The development of structure-switching electrochemical, aptamer-based sensors over the past ~10 years has lead to a variety of reagentless sensors capable of analytical detection in a range of sample matrices. The crux of this methodology is the coupling of target-induced conformation changes of a redox-labeled aptamer with electrochemical detection of the resulting altered charge transfer rate between the redox molecule and electrode surface. Using aptamer recognition expands the highly sensitive detection ability of electrochemistry to a range of previously inaccessible analytes. In this review, we focus on the methods of sensor fabrication and how sensor signaling is affected by fabrication parameters. We then discuss the fundamentals of sensor signaling, as well as quantitative characterization of the analytical performance of electrochemical, aptamer-based sensors. Using illustrative examples, we highlight recent advances in the field that impact important areas of analytical chemistry. Finally, we discuss challenges and prospects for this class of sensors.

Aptamers have emerged as promising biorecognition elements in the development of electrochemical-based sensors. Aptamers, short oligonucleotides of DNA or RNA selected in vitro to bind a specific target, boast comparable binding affinities to antibodies, but possess superior chemical and biochemical stabilities. In addition, they require relatively simple synthesis protocols compared to the in vivo development of antibodies. Coupling the specific recognition abilities of aptamers with the selective and sensitive detection abilities of electrochemical signal transduction enables an almost unlimited number of strategies for generating biosensor architectures. Here we present a critical review of electrochemical sensing strategies employing aptamers through a presentation of the various signal transduction mechanisms employed. We find that, while there are numerous examples of aptamer-based sensors, the penetration of these platforms beyond the academic laboratory and proof-of-concept remains limited. We believe that aptamers will continue to be a focus in the development electrochemical sensors, although the limited number of proven aptamers hinders the progress of electrochemical, aptamer-based sensors from impacting fields beyond the electrochemical laboratory. However, considering the historical success of electrochemical-based sensors as well as the promise of selecting aptamers for virtually any target, we believe the future is bright for aptamers in electroanalysis and beyond.Electrochemistry has maintained a strong presence in the field of chemical and biological sensors. 1-6 This is because electrochemical readout mechanisms can provide a selective way to quantify the interaction between a recognition element and a target analyte. One of the most prolific examples of an electrochemical sensor is the glucose sensor, which incorporates an immobilized enzyme as a specific biorecognition element. The enzymatic conversion of glucose is monitored amperometrically using a wide variety of detection schemes (for a review see ref. 4). Regardless of the scheme, biology provides the specific recognition abilities of this sensing platform because the enzyme, glucose oxidase for example, has evolved to react only with glucose. Electrochemical readout provides the selective measurement capabilities. The known redox potential of the product or mediator and the relatively few electrochemically active interferents in biological medium allow this detection to be performed directly in undiluted human blood. 4 The numerous examples of enzyme-based glucose sensors demonstrate the utility of coupling specific recognition with electrochemical signal transduction.In addition to the enzyme-based sensor described above, electrochemical detection has seen widespread use in endless examples of chemical and biosensors. For example, electrochemical readout is utilized in the development of enzyme-linked immunosorbent assays or ELISAs. In this class of sensors, the specificity of antibody-antigen interactions provides target recognition, ...

The utility of stochastic single-molecule detection using protein nanopores has found widespread application in bioanalytical sensing as a result of the inherent signal amplification of the resistive pulse method. Integration of protein nanopores with high-resolution scanning ion conductance microscopy (SICM) extends the utility of SICM by enabling selective chemical imaging of specific target molecules, while simultaneously providing topographical information about the net ion flux through a pore under a concentration gradient. In this study, we describe the development of a bioinspired scanning ion conductance microscopy (bio-SICM) approach that couples the imaging ability of SICM with the sensitivity and chemical selectivity of protein channels to perform simultaneous pore imaging and specific molecule mapping. To establish the framework of the bio-SICM platform, we utilize the well-studied protein channel α-hemolysin (αHL) to map the presence of β-cyclodextrin (βCD) at a substrate pore opening. We demonstrate concurrent pore and specific molecule imaging by raster scanning an αHL-based probe over a glass membrane containing a single 25-μm-diameter glass pore while recording the lateral positions of the probe and channel activity via ionic current. We use the average channel current to create a conductance image and the raw current–time traces to determine spatial localization of βCD. With further optimization, we believe that the bio-SICM platform will provide a powerful analytical methodology that is generalizable, and thus offers significant utility in a myriad of bioanalytical applications.

The utility of biological nanopores for the development of sensors has become a growing area of interest in analytical chemistry. Their emerging use in chemical analysis is a result of several ideal characteristics. First, they provide reproducible control over nanoscale pore sizes with an atomic level of precision. Second, they are amenable to resistive-pulse type measurement systems when embedded into an artificial lipid bilayer. A single binding event causes a change in the flow of millions of ions across the membrane per second that is readily measured as a change in current with excellent signal-to-noise ratio. To date, ion channel-based biosensors have been limited to well-behaved proteins. Most demonstrations of using ion channels as sensors have been limited to proteins that remain in the open, conducting state, unless occupied by an analyte of interest. Furthermore, these proteins are nonspecific, requiring chemical, biochemical, or genetic manipulations to impart chemical specificity. Here, we report on the use of the pore-forming abilities of heat shock cognate 70 (Hsc70) to quantify a specific analyte. Hsc70 reconstitutes into phospholipid membranes and opens to form multiple conductance states specifically in the presence of ATP. We introduce the measurement of “charge flux” to characterize the ATP-regulated multiconductance nature of Hsc70, which enables sensitive quantification of ATP (100 μM–4 mM). We believe that monitoring protein-induced charge flux across a bilayer membrane represents a universal method for quantitatively monitoring ion-channel activity. This measurement has the potential to broaden the library of usable proteins in the development of nanopore-based biosensors.

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The development of a hybrid, tri-catalytic architecture is demonstrated by immobilizing MWCNTs, TEMPO-modified linear poly(ethylenimine) and oxalate decarboxylase on an electrode to enable enhanced electrochemical oxidation of glycerol. This immobilized, hybrid catalytic motif results in a synergistic 3.3-fold enhancement of glycerol oxidation and collects up to 14 electrons per molecule of glycerol.

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