4 research outputs found
Selection and Identification of DNA Aptamers against Okadaic Acid for Biosensing Application
This work describes the selection
and identification of DNA aptamers
that bind with high affinity and specificity to okadaic acid (OA),
a lipophilic marine biotoxin that accumulates in shellfish. The aptamers
selected using systematic evolution of ligands by exponential enrichment
(SELEX) exhibited dissociation constants in the nanomolar range. The
aptamer with the highest affinity was then used for the fabrication
of a label-free electrochemical biosensor for okadaic acid detection.
The aptamer was first immobilized on the gold electrode by a self-assembly
approach through AuāS interaction. The binding of okadaic acid
to the aptamer immobilized on the electrode surface induces an alteration
of the aptamer conformation causing a significant decrease in the
electron-transfer resistance monitored by electrochemical impedance
spectroscopy. The aptasensor showed a linear range for the concentrations
of OA between 100 pg/mL and 60 ng/mL with a detection limit of 70
pg/mL. The dissociation constant of okadaic acid with the aptamer
immobilized on the electrode surface showed good agreement with that
determined using fluorescence assay in solution. Moreover, the aptasensor
did not show cross-reactivity toward toxins with structures similar
to okadaic acid such as dinophysis toxin-1 and 2 (DTX-1, DTX-2). Further
biosensing applications of the selected aptamers are expected to offer
promising alternatives to the traditional analytical and immunological
methods for OA detection
Selection, Characterization, and Biosensing Application of High Affinity Congener-Specific Microcystin-Targeting Aptamers
The efficiency of current microcystin detection methods
has been
hampered by the low detection limits required in drinking water and
that routine detection is restricted to a few of the congeners with
high degree of undesired cross-reactivity. Here, we report the development
of novel microcystin-targeting molecules and their application in
microcystin detection. We have selected DNA aptamers from a diverse
random library that exhibit high affinity and specificity to microcystin-LR,
-YR, and -LA. We obtained aptamers that bind to all chosen congeners
with high affinity with <i>K</i><sub>D</sub> ranging from
28 to 60 nM. More importantly, we also obtained aptamers that are
selective among the different congeners, with selectivity from 3-folds
difference in binding affinity to total discrimination (<i>K</i><sub>D</sub> of 50 nM versus nonspecific binding). Electrochemical
aptasensors constructed with the selected aptamers were able to achieve
sensitive and congener-specific microcystin detection with detection
limit as low as 10 pM
Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone
Rising progesterone (P4) levels in
humans due to its overconsumption
through hormonal therapy, food products, or drinking water can lead
to many negative health effects. Thus, the simple and accurate assessment
of P4 in both environmental and clinical samples is highly important
to protect public health. In this work, we present the selection,
identification, and characterization of ssDNA aptamers with high binding
affinity to P4. The aptamers were selected in vitro from a single-stranded
DNA library of 1.8 Ć 10<sup>15</sup> oligonucleotides showing
dissociation constants (<i>K</i><sub>D</sub>) in the low
nanomolar range. The dissociation constant of the best aptamer, designated
as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy
(EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did
not show cross-reactivity to analogues similar to progesterone such
as 17Ī²-estradiol (E2) and norethisterone (NET). An impedimetric
aptasensor for progesterone was then fabricated based on the conformational
change of P4G13 aptamer, immobilized on the gold electrode by self-assembly,
upon binding to P4, which results in an increase in electron transfer
resistance. Aptamerācomplementary DNA (cDNA) oligonucleotides
were tested to maximize the signal gain of the aptasensor after binding
with progesterone. Significant signal enhancement was observed when
the aptamer hybridized with a short complementary sequence at specific
site was used instead of pure aptamer. This signal gain is likely
due to the more significant conformational change of the aptamerācDNA
than the pure aptamer upon binding with P4, as confirmed by circular
dichroism (CD) spectroscopy. The developed aptasensor exhibited a
linear range for concentrations of P4 from 10 to 60 ng/mL with a detection
limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked
tap water samples and showed good recovery percentages. The new selected
progesterone aptamers can be exploited in further biosensing applications
for environmental, clinical, and medical diagnostic purposes
Sensitive Detection of ssDNA Using an LRET-Based Upconverting Nanohybrid Material
Water-dispersible,
optical hybrid nanoparticles are preferred materials
for DNA biosensing due to their biocompatibility. Upconverting nanoparticles
are highly desirable optical probes in sensors and bioimaging owing
to their sharp emission intensity in the visible region. We herein
report a highly sensitive ss-DNA detection based on an energy transfer
system that uses a nanohybrid material synthesized by doping NaYF<sub>4</sub>:Tm<sup>3+</sup>/Yb<sup>3+</sup> upconverting nanoparticles
(UCNPs) on silica coated polystyrene-<i>co</i>-acrylic acid
(PSA) nanoparticles (PSA/SiO<sub>2</sub>) as the donor, and gold nanoparticles
(AuNPs) decorated with IrĀ(III) complex as the acceptor. UCNPs tagged
on PSA/SiO<sub>2</sub> and the cyclometalated IrĀ(III)/AuNP conjugates
were then linked through the ss-DNA sequence. Sequential addition
of the target DNA to the probe molecular beacon complex resulted in
the separation of the optical nanohybrid material and the quencher,
leading to a measurable increase in the blue fluorescence emission
intensity. Our results have shown a linear relationship between the
fluorescence intensity and target DNA concentration down to the picomolar