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Hybridising inorganic materials with fluorescent BOPHY dyes: A structural and optical comparative study.

ABSTRACT: This study presents the design and characterization of new monochromatic light-harvesting systems based on inorganic porous materials hybridized with organic dye molecules within their structure. A new fluorescent BOPHY dye was prepared, characterized optically and used as both reference and synthetic precursor for two alkoxysilane derivatives that were incorporated separately within a silica structure. The dyes, one bearing one alkoxysilane group and the other one two, were co-condensed with tetraethyl orthosilicate to form a hybrid organo-silica framework, where they are found at specific locations. The structure of the new materials was analysed by powder XRD and TEM, which confirmed the presence of the hexagonal pore arrangement typical of mesoporous MCM-41 silica particles. The steady-state and time-resolved analysis showed that the particles where the dyes are most dispersed within the framework retain the highest fluorescence quantum yield, up to 0.63, in the green-yellow region of the visible spectrum. On the other hand, increasing the content of BOPHY units in the solid matrix seem to favour non-radiative deactivation pathways and aggregation phenomena, which lower the efficiency of light emission. The materials also exhibit interesting properties, such as a dual excited-state decay and fluorescence anisotropy. The short fluorescence lifetime, about 2 ns, matches the typical singlet lifetime of BOPHY dyes, whereas the long component, up to 20 ns, is attributed to delayed fluorescence, which could take place via charge recombination. Optical anisotropy experiments revealed that all materials show polarised light emission to a significant extent and, for most samples, it was also possible to determine a polarisation transfer decay trace, from 400 to 800 ps This is ascribed to the occurrence of energy migration between neighbouring dye units within the silica structure.

SUBMITTER: Sani U

PROVIDER: S-EPMC9274302 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA. Keywords: Comparison of fluorescent dyes Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light). Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen). 3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2Âµg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, RÃ¸dovre, Denmark). Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5Âµl cDNA from each synthesis, 0.5Âµl Salmon sperm (10Âµg/Âµl) and 13.5Âµl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29Âµl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5Âµl of each Capture reagent and 26Âµl SDS-Based hybridization buffer was mixed to a final volume of 52Âµl. 26Âµl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).

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Hybridising inorganic materials with fluorescent BOPHY dyes: A structural and optical comparative study.

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