pH In Living Cells Research Articles

A hemicyanine-based fluorescent probe for detection of pH and its applications in live cells imaging and food monitoring

pH as an important parameter is closely related to many pathological processes and food freshness. In this paper, a new pH-sensitive fluorescent probe 1 based on hemicyanine dye was synthesized and used for the detection of pH in live cells and food spoilage. Under neutral and alkaline conditions, the phenolic hydroxyl group of the probe deprotonates, forming a closed spirocyclic structure and resulting in fluorescence quenching. Under acidic conditions, the probe maintains an open-loop structure and has intense fluorescence. The probe possessing good selectivity and stability can target lysosomes and visualize the exogenous pH in live cells. Furthermore, by using this probe, we successfully achieved the monitoring of pH in metamorphic process of shrimp and pork. This probe is expected to provide a good imaging tool for real-time detection of pH in live cells and food monitoring.

Carbon Nanotube-Based Nanosensor Reports Lysosomal pH and Autophagy Activation in vivo

One of the major challenges in studying biological systems is the dearth of robust tools available to monitor dynamic biological processes in live cells and animals. For example, current techniques to measure autophagy are either ex vivo or involve conventional fluorophores/fluorescent proteins, which can be used for in vitro measurements but present problems in vivo. In this presentation, we show the development of optical nanosensors that non-invasively monitor autophagy-associated lysosomal pH changes. The sensor technology is based on quantum-defect-modified fluorescent carbon nanotubes, often called organic color centers (OCCs). OCC fluorescence is bright, photostable, and optically tunable by the chemical nature of the color center. We designed the OCC-nanosensors to be sensitive and selective to endolysosomal pH. Their unique chemical and photophysical properties enabled dynamic and quantitative measurements of lysosomal pH in live cells and in vivo. We will discuss how we utilize these capabilities to facilitate previously inaccessible mechanistic insights in the fields of cancer biology.

A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range.

The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.5-9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range.

Carbon Nanotube Quantum Defect Photoluminescence Modulation for Biosensors

Covalent sp3 defects on single-walled carbon nanotubes (SWCNTs), often called organic color centers (OCCs) have been explored for their photoluminescent properties, including the potential for introducing new emission bands, increasing quantum yield, and introducing new chemical sensitivities. The application of OCCs for biomedical applications requires new methods to synthesize them via covalent chemistry, to control their biological interactions/biocompatibility via supramolecular interactions with excipients, and to measure their emission in biological environments. We have found specific utilities of OCCs for the detection of chemical phenomena where SWCNT photoluminescence is normally relatively insensitive, such pH changes in the physiologic range. We developed OCCs to measure lysosomal pH in live cells and in vivo, enabling new biological discoveries. We also developed OCC-based biosensors for cancer detection. We believe that OCCs will facilitate the development of an important class of sensors for biomedical applications.

Cascade C-H-Activated Polyannulations toward Ring-Fused Heteroaromatic Polymers for Intracellular pH Mapping and Cancer Cell Killing.

The development of straightforward and efficient synthetic methods toward ring-fused heteroaromatic polymers with attractive functionalities has great significance in both chemistry and materials science. Herein, we develop a facile cascade C-H-activated polyannulation route that can in situ generate multiple ring-fused aza-heteroaromatic polymers from readily available monomers in an atom-economical manner. A series of complex polybenzimidazole derivatives with high absolute molecular weights of up to 24 000 are efficiently produced in high yields within 2 h. Benefiting from their unique imidazole-containing ring-fused structures with multiple aryl pendants, the obtained polymers show excellent thermal and morphological stability, good solution processability, high refractive index, small chromic dispersion, as well as remarkable acid-base-responsive fluorescence. Taking advantage of the ratiometric fluorescence response of the triphenylamine-substituted heteroaromatic polymer to pH variations, we successfully apply it as a sensitive fluorescence probe for the mapping and quantitative analysis of intracellular pH in live cells. Furthermore, through the simple N-methylation reaction of the ring-fused polybenzimidazoles, diverse azonia-containing polyelectrolytes are readily produced, which can efficiently kill cancer cells via the synergistic effects of dark toxicity and phototoxicity.

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

(Invited) Organic Color Center Photoluminescence Modulation for Biomedical Applications

The modulation of single-walled carbon nanotube (SWCNT) photoluminescence has been under investigation for the unique potential to develop biosensors to detect many classes of analytes. Covalent sp3 defects on SWCNTs, dubbed organic color centers (OCCs), have been explored for their photoluminescent properties, including increasing quantum yield and introducing new chemical sensitivities. The application of OCCs for biomedical applications requires new methods to control SWCNT surface chemistry and the resulting biological interactions/biocompatibility via supramolecular interactions with polymers and other excipients. We have found specific utilities of OCCs for the detection of chemical phenomena where SWCNT photoluminescence is normally relatively insensitive, such as pH changes in the physiologic range of 4.5 - 8. We developed OCC-based sensors to measure pH in live cells and in vivo, as well as other emerging applications. We believe that OCCs will facilitate the development of biosensors for disease detection and biomedical research.

One probe for multiple targets: A NIR fluorescent rhodamine-based probe for ONOO− and lysosomal pH detection in live cells

• A new rhodamine-based probe ONOO-LysopH was developed. • ONOO-LysopH responds to multiple targets: ONOO − and acidic pH. • ONOO-LysopH shows NIR fluorescence changes with a large Stokes shift. • ONOO-LysopH has a pKa of 5.41 and good lysosome-targeting ability. • ONOO-LysopH is applicable for cell imaging ONOO − and tracking lysosomal pH changes. Peroxynitrite and lysosomes are important biologically reactive oxygen species and subcellular organelles, respectively. The level of peroxynitrite and the value change of pH in lysosomes have a significant impact on living cells. Thus, the detection of peroxynitrite and lysosomal pH in live cells is very important. Herein, a novel probe ( ONOO-LysopH ) was developed, which not only can detect peroxynitrite in lysosomes, but also can be used to track the lysosomal pH changes. ONOO-LysopH is based on a new rhodamine derivate with near-infrared (NIR) emission and large Stokes shift. ONOO-LysopH is sensitive to acid and shows an instantaneous and reversible response of NIR emission around 686 nm with a pKa value of 5.41. ONOO-LysopH also exhibits a quick, sensitive, and selective response to peroxynitrite at physiological pH with a largely enhanced NIR fluorescence signal at 678 nm. The application of ONOO-LysopH to track lysosomal pH changes and image both endogenous and exogenous peroxynitrite in live cells was demonstrated. Thus, this work provided a potential multifunctional probe for peroxynitrite detection and lysosomal pH change tracking in live systems.

Near-Infrared Ratiometric Two-Photon Probe for pH Measurement in Human Stomach Cancer Tissue.

Stomach cancer is a global health issue because of its incidence and mortality rates worldwide. We developed a near-infrared (NIR) emissive ratiometric two-photon (TP) probe (HCC1) for the quantitative analysis of pH in live cells and human stomach tissues. The probe design is based on a restrained hemicyanine core that controls the intramolecular charge transfer from 2-naphthol, with a suitable pKa value (7.50) under physiological conditions. The probe exhibited improved quantum yield, stability, and TP activity under physiological conditions. In addition, intracellular pH titration (pH 4.0 to 10.0) of HCC1 revealed an ideal intracellular pKa of approximately 7.2, negligible cytotoxicity, and TP excited fluorescence in situ, thereby allowing direct imaging of the cellular pH in live cells and tissues. Ratiometric two-photon microscope imaging with HCC1 of human stomach tissue revealed a clear intratissue pH variation among normal, adenoma, and cancer tissues. Our results demonstrate that HCC1 is useful as an NIR imaging probe for in situ pH-related studies and in cancer research.

Real-Time, In Vivo Monitoring of Pharmacodynamics in Solid Tumors Using Organic Color Centers

Covalent bonding of organic functional groups to the sp2 carbon lattice can produce tunable sp3 quantum defects that fluoresce brightly in the second near-infrared (NIR-II, 1000-1700 nm). Such synthetic defects, or ‘organic color centers’ (OCCs), provide a molecular focal point that sensitively and selectively responds to the local dielectric environments. To benefit from the unique electronic and optical properties of OCCs in biomedical applications, we present a new method that controls the biological interactions/biocompatibility of OCC-based nanosensors via their supramolecular interactions with polymers and other excipients. The resultig OCC-based nanosensor measures endolysosomal pH in live cells and in vivo. We discuss the utility of the nanosensor to real-time monitor the pharmacodynamics of anticancer drugs in solid tumors.

Organic Color Center Photoluminescence Modulation for Biomedical Applications

The modulation of single-walled carbon nanotube (SWCNT) photoluminescence has been under investigation for the unique potential to develop biosensors capable of detecting many classes of analytes. Covalent sp3 defects have introduced emissive color centers on SWCNTs, significantly enhancing photoluminescence quantum yield and chemical selectivity/sensitivity to certain analytes. The application of these organic color centers (OCCs) for biomedical applications requires new methods to both conduct covalent chemistry and to concomitantly control the surface chemistry and the resulting biological interactions/biocompatibility via their supramolecular interactions with polymers and other excipients. We will report specific utilities of OCCs for the detection of chemical phenomena where SWCNT photoluminescence is normally relatively insensitive, such as pH changes in the physiologic range of 4.5 - 8. We have developed OCCs to measure endolysosomal pH in live cells and in vivo, as well as other emerging applications. The results support the feasibility of using OCCs as biosensors for disease detection and biomedical research.

Real-time monitoring of intracellular pH in live cells with fluorescent ionic liquid

Real-time monitoring of intracellular pH is of great significance due to its essential role in physiological and pathological processes. In present work, the ionic liquid (IL) N-methyl-6-hydroxyquinolinium bis(trifluoromethylsulfonyl) imide ([6MQc][NTf2]) is proposed as a fluorescence probe for the quantitative imaging of intracellular pH in response to external stimuli. The fluorescence of the IL [6MQc][NTf2] exhibits a sensitive response to pH variations, as the deprotonation of [6MQc][NTf2] generates the highly fluorescent zwitterionic product [6MQz]. pH fluctuations in the range of 6.0–7.5 can be accurately sensed by monitoring the fluorescence change at 555 nm. Moreover, this IL probe exhibits favorable biocompatibility, excellent anti-photobleaching properties, and high tolerance to ionic strength. Using the IL probe, real-time sensing of hypoxia- and drug-induced intracellular pH changes in MCF-7 cells is achieved.

Time-Resolved Activation of pH Sensing and Imaging in Vivo by a Remotely Controllable DNA Nanomachine.

Construction of probes or nanodevices capable of sensing pH with high spatial and temporal precision remains a challenge, despite their importance in monitoring of diverse physiological and pathological processes. Here we disclose the first remotely and noninvasively controlled DNA nanomachine that can monitor pH in live cells and animals in a temporally programmable manner. The nanomachine is designed by rational engineering of the DNA motif with a light-responsive element and further combination with an upconversion nanoparticle that works as a transducer to manipulate the nanomachine with the high precision of NIR light. The nanomachine not only allows for activated fluorescent imaging of intracellular pH, but it also can exert spatiotemporal control over its pH sensing activity in tumor-bearing mice by NIR light irradiation at a chosen time and place. This work illustrates the potential of combining DNA nanotechnology with upconversion tools to yield a precisely controlled nanomachine for temporally resolved pH sensing and imaging.

Dual-Monitoring Glycosylation and Local pH in Live Cells by Metabolic Oligosaccharide Engineering with a Ratiometric Fluorescent Tag.

Glycosylation and local pH change play vital roles in many biological processes. Monitoring the distribution of glycosylated molecules and their local nanoenvironment pH in live cells would provide deep insights into cellular metabolism. In this paper, we have reported a new cyclooctyne-linked fluorescent tag, ANCO, which can freely diffuse into cells and selectively label azide-modified glycans by a bioorthogonal copper-free click reaction. Combining with metabolic oligosaccharide engineering, ANCO could help to visualize the dynamic distribution of glycans as well as their local pH by ratiometric fluorescence signals. Using this strategy, we simultaneously observed the glycosylation inhibition and the local pH change of glycans in live cells after treatment with tunicamycin, a glycosylation inhibitor. ANCO showed great promise as a universal tool for dual-monitoring the distribution of azido-metabolic molecules and their local nanoenvironment pH in live cells.

A novel water-soluble fluorescent probe with ultra-sensitivity over a wider pH range and its application for differentiating cancer cells from normal cells.

A novel pH-sensitive fluorescent probe has been designed and synthesized for sensing intracellular pH. This probe showed excellent water solubility, it was sensitive toward the pH range from 4 to 12, and it was especially sensitive in alkaline environments. During the pH changes from acidic to alkaline environments, the color of the solution turned from yellow to purple, thus the difference in color can be used to distinguish between acidic and alkaline systems. The other major features of probe pH-DCN including high selectivity, low toxicity, good reversibility and stability allowed pH-DCN to visualize fluctuations of the pH in live cells. Moreover, probe pH-DCN has successfully discriminated cancer cells from normal cells by monitoring their different intracellular pH levels.

A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging of Acid pH in Living Cells and Tissues and Early Detection of Tumor in Mouse Model.

Acid-base disorders disrupt proper cellular functions, which are associated with diverse diseases. Development of highly sensitive pH probes being capable of detecting and monitoring the minor changes of pH environment in living systems is of considerable interest to diagnose disease as well as investigate biochemical processes in vivo. We report herein two novel high-resolution ratiometric two-photon (TP) fluorescent probes, namely, PSIOH and PSIBOH derived from carbazole-oxazolidine π-conjugated system for effective sensing and monitoring acid pH in a biological system. Remarkably, PSIOH exhibited the largest emission shift of ∼169 nm from 435 to 604 nm upon pH changing from basic to acidic with an ideal p Ka value of 6.6 within a linear pH variation range of 6.2-7.0, which is highly desirable for high-resolution tracking and imaging the minor fluctuation of pH in live cells and tissues. PSIOH also exhibits high pH sensitivity, excellent photostability, and reversibility as well as low cytotoxicity. More importantly, this probe was successfully applied to (i) sense and visualize the pH alteration in HeLa cells caused by various types of exogenous stimulation and (ii) detect and differentiate cancer and tumors in liver tissues and a mouse model, realizing its practical in vitro and in vivo applications.

A two-photon ratiometric fluorescent probe for effective monitoring of lysosomal pH in live cells and cancer tissues

Lysosomes are ubiquitous acidic organelles responsible for breaking down of biological substances. Acidic pH is a characteristic feature of lysosomes whereas aberrant lysosomal pH has been manifested in myriad diseases. Thus, being able to perform real-time monitoring of lysosomal pH is crucial to understand and investigate pathological and physiological processes. Herein, we report a novel and efficient carbazole-benzimidazole-based two-photon ratiometric fluorescence probe, namely BIMC for fast-response lysosomal pH detection and imaging. In this probe, the methylcarbitol substituent serves as a targeting unit for lysosomes and the benzimidazole moiety acts as a proton reaction site. BIMC exhibits an ideal pKa value of 4.46, which is found useful for quantitative detection and imaging of lysosomal pH change in the linearly proportional range of 3.8–5.0. Two-photon ratiometric imaging studies shows that BIMC can selectively monitor pH with excellent photostability and low cytotoxicity in the lysosomes of live cells and in living cancer tissues. All these results demonstrate that BIMC is a promising two-photon fluorescent probe for a practical use in investigating lysosome-associated pathological and physiological processes.

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.

Fluorescent Silicon Nanorods-Based Ratiometric Sensors for Long-Term and Real-Time Measurements of Intracellular pH in Live Cells.

Long-term and real-time investigation of the dynamic process of pHi changes is critically significant for understanding the related pathogenesis of diseases and the design of intracellular drug delivery systems. Herein, we present a one-step synthetic strategy to construct ratiometric pH sensors, which are made of europium (Eu)-doped one-dimensional silicon nanorods (Eu@SiNRs). The as-prepared Eu@SiNRs have distinct emission maxima peaks at 470 and 620 nm under 405 nm excitation. Of particular note, the fluorescence emission intensity at 470 nm decreases along with the increase of pH, while the one at 620 nm is nearly unaffected by pH changes, making Eu@SiNRs a feasible probe for pH sensing ratiometrically. Moreover, Eu@SiNRs are found to be responsive to a broad pH range (ca. 3-9), biocompatible (e.g., ∼100% of cell viability during 24 h treatment) and photostable (e.g., ∼10% loss of intensity after 40 min continuous UV irradiation). Taking advantages of these merits, we employ Eu@SiNRs for the visualization of the cytoplasmic alkalization process mediated by nigericin in living cells, for around 30 min without interruption, revealing important information for understanding the dynamic process of pHi fluctuations.

A new ratiometric two-photon fluorescent probe for imaging of lysosomes in living cells and tissues

Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments demonstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging resolution and deep-tissue imaging depth over 180 μm, we thus may expect the new platform prompt the development of a wide variety of ratiometric two-photon fluorescent probes application in biological systems.