Hybrid bilayers composed of the lipid phosphatidylcholine (PC) and a submonolayer of 1-decanethiol bound to gold nanoparticles are very stable to potassium cyanide.

We report on a competitive electrochemical detection system that is free of wash-steps and enables the real-time monitoring of adenosine triphosphate (ATP) in a quantitative manner over a five-log concentration range. The system utilizes a recognition surface based on ATP aptamer (ATPA) capture probes pre-bound to electro-active Flavin adenine dinucleotide (FAD) molecules, and a signaling surface utilizing graphene (Gr) and gold nanoparticle (AuNP) modified carbon paste electrode (Gr-AuNP-CPE) that is optimized to enhance electron transfer kinetics and signal sensitivity. Binding of ATP to ATPA at the recognition surface causes the release of an equivalent concentration of FAD that can be quantitatively monitored in real-time at the signaling surface, thereby enabling a wide linear working range (1.14 ×10−10–3.0×10−5M), a low detection limit (2.01×10−11M using graphene and AuNP modified glassy carbon), and fast target binding kinetics (steady-state signal within 12 minutes at detection limit). Unlike assays based on capture probe-immobilized electrodes, this double-surface competitive assay offers the ability to speed-up target binding kinetics by increasing the capture probe concentration, with no limitations due to intermolecular coulombic interactions and non-specific binding. We utilize the real-time monitoring capability to compute kinetic parameters for target binding and to make quantitative distinctions on degree of base-pair mismatch through monitoring target binding kinetics over a wide concentration range. Based on simplicity of the assay chemistry and the quantitative detection of ATP within fruit and serum media, as demonstrated by comparison of ATP levels against those determined using a standard HPLC-UV absorbance method, we envision a versatile detection platform for applications requiring real-time monitoring over a wide target concentration range.

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Aptamer based ATP binding leads to the release of the cofactor FAD, which acts as a trigger to ‘turn-on’ the activity of apo-GOx and thus generate a measureable response.

Modular allosteric aptamers with discrete recognition and signaling regions provide a facile method of carrying out label-free detection by forgoing complex target labeling requirements. Herein, we describe the design and function of an aptamer scaffold capable of forming a hairpin loop in the presence of FAD (the signaling trigger). The aptamer includes a recognition region for the microRNA (miR) Let-7i. Upon selective miR hybridization, the aptamer undergoes a conformational shift to release FAD and thus produce a measurable response. As a result, the described method can sensitively and selectively detect miR Let-7i with a wide linear range of 0.1 pM to 1 μM and a detection limit of 150 fM. Additionally, this strategy was able to selectively discriminate between sequences with 1-and 2-nucleotide (nt) differences.