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Aptamers have shown considerable promise as chemical recognition elements in biosensor systems because they can be chemically synthesized and can deliver high stability, sensitivity, and specificity. However, since native aptamers do not inherently exhibit signaling activity upon target recognition, engineering approaches are needed to introduce signal transduction functionality so that they can generate a measurable output signal upon target binding. A number of signaling modalities have been explored to date including electrochemical aptamer-based (E-AB) sensors, which measure changes in current that result from target-binding-induced conformational changes in redox-tagged aptamer molecules. E-AB sensors have repeatedly demonstrated the potential for continuous detection over extended periods of time in vivo with high specificity and stability . However, the adaptation of existing aptamers for electrochemical platforms remains a bottleneck7, since aptamers identified through SELEX-based strategies will not necessarily undergo a meaningful conformational change upon target binding, and there is no guarantee that a promising aptamer can be readily engineered to generate an E-AB sensor that yields a sufficient signal response within the desired target concentration range. This limitation is one of the reasons why E-AB sensor implementation has been largely limited to a few target molecules.
Fluorescence-based optical readouts are of particular interest as they offer the capability to achieve single-photon sensitivity with appropriate detection hardware, can readily be multiplexed through fluorophores that emit at different wavelengths, and can accelerate the transition from aptamer selection to sensor development. Aptamer beacons are a popular design strategy for fluorescence-based detection, in which an aptamer is labeled with a fluorophore and then combined with a separate, quencher-functionalized strand that directly competes with target binding, such that a fluorescence signal is only generated in response to target binding. However, this method suffers from a major drawback in the context of biosensor applications in that it cannot be used for continuous measurements, as the quenching sequence is unlikely to rehybridize once released. As an alternative, Tang et al. used a polyethylene glycol (PEG) moiety to couple aptamer sequences to a short complementary sequence, which were respectively labeled with a fluorophore and quencher. More recently, our lab devised another effective fluorescent sensor design based on intramolecular strand displacement (ISD) molecular switches, in which the complementary strand is physically coupled to the aptamer strand via a flexible linker. This strategy enables reversible switching and by modulating the lengths of the linker and the complementary region, one can achieve independent tuning of both the kinetics and thermodynamics of the resulting sensor. However, both approaches face important limitations in that they require considerable trial and error optimization to achieve an ideal balance between minimizing the background signal and ensuring efficient target binding-induced displacement of the complementary displacement strand. Optimization typically requires detailed characterization of the aptamer’s structure because post-selection engineering of the aptamer often compromises its affinity or specificity. For these reasons, most aptamer switch designs to date have been prototyped with a handful of well-characterized aptamers. Multiple rational or semi-rational approaches have been developed to date to engineer aptamers to undergo a binding-induced conformational change, including enzymatic5 and truncation approaches. For newly discovered aptamers, however, these engineering processes remain challenging as it does not always yield a sensor with a sufficient signal response across the desired target concentration range. There is a need for a universal and systematic approach for reliably endowing switching functionality into aptamers to enable the efficient generation of biosensors for diverse molecular targets.
In this work, we describe a novel duplex-bubble switch (DBS) aptamer switch architecture, wherein any aptamer switch generated via Capture-SELEXcan be engineered in order to tune its kinetic and thermodynamic response without a priori knowledge of its structure or binding domain. Importantly, the DBS enables reversible switching, and we demonstrate that the architecture is suitable for use in continuous detection applications, achieving a rapid and reversible binding-induced fluorescent response at a time-scale of seconds even in complex biological matrices. We first show mathematically and experimentally that we can fine-tune the DBS design to precisely adjust the thermodynamics and kinetics of the resulting aptamer switches, both in solution and in a surface-coupled assay format. We then show that we can incorporate these DBS constructs onto a fiber-optic probe integrated with single-photon-counting hardware, exploiting a sensor design that allows us to achieve direct measurement of analytes in complex biological matrices while rejecting background autofluorescence from interferents and minimizing the impact of fouling. Specifically, we demonstrate continuous detection of dopamine in both buffer and artificial cerebrospinal fluid (aCSF) for more than a day with fast time resolution (< 3 s) and a dynamic range spanning 500 nM–500 μM. We subsequently developed a second, cortisol-specific DBS probe, achieving continuous cortisol detection in undiluted human plasma with nanomolar sensitivity and a dynamic range suitable for detecting physiologically relevant concentrations (200 nM–100 μM) over the course of multiple hours in undiluted human plasma. The DBS design thus offers a generalizable approach to accelerate the transition from aptamer selection to sensor development, and to rapidly design aptamer-based sensors for the sensitive and specific continuous optical detection of diverse small-molecule analytes in a range of biomedical applications.
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