Rapid Two-Millisecond Interrogation of Electrochemical, Aptamer-Based Sensor Response Using Intermittent Pulse Amperometry

Abstract

In this manuscript, we employ the technique intermittent pulse amperometry (IPA) to interrogate equilibrium and kinetic target binding to the surface of electrochemical, aptamer-based (E-AB) sensors, achieving as fast as 2 ms time resolution. E-AB sensors comprise an electrode surface modified with a flexible nucleic acid aptamer tethered at the 3'-terminus with a redox-active molecule. The introduction of a target changes the conformation and flexibility of the nucleic acid, which alters the charge transfer rate of the appended redox molecule. Typically, changes in charge transfer rate within this class of sensor are monitored via voltammetric methods. Here, we demonstrate that the use of IPA enables the detection of changes in charge transfer rates (i.e., current) at times <100 μs after the application of a potential pulse. Changes in sensor current are quantitatively related to target analyte concentration and can be used to create binding isotherms. Furthermore, the application of IPA enables rapid probing of the electrochemical surface with a time resolution equivalent to as low as twice the applied potential pulse width, not previously demonstrated with traditional voltammetric techniques employed with E-AB sensors (alternating current, square wave, cyclic). To visualize binding, we developed false-color plots analogous to those used in the field of fast-scan cyclic voltammetry. The use of IPA is universal, as demonstrated with two representative small molecule E-AB sensors directed against the aminoglycoside antibiotic tobramycin and adenosine triphosphate (ATP). Intermittent pulse amperometry exhibits an unprecedented sub-microsecond temporal response and is a general method for measuring rapid sensor performance.

Keywords: aptamers; binding kinetics; electrochemistry; intermittent pulse amperometry; sensors.

Figure 1.

Intermittent pulse amperometry (IPA) involves (top left) the application of repetitive potential pulses to the working electrode, alternating between a potential sufficient to reduce/oxide a redox-active molecule and a potential to reversibly oxidize/reduce the molecule (i.e. either side of the standard electrode potential E°). (Bottom left) The resulting continuous amperometric time trace is represented as a series of forward and reverse current pulses. In sampling E-AB sensors, we sample current at a given time after the pulse (dt). (Top right) If the rates of charge transfer (k_et) differ (e.g., free aptamer without or with the target), the current sampled (Di) will be different, thus provides the basis for signal transduction of a target binding. (Bottom right) A plot of sampled-current vs. time yields a “sampled” current-time, or amperometric trace.

Figure 2.

Comparison of the amperometric current-time trace with and without target (tobramycin) reveals a difference in absolute current measured. (Left) The current-time response using a representative forward pulse of a tobramycin E-AB sensor with and without 1 mM tobramycin present exhibits notable changes in current. These data represent the current-time response with the application of a potential pulse from 0.0 V to −0.4 V (vs. Ag/AgCl) at dt = 0. (Right) Current plotted on a log-log scale better illustrates the difference in current with and without target analyte. The current-time response in both cases appears to follow a double exponential decay.

Figure 3.

Differences in current are observed at both the forward and reverse potential pulse. Current response after both a (top) forward and (bottom) reverse pulse show a different response to the presence and absence of target analyte. E-AB sensors for the detection of tobramycin show marked differences in absolute current and decay rates for both (top) forward (−0.4V vs. Ag/AgCl) and (bottom) reverse (0.0 V vs. Ag/AgCl) pulses. Plots on the right show the zoomed-in regions of interest from the pulses on the left, where this difference in current is most apparent.

Figure 4.

Amperometric current response is quantitatively related to the concentration of tobramycin present, and the magnitude and polarity of that response is a function of when the current is measured after the application of a pulse (dt) for both forward (left) and reverse (right) pulses. (Top) Evaluation of current after a potential pulse shows a significant signal change in times as fast as 50 μs upon target addition, with the most significant changes occurring at 84 μs after the potential pulse. (Middle) Zoom-ins of the shaded regions show the sampling times that give greatest percent signal change. (Bottom) Plotting percent signal change at different dt values creates quantitative binding curves that can be fitted to Langmuir-like isotherms for quantitation of target concentrations. The current was sampled at three dt values, marked as dashed lines in the top plots.

Figure 5.

The use of IPA interrogation is a general means of probing E-ABs. (Left) ATP-specific E-AB sensor interrogated using IPA exhibited sensitive target-induced responses within tens of μs timeframe. Changes in current are dependent on ATP concentration as well as dt with an optimal sensor response at dt values between 10 – 140 μs. (Right) Data obtained at multiple dt values (dashed lines on the left plot) fit Langmuir-like isotherms for quantitative analysis of ATP concentration.

Figure 6.

False color plots allow visualization of IPA current response with the addition of target analyte (tobramycin and ATP). 2D and 3D color plots (zoom-in region from false color plots) align each forward pulse along the x-axis, dt along the y-axis, and the change in current (D Current) in the out of plane, or z-axis. Δ Current is plotted as positive (signal-on) for both ATP and Tobramycin (where the sign of the Tobramycin change has been inverted), so that the two sensors can more be more easily compared. Line scans, indicated by the white dashed lines, provide sampled-current time traces with 2 ms time resolution or box-car averaged to yield 10 ms time resolution. Consistent with equilibrium binding data, the sensitivity of the measurement is a function of which dt value is used.

Figure 7.

E-AB sensor response follows a single exponential rise as illustrated by sampled-current time traces after the addition of 200 μM tobramycin (left) and ATP (right). Tobramycin sensors respond with a k_obs = 0.72 ± 0.04 s⁻¹ whereas the ATP sensor responds in about half the time with a k_obs = 1.4 ± 0.4 s⁻¹. Data shown are boxcar averaged and thus represent 10 ms/point time resolution.

Scheme 1.

Electrochemical, aptamer-based sensors employ target-induced conformational changes of a redox active-labeled aptamer sequence to quantitatively report on the presence of a specific molecular analyte. Changes in charge transfer rates (k_et) between the redox moiety with (k’_et) and without (k_et) target lead to changes in measured current.