5 research outputs found

    An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming

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    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

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    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

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    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 μM 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

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    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

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    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
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