Dataset Information

Fluorogenic Templated Reaction Cascades for RNA Detection.

ABSTRACT: Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal amplification, which hinders the detection of small amounts of genetic material. To address this limitation, we tested a new strategy for attaining higher-order signal amplification, in which a target sequence templates a chemical ligation, and the product of this reaction is in turn detected with a second templated reaction. The method is nonenzymatic, isothermal, and fluorogenic, allowing the direct detection of nucleic acids in complex matrices. Using this approach, as little as 500 attomoles (10 pM) could be detected with single nucleotide resolution. In a test of selectivity, single nucleotide substitutions and deletions could successfully be detected, including a deletion that is associated with tetracycline resistance in Helicobacter pylori. Compatibility with biological matrices was demonstrated by the direct detection of rRNA in bacterial lysate. Imaging and detection of target sequences on a solid support further illustrates the potential of the new approach for high-throughput analysis.

SUBMITTER: Velema WA

PROVIDER: S-EPMC5568022 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Fluorogenic Templated Reaction Cascades for RNA Detection.

Velema Willem A WA Kool Eric T ET

Journal of the American Chemical Society 20170405 15

Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal amplification, which hinders the detection of small amounts of genetic material. To address this limitation, we tested a new strategy for attaining high ...[more]

PMID: 28345912

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Project description:Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually are carried out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 x 10(4) M(-1)s(-1). The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation, and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of beta-NADPH (beta-nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half-maximum (fwhm) for the internal standard were 2.5%, 2.0%, and 1.0%, respectively, with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and approximately 10(-18) mol, respectively. The Michaelis constants (K(m)) for GSSG and beta-NADPH were found to be 40 +/- 11 and 4.4 +/- 0.6 muM, respectively, comparable with those obtained from UV/vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation, and detection for continuous GSH determinations.

Dataset Information

Fluorogenic Templated Reaction Cascades for RNA Detection.

Publications

Fluorogenic Templated Reaction Cascades for RNA Detection.

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