Haiying Liu

Three BODIPY-based fluorescent probes, AH⁺, BH⁺, and CH⁺, were synthesized for ratiometric pH sensing in living cells and fruit fly larvae. These probes were designed to target mitochondrial environments by functionalizing the BODIPY core with various substituent groups that tune their pH sensitivity. The probes exhibited strong ratiometric fluorescence changes with pK_a values (AH⁺, 7.3; BH⁺, 7.5; CH⁺, 7.2) suitable for mitochondrial pH detection. Theoretical calculations supported these findings by establishing the geometries and electronic transitions and also resulted in the derivation of their pK_a values. Confocal imaging confirmed mitochondrial accumulation of these probes in HeLa cells, facilitating a broad-range pH monitoring across a wide pH spectrum (3.5 to 9.1). These ratiometric pH sensors display good reversibility and response time under varying pH conditions. In application, the probe AH⁺ was utilized to monitor pH fluctuations under conditions of oxidative stress and starvation conditions. Dual-channel cell imaging revealed a pH-dependent fluorescence shift with precise transitions, demonstrating the feasibility of real-time monitoring of mitochondrial pH changes in living cells. Furthermore, the probe AH⁺ effectively visualized pH changes in Drosophila melanogaster larvae, further supporting its applicability across diverse biological systems. We demonstrate that a fluorescence ratiometric intensity graph for probe AH⁺ can be effectively employed to determine pH values within the mitochondria of HeLa cells.

We introduce a ratiometric sensor designed for sensitive and specific detection of NAD(P)H in living cells, tissues, and whole organisms.

Monitoring the dynamic fluctuations of NADH and NADPH, key coenzymes in intracellular redox homeostasis and bioenergetics, is essential for understanding both normal physiology and disease pathology. However, precise, real-time imaging of NAD(P)H levels in complex biological systems remains a major challenge due to interference from endogenous fluorescence and the limitations of intensity-based sensors. We present a newly engineered dual-emission fluorescent sensor, sensor A, which overcomes these obstacles by offering ratiometric detection with high specificity and sensitivity. The sensor features a 3-quinolinium moiety conjugated to a cyanine dye scaffold via an amine linker, enabling selective reactivity toward NAD(P)H. Upon reduction, the sensor displays a pronounced fluorescence shift with visible emission enhancement at 511 nm and a simultaneous near-infrared emission decrease at 711 nm. This ratiometric response provides a self-calibrating readout, minimizing errors from sensor concentration or excitation variability. Sensor A was validated in diverse biological contexts. In live cancer cell lines (HeLa and MD-MB453), it sensitively tracked NAD(P)H alterations induced by metabolic stimulation and chemotherapy. In Drosophila melanogaster larvae, the sensor enabled in vivo imaging of redox changes under nutrient deprivation and drug exposure. Remarkably, sensor A also distinguished between healthy and diseased human kidney tissues, revealing elevated NADH levels in polycystic kidney disease samples. These results demonstrate the broad utility of sensor A for investigating NAD(P)H-related processes in both research and clinical diagnostics. Its dual-emission, ratiometric output, and compatibility with live cell and in vivo imaging make it a valuable platform for probing redox biology across multiple model systems.

Three BODIPY-based fluorescent probes, AH+, BH+, and CH+, were synthesized for ratiometric pH sensing in living cells, fruit flies, and zebrafish larvae. These probes were designed to target mitochondrial environments by functionalizing the BODIPY core with various substituent groups that tune the pH sensitivity. The probes exhibited strong ratiometric fluorescence changes with pKa values (AH+, 7.3; BH+, 7.5; CH+, 7.2) suitable for mitochondrial pH detection. Theoretical calculations supported these findings by establishing the geometries and electronic transitions and also resulted in the derivation of their pKa values. Confocal imaging confirmed mitochondrial accumulation of these probes in HeLa cells, facilitating broad-range pH monitoring across a wide pH spectrum (3.5 to 9.1). These ratiometric pH sensors display good reversibility and response times under varying pH conditions. In application, the probe AH+ was employed to monitor pH fluctuations under conditions of oxidative stress and nutrient deprivation. Dual-channel cell imaging revealed a pH-dependent fluorescence shift with precise transitions, demonstrating the feasibility of real-time monitoring of the mitochondrial membrane potential in living cells. Furthermore, the probe AH+ effectively visualized pH changes in Drosophila melanogaster and zebrafish larvae, further supporting its applicability across diverse biological systems. We demonstrate that a fluorescence ratiometric intensity graph for probe AH+ can be effectively employed to determine pH values within the mitochondria of HeLa cells.

Two rhodamine-based probes were developed for ratiometric pH detection. Probe A was effective at pH sensing in HeLa cells and fruit fly larvae due to the pH- and viscosity-dependent ring-opening behavior of the spirolactam switch.

We developed two deep-red cyanine chromophores, probes A and B, for selective mitochondrial NAD(P)H detection in live cells. Probe A features a 1,2,3,3-tetramethyl-3H-indolium core, while probe B incorporates a 1,1,2,3-tetramethyl-1H-benzo[e]indol-3-ium moiety, both linked to quinolinium via a vinyl bond to enable fluorescence modulation upon NAD(P)H reduction of probes A and B. To explore the role of electron-withdrawing groups in probe sensitivity, we synthesized three additional cyanine dyes (probes C, D, and E) via condensation of 6-quinolinecarboxaldehyde with 2,3-dimethyl-1,3-benzothiazolium acceptor and malononitrile derivatives, followed by methylation. Under NAD(P)H-deficient conditions, probe A showed absorption at 382 nm with weak fluorescence at 636 nm, while probe B absorbed at 443 nm with weak fluorescence at 618 nm. Upon NAD(P)H reduction, probe A exhibited red-shifted absorption at 520 nm with enhanced emission at 589 nm, and probe B at 550 nm with strong emission at 610 nm. Probe C showed absorption at 524 nm with enhanced emission at 586 nm, while probes D and E exhibited no detectable NAD(P)H response, highlighting the critical role of quinolinium acceptors. Probe B demonstrated superior sensitivity, successfully tracking NAD(P)H fluctuations in HeLa cells under glycolysis stimulation (glucose, lactate, pyruvate) and treatments with LPS and methotrexate. It also visualized NAD(P)H in Drosophila larvae, revealing increased levels after drug treatments. Notably, probe B distinguished between healthy and diseased human kidney tissues, detecting significantly elevated NADH levels in autosomal dominant polycystic kidney disease (ADPKD) samples, emphasizing its diagnostic potential. This study introduces probe B as a versatile and reliable NAD(P)H sensor for metabolic research and disease diagnostics, offering valuable insights into redox processes in live cells, organisms, and clinical samples.