5 research outputs found
An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming
The
monitoring and control of toxic cyanobacterial strains, which
can produce microcystins, is critical to protect human and ecological
health. We herein reported an optical-biosensor-based quantification
of the microcystin synthetase A (mcyA) gene so as to discriminate
microcystin-producing strains from nonproducing strains. In this assay,
the mcyA-specific ssDNA probes were designed in silico with an on-line
tool and then synthesized to be covalently immobilized on an optical-fiber
surface. Production of fluorescently modified target DNA fragment
amplicons was accomplished through the use of Cy5-tagged deoxycytidine
triphosphates (dCTPs) in the polymerase chain reaction (PCR) method,
which resulted in copies with internally labeled multiple sites per
DNA molecule and delivered great sensitivity. With a facile surface-based
hybridization process, the PCR amplicons were captured on the optical-fiber
surface and were induced by an evanescent-wave field into fluorescence
emission. Under the optimum conditions, the detection limit was found
to be 10 pM (S/N ratio = 3) and equaled 10<sup>3</sup> gene copies/mL.
The assay was triumphantly demonstrated for PCR amplicons of mcyA
detection and showed satisfactory stability and reproducibility. Moreover,
the sensing system exhibited excellent selectivity with quantitative
spike recoveries from 87 to 102% for <i>M. aeruginosa</i> species in the mixed samples. There results confirmed that the method
would serve as an accurate, cost-effective, and rapid technique for
in-field testing of toxic <i>Microcystis</i> sp. in water,
giving early information for water quality monitoring against microcystin-producing
cyanobacteria
Free-Energy-Driven Lock/Open Assembly-Based Optical DNA Sensor for Cancer-Related microRNA Detection with a Shortened Time-to-Result
Quantification
of cancer biomarker microRNAs (miRs) by exquisitely designed biosensors
with a short time-to-result is of great clinical significance. With
immobilized capture probes (CPs) and fluorescent-labeled signal probes
(SPs), surface-involved sandwich-type (SST) biosensors serve as powerful
tools for rapid, highly sensitive, and selective detection of miR
in complex matrices as opposed to the conventional techniques. One
key challenge for such SST biosensors is the existence of false-negative
signals when the amount of miRs exceeds SPs in solution phase for
a surface with a limited number of CP. To meet this challenge, a dynamic
lock/open DNA assembly was designed to rationally program the pathway
for miR/SP hybrids. Based on secondary structure analysis and free-energy
assessment, a “locker” strand that partially hybridizes
with target miR by two separated short arms was designed to stabilize
target miR, preventing possible false-negative signals. The strategy
was demonstrated on a fiber-based fluorescent DNA-sensing platform.
CP/miR/SP sandwiches formed on the fiber surface would generate fluorescent
signals for quantitative analysis. The developed SST biosensor was
able to detect miR Hsa <i>let-7a</i> with a detection limit
of 24 pM. The applicability of this free-energy-driven lock/open assembly-based
optical DNA sensor was further confirmed with spiked human urine and
serum samples
Quantum Dot/Carrier–Protein/Haptens Conjugate as a Detection Nanobioprobe for FRET-Based Immunoassay of Small Analytes with All-Fiber Microfluidic Biosensing Platform
This study demonstrates the use of carrier-protein/haptens
conjugate
(e.g., BSA/2,4-dichlorophenoxyacetic acid, 2,4-D-BSA) for biological
modification of quantum dots (QDs) for the detection of small analytes.
Bioconjugated QDs, which are used as a detection nanoimmunoprobe,
were prepared through conjugating carboxyl QDs with 2,4-D-BSA conjugate.
Based on the principle of quantum dot–fluorescence resonance
energy transfer (QD-FRET), an all-fiber microfluidic biosensing platform
has been developed for investigating FRET efficiency, immunoassay
mechanism and format, and binding kinetics between QD immunoprobe
and fluorescence labeled anti-2,4-D monoclonal antibody. The structure
of multiplex-haptens/BSA conjugate coupling to QD greatly improves
the FRET efficiency and the sensitivity of the nanosensor. With a
competitive detection mode, samples containing different concentrations
of 2,4-D were incubated with a given concentration of QD immunoprobe
and fluorescence-labeled antibody, and then detected by the all-fiber
microfluidic biosensing platform. A higher concentration of 2,4-D
led to less fluorescence-labeled anti-2,4-D antibody bound to the
QD immunoprobe surface and, thus, a lower fluorescence signal. The
quantification of 2,4-D over concentration ranges from 0.5 nM to 3
ÎĽM with a detection limit determined as 0.5 nM. The performance
of the nanosensor with spiked real water samples showed good recovery,
precision, and accuracy, indicating that it was less suspectable to
water matrix effects. With the use of different QD nanobioprobes modified
by other carrier-protein/haptens conjugates, this biosensing protocol
based on QD-FRET can be potentially applied for on-site, real-time,
inexpensive, and easy-to-use monitoring of other trace analytes
Isoelectric Bovine Serum Albumin: Robust Blocking Agent for Enhanced Performance in Optical-Fiber Based DNA Sensing
Surface
blocking is a well-known process for reducing unwanted
nonspecific adsorption in sensor fabrication, especially important
in the emerging field where DNA/RNA applied. Bovine serum albumin
(BSA) is one of the most popular blocking agents with an isoelectric
point at pH 4.6. Although it is widely recognized that the adsorption
of a blocking agent is strongly affected by its net charge and the
maximum adsorption is often observed under its isoelectric form, BSA
has long been perfunctorily used for blocking merely in neutral solution,
showing poor blocking performances in the optical-fiber evanescent
wave (OFEW) based sensing toward DNA target. To meet this challenge,
we first put forward the view that isoelectric BSA (iep-BSA) has the
best blocking performance and use an OFEW sensor platform to demonstrate
this concept. An optical-fiber was covalently modified with amino-DNA,
and further coupled with the optical system to detect fluorophore
labeled complementary DNA within the evanescent field. A dramatic
improvement in the reusability of this DNA modified sensing surface
was achieved with 120 stable detection cycles, which ensured accurate
quantitative bioassay. As expected, the iep-BSA blocked OFEW system
showed enhanced sensing performance toward target DNA with a detection
limit of 125 pM. To the best of our knowledge, this is the highest
number of regeneration cycles ever reported for a DNA immobilized
optical-fiber surface. This study can also serve as a good reference
and provide important implications for developing similar DNA-directed
surface biosensors
Screening Criteria for Qualified Antibiotic Targets in Unmodified Gold Nanoparticles-Based Aptasensing
In
designing unmodified gold nanoparticles-based aptasensing (uGA) assays
for antibiotics, we find that some antibiotics can adsorb directly
on gold nanoparticles (GNP) regardless of the presence of aptamers,
which have been long overlooked in the past. Some adsorptions, however,
would strongly disturb the charge distribution on the GNP surface,
break up the static colloidal profile, and thus generate false positive
colorimetric signals. To identify antibiotics qualified for uGA assays,
we established two rational screening criteria for antibiotic targets
relying on their oil–water partition coefficients (log <i>P</i> values) and net physiological charges: log <i>P</i> > 0 and charge ≤0. A good agreement of the GNP color change
was obtained between the two criteria-based predictions and the actual
tests using six representative antibiotics. The proposed criteria
help to shed light on GNP–target interactions, which is significant
for developing novel GNP-based colorimetric assays with high reliability