Sensing of intracellular NAD(P)H is important for numerous applications ranging from diagnostic assays to drug discovery. Up to now, investigation of NAD(P)H-dependent pathways in live cells has been restrained by the lack of efficient tools. Toward this goal, we developed a small molecule indicator, which allows both colorimetric and fluorescent NAD(P)H detection in biological samples. The design is based on a cyanine dye scaffold and utilizes a novel two-acceptor “turn-on” mechanism. Consequently, this indicator features unprecedented sensitivity and rapid response toward NAD(P)H at low micromolar levels under physiological conditions. First, we demonstrated the value of this reagent in a diagnostic assay of glucose, through the enzyme-coupling reaction of NAD(P)+-dependent glucose dehydrogenase (GDH). Second, we showed the utility of our indicator for NAD(P)H imaging in live cells. We confirmed its ability to reflect different NAD(P)H levels using the human colon cancer cell line deficient on mitochondrial respiration. Expanding the use of this indicator to advanced tissue models, we demonstrated its ability to visualize different metabolic states in the hypoxic core of tumor spheroids. This study demonstrates that small molecule indicators could serve as a valuable tool for the specific analysis of redox states at the single-cell level and may be well suitable for high-throughput metabolic screening of antitumor agents.
Capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) has become a key method in high-throughput glycan analysis. At present, CGE-LIF relies on the green fluorophore 8-aminopyrene-1,3,6-trisulfonic acid (APTS). However, APTS has moderate reactivity in labeling of glycans and a fixed selectivity profile. Here, we report synthesis of red-emitting and highly reactive fluorescent tags for glycan derivatization. The design is based on a 9-aminoacridine scaffold with various acceptor groups at C-2 (CN, SO 2 R) and a primary amino group at C-7 for conjugation via reductive amination. These reactive dyes exhibit absorption maxima close to 450 nm and emission above 600 nm. They readily undergo conjugation with reducing sugars at the desired 1:1 stoichiometry. The red emission of conjugates with a maximum at 610−630 nm can be observed under excitation with 488 nm light and detected separately from the APTS-labeled oligosaccharides. Phosphorylated 7,9-diaminoacridine-2-SO 2 R derivatives with variable amounts of negative charges provide high mobilities of glycoconjugates on polyacrylamide gel electrophoresis (PAGE), as compared with those of APTS. We further demonstrate their utility by labeling and separating a maltodextrin ladder and sialyllactose isomers. The new dyes are expected to cross-validate and increase the glycan identification precision in CGE-LIF and help to reveal "heavy" glycans, yet undetectable with the APTS label.
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