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 10³ 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

TEM image of AuNPs synthesized by sodium citrate.

<p>TEM image of AuNPs synthesized by sodium citrate.</p

Photographic image of the multiplex aptasensor using different targets at different concentrations.

<p>Photographic image of the multiplex aptasensor using different targets at different concentrations.</p

A schematic depicting the proposed multiplex detection using a colorimetric aptasensor.

<p>A schematic depicting the proposed multiplex detection using a colorimetric aptasensor.</p

Absorption spectra of AuNPs solutions in the presence of different targets, each with a concentration of 500 ng/mL (A) and the corresponding ratio of 620 nm to 520 nm (B).

<p>Absorption spectra of AuNPs solutions in the presence of different targets, each with a concentration of 500 ng/mL (A) and the corresponding ratio of 620 nm to 520 nm (B).</p

Absorption spectra of a single-detection aptasensor in the presence of various concentrations of corresponding targets: SDM (A), ADE (B), KAN(C).

<p>Corresponding photographic images (D).</p

Annealing temperatures.

*<p>ISAG-FAO recommended microsatellite markers for cattle.</p

Comparison of amplification results of DNA extracted from hair shafts using the proposed method and that extracted from liver using commercial Genomic DNA Purification Kit (on-chip-electrophoresis results).

<p>The above panel is <i>INRA035</i> comparison result, sample 6 is amplification result of hair shaft DNA from pure bred Luxi cattle and sample 3 is amplification result of liver DNA from beef cattle; The below panel is <i>CSRM60</i> comparison result, sample 2 is amplification result of hair shaft DNA from pure bred Luxi cattle and sample 11 is amplification result of liver DNA from beef cattle.</p

Experimental design of DNA extraction for real-time PCR

*<p>ISAG-FAO recommended microsatellite markers for cattle. D: Diao™ enzymatic laundry powder. K: Keon™ enzymatic laundry powder. O: OMO™ enzymatic laundry powder. Each sample weight group has 3 extraction reagent groups (8 cattle for each extraction reagent group). For PCR, each sample was set 6 PCR template amount groups in 1^st round.</p

Results after two rounds of Real-time PCR using different enzymatic laundry powder, sample amounts and template volume in the first round PCR.

<p>√ Amplified successfully; × Failed amplification. Neg, reagent blanks. D: Diao™ enzymatic laundry powder. K: Keon™ enzymatic laundry powder. O: OMO™ enzymatic laundry powder.</p