Dual-Purpose Aptamer-Based Sensors for Real-Time, Multiplexable Monitoring of Metabolites in Cell Culture Media.

The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in blood and tissues.

Effects of storage conditions on the performance of an electrochemical aptamer-based sensor.

The electrochemical aptamer-based (EAB) sensor platform is the only molecular monitoring approach yet reported that is (1) real time and effectively continuous, (2) selective enough to deploy in the living body, and (3) independent of the chemical or enzymatic reactivity of its target, rendering it adaptable to a wide range of analytes. These attributes suggest the EAB platform will prove to be an important tool in both biomedical research and clinical practice. To advance this possibility, here we have explored the stability of EAB sensors upon storage, using retention of the target recognizing aptamer, the sensor's signal gain, and the affinity of the aptamer as our performance metrics.

The Use of Xenonucleic Acids Significantly Reduces the In Vivo Drift of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision.

Calibration-Free, Seconds-Resolved In Vivo Molecular Measurements using Fourier-Transform Impedance Spectroscopy Interrogation of Electrochemical Aptamer Sensors.

Electrochemical aptamer-based (EAB) sensors are capable of measuring the concentrations of specific molecules in vivo, in real time, and with a few-second time resolution. For their signal transduction mechanism, these sensors utilize a binding-induced conformational change in their target-recognizing, redox-reporter-modified aptamer to alter the rate of electron transfer between the reporter and the supporting electrode. While a variety of voltammetric techniques have been used to monitor this change in kinetics, they suffer from various drawbacks, including time resolution limited to several seconds and sensor-to-sensor variation that requires calibration to remove.

A tight squeeze: geometric effects on the performance of three-electrode electrochemical-aptamer based sensors in constrained, placements.

Electrochemical, aptamer-based (EAB) sensors are the first molecular monitoring technology that is (1) based on receptor binding and not the reactivity of the target, rendering it fairly general, and (2) able to support high-frequency, real-time measurements in the living body. To date, EAB-derived measurements have largely been performed using three electrodes (working, reference, counter) bundled together within a catheter for insertion into the rat jugular. Exploring this architecture, here we show that the placement of these electrodes inside or outside of the lumen of the catheter significantly impacts sensor performance.

Improved calibration of electrochemical aptamer-based sensors.

Electrochemical aptamer-based (EAB) sensors support the real-time, high frequency measurement of pharmaceuticals and metabolites in-situ in the living body, rendering them a potentially powerful technology for both research and clinical applications. Here we explore quantification using EAB sensors, examining the impact of media selection and temperature on measurement performance. Using freshly-collected, undiluted whole blood at body temperature as both our calibration and measurement conditions, we demonstrate accuracy of better than ± 10% for the measurement of our test bed drug, vancomycin.

Elucidating the Mechanisms Underlying the Signal Drift of Electrochemical Aptamer-Based Sensors in Whole Blood.

The ability to monitor drugs, metabolites, hormones, and other biomarkers in situ in the body would greatly advance both clinical practice and biomedical research. To this end, we are developing electrochemical aptamer-based (EAB) sensors, a platform technology able to perform real-time, in vivo monitoring of specific molecules irrespective of their chemical or enzymatic reactivity. An important obstacle to the deployment of EAB sensors in the challenging environments found in the living body is signal drift, whereby the sensor signal decreases over time.

Measuring and Controlling the Local Environment of Surface-Bound DNA in Self-Assembled Monolayers on Gold When Prepared Using Potential-Assisted Deposition.

DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single-crystal bead electrodes under a number of conditions (constant or square-wave potential perturbations in TRIS or phosphate immobilization buffers with and without Cl). The local environment around the fluorophore-labeled DNA tethered to the electrode surface was characterized using in situ fluorescence microscopy during electrochemical measurements as a function of the underlying surface crystallography. Potential-assisted deposition from a TRIS buffer containing Cl created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography.

Electrodepositing DNA Self-Assembled Monolayers on Au: Detailing the Influence of Electrical Potential Perturbation and Surface Crystallography.

The preparation of DNA self-assembled monolayers (SAMs) on single-crystal gold bead electrodes using an applied potential is evaluated with in situ electrochemical fluorescence microscopy. Applying a constant deposition potential or a square-wave potential perturbation during the formation of DNA SAMs is compared for two different modification methods: DNA SAM formation on a clean gold surface followed by alkythiol backfilling (as is typically done in the literature) or via thiol-exchange on an alkylthiol-modified gold surface. DNA SAMs prepared from a chloride-containing deposition buffer were not significantly different when using either square-wave potential perturbation or at a constant applied potential even when considering different surface crystallographies.

Quantifying the Selective Modification of Au(111) Facets via Electrochemical and Electroless Treatments for Manipulating Gold Nanorod Surface Composition.

Manipulating the composition of a mixed alkylthiol self-assembled monolayer (SAM) modified gold surface using both electrochemical and electroless methods is demonstrated. Through the use of fluorophore labeled thiolated DNA and in situ fluorescence microscopy with a gold single crystal bead electrode, a procedure was developed to study and quantify the selective desorption of an alkylthiolate SAM. This method enabled a self-consistent measurement of the removal of the SAM from the 111 surface compared to the 100 surface region at various potentials.