Knowledge of drug concentrations in the brains of behaving subjects remains constrained on a number of dimensions, including poor temporal resolution and lack of real-time data. Here, however, we demonstrate the ability of electrochemical aptamer-based sensors to support seconds-resolved, real-time measurements of drug concentrations in the brains of freely moving rats. Specifically, using such sensors, we achieve 1.5 hours. These results demonstrate the utility of such sensors in (i) the determination of the site-specific, seconds-resolved neuropharmacokinetics, (ii) enabling the study of individual subject neuropharmacokinetics and concentration-response relations, and (iii) performing high-precision control over intracranial drug levels.

Electrochemical aptamer-based sensors support real-time, in vivo molecular measurements. Here we compare the relative merits of square-wave, alternating current, and differential pulse voltammetry in the interrogation of such 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. Here, however, we show that the use of fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate EAB sensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. To showcase these benefits, we demonstrate the approach's ability to perform real-time molecular measurements in the veins of living rats.

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 situ 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. Doing so we find that low-temperature (−20 °C) storage is sufficient to preserve sensor functionality for at least six months without the need for exogenous preservatives.