Biocompatible fluorescent probe for detecting mitochondrial alkaline phosphatase activity in live cells.

Alkaline phosphatase (ALP) is an enzyme that actively plays a significant role in the various metabolic processes by transferring a phosphate group to the protein, nucleic acid, etc. The elevated level of ALP in blood plasma is the hallmark of inflammation/cancer. The hyperactive mitochondria in cancer cells produce an excess of ATP to fulfill the high energy demand. Thus, we have developed a fluorescent probe Mito-Phos for ALP, which can detect phosphatase expression in mitochondria in live cells. The probe Mito-Phos has shown ~15-fold fluorescence intensity increments at 450 nm in the presence of 500 ng/mL of ALP. It takes about 60 min to consume the whole amount of ALP (500 ng/mL) in physiological buffer saline. It can selectively react with ALP even in the presence of other probable cellular reactive components. It is highly biocompatible and nontoxic to the live cells. It has shown ALP expression in a dose-dependent manner by providing concomitant fluorescence images in the blue-channel region. It has localized exclusively in the mitochondria in live cells. The probe Mito-Phos is highly biocompatible with the ability to assess ALP expression in mitochondria in live cells.

Highly Chemoselective Self-Calibrated Fluorescent Probe Monitors Glutathione Dynamics in Nucleolus in Live Cells.

The redox-regulator glutathione (GSH) maintains a specific redox potential to sustain routine cellular activity from oxidative damage. In the early stage of the cell cycle process, the glutathione levels increase in the nuclei for protecting the DNA replication process from reactive oxygen species (ROS). In the first attempt, we developed a new ratiometric fluorescent probe that has provided information about glutathione levels in the nuclei. The UV-vis. absorption of probe GScp has shown a hypsochromic shift from 410 to 350 nm in the presence of GSH. In fluorescence titration, we observed that fluorescence emission of the GScp switched from 510 to 460 nm in the presence of GSH. The self-calibrated probe GScp has shown nearly optimal reversibility in GSH redox dynamics with the dissociation constant 2.47 mM. The probe is ideal for GSH tracking in live cells, as its toxicity has within the safe zone. The probe GScp has validated GSH levels in nucleoli by providing fluorescence images in blue-channel. This finding inspires us to use for validation of GSH dynamics in the nucleoli in the cell cycle process.

Highly chemoselective turn-on fluorescent probe for ferrous (Fe2+) ion detection in cosmetics and live cells.

In modern society, the use of cosmetics has increased extensively; unfortunately, so-called several toxic metal salts are present as the colorant or filler in cosmetics. The ferrous ion (Fe2+) is one of the metal ions used in cosmetics as a colorant. Ferrous ion (Fe2+) is a vital component in live cells. Considering the adverse effect of high doses of ferrous ions in cosmetics and live cells, we developed a turn-on fluorescent probe PFe(II) for quantitative estimation of ferrous ion (Fe2+) in cosmetics and monitoring of labile ferrous (Fe2+) ion in live cells. The fluorescent probe PFe(II) showed a visual color change from colorless to orange in the presence of ferrous ion (Fe2+) in the cosmetics. We observed that UV-absorption increased at 390 nm upon incubation with ferrous ion (Fe2+). The probe PFe(II) has provided quantitative information on ferrous ion (Fe2+) in various cosmetics, kajol, lip balm, face foundation, mascara, eyeliner, lipliner, face makeup, sindoor, lipstick, nail polish in ppm level through the fluorescence signaling at 460 nm.The probe PFe(II) provided information on labile Fe2+ ion pool via a fluorescence imaging. It is a new addition to the diagnostic inventory for detecting ferrous ion in live cells and cosmetics.

Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues.

Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson's correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.

A rhodamine based fluorescent probe validates substrate and cellular hypoxia specific NADH expression.

Herein, we report a rhodamine-based redox probe (MQR) to visualize cytosolic NADH in the cellular milieu. Its high sensitivity and selectivity allowed it to track the alteration of the NADH level under metabolic perturbation, suggesting its potential as a useful tool to study the association between the NADH level and metabolic abnormalities with clinical significance.

A 'turn-on' fluorescent probe for lysosomal phosphatase: a comparative study for labeling of cancer cells.

We have described the ability of a newly synthesized fluorescent probe (LP1) to detect phosphatase activity in lysosomes in cancer cells. Probe LP1 displayed a 33-fold fluorescence intensity enhancement at λem 532 nm in the presence of phosphatase in HEPES buffer (pH 4.5). The quantum yield of probe LP1 was increased by ∼21-fold upon exposure to phosphatase at acidic pH. The probe LP1 is highly chemoselective toward phosphatase (ALP/ACP) and is insensitive to interference by ubiquitous biological analytes. The high cell adhesion property and cell viability of LP1 indicate that LP1 is biocompatible and nontoxic; these two characteristic features make it a suitable candidate for phosphatase tracking in living cells. LP1 dose-dependent fluorescence images in living cells suggested that the expression of phosphatase in cancer cells (HeLa) is 2-fold higher as compared to the normal NIH-3T3 cells. The colocalization images confirmed that LP1 was exclusively localized in lysosomes. We envision that LP1 could be a potential tool in clinical diagnosis for discriminating cancer cells from normal cells depending on the expression of phosphatase in lysosomes.