Monitoring of Cardiac Disease with Reagent-free Molecular Pendulum Aptasensors

Abstract

The development of self-contained analytical devices for accurate and rapid quantification of molecular analytes in unprocessed biological fluids holds great promise for next-generation precision medicine [1]. Since reagentless electrochemical approaches are ideal candidates for building fully integrated testing devices with optimal user-friendliness, several sensors based on redox-active enzymes, and affinity-binding assays with different configurations containing recognition elements have been applied broadly to biomolecular detection. These advancements have addressed a variety of challenging problems, including in vivo sensing of small molecules and single-step, quantitative detection of multiple antibodies, however, most of the existing affinity-based sensors are fundamentally limited to recognition agents based on small molecules, peptide epitopes, and small proteins. Moreover, their compromised sensitivity (sub-nanomolar to hundreds of nanomolar) in unprocessed biofluids greatly limits their clinical usability [2].Recently, we have developed a new class of biomolecular sensors using molecular pendulum (MP) [3], which is a general approach for the development of reagentless sensors to monitor physiologically relevant proteins directly in body fluids. The sensing strategy is based on the motion of an inverted MPs tethered to an electrode surface that exhibits field-induced transport modulated by the presence of a bound analyte.This powerful and versatile sensing strategy, using antibody as the bioreceptor unit, demonstrated to detect as low as 1 pgml−1 (~40 fM) of a target protein, cardiac troponin I (cTnI), in several bio-fluids including blood and saliva. Since aptamers, in addition to having the molecular recognition properties of antibodies, such as high affinity and selectivity [4], have unique compelling features for high-throughput applications in diagnostics including small physical size, quick chemical production with low cost and minimum batch-to-batch variability, high stability and long shelf-life [5], we sought out to theoretically investigate and experimentally explore the feasibility to employ aptamers to develop novel and robust MP aptasensors. Here we describe novel reagent-free MP aptasensors that quantify different heart failure biomarkers including BNP and NT-pro BNP directly in whole human blood using only a sensor-modified electrode chip by leveraging and upgrading the MP approach. The electrode-tethered aptasensors (Fig. 1a) are constructed by anchoring a negatively charged double stranded DNA linker on to an electrode; one of the DNA strands is extended to form an analyte-binding aptamer and the other strand features a tethered redox probe. Using chronoamperometry, which enables the sensor transportation to the electrode surface, the presence of an analyte bound to our sensor complex can be detected and subsequently quantified as these species increase the hydrodynamic drag on the sensor. To explore the feasibility of this approach, we modeled the behavior of an MP aptasensor, and thanks to the intrinsic negative charge and small size of aptamers, our model yielded a higher Δτ, which is difference in the fall time of the sensors to the electrode surface between its unbound and bound state to its target analyte, for the aptasensors compared to its antibody-based counterpart; and a similar trend was observed when we explored these phenomena experimentally (Fig. 1b). We then systematically investigated the analytical strength of the MP aptasensors in lab buffer and in whole human blood, and the results show excellent performance matrices, such as a wide dynamic range (10 fg/mL to 10 ng/mL of BNP), a LOD of ~0.67fM BNP, and high specificity, and long-term stability of the sensors (Fig 1c-f).The performance of the MP aptasensors to quantify BNP and NT-pro BNP in whole human blood has been evaluated and the data warrant potential applicability of these new class of sensors for earlier identification and better risk stratification of patients with chronic heart failure.