The ability to measure drugs in the body rapidly and in real time would advance both our understanding of pharmacokinetics and our ability to optimally dose and deliver pharmacological therapies. To this end, we are developing electrochemical aptamer-based (E-AB) sensors, a seconds-resolved platform technology that, as critical for performing measurements in vivo, is reagentless, reversible, and selective enough to work when placed directly in bodily fluids. Here we describe the development of an E-AB sensor against irinotecan, a member of the camptothecin family of cancer chemotherapeutics, and its adaptation to in vivo sensing. To achieve this we first re-engineered (via truncation) a previously reported DNA aptamer against the camptothecins to support high-gain E-AB signaling. We then co-deposited the modified aptamer with an unstructured, redox-reporter-modified DNA sequence whose output was independent of target concentration, rendering the sensor's signal gain a sufficiently strong function of square-wave frequency to support kinetic-differential-measurement drift correction. The resultant, 200 μm-diameter, 3 mm-long sensor achieves 20 s-resolved, multi-hour measurements of plasma irinotecan when emplaced in the jugular veins of live rats, thus providing an unprecedentedly high-precision view into the pharmacokinetics of this class of chemotherapeutics.

Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating base-modified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of ∼10⁷ per hour. Our "click-particle display (PD)" strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially available polymerase. Second, click-PD can screen vast numbers of individual aptamers on the basis of quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, the increasing availability of FACS instrumentation in academia and industry allows for easy adoption of click-PD in a broader scientific community. Using click-PD, we generated a boronic acid-modified aptamer with ∼1 μM affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (Con A). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our Con A aptamer exhibited exceptional specificity, with minimal binding to other structurally similar lectins. Finally, we show that our aptamer has remarkable biological activity. Indeed, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.