The development of a technology capable of tracking the levels of drugs, metabolites, and biomarkers in the body continuously and in real time would advance our understanding of health and our ability to detect and treat disease. It would, for example, enable therapies guided by high-resolution, patient-specific pharmacokinetics (including feedback-controlled drug delivery), opening new dimensions in personalized medicine. In response, we demonstrate here the ability of electrochemical aptamer-based (E-AB) sensors to support continuous, real-time, multihour measurements when emplaced directly in the circulatory systems of living animals. Specifically, we have used E-AB sensors to perform the multihour, real-time measurement of four drugs in the bloodstream of even awake, ambulatory rats, achieving precise molecular measurements at clinically relevant detection limits and high (3 s) temporal resolution, attributes suggesting that the approach could provide an important window into the study of physiology and pharmacokinetics.aptamer | square-wave voltammetry | in vivo | E-DNA | precision medicine

The real-time monitoring of specific analytes in situ in the living body would greatly advance our understanding of physiology and the development of personalized medicine. Because they are continuous (wash-free and reagentless) and are able to work in complex media (e.g., undiluted serum), electrochemical aptamer-based (E-AB) sensors are promising candidates to fill this role. E-AB sensors suffer, however, from often-severe baseline drift when deployed in undiluted whole blood either in vitro or in vivo. We demonstrate that cell-membrane-mimicking phosphatidylcholine (PC)-terminated monolayers improve the performance of E-AB sensors, reducing the baseline drift from around 70% to just a few percent after several hours in flowing whole blood in vitro. With this improvement comes the ability to deploy E-AB sensors directly in situ in the veins of live animals, achieving micromolar precision over many hours without the use of physical barriers or active drift-correction algorithms.

A wide range of new devices aimed at in vivo molecular detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics associated with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approximately −2.2. Such behavior is consistent with a diffusioncontrolled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the observed rates are, at 5−400 s −1 , orders of magnitude slower than the intramolecular dynamics of single-stranded oligonucleotides when free in solution. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equivalent terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is negatively charged at the redox potential of methylene blue. Consistent with this, changing monolayer composition so as to increase the negative charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theoretical models and computer simulations performed in support of our experimental studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.

The ability to monitor specific molecules in real-time directly in a flowing sample stream and in a manner that does not adulterate that stream could greatly augment quality control in, for example, food processing and pharmaceutical manufacturing. Because they are continuous, reagentless, and able to work directly in complex samples, electrochemical DNA-based (E-DNA) sensors, a modular and, thus, general sensing platform, are promising candidates to fill this role. In support, we describe here an E-DNA sensor supporting the continuous, real-time measurement of melamine in flowing milk. Using target-driven DNA triplex formation to generate an electrochemical output, the sensor responds to rising and falling melamine concentration in seconds without contaminating the product stream. The continuous, autonomous, real-time operation of sensors such as this could provide unprecedented safety, convenience, and cost-effectiveness relative to the batch processes historically employed in molecular quality control.

The real-time monitoring of specific analytes in situ in the living body would greatly advance our understanding of physiology and the development of personalized medicine. Because they are continuous (wash-free and reagentless) and are able to work in complex media (e.g., undiluted serum), electrochemical aptamer-based (E-AB) sensors are promising candidates to fill this role.E-AB sensors suffer,however,from often-severe baseline drift when deployed in undiluted whole blood either in vitro or in vivo.W ed emonstrate that cellmembrane-mimicking phosphatidylcholine (PC)-terminated monolayers improve the performance of E-AB sensors, reducing the baseline drift from around 70 %t oj ust af ew percent after several hours in flowing whole blood in vitro. With this improvement comes the ability to deploy E-AB sensors directly in situ in the veins of live animals,a chieving micromolar precision over many hours without the use of physical barriers or active drift-correction algorithms.