Real-Time
Electrochemical Monitoring of Adenosine
Triphosphate in the Picomolar to Micromolar Range Using Graphene-Modified
Electrodes
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Abstract
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 prebound to electroactive 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<sup>–10</sup> to
3.0 × 10<sup>–5</sup> M), a low detection limit (2.01
× 10<sup>–11</sup> M using graphene and AuNP modified
glassy carbon), and fast target binding kinetics (steady-state signal
within 12 min 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 nonspecific 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. On the basis of the 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
high-performance liquid chromatography (HPLC)-UV absorbance method,
we envision a versatile detection platform for applications requiring
real-time monitoring over a wide target concentration range