A Ratiometric Fluorescent Biosensor Based on Exonuclease ⅢAmplification Strategy for Pathogenic Bacterial Gene Detection / 分析化学

Food-borne pathogenic bacteria seriously threaten public health. Based on the mechanism of fluorescence resonance energy transfer (FRET), a ratiometric fluorescence biosensor was constructed by integration of Exo III-based signal amplification strategy. The Cy3 labeled R1-DNA firstly hybridized with Cy5 labeled R2-DNA to form duplex of R1/R2. Cy3 showed a low fluorescence response while Cy5 showed a high fluorescence response. The addition of target pathogenic bacterial gene (Lac Z gene) could de-hybridize the R1/R2,resulting in the fluorescence decreasing of Cy5 and the fluorescence recovering of Cy3. Under the assistance of Exo III, the signal change was further amplified. The detection of limit reached as low as 5.29 pmol/L. The linear detection range was from 10 pmol/L to 2000 pmol/L. The developed ratiomtric detection strategy significantly reduced the possibility of false-positive and false-negative detection results.

A F?rster Resonance Energy Transfer Ratiometric Probe Based on Quantum Dot-Cresyl Violet for Imaging Hydrogen Sulfide in Living Cells / 分析化学

Hydrogen sulfide ( H2S ) has been confirmed as a significant endogenous gaseous signaling molecule involved in various physiological processes.To monitor H2S in living cells, a F?rster resonance energy transfer ( FRET) ratiometric probe based on quantum dot-cresyl violet was developed.In this work, quantum dot nanospheres ( QDS) were firstly synthesiZed via a facile ultrasonication emulsion strategy, and the mixture chloroform solution containing hydrophobic quantum dots and COOH-functionaliZed amphiphilic polymer were successfully transferred into the oil-in-water micelle.The negatively charged quantum dot nanospheres with quantum dots embedded in the polymer matrixes were successfully fabricated after the evaporation of chloroform.And then, these quantum dot nanospheres were condensed with positively charged cresyl violet-aZide ( CV-N3 ) via electrostatic interaction to obtain the QDS-N3 complexes.The as-prepared QDS-N3 complexes were monodispersed nanospheres with an average diameter of about 120 nm.These complexes were taken up by the cell through endocytosis, and they were still stable even in wide pH range.In addition, the QDS-N3 complexes exhibited no cellular toxicity which was verified by MTT assay.In this ratiometric probe, CV-N3 as a FRET acceptor was conjugated to quantum dot nanospheres.The quantum dots emitted at 591 nm and served as the FRET donor;once the aryl aZide on the CV-N3 was reduced to aniline by H2S, the probe emitted at 620 nm.The ratiometric probe allowed the elimination of interference of excitation intensity, intracellular environment and other factors.Furthermore, this method also offered a general protocol for preparing nanosensors for monitoring various small molecular in living cells.