Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo.

The measurement of serum vancomycin levels at the clinic is critical to optimizing dosing given the narrow therapeutic window of this antibiotic. Current approaches to quantitate serum vancomycin levels are based on immunoassays, which are multistep methods requiring extensive processing of patient samples. As an alternative, vancomycin-binding electrochemical, aptamer-based sensors (E-ABs) were developed to simplify the workflow of vancomycin monitoring. E-ABs enable the instantaneous measurement of serum vancomycin concentrations without the need for sample dilution or other processing steps. However, the originally reported vancomycin-binding E-ABs had a dissociation constant of 45 µM, which is approximately 1 order of magnitude higher than the recommended trough concentrations of vancomycin measured in patients. This limited sensitivity hinders the ability of E-ABs to accurately support vancomycin monitoring. To overcome this problem, here we sought to optimize the length of the vancomycin-binding aptamer sequence to enable a broader dynamic range in the E-AB platform. Our results demonstrate, via isothermal calorimetry and E-AB calibrations in undiluted serum, that superior affinity and near-equal sensor gain in vitro can be achieved using a one-base-pair-longer aptamer than the truncated sequence originally reported. We validate the impact of the improved binding affinity in vivo by monitoring vancomycin levels in the brain cortex of live mice following intravenous administration. While the original sequence fails to resolve vancomycin concentrations from baseline noise (SNR = 1.03), our newly reported sequence provides an SNR of 1.62 at the same dose.

High frequency, real-time neurochemical and neuropharmacological measurements in situ in the living body.

The beautiful and complex brain machinery is perfectly synchronized, and our bodies have evolved to protect it against a myriad of potential threats. Shielded physically by the skull and chemically by the blood brain barrier, the brain processes internal and external information so that we can efficiently relate to the world that surrounds us while simultaneously and unconsciously controlling our vital functions. When coupled with the brittle nature of its internal chemical and electric signals, the brain's "armor" render accessing it a challenging and delicate endeavor that has historically limited our understanding of its structural and neurochemical intricacies. In this review, we briefly summarize the advancements made over the past 10 years to decode the brain's neurochemistry and neuropharmacology in situ, at the site of interest in the brain, with special focus on what we consider game-changing emerging technologies (eg, genetically encoded indicators and electrochemical aptamer-based sensors) and the challenges these must overcome before chronic, in situ chemosensing measurements become routine.

Fluorescence-Based Observation of Transient Electrochemical and Electrokinetic Effects at Nanoconfined Bipolar Electrodes.

Bipolar electrodes (BPEs) are conductors that, when exposed to an electric field, polarize and promote the accumulation of counterionic charge near their poles. The rich physics of electrokinetic behavior near BPEs has not yet been rigorously studied, with our current understanding of such bipolar effects being restricted to steady-state conditions (under constant applied fields). Here, we reveal the dynamic electrokinetic and electrochemical phenomena that occur near nanoconfined BPEs throughout all stages of a reaction. Specifically, we demonstrate, both experimentally and through numerical modeling, that the removal of an electric field produces solution-phase charge imbalances in the vicinity of the BPE poles. These imbalances induce intense and short-lived nonequilibrium electric fields that drive the rapid transport of ions toward specific BPE locations. To determine the origin of these electrokinetic effects, we monitored the movement and fluorescent behavior (enhancement or quenching) of charged fluorophores within well-defined nanofluidic architectures via real-time optical detection. By systematically varying the nature of the fluorophore, the concentration of the electrolyte, the strength of the applied field, and oxide growth on the BPE surface, we dissect the ion transport events that occur in the aftermath of field-induced polarization. The results contained in this work provide new insights into transient bipolar electrokinetics that improve our understanding of current analytical platforms and can drive the development of new micro- and nanoelectrochemical systems.

High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors.

The electrochemical, aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific molecular targets (irrespective of their chemical reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model we employ, entails devices less than 200 µm in diameter. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response we have developed an electrochemical roughening approach that enhances the signaling of small electrochemical sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, our method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufacturing. Using these high surface area electrodes, we demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the determination of drug disposition parameters.

Quantitative electrochemical metalloimmunoassay for TFF3 in urine using a paper analytical device.

We report a paper-based assay platform for the detection of the kidney disease marker Trefoil Factor 3 (TFF3) in human urine. The sensor is based on a quantitative metalloimmunoassay that can determine TFF3 concentrations via electrochemical detection of environmentally stable silver nanoparticle (AgNP) labels attached to magnetic microbeads via a TFF3 immunosandwich. The paper electroanalytical device incorporates two preconcentration steps that make it possible to detect concentrations of TFF3 in human urine at the low end of the target TFF3 concentration range (0.03-7.0 µg mL(-1)). Importantly, the paper device provides a level of accuracy for TFF3 determination in human urine equivalent to that of a commercial kit. The paper sensor has a dynamic range of ∼2.5 orders of magnitude, only requires a simple, one-step incubation protocol, and is fast, requiring only 10 min to complete. The cost of the materials at the prototypic laboratory scale, excluding reagents, is just US$0.42.

Detection of hepatitis B virus DNA with a paper electrochemical sensor.

Here we show that a simple paper-based electrochemical sensor, fabricated by paper folding, is able to detect a 30-base nucleotide sequence characteristic of DNA from the hepatitis B virus (HBV) with a detection limit of 85 pM. This device is based on design principles we have reported previously for detecting proteins via a metalloimmunoassay. It has four desirable attributes. First, its design combines simple origami (paper folding) assembly, the open structure of a hollow-channel paper analytical device to accommodate micrometer-scale particles, and a convenient slip layer for timing incubation steps. Second, two stages of amplification are achieved: silver nanoparticle labels provide a maximum amplification factor of 250 000 and magnetic microbeads, which are mobile solid-phase supports for the capture probes, are concentrated at a detection electrode and provide an additional ∼25-fold amplification. Third, there are no enzymes or antibodies used in the assay, thereby increasing its speed, stability, and robustness. Fourth, only a single sample incubation step is required before detection is initiated.

Paper diagnostic device for quantitative electrochemical detection of ricin at picomolar levels.

We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min.

Simple, sensitive, and quantitative electrochemical detection method for paper analytical devices.

We report a new type of paper analytical device that provides quantitative electrochemical output and detects concentrations as low as 767 fM. The model analyte is labeled with silver nanoparticles (AgNPs), which provide 250,000-fold amplification. AgNPs eliminate the need for enzymatic amplification, thereby improving device stability and response time. The use of magnetic beads to preconcentrate the AgNPs at the detection electrode further improves sensitivity. Response time is improved by incorporation of a hollow channel, which increases the flow rate in the device by a factor of 7 and facilitates the use of magnetic beads. A key reaction necessary for label detection is made possible by the presence of a slip layer, a fluidic switch that can be actuated by manually slipping a piece of paper. The design of the device is versatile and should be useful for detection of proteins, nucleic acids, and microbes.

DNA detection using origami paper analytical devices.

We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.

Simultaneous solid phase extraction and derivatization of aliphatic primary amines prior to separation and UV-absorbance detection.

To overcome the problem of poor sensitivity of capillary electrophoresis-UV absorbance for the detection of aliphatic amines, a solid phase extraction and derivatization scheme was developed. This work demonstrates successful coupling of amines to a chromophore immobilized on a solid phase and subsequent cleavage and analysis. Although the analysis of many types of amines is relevant for myriad applications, this paper focuses on the derivatization and separation of amines with environmental relevance. This work aims to provide the foundations for future developments of an integrated sample preparation microreactor capable of performing simultaneous derivatization, preconcentration, and sample cleanup for sensitive analysis of primary amines.

Bipolar electrochemistry.

A bipolar electrode (BPE) is an electrically conductive material that promotes electrochemical reactions at its extremities (poles) even in the absence of a direct ohmic contact. More specifically, when sufficient voltage is applied to an electrolyte solution in which a BPE is immersed, the potential difference between the BPE and the solution drives oxidation and reduction reactions. Because no direct electrical connection is required to activate redox reactions, large arrays of electrodes can be controlled with just a single DC power supply or even a battery. The wireless aspect of BPEs also makes it possible to electrosynthesize and screen novel materials for a wide variety of applications. Finally, bipolar electrochemistry enables mobile electrodes, dubbed microswimmers, that are able to move freely in solution.

Electrochemically-gated delivery of analyte bands in microfluidic devices using bipolar electrodes.

A method for controlling enrichment, separation, and delivery of analytes into different secondary microchannels using simple microfluidic architecture is described. The approach, which is based on bipolar electrochemistry, requires only easily fabricated electrodes and a low-voltage DC power supply: no pumps or valves are necessary. Upon application of a voltage between two driving electrodes, passive bipolar electrodes (BPEs) are activated that result in formation of a local electric field gradient. This gradient leads to separation and enrichment of a pair of fluorescent analytes within a primary microfluidic channel. Subsequently, other passive BPEs can be activated to deliver the enriched tracers to separate secondary microchannels. The principles and performance underpinning the method are described.

Recent applications of carbon-based nanomaterials in analytical chemistry: critical review.

The objective of this review is to provide a broad overview of the advantages and limitations of carbon-based nanomaterials with respect to analytical chemistry. Aiming to illustrate the impact of nanomaterials on the development of novel analytical applications, developments reported in the 2005-2010 period have been included and divided into sample preparation, separation, and detection. Within each section, fullerenes, carbon nanotubes, graphene, and composite materials will be addressed specifically. Although only briefly discussed, included is a section highlighting nanomaterials with interesting catalytic properties that can be used in the design of future devices for analytical chemistry.