Rapid and Quantitative Detection of Lung Cancer Biomarker ENOX2 Using a Novel Aptamer in an Electrochemical DNA-Based (E-DNA) Biosensor.

To overcome early cancer detection challenges, diagnostic tools enabling more sensitive, rapid, and noninvasive detection are necessary. An attractive cancer target for diagnostic blood tests is human Ecto-NOX disulfide-thiol exchanger 2 (ENOX2), expressed in most human cancer types and regularly shed into blood sera. Here, we developed an electrochemical DNA-based (E-DNA) biosensor that rapidly detects physiologically relevant levels of ENOX2. To identify ENOX2-binding aptamers that could potentially be used in a biosensor, recombinantly expressed ENOX2 was used as a binding target in an oligonucleotide library pull-down that generated a highly enriched ENOX2-binding aptamer. This candidate aptamer sensitively bound ENOX2 via gel mobility shift assays. To enable this aptamer to function in an ENOX2 E-DNA biosensor, the aptamer sequence was modified to adopt two conformations, one capable of ENOX2 binding, and one with disrupted ENOX2 binding. Upon ENOX2 introduction, a conformational shift to the ENOX2 binding state resulted in changed dynamics of a redox reporter molecule, which generated a rapid, significant, and target-specific electrical current readout change. ENOX2 biosensor sensitivity was at or below the diagnostic range. The ENOX2 E-DNA biosensor design presented here may enable the development of more sensitive, rapid, diagnostic tools for early cancer detection.

A tight squeeze: geometric effects on the performance of three-electrode electrochemical-aptamer based sensors in constrained, in vivo 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 situ in the living body. To date, EAB-derived in vivo 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. Specifically, we find that retaining the counter electrode within the catheter increases the resistance between it and the working electrode, increasing the capacitive background. In contrast, extending the counter electrode outside the lumen of the catheter reduces this effect, significantly enhancing the signal-to-noise of intravenous molecular measurements. Exploring counter electrode geometries further, we find that they need not be larger than the working electrode. Putting these observations together, we have developed a new intravenous EAB architecture that achieves improved performance while remaining short enough to safely emplace in the rat jugular. These findings, though explored here with EAB sensors may prove important for the design of many electrochemical biosensors.

Real-Time, Seconds-Resolved Measurements of Plasma Methotrexate In Situ in the Living Body.

Dose-limiting toxicity and significant patient-to-patient pharmacokinetic variability often render it difficult to achieve the safe and effective dosing of drugs. This is further compounded by the slow, cumbersome nature of the analytical methods used to monitor patient-specific pharmacokinetics, which inevitably rely on blood draws followed by post-facto laboratory analysis. Motivated by the pressing need for improved "therapeutic drug monitoring", we are developing electrochemical aptamer-based (EAB) sensors, a minimally invasive biosensor architecture that can provide real-time, seconds-resolved measurements of drug levels in situ in the living body. A key advantage of EAB sensors is that they are generalizable to the detection of a wide range of therapeutic agents because they are independent of the chemical or enzymatic reactivity of their targets. Three of the four therapeutic drug classes that have, to date, been shown measurable using in vivo EAB sensors, however, bind to nucleic acids as part of their mode of action, leaving open questions regarding the extent to which the approach can be generalized to therapeutics that do not. Here, we demonstrate real-time, in vivo measurements of plasma methotrexate, an antimetabolite (a mode of action not reliant on DNA binding) chemotherapeutic, following human-relevant dosing in a live rat animal model. By providing hundreds of drug concentration values, the resulting seconds-resolved measurements succeed in defining key pharmacokinetic parameters, including the drug's elimination rate, peak plasma concentration, and exposure (area under the curve), with unprecedented 5 to 10% precision. With this level of precision, we easily identify significant (>2-fold) differences in drug exposure occurring between even healthy rats given the same mass-adjusted methotrexate dose. By providing a real-time, seconds-resolved window into methotrexate pharmacokinetics, such measurements can be used to precisely "individualize" the dosing of this significantly toxic yet vitally important chemotherapeutic.

Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

Electrochemical aptamer scaffold biosensors for detection of botulism and ricin toxins.

Protein toxins present considerable health risks, but detection often requires laborious analysis. Here, we developed electrochemical aptamer biosensors for ricin and botulinum neurotoxins, which display robust and specific signal at nanomolar concentrations and function in dilute serum. These biosensors may aid future efforts for the rapid diagnosis of toxins.