Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor

Abstract

The development of a biosensor system capable of continuous, real-time measurement of smallmolecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Towards a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (~1 minute time resolution) detection of the small molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the ground work for the realtime, point-of-care detection of a wide variety of molecular targets.The capability to perform in situ, continuous, real-time monitoring of specific small molecules in complex, unprocessed aqueous samples is important for a broad spectrum of applications ranging from medical diagnostics to environmental monitoring. 1 However, successful implementation of such strategies has proven elusive, and thus far has been achieved for only a few, specialized targets (e.g., neurotransmitters, such as 5-hydroxytryptamine, 2 hydrogen ions 3 and glucose 4 ). The challenge of real-time sensing arises from the fact that, to meet this demanding application, a sensor needs not only be sensitive, stable and selective enough to deploy directly in complex sample matrices, but it also needs to be reagentless, regenerable and able to respond rapidly relative to the timescale with which the target concentration fluctuates.Previously-developed strategies for the real-time detection of small-molecule analytes have typically made use of sensors that measure changes in mass, 5 index of refraction 6 or charge 7 that occur when the target molecules bind to the sensor surface. Such measurements, however, suffer from often severe false positives; for example, it has proven difficult to discriminate between changes in mass arising from the binding of authentic target molecules and those *To whom all correspondence should be addressed. arising due to the non-specific adsorption of contaminants, which has significantly hindered the application of these adsorption-based sensors in complex samples, such as unprocessed clinical or environmental materials. [5][6][7] In contrast, sensors based on binding-induced conformational changes in a bimolecular probe [8][9] have proven more effective in rejecting such false positives. An example is the electrochemical, aptamer-based (E-AB) sensor platform, which operates via the target binding-induced folding of DNA and RNA aptamers and has been shown to work directly in blood serum, 10-12 crude cellular extracts, 13 soil extracts 14 and foodstuffs. 15 The high ...

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