3 research outputs found
Electrokinetic Preconcentration and Detection of Neuropeptides at Patterned Graphene-Modified Electrodes in a Nanochannel
Neuropeptides
are vital to the transmission and modulation of neurological
signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic
information on stress, depression, and neurotrauma. NPY is an especially
significant biomarker, since it can be noninvasively collected from
sweat, but its detection has been limited by poor sensitivity, long
assay times, and the inability to scale-down sample volumes. Herein,
we apply electrokinetic preconcentration of the neuropeptide onto
patterned graphene-modified electrodes in a nanochannel by frequency-selective
dielectrophoresis for 10 s or by electrochemical adsorptive accumulation
for 300 s, to enable the electrochemical detection of NPY and OXA
at picomolar levels from subnanoliter samples, with sufficient signal
sensitivity to avoid interferences from high levels of dopamine and
ascorbic acid within biological matrices. Given the high sensitivity
of the methodology within small volume samples, we envision its utility
toward off-line detection from droplets collected by microdialysis
for the eventual measurement of neuropeptides at high spatial and
temporal resolutions
Structured DNA Aptamer Interactions with Gold Nanoparticles
DNA
aptamers that bind biomolecular targets are of interest as
the recognition element in colorimetric sensors based on gold nanoparticles
(AuNP), where sensor functionality is related to changes in AuNP colloidal
stability upon target binding. In order to understand the role of
target binding on DNA–AuNP colloidal stability, we have used
high-resolution NMR to characterize the interactions of the 36 nucleotide
cocaine-binding aptamer (MN4) and related aptamers with AuNPs, cocaine,
and cocaine metabolites. Changes in the aptamer imino proton NMR spectra
with low (20 nM) concentrations of AuNP show that the aptamers undergo
fast-exchange adsorption on the nanoparticle surface. An analysis
of the spectral changes and the comparison with modified MN4 aptamers
shows that the AuNP binding domain is localized on stem two of the
three-stemmed aptamer. The identification of an AuNP recognition domain
allows for the incorporation of AuNP binding functionality into a
wide variety of aptamers. AuNP-induced spectral changes are not observed
for the aptamer–AuNP mixtures in the presence of cocaine, demonstrating
that aptamer absorption on the AuNP surface is modulated by aptamer–target
interactions. The data also show that the DNA–AuNP interactions
and sensor functionality are critically dependent on aptamer folding
Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications
The utilization of
structure-switching aptamers (SSAs) has enabled
the development of novel sensing platforms for the sensitive and continuous
detection of molecules. De novo development of SSAs,
however, is complex and laborious. Here we describe a rational approach
to SSA optimization that simultaneously improves aptamer binding affinity
and introduces target-dependent conformation-switching for compatibility
with real-world biosensor applications. Key structural features identified
from NMR and computational modeling were used to optimize conformational
switching in the presence of target, while large-scale, microarray-based
mutation analysis was used to map regions of the aptamer permissive
to mutation and identify combinations of mutations with stronger binding
affinity. Optimizations were carried out in a relevant biofluid to
ensure a seamless transition of the aptamer to a biosensing platform.
Initial proof-of-concept for this approach is demonstrated with a
cortisol binding aptamer but can easily be translated to other relevant
aptamers. Cortisol is a hormone correlated with the stress response
that has been associated with various medical conditions and is present
at quantifiable levels in accessible biofluids. The ability to continuously
track levels of stress in real-time via cortisol monitoring, which
can be enabled by the aptamers reported here, is crucial for assessing
human health and performance