A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

NADH-Activated Dual-Channel Fluorescent Probes for Multicolor Labeling of Live Cells and Tumor Mimic Spheroids.

The 1,4-dihydronicotinamide adenine dinucleotide (NADH) is one of the key coenzymes that participates in various metabolic processes including maintaining the redox balance. Early information on the imbalance of NADH is crucial in the context of diagnosing the pathogenic conditions. Thus, a dual-channel fluorescent probe (MQN) is developed for tracking of NADH/NAD(P)H in live cells. In the presence of NADH, only it showed emission signals at 460 and 550 nm upon excitation at 390 and 450 nm, respectively. The probe could provide accurate information on NADH levels in cancer cells (HeLa) and normal cells (WI-38). We observed that the NADH level in cancer cells (HeLa) is relatively higher than that in normal WI-38 cells. We received similar information on NADH upon calibrating with a commercial NADH kit. Moreover, we evaluated substrate-specific NADH expression in the glycolysis pathway and oxidative phosphorylation process. Also, the dual-channel probe MQN has visualized NADH manipulation in the course of depletion of GSH to maintain cellular redox balance. This dual-channel molecular probe MQN comes out as a new detection tool for NADH levels in live cells and tumor mimic spheroids.

Polarity-Driven Two-Photon Fluorescent Probe for Monitoring the Perturbation in Lipid Droplet Levels during Mitochondrial Dysfunction and Acute Pancreatitis.

Lipid droplets (LDs) act as an energy reservoir in cancer cells; on the other hand, mitochondria are hyperactive to fulfill the energy demand to accelerate cell proliferation. We are interested in unfolding the relationship between the cellular energy reservoir and energy producer through fluorescence labeling. Thus, a dual organelle-targeted fluorescent probe MLD-1 has been rationally developed. It visualized the crosstalk between mitochondrial dysfunction and the fluctuation of LDs in live cells. Its two-photon ability allowed us to acquire deep tissue images. For the first time, we have shown that the probe has the ability to track the accumulation of LDs in different mouse organs during pancreatic inflammation. MLD-1, being a selectively polarity-driven, chemo- and photostable LD probe, may offer great possibilities for studying LD-associated biology in due course.

Monitoring glutathione dynamics in DNA replication (S-phase) using a two-photon reversible ratiometric fluorescent probe.

The redox regulator glutathione (GSH) migrates to the nucleus to give a safeguard to DNA replication in the S-phase. The fluctuation of GSH dynamics in the cell cycle process may help to understand cancerogenesis or other abnormalities in DNA replication. For the first time, we attempted to track the time-dependent S-phase change using the newly developed ratiometric fluorescent probe Nu-GSH. This probe is highly chemoselective towards glutathione and shows an emission intensity shift from 515 nm to 455 nm. It has shown fluorescence reversibility from blue to green channels while scavenging reactive oxygen species H2O2. Both ratiometric fluorescence images and FACS analysis have provided quantitative information on the GSH levels in the nucleoli during DNA replication in the S-phase. Furthermore, GSH fluctuation reciprocated the decay of the S-phase on a time scale. Additionally, its two-photon ability guaranteed its capability to study GSH dynamics in live cells/tissues noninvasively. We envision that the probe Nu-GSH can be used to get high-throughput quantitative information on glutathione dynamics and give an opportunity to monitor its perturbation during the course of cell division.

Direct Readout Hypoxia Tumor Suppression In Vivo through NIR-Theranostic Activation.

Urgency in finding a suitable therapy in tumor hypoxia strives to develop hypoxia-targeted activatable theranostic. A strategic theranostic prodrug (Azo-M) has been synthesized. Its azo-linker scission under the hypoxia condition has released an near-infrared (NIR)-reporter to determine the extent of chemotherapeutic (melphalan analogue) activation. Under an artificial hypoxia condition, a large shift from 520 to 590 nm in UV absorption was observed in Azo-M. Alongside, the emission maxima had appeared at 625 nm under the said condition. The Azo-M post-incubated HeLa cells have shown upregulation of various apoptotic factors under oxygen deprivation (3%) condition. Azo-M has shown antiproliferative activity under hypoxia conditions in various cancer cells. An ex-vivo biodistribution study indicated that theranostic Azo-M only activated in tumor tissue and to some extent in the liver. The therapeutic activity study in vivo indicated that Azo-M effectively reduced the tumor size and volume (about 2-fold) without the change of bodyweight of mice. The theranostic Azo-M can be a cornerstone to suppress tumor hypoxia and tracking its extent of suppression.

Amphiphilic fluorescent probe self-encored in plasma to detect pH fluctuations in cancer cell membranes.

We have developed an amphiphilic pH probe (P1CS) to detect pH levels in the plasma membrane in cancer cells. An elevated fluorescence signal at 550 nm at the cell surface of cancer cells (MDA-MB-231, HeLa cells) prompted the application of P1CS as a pH marker for the cancer cell surface, discriminating it from normal cells (WI-38). Moreover, the probe enables labeling of the surface of multilayered tumor spheroids, which promotes its use as a marker for the surface of tumor tissue.

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.

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.