18 research outputs found
Antibody-Bridged Beacon for Homogeneous Detection of Small Molecules
In
conventional competitive immunoassays for small molecules (SM),
antibodies are either immobilized to solid phases or labeled with
magnetic particles or probes. The former involves laborious blocking
and washing steps, whereas the latter requires complicated labeling
and purification steps. To circumvent these limitations, we describe
here a new type of molecular beacon, termed antibody-bridged beacon
(AbB), enabling homogeneous detection of SM without any immobilization
or labeling of the antibody. The AbB is formed by the binding of an
antibody to a pair of SM-labeled oligonucleotide probes that each
comprise a stem sequence conjugated by either a fluorophore or a quencher.
Competitive binding of the SM target to the antibody destructs the
stem-loop structure of AbB, restoring the quenched fluorescence. A
minimum binding energy of stem sequences is required for efficient
formation of the desired stem-loop structure of AbB. A systematic
study of the impact of stem sequences on the fluorescence background
and quenching efficiency provided useful benchmarks, e.g., binding
energy of −11 kcal/mol, for the construction of AbB. The optimized
AbB showed fast signal responses, as demonstrated in the analyses
of two small molecule targets, biotin and digoxin. Low nanomolar limits
of detection were achieved. The novel AbB strategy, along with the
guidelines established for the construction and application of AbB,
offers a promising approach for homogeneous detection of small molecules,
obviating immobilization or labeling of antibodies as required by
other competitive immunoassays
Binding-Induced Formation of DNA Three-Way Junctions and Its Application to Protein Detection and DNA Strand Displacement
DNA
three-way junctions (DNA TWJs) are important building blocks
to construct DNA architectures and dynamic assemblies. We describe
here a binding-induced DNA TWJ strategy that is able to convert protein
bindings to the formation of DNA TWJ. The binding-induced DNA TWJ
makes use of two DNA motifs each conjugated to an affinity ligand.
The binding of two affinity ligands to the target molecule triggers
assembly of the DNA motifs and initiates the subsequent DNA strand
displacement, resulting in a binding-induced TWJ. Real-time fluorescence
monitoring of the binding-induced TWJ enables detection of the specific
protein targets. A detection limit of 2.8 ng/mL was achieved for prostate-specific
antigen. The binding-induced TWJ approach compares favorably with
the toehold-mediated DNA strand-displacement, the associative (combinative)
toehold-mediated DNA strand-displacement, and the binding-induced
DNA strand-displacement. Importantly, the binding-induced TWJ broadens
the scope of dynamic DNA assemblies and provides a new strategy to
design protein-responsive DNA devices and assemblies
Binding-Induced DNA Assembly and Its Application to Yoctomole Detection of Proteins
We describe the binding-induced DNA assembly principle
and strategy
that enable ultrasensitive detection of molecular targets and potential
construction of unique nanostructures/nanoreactors. Two DNA motifs
that are conjugated to specific affinity ligands assemble preferentially
only when a specific target triggers a binding event. The binding-induced
assembly of the DNA motifs results in the formation of a highly stable
closed-loop structure, raising the melting temperature (<i>T</i><sub>m</sub>) of the hybrid by >30 °C and enabling effective
differentiation of the target-specific assembly from the background.
The ability to detect as few as a hundred molecules (yoctomole) of
streptavidin, platelet derived growth factor, and prostate specific
antigen represents an improvement of detection limits by 10<sup>3</sup>–10<sup>5</sup>-fold over traditional immunoassays. The assay
is performed in a single tube, eliminating separation, immobilization,
and washing steps of conventional assays. By incorporating unique
signaling and structural features into the DNA motifs, we envision
diverse applications in biosensing and nanotechnology
Aptamer Capturing of Enzymes on Magnetic Beads to Enhance Assay Specificity and Sensitivity
Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers
Accumulation and Transport of Roxarsone, Arsenobetaine, and Inorganic Arsenic Using the Human Immortalized Caco‑2 Cell Line
Roxarsone (Rox), an organoarsenic
compound, served as a feed additive
in the poultry industry for more than 60 years. Residual amounts of
Rox present in chicken meat could give rise to potential human exposure
to Rox. However, studies on the bioavailability of Rox in humans are
scarce. We report here the accumulation and transepithelial transport
of Rox using the human colon-derived adenocarcinoma cell line (Caco-2)
model. The cellular accumulation and transepithelial passage of Rox
in Caco-2 cells were evaluated and compared to those of arsenobetaine
(AsB), arsenite (As<sup>III</sup>), and arsenate (As<sup>V</sup>).
When Caco-2 cells were exposed to 3 μM Rox, AsB, and As<sup>III</sup> separately for 24 h, the maximum accumulation was reached
at 12 h. After 24-h exposure, the accumulated Rox was 6–20
times less than AsB and As<sup>III</sup>. The permeability of Rox
from the apical to basolateral side of Caco-2 monolayers was similar
to As<sup>V</sup> but less than As<sup>III</sup> and AsB. The results
of lower bioavailability of Rox are consistent with previous observations
of relatively lower amounts of Rox retained in the breast meat of
Rox-fed chickens. These data provide useful information for assessing
human exposure to and intestinal bioavailability of Roxarsone
Thermal Stability of DNA Functionalized Gold Nanoparticles
Therapeutic
uses of DNA functionalized gold nanoparticles (DNA-AuNPs)
have shown great potential and exciting opportunities for disease
diagnostics and treatment. Maintaining stable conjugation between
DNA oligonucleotides and gold nanoparticles under thermally stressed
conditions is one of the critical aspects for any of the practical
applications. We systematically studied the thermal stability of DNA-AuNPs
as affected by organosulfur anchor groups and packing densities. Using
a fluorescence assay to determine the kinetics of releasing DNA molecules
from DNA-AuNPs, we observed an opposite trend between the temperature-induced
and chemical-induced release of DNA from DNA-AuNPs when comparing
the DNA-AuNPs that were constructed with different anchor groups.
Specifically, the bidentate Au–S bond formed with cyclic disulfide
was thermally less stable than those formed with thiol or acyclic
disulfide. However, the same bidentate Au–S bond was chemically
more stable under the treatment of competing thiols (mercaptohexanol
or dithiothreitol). DNA packing density on AuNPs influenced the thermal
stability of DNA-AuNPs at 37 °C, but this effect was minimum
as temperature increased to 85 °C. With the improved understanding
from these results, we were able to design a strategy to enhance the
stability of DNA-AuNPs by conjugating double-stranded DNA to AuNPs
through multiple thiol anchors
Binding-Induced Fluorescence Turn-On Assay Using Aptamer-Functionalized Silver Nanocluster DNA Probes
We present here a binding-induced fluorescence turn-on
assay for
protein detection. Key features of this assay include affinity binding-induced
DNA hybridization and fluorescence enhancement of silver nanoclusters
(Ag NCs) using guanine-rich DNA sequences. In an example of an assay
for human α-thrombin, two aptamers (Apt15 and Apt29) were used
and were modified by including additional sequence elements. A 12-nucleotide
(nt) sequence was used to link the first aptamer with a nanocluster
nucleation sequence at the 5′-end. The second aptamer was linked
through a complementary sequence (12-nt) to a G-rich overhang at the
3′-end. Binding of the two aptamer probes to the target protein
initiates hybridization between the complementary linker sequences
attached to each aptamer and thereby bring the end of the G-rich overhang
to close proximity to Ag NCs, resulting in a significant fluorescence
enhancement. With this approach, a detection limit of 1 nM and a linear
dynamic range of 5 nM–2 μM were achieved for human α-thrombin.
This fluorescence assay is performed in a single tube, and it does
not require washing or separation steps. The principle of the binding-induced
DNA hybridization and fluorescence enhancement of Ag NCs can be extended
to other homogeneous assay applications provided that two appropriate
probes are available to bind with the same target molecule
Identification of Methylated Dithioarsenicals in the Urine of Rats Fed with Sodium Arsenite
Biotransformation
of inorganic arsenic results in the formation of methylarsenicals
of both oxygen and sulfur analogues. Aiming to improve our understanding
of metabolism of inorganic arsenic in animals, we conducted an animal
feeding study with an emphasis on identifying new arsenic metabolites.
Female F344 rats were given 0, 1, 10, 25, 50, and 100 μg/g of
arsenite (iAs<sup>III</sup>) in the diet. Arsenic species in rat urine
were determined using high performance liquid chromatography (HPLC)
separation and inductive coupled plasma mass spectrometry (ICPMS)
and electrospray ionization tandem mass spectrometry (ESI MS/MS) detection.
Nine arsenic species were detected in the urine of the iAs<sup>III</sup>-dosed rats. Seven of these arsenic species were consistent with
previous reports, including iAs<sup>III</sup>, arsenate, monomethyarsonic
acid, dimethylarsinic acid, trimethylarsine oxide, monomethylmonothioarsonic acid,
and dimethylmonothioarsinic acid. Two new methyldithioarsencals, monomethyldithioarsonic
acid (MMDTA<sup>V</sup>) and dimethyldithioarsinic acid (DMDTA<sup>V</sup>), were identified for the first time in the urine of rats
treated with iAs<sup>III</sup>. The concentrations of both MMDTA<sup>V</sup> and DMDTA<sup>V</sup> in rat urine were dependent on the
dosage of iAs<sup>III</sup> in diet. The concentration of DMDTA<sup>V</sup> was approximately 5 times higher than that of MMDTA<sup>V</sup>. MMDTA<sup>V</sup> has not been identified in any biological samples
of animals, and DMDTA<sup>V</sup> has not been reported as a metabolite
of inorganic arsenic in the rats. The identification of novel methylated
dithioarsenicals as metabolites of inorganic arsenic in the rat urine
provided further insights into the understanding of the metabolism
of arsenic
Kinetics of Proximity-Induced Intramolecular DNA Strand Displacement
Proximity-induced
intramolecular DNA strand displacement (PiDSD)
is one of the key mechanisms involved in many DNA-mediated proximity
assays and protein-responsive DNA devices. However, the kinetic profile
of PiDSD has never been systematically examined before. Herein, we
report a systematic study to explore the kinetics of PiDSD by combining
the uses of three DNA strand displacement techniques, including a
binding-induced DNA strand displacement to generate PiDSD, an intermolecular
DNA strand-exchange strategy to measure a set of key kinetic parameters
for PiDSD, and a toehold-mediated DNA strand displacement to generate
fluorescence signals for the real-time monitoring of PiDSD. By using
this approach, we have successfully revealed the kinetic profiles
of PiDSD, determined the enhanced local effective concentrations of
DNA probes that are involved in PiDSD, and identified a number of
key factors that influence the kinetics of PiDSD. Our study on PiDSD
establishes knowledge and strategies that can be used to guide the
design and operation of various DNA-mediated proximity assays and
protein-triggered DNA devices