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Silicon-Based Nanoscale Probes for Biological Cells
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Youjin Lee, Andrew W.Phillips, Bozhi Tian
Modulating biochemical and biophysical processes naturally requires a method to sense changes to these natural mechanisms. The biological effect should be considered, particularly at the level where cellular regulation it occurs. For instance, if an induced change to cellular physiology is caused by an alteration of gene expression, this result could likely be assayed at the level of gene expression by quantifying changes in messenger RNA by methods such as real-time quantitative polymerase chain reaction (RT-PCR) or RNA sequencing (RNA-Seq). Alternatively, if the modulated gene encodes for a protein, this could be used to quantify the effect induced on the cell via protein quantification methods like Western blotting, or more sophisticated proteomics methods like stable isotope labeling by/with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantitation (iTRAQ) (Bantscheff et al. 2007). Proteomics methods and Western blotting are typically destructive to the cell. Noninvasive methods for protein detection and quantification include using fluorescently labeled antibodies specific for the protein of interest. Permeabilizing and staining the cells can allow for protein quantification while preserving the cell. Nucleic acids can similarly be quantified using complementary nucleic acid hairpin structures containing a fluorescent dye and a quencher. These probes exhibit turn-on fluorescence when the complementary nucleic acid sequence binds the probe and subsequently spatially separate the fluorophore and quencher, turning off Förster resonance energy transfer from the dye to the quencher, restoring fluorescence to the dye. A variety of small molecule fluorophores that have a specific interaction with an analyte, resulting in a spectral change, can also be used for live-cell imaging. Calcium imaging using Fura-2 is a popular method to visualize calcium fluxes in live cells, which can be correlated with electrical activity (Williams et al. 1985).
Introduction to Fluorescence and Photophysics
Published in Mary-Ann Mycek, Brian W. Pogue, Handbook of Biomedical Fluorescence, 2003
The quantum yield of fluorescence is an important parameter in photophysics. Often a measurement of the quantum yield of fluorescence of a fluorophore will give information regarding its biological environment. The photophysical deactivation pathways can be greatly influenced by the immediate environment. One example is the difference in fluorescence observed between DP in aqueous solution and when bound to proteins such as serum albumin. We have already demonstrated that aggregation of DP occurs in aqueous solution, leading to changes in absorption spectrum (Fig. 8). In the presence of bovine serum albumin (BSA), protein binding effectively disaggregates the DP to give fluorescent monomers. Figure 10 shows the change in the Soret band of the absorption spectrum from aggregated to monomeric form and concomitant increase in fluorescence emission as the monomer concentration increases with serum albumin concentration. Other examples of biological interest include ethidium bromide where the restriction on molecular motion that results from intercalation in DNA inhibits radiationless deactivation and produces a 20- to 30-fold increase in the fluorescence quantum yield and a 15-nm blue shift in the fluorescence emission spectra. Acridine orange (AO) is taken up in both the lysosomes and the nucleus in cells, but the resulting fluorescence is organelle dependent. In the acidic environment of the lysosome AO is protonated and emits a red fluorescence, but when intercalated in DNA the unprotonated form emits a strong green fluorescence. Another example of biological influence is seen in the calcium probe Fura-2. In the absence of Ca2+, Fura-2 has an absorption maximum at 363 nm, with a fluorescence emission maximum of 512 nm and a Φf of 0.23. However, when bound to Ca2+ the absorption maximum is shifted to 335 nm and the Φf increases more than twofold. Fura-2 is used as a ratiometric calcium indicator in cells as the calcium concentration can be accurately determined from the ratio of the 340 nm/380 nm signals in the fluorescence excitation spectrum (emission set at 510 nm).
27-Deoxyactein prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cellular damage in MC3T3-E1 osteoblastic cells
Published in Journal of Environmental Science and Health, Part A, 2018
Kwang Sik Suh, Eun Mi Choi, Woon-Won Jung, So Young Park, Sang Ouk Chin, Sang Youl Rhee, Youngmi Kim Pak, Suk Chon
To determine the most relevant targets of TCDD cytotoxicity, the mechanisms responsible for the protective effects of 27-deoxyactein were investigated. Because intracellular Ca2+ concentrations ([Ca2+]i) play a role in cell death, changes in [Ca2+]i were measured using the Fura-2 fluorescence technique. In the TCDD-treated group, there was an increase in [Ca2+]i concentrations relative to the control (Fig. 2). However, treatment with 27-deoxyactein (0.01–1 μM) significantly reduced [Ca2+]i levels in the TCDD-treated cells. TCDD-mediated oxidative stress is also responsible for mitochondrial damage and, thus, apoptosis.[30,31] To quantify the changes in levels of intracellular ROS induced by TCDD, MC3T3-E1 osteoblastic cells were treated with DCF-DA, which passively diffuses into cells and is oxidized by ROS to create a fluorescent form. Compared to the control, there was an increase in fluorescent intensity following treatment with 100 nM of TCDD for 48 h (Fig. 3). This finding is consistent with those of earlier studies that demonstrated the induction of ROS by TCDD in human endothelial cells [32] and human neuroblastoma SH-SY5Y cells.[24] Therefore, TCDD-induced cytotoxicity may be due to higher levels of ROS that lead to oxidative damage and alterations in the structural integrity of the cell. These findings are supported by those of Aly and Domenech,[33] who found that TCDD significantly induces H2O2 production, ROS generation, and cytotoxicity in rat hepatocytes. In the present study, the group that was pre-treated with 27-deoxyactein (0.01–1 μM) exhibited a decrease in ROS production in MC3T3-E1 osteoblastic cells compared to the TCDD-treated group. Thus, the increased survival rate of cells in the 27-deoxyactein pre-treated group was likely due to the prevention of ROS accumulation and the protection of cellular integrity.