Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine

Abstract

Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 μM and a 0.3 μM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the possibility to distinguish between the binding mechanisms of structure-switching (MN6) vs rigid (MN4) aptameric probes, an ability that could allow the EMPAS to provide a more universal aptasensing platform than what is ordinarily observed in the literature.

Aptamer adaptive binding assessed by stilbene photoisomerization towards regenerating aptasensors

“…As candidates for replacing antibodies, aptamers possess advantages of relatively simple synthetic preparation, low batch-to-batch variability, facile modification, low immunogenicity, and structural robustness [7][8][9][10]. In addition, it has been widely reported that aptamers have unique properties of conformational change/adaptive binding, making them more versatile as molecular recognition elements [11][12][13][14][15]. Thus, they have been applied in diagnosis, biosensing, bio-imaging, drug delivery, and drug discovery [16][17][18].…”

Aptamers for Diagnostics with Applications for Infectious Diseases

“…The sensing interface consists of a S-(11-trichlorosilyl-undecanyl) benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer was immobilized, completing the preparation of the MN4 cocaine aptamer with an apparent Kd of 45 ± 12 μM and limit of detection of 0.9 μM. The same group developed an MN6 cocaine aptasensor using an EMPAS platform that had apparent Kd of 27 ± 6 and a 0.3 μM detection limit [54].…”

Design and optimisation of Photochrome Aptamer Switch Assay (PHASA)

“…The PHASA combines the advantages of the aptamer adaptive binding assay [5,6,[10][11][12] and fluorescence-photochrome assay based on stilbene photoisomerisation [13][14][15][16][17][18]. The PHASA relies on the photoisomerisation of a fluorescent trans-stilbene to a non-fluorescent cis-stilbene -an intramolecular process taking less than several nanoseconds.…”

“…By rearranging Equation [12], the concentration of the formed ligand-aptamer complex can then be expressed as:…”