Single Voltammetric Sweep Calibration-Free Interrogation of Electrochemical Aptamer-Based Sensors Employing Continuous Square Wave Voltammetry.

Continuous square wave voltammetry (cSWV) is a technique that enables the continuous collection of current data (at 100 kHz) to maximize the information content obtainable from a single voltammetric sweep. This data collection procedure results in the generation of multiple voltammograms corresponding to different effective square wave frequencies. The application of cSWV brings significant benefits to electrochemical aptamer-based (E-AB) sensors. The E-AB sensor platform permits continuous real-time monitoring of small biological molecules. Traditionally, E-AB sensors report only on changes in analyte concentration rather than absolute quantification in matrices when basal concentrations are not known a priori. This is because they exhibit a voltammetric peak current even in the absence of a target. However, using a dual-frequency approach, calibration-free sensing can be performed effectively, eliminating the sensor-to-sensor variation by taking ratiometric current responses obtained at two different frequencies from two different voltammetric sweeps. In employing our approach, cSWV provides a great advantage over the conventionally used square wave voltammetry since the required voltammograms are collected with a single sweep, which improves the temporal resolution of the measurement when considering the current at multiple frequencies for improved accuracy and reduced surface interrogation. Moreover, we show here that using cSWV provides significantly improved concentration predictions. E-AB sensors sensitive to ATP and tobramycin were interrogated across a wide range of concentrations. With this approach, cSWV allowed us to estimate the target concentration, retaining up to an ±5% error of the expected concentration when tested in buffer and complex media.

Microscale, Electrochemical, Aptamer-Based Sensors for Enhanced Small-Molecule Detection at Millisecond Time Scales.

Microscale electrodes offer the advantages of increased mass transport rates, high sensitivity, and rapid measurement capabilities. Fabricating electrochemical aptamer-based (E-AB) sensors on these electrode platforms opens new applications to chemical and biological sensing but has remained challenging due to low signal-to-noise ratios and monolayer instability. In this article, we report the development and characterization of E-AB sensors on a gold microelectrode platform (∼500 nm radius). To overcome the small current response, we modified the electrodes by growing nanostructures via electrodeposition. We interrogated the sensors with two different electroanalytical techniques, square wave voltammetry (SWV) and intermittent pulse voltammetry (IPA), to measure the representative response of an ATP sensor and determine aptamer-target binding and dissociation time scales. We find robust and stable sensor performance with an increased response rate over sensors fabricated on macroscale electrodes. These results demonstrate that sensors developed on this microelectrode platform can be employed for enhanced spatiotemporal resolution measurements in chemical and biological environments.

Development of an Electrochemical, Aptamer-Based Sensor for Dynamic Detection of Neuropeptide Y.

The ability to monitor dynamic changes in neuropeptide Y (NPY) levels in complex environments can have an impact on many fields, including neuroscience and immunology. Here, we describe the development of an electrochemical, aptamer-based (E-AB) sensor for the dynamic (reversible) measurement of physiologically relevant (nanomolar) concentrations of neuropeptide Y. The E-AB sensors are fabricated using a previously described 80 nucleotide aptamer1 reported to specifically bind NPY with a binding affinity Kd = 0.3 ± 0.2 uM. We investigated two redox tag placement locations on the aptamer sequence (terminal vs internal) and various sensor fabrication and interrogation parameters to tune the performance of the resulting sensor. The best-performing sensor architecture displayed a physiologically relevant dynamic range (nM) and low limit of detection and is selective among competitors and similar molecules. The development of this sensor accomplishes two breakthroughs: first, the development of a nonmicrofluidic aptamer-based electrochemical sensor that can detect NPY on a physiologically relevant (seconds to minutes) time scale and across a relevant concentration range; second, the expansion of the range of molecules for which an electrochemical, aptamer-based sensor can be used.

Continuous Square Wave Voltammetry for High Information Content Interrogation of Conformation Switching Sensors.

Square wave voltammetry (SWV) is a voltammetric technique for measuring Faradaic current while minimizing contributions from non-Faradaic processes. In square wave voltammetry, the potential waveform applied to a working electrode and the current sampling protocols followed are designed to minimize contributions from non-Faradaic processes (i.e., double layer charging) to improve voltammetric sensitivity. To achieve this, the current is measured at the end of each forward and reverse potential pulse after allowing time for non-Faradaic currents to decay exponentially. A consequence of sampling current at the end of a potential pulse is that the current data from the preceding time of the potential pulse are discarded. These discarded data can provide information about the non-Faradaic contributions as well as information about the redox system including charge transfer rates. In this paper, we introduce continuous square wave voltammetry (cSWV), which utilizes the continuous collection of current to maximize the information content obtainable from a single voltammetry sweep eliminating the need for multiple scans. cSWV enables acquiring a multitude of voltammograms corresponding to various frequencies and, thus, different scan rates from a single sweep. An application that benefits significantly from cSWV is conformation switching, functional nucleic acid sensors. We demonstrate the utility of cSWV on two representative small molecules targeting electrochemical, aptamer-based sensors. Moreover, we show that cSWV provides comparable results to those obtained from traditional square wave voltammetry, but with cSWV, we are able to acquire dynamic information about the sensor surfaces enabling rapid calibration and optimization of sensing performance. We also demonstrate cSWV on soluble redox markers. cSWV can potentially become a mainstay technique in the field of conformation switching sensors.