotes the surface charge. Each data point represents the mean of the conductance values calculated over 10-min windows of each pH measurement. The error bars represent the standard deviation of the conductance calculations over the 10-min windows. The shaded region shows the first ten minutes of the measurement where electrolyte was being flowed to exchange for water. Measurements were carried out in 0.1M KCl with an acquisition rate of 10 kHz and a low-pass Bessel filter set to 1 kHz.

FIG. 6.

Positive-going capacitive decay fit time constant, τ, vs pH for two nanopore surfaces over the 10- to 20-min window (red) and the final 50- to 60-min window (black) across a range of pHs for (a) a native SiN_x nanopore and (b) a 4-pentenoic-acid-coated nanopore. Each data point is the average of 10 min of voltage cycles from 0 to +100 mV or 23 positive voltage capacitive spike time constants. The error bars represent the standard deviation over each window. The measurements were performed in 0.1M KCl with an acquisition rate of 10 kHz and a low-pass Bessel filter set to 1 kHz.