Redox Reporter - Ligand Competition to Support Signaling in the Cocaine-Binding Electrochemical Aptamer-Based Biosensor.

Electrochemical aptamer-based (E-AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode-bound, redox-reporter-modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding-induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance-dependent change in electron transfer rates in E-AB sensors. In response, we investigate here the signal transduction of the well-studied cocaine-binding aptamer with different analytical methods and found that this sensor relies on a redox-reporter - ligand competition mechanism rather than a ligand-induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine-binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine-binding aptamer, given the prevalence of methylene blue in E-AB sensors, a similar competition-based may occur in other systems.

Finding the Lost Dissociation Constant of Electrochemical Aptamer-Based Biosensors.

Electrochemical aptamer-based (E-AB) biosensors afford real-time measurements of the concentrations of molecules directly in complex matrices and in the body, offering alternative strategies to develop innovative personalized medicine tools. While different electroanalytical techniques have been used to interrogate E-AB sensors (i.e., cyclic voltammetry, electrochemical impedance spectroscopy, and chronoamperometry) to resolve the change in electron transfer of the aptamer's covalently attached redox reporter, square-wave voltammetry remains a widely used technique due to its ability to maximize the redox reporter's faradic contribution to the measured current. Several E-AB sensors interrogated with this technique, however, show lower aptamer affinity (i.e., µM-mM) even in the face of employing aptamers that have high affinities (i.e., nM-µM) when characterized using solution techniques such as isothermal titration calorimetry (ITC) or fluorescence spectroscopy. Given past reports showing that E-AB sensor's response is dependent on square-wave interrogation parameters (i.e., frequency and amplitude), we hypothesized that the difference in dissociation constants measured with solution techniques stemmed from the electrochemical interrogation technique itself. In response, we decided to compare six dissociation constants of aptamers when characterized in solution with ITC and when interrogated on electrodes with electrochemical impedance spectroscopy, a technique able to, in contrast to square-wave voltammetry, deconvolute and quantify E-AB sensors' contributions to the measured current. In doing so, we found that we were able to measure dissociation constants that were either separated by 2-3-fold or within experimental errors. These results are in contrast with square-wave voltammetry-measured dissociation constants that are at the most separated by 2-3 orders of magnitude from ones measured by ITC. We thus envision that the versatility and time scales covered by electrochemical impedance spectroscopy offer the highest sensitivity to measure target binding in electrochemical biosensors relying on changes in electron-transfer rates.

Recent advances in functionalization of plasmonic nanostructures for optical sensing.

This review summarizes the progress that has been made in the use of nanostructured SPR-based chemical sensors and biosensors. Following an introduction into the field, a first large section covers principles of nanomaterial-based SPR sensing, mainly on methods using noble metal nanoparticles (spheres, cubes, triangular plates, etc.). The next section covers methods for functionalization of plasmonic nanostructures, with subsections on functionalization using (a) amino acids and proteins; (b) oligonucleotides, (c) organic polymers, and (d) organic compounds. Several tables are presented that give an overview on the wealth of methods and materials published. A concluding section summarizes the current status, addresses current challenges, and gives an outlook on potential future trends. This review is not intended to be a comprehensive compilation of the literature in the field but rather is a systematic overview of the state of the art in surface chemistry of plasmonic nanostructures. The ability of various ligands and receptors for functionalization of nanoparticles as well as their sensing capability is discussed.