Advances in pH Sensing: From Traditional Approaches to Next-Generation Sensors in Biological Contexts.

pH has been considered one of the paramount factors in bodily functions because most cellular tasks exclusively rely on precise pH values. In this context, the current techniques for pH sensing provide us with the futuristic insight to further design therapeutic and diagnostic tools. Thus, pH-sensing (electrochemically and optically) is rapidly evolving toward exciting new applications and expanding researchers' interests in many chemical contexts, especially in biomedical applications. The adaptation of cutting-edge technology is subsequently producing the modest form of these biosensors as wearable devices, which are providing us the opportunity to target the real-time collection of vital parameters, including pH for improved healthcare systems. The motif of this review is to provide insight into trending tech-based systems employed in real-time or in-vivo pH-responsive monitoring. Herein, we briefly go through the pH regulation in the human body to help the beginners and scientific community with quick background knowledge, recent advances in the field, and pH detection in real-time biological applications. In the end, we summarize our review by providing an outlook; challenges that need to be addressed, and prospective integration of various pH in vivo platforms with modern electronics that can open new avenues of cutting-edge techniques for disease diagnostics and prevention.

Hell's Gate Globin-I from Methylacidiphilum infernorum Displays a Unique Temperature-Independent pH Sensing Mechanism Utililized a Lipid-Induced Conformational Change.

Hell's Gate globin-I (HGb-I) is a thermally stable globin from the aerobic methanotroph Methylacidiphilium infernorum. Here we report that HGb-I interacts with lipids stoichiometrically to induce structural changes in the heme pocket, changing the heme iron distal ligation coordination from hexacoordinate to pentacoordinate. Such changes in heme geometry have only been previously reported for cytochrome c and cytoglobin, linked to apoptosis regulation and enhanced lipid peroxidation activity, respectively. However, unlike cytoglobin and cytochrome c, the heme iron of HGb-I is altered by lipids in ferrous as well as ferric oxidation states. The apparent affinity for lipids in this thermally stable globin is highly pH-dependent but essentially temperature-independent within the range of 20-60 °C. We propose a mechanism to explain these observations, in which lipid binding and stability of the distal endogenous ligand are juxtaposed as a function of temperature. Additionally, we propose that these coupled equilibria may constitute a mechanism through which this acidophilic thermophile senses the pH of its environment.

Aspartyl peptidase May1 induces host inflammatory response by altering cell wall composition in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3. IMPORTANCE: The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

pH Modulation of Super-Assembled Heteromembranes for Sustainable Chiral Sensing.

Enantioselective sensing and separation represent formidable challenges across a diverse range of scientific domains. The advent of hybrid chiral membranes offers a promising avenue to address these challenges, capitalizing on their unique characteristics, including their heterogeneous structure, porosity, and abundance of chiral surfaces. However, the prevailing fabrication methods typically involve the initial preparation of achiral porous membranes followed by subsequent modification with chiral molecules, limiting their synthesis flexibility and controllability. Moreover, existing chiral membranes struggle to achieve coupled-accelerated enantioseparation (CAE). Here, we report a replacement strategy to controllably produce mesoscale and chiral silica-carbon (MCSC) hybrid membranes that comprise chiral pores by interfacial superassembly on a macroporous alumina (AAO) membrane, in which both ion- and enantiomers can be effectively and selectively transported across the membrane. As a result, the heterostructured hybrid membrane (MCSC/AAO) exhibits enhanced selectivity for cations and enantiomers of amino acids, achieving CAE for amino acids with an isoelectric point (pI) exceeding 7. Interestingly, the MCSC/AAO system demonstrates enhanced pH-sensitive enantioseparation compared to chiral mesoporous silica/AAO (CMS/AAO) with significant improvements of 78.14, 65.37, and 14.29% in the separation efficiency, separation factor, and permeate flux, respectively. This work promises to advance the synthesis of two or more component-integrated chiral nanochannels with multifunctional properties and allows a better understanding of the origins of the homochiral hybrid membranes.

Green Synthesis of Highly Fluorescent NCQDs: A Comprehensive Study on Synthesis, Characterization, Photophysical Properties, pH Sensing, Heavy Metal Detection, and Solvatochromic Behavior through Hydrothermal Method.

Solvatochromic studies in conjunction with NCQDs and analysis of material at different pH levels provide valuable insights about the process of metal ion sensing. Metal ion sensing holds significant importance in various fields like environment monitoring, biomedical diagnostics and various industrial purpose. The detection of metal ions by mixing the nitrogen-doped quantum dots (NCQDs) in the solvent at different pH levels for the analysis of the photoluminescence spectra is the unique property to achieve selective metal ion detection. In present study, the synthesis of NCQDs was performed by the use of flowers of Tecoma stans. The synthesis of NCQDs to best of our knowledge using flowers of Tecoma stans as natural carbon source via hydrothermal process has been done for the first time. The NCQDs exhibit absorption bands ranging from 190 to 450 nm, with the energy band gap varying from 3.55 to 5.42 eV when mixed with different solvent such as, 1-4 dioxane, acetone, acetonitrile, ethyl- acetate, ethanol, methanol and toluene. The fluorescence spectra exhibited highly intense range from approximately 390 to 680 nm across various solvents. XRD analysis further confirmed the crystalline nature of the particles with an average size of 6.96 nm. Different peak positions of the FTIR spectra support functional groups having C-H stretching, C = O (carbonyl) stretching, and C = C stretching vibrations. In the study a notable solvatochromic shift was observed, indicating sensitivity to change in solvent polarity. Additionally, the investigation of the ratio of ground to excited state dipole moment based on solvatochromic shift yielded a value of 3.30. This provide valuable information about optical and electronic properties of NCQDs. Overall, our study sheds light on the unique properties of NCQDs synthesized from Tecoma stans flowers and their potential applications in metal ion sensing, pH probing, and solvent polarity studies.

Automatic segmentation of lysosomes and analysis of intracellular pH with Radachlorin photosensitizer and FLIM.

We report application of the fluorescence lifetime imaging microscopy (FLIM) for analysis of distributions of intracellular acidity using a chlorin-e6 based photosensitizer Radachlorin. An almost two-fold increase of the photosensitizer fluorescence lifetime in alkaline microenvironments as compared to acidic ones allowed for clear distinguishing between acidic and alkaline intracellular structures. Clusterization of a phasor plot calculated from fits of the FLIM raw data by two Gaussian distributions provided accurate automatic segmentation of lysosomes featuring acidic contents. The approach was validated in colocalization experiments with LysoTracker fluorescence in living cells of four established lines. The dependence of photosensitizer fluorescence lifetime on microenvironment acidity allowed for estimation of pH inside the cells, except for the nuclei, where photosensitizer does not penetrate. The developed method is promising for combined application of the photosensitizer for both photodynamic treatment and diagnostics.

Role of pH-sensing receptors in colitis.

Low pH in the gut is associated with severe inflammation, fibrosis, and colorectal cancer (CRC) and is a hallmark of active inflammatory bowel disease (IBD). Subsequently, pH-sensing mechanisms are of interest for the understanding of IBD pathophysiology. Tissue hypoxia and acidosis-two contributing factors to disease pathophysiology-are linked to IBD, and understanding their interplay is highly relevant for the development of new therapeutic options. One member of the proton-sensing G protein-coupled receptor (GPCR) family, GPR65 (T-cell death-associated gene 8, TDAG8), was identified as a susceptibility gene for IBD in a large genome-wide association study. In response to acidic extracellular pH, GPR65 induces an anti-inflammatory response, whereas the two other proton-sensing receptors, GPR4 and GPR68 (ovarian cancer G protein-coupled receptor 1, OGR1), mediate pro-inflammatory responses. Here, we review the current knowledge on the role of these proton-sensing receptors in IBD and IBD-associated fibrosis and cancer, as well as colitis-associated cancer (CAC). We also describe emerging small molecule modulators of these receptors as therapeutic opportunities for the treatment of IBD.

In situ self-referenced intracellular two-electrode system for enhanced accuracy in single-cell analysis.

Since the emergence of single-cell electroanalysis, the two-electrode system has become the predominant electrochemical system for real-time behavioral analysis of single-cell and multicellular populations. However, due to the transmembrane placement of the two electrodes, cellular activities can be interrupted by the transmembrane potentials, and the test results are susceptible to influences from factors such as intracellular solution, membrane, and bulk solution. These limitations impede the advancement of single-cell analysis. Here, we propose a highly miniaturized and integrated in situ self-referenced intracellular two-electrode system (IS-SRITES), wherein both the working and reference electrodes are positioned inside the cell. Additionally, we demonstrated the stability (0.28 mV/h) of the solid-contact in situ Ag/AgCl reference electrode and the ability of the system to conduct standard electrochemical testing in a wide pH range (pH 6.0-8.0). Cell experiments confirmed the non-destructive performance of the electrode system towards cells and its capacity for real-time monitoring of intra- and extracellular pH values. Moreover, through equivalent circuits, finite element simulations, and drug delivery experiments, we illustrated that the IS-SRITES can yield more accurate test results and exhibit enhanced resistance to interference from the extracellular environment. Our proposed system holds the potential to enable the precise detection of intracellular substances and optimize the existing model of the electrode system for intracellular signal detection, thereby spearheading advancements in single-cell analysis.

Multi-functional ratiometric detection based on dual-emitting N-doped carbon dots.

Ratiometric fluorescence probes based on multi-emission carbon dots improve accuracy and sensitivity on detecting various environment issues. Herein, a novel dual-emitting N-doped carbon dots (N-CDs) was synthesized from citric acid and urea via a solvothermal method in N,N-dimethylformamide (DMF). The blue and orange emissions of N-CDs in water were modulated, and pure white light-emitting with Commission Internationale de L'Eclairage (CIE) coordinates of (0.33, 0.33) was achieved. The two PL centers behaved differently for Fe3+, Cu2+ and Ag+ ions, with the limit of detection (LOD) of ppm as fluorescence probes. Additionally, N-CDs displayed unique solvatochromism phenomenon. A new green emission appeared in organic solvents and gradually quenched with the increase of solvent polarity. The ratiometric PL displayed an excellent linear response for detecting water, and the LOD was between 0.003 % and 0.3 % in DMF, ethanol, isopropanol and N-methylpyrrolidone. Furthermore, N-CDs exhibited pH-sensitive response in the range of 4.0-7.0 and temperature-dependent response during heating-cooling cycles between 15 and 70 °C. A simple, efficient and reliable multi-functional ratiometric probe for detecting metal ions, water content, pH and temperature simultaneously was realized. However, there is a need for future application research to overcome the limitation imposed by the excitation wavelength of 330 nm.

Fluorescence Spectra of Prototropic Forms of Fluorescein and Some Derivatives and Their Potential Use for Calibration-Free pH Sensing.

Fluorescence pH sensing has proven to be efficient but with the drawback that molecules photobleach, requiring frequent calibrations. Double-emission peak molecules allow ratiometric measurements and theoretically avoid calibration. However, they are often expensive and fragile and usually have very low quantum yields. Single emission peaks such as fluorescein and derivatives are inexpensive and have very high quantum yields. Because they are single emission peaks, the pH is assumed to be derived from the ratio of emitted intensities at measured pH and at high pH values, i.e., they require frequent calibration. However, the shape of their single emitted peak evolves slightly with pH. In this paper, we first demonstrate a simple method to calculate the emission spectrum shape of each prototropic form of fluorescein (and derivatives) as well as the values of the pKas. A complete model of the evolution of the emission spectrum shape with pH is then constructed. Second, we evaluate the potential of these molecules for pH sensing by fitting the experimental spectra with the complete emission model. The method is applied to fluorescein, FITC and FAM. Depending on the molecule, pH can be measured from pH 1.9 to pH 7.3 with standard deviations between 0.06 and 0.08 pH units. Estimating pH and pKas from shape instead of intensity allows calibration-free measurements even with single-emission peak molecules.

Comparing solution-gate and bottom-gate nanowire field-effect transistors on pH sensing with different salt concentrations and surface modifications.

Field-effect transistors (FETs) have been developed as pH sensors by using various device structures, fabrication technologies, and sensing film materials. Different transistor structures, like extended-gate (EG) FETs, floating-gate FET sensors, and dual-gate (DG) FETs, can enhance the sensor performance. In this article, we report the effects of using solution-gate and bottom-gate FET configurations on pH sensing and investigate the influence of different ionic concentrations of buffers in the measured signals. The surface charge of hafnium dioxide (HfO2) affected by the buffer pH, with/without the modification of polyethylene glycol (PEG) terminated with hydroxyl groups, and the location of applied gate voltage are vital factors to the sensor performance in pH sensing. Based on the results, the solution-gate FET exhibits good pH sensitivity even in the high ionic strength solutions of bis-tris propane (BTP), and these values of pH sensitivity are close to the Nernst limit (59.2 mV/pH). In general, silane-PEG-OH modification can reduce the deviations of measured signals in pH sensing. The performance of bottom-gate FET is inferior in the BTP buffers with high ionic solutions but suitable to be operated in low ionic concentrations, such as 0.1, 1, and 10 mM BTP buffers. The size of the ions was also studied and discussed. The solution-gate FET demonstrates excellent performance under high ionic strengths, meaning a more significant potential for detecting biological molecules under physiological conditions.

Carbon Dots for Multiuse Platform: Intracellular pH Sensing and Complementary Intensified T1-T2 Dual Imaging Contrast Nanoprobes.

Measurement of pH in living cells is a great and decisive factor for providing an early and accurate diagnosis factor. Along with this, the multimodal transverse and longitudinal relaxivity enhancement potentiality over single modality within a single platform in the magnetic resonance imaging (MRI) field is a very challenging issue for diagnostic purposes in the biomedical field of application. Therefore, this work aims to design a versatile platform by fabricating a novel nanoprobe through holmium- and manganese-ion doping in carbon quantum dots (Ho-Mn-CQDs), which can show nearly neutral intracellular pH sensing and MRI imaging at the same time. These manufactured Ho-Mn-CQDs acted as excellent pH sensors in the near-neutral range (4.01-8.01) with the linearity between 6.01 and 8.01, which could be useful for the intracellular pH-sensing capability. An innumerable number of carboxyl and amino groups are present on the surface of the prepared nanoprobe, making it an excellent candidate for pH sensing through fluorescence intensity quenching phenomena. Cellular uptake and cell viability experiments were also executed to affirm the intracellular accepting ability of Ho-Mn-CQDs. Furthermore, with this pH-sensing quality, these Ho-Mn-CQDs are also capable of acting as T1-T2 dual modal imaging contrast agents in comparison with pristine Ho-doped and Mn-doped CQDs. The Ho-Mn-CQDs showed an increment of r1 and r2 relaxivity values simultaneously compared with only the negative contrast agent, holmium in holmium-doped CQDs, and the positive contrast agent, manganese in manganese-doped CQDs. The above-mentioned observations elucidate that its tiny size, excitation dependence of fluorescence behavior, low cytotoxicity, and dual modal contrast imaging capability make it an ideal candidate for pH monitoring in the near-neutral range and also as a dual modal MRI imaging contrast enhancement nanoprobe at the same time.

A high-precision and wide-range pH monitoring system based on broadband cavity-enhanced absorption spectrum.

A high-precision pH monitoring system over a wide pH range is introduced. The system comprises a cavity-enhancement module constructed by two high-reflectivity mirrors, a microfluidic pH sensing chip based on a binary-indicator membrane of Congo red and m-cresol purple, and a hyperspectral transmission module. This structure extends the effective absorption optical path of the sensing chip, significantly amplifying the spectral differences at various pH values. The spectrum of the transmitted light is recorded by a self-developed hyperspectral module and then converted to broadband cavity-enhanced absorption spectrum (BBCEAS) via the Beer-Lambert law. An artificial neural network (ANN) is employed to predict pH values of the solution. With such a design, this system exhibits a wide detecting range of 2 M [H+] - 2 M [OH-] (corresponding to pH -0.3-14.3) with a response time of about 120 s. The system can achieve a higher detection accuracy with root mean square error (RMSE) of 0.073, as compared to 0.137 without the cavity enhancement. The system also possesses good properties of repeatability, long-term stability, ion resistance, and organic corrosion resistance. These excellent properties make the proposed system a promising candidate technology for harsh environments, such as seawater acidification warning, chemical plant sewage monitoring, and biological sample detection.

Protein Phosphorylation Orchestrates Acclimations of Arabidopsis Plants to Environmental pH.

Environment pH (pHe) is a key parameter dictating a surfeit of conditions critical to plant survival and fitness. To elucidate the mechanisms that recalibrate cytoplasmic and apoplastic pH homeostasis, we conducted a comprehensive proteomic/phosphoproteomic inventory of plants subjected to transient exposure to acidic or alkaline pH, an approach that covered the majority of protein-coding genes of the reference plant Arabidopsis thaliana. Our survey revealed a large set-of so far undocumented pHe-dependent phospho-sites, indicative of extensive post-translational regulation of proteins involved in the acclimation to pHe. Changes in pHe altered both electrogenic H+ pumping via P-type ATPases and H+/anion co-transport processes, putatively leading to altered net trans-plasma membrane translocation of H+ ions. In pH 7.5 plants, the transport (but not the assimilation) of nitrogen via NRT2-type nitrate and AMT1-type ammonium transporters was induced, conceivably to increase the cytosolic H+ concentration. Exposure to both acidic and alkaline pH resulted in a marked repression of primary root elongation. No such cessation was observed in nrt2.1 mutants. Alkaline pH decreased the number of root hairs in the wild type but not in nrt2.1 plants, supporting a role of NRT2.1 in developmental signaling. Sequestration of iron into the vacuole via alterations in protein abundance of the vacuolar iron transporter VTL5 was inversely regulated in response to high and low pHe, presumptively in anticipation of associated changes in iron availability. A pH-dependent phospho-switch was also observed for the ABC transporter PDR7, suggesting changes in activity and, possibly, substrate specificity. Unexpectedly, the effect of pHe was not restricted to roots and provoked pronounced changes in the shoot proteome. In both roots and shoots, the plant-specific TPLATE complex components AtEH1 and AtEH2-essential for clathrin-mediated endocytosis-were differentially phosphorylated at multiple sites in response to pHe, indicating that the endocytic cargo protein trafficking is orchestrated by pHe.

A novel anthocyanin indicator film with rosmarinic acid copigmentation having enhanced stability and pH indicator ability for monitoring pork freshness.

BACKGROUND: Anthocyanin-based pH-sensing films have been widely fabricated for potential application in monitoring food freshness. However, the color fading of anthocyanins limits their application for the food industry due to their low stability. In addition, the color sensitivity and pH indicator ability of anthocyanin-based films currently available are not satisfied and need to be improved. RESULTS: Chitosan/xanthan gum (CX)-based colorimetric films with addition of purple cabbage anthocyanin (PAN) and different amounts of rosmarinic acid (RA) were fabricated. RA copigmentation in chitosan/xanthan gum-purple cabbage anthocyanin-rosmarinic acid (CX-P-RA) films significantly improved the stability and pH response sensitivity of PAN, and the combined copigmentation of RA and xanthan gum exhibited an additive effect. The addition of RA significantly improved the tensile strength and elongation at break, thermal stability, antioxidant and antibacterial activities of CX-P-RA films. Moreover, addition of RA enhanced the pH sensitivity and colorimetry of CX-P-RA films, which exhibited a good response to different pH values. CX-P-RA2 film was tested to monitor the freshness of pork. It showed visible color changes during the storage of pork. In addition, the ∆E of CX-P-RA2 film was highly correlated with changes in total volatile basic nitrogen in pork (R2 = 0.951). CONCLUSION: These results indicated that CX-P-RA2 film can be used as a pH-sensing indicator with good stability and high sensitivity for real-time monitoring of pork freshness. © 2023 Society of Chemical Industry.

TCR signaling promotes formation of an STS1-Cbl-b complex with pH-sensitive phosphatase activity that suppresses T cell function in acidic environments.

T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.

Liposomes Containing Zinc-Based Chemotherapeutic Drug Block Proliferation and Trigger Apoptosis in Breast Cancer Cells.

Platinum-based chemotherapeutic drugs are effective in killing malignant cells but often trigger drug resistance or off-target side effects. Unlike platinum, zinc is used as an endogenous cofactor for several cellular enzymes and may, thus, display increased biocompatibility. In this present study, we have rationally designed and synthesized two substituted phenanthro[9,10-d]imidazole-based ligands L1 and L2 with pyridine and quinoline substitution at the 2 position and their corresponding Zn(II) complexes; (L1)2Zn and (L2)2Zn, which are characterized by standard analytical and spectroscopic methods. (L2)2Zn, but not (L1)2Zn has intrinsic fluorescence, indicating its potential utility in imaging applications. To facilitate cellular uptake, we generated liposomal formations with a phospholipid DMPC (1,2-Dimyristoyl-sn-glycero-3-phosphocholine) through molecular self-assembly. These liposomal formulations Lip-(L1)2Zn and Lip-(L2)2Zn were able to enter breast cancer cells, induce DNA fragmentation, arrest the cell cycle at the G0/G1 phase, decrease proliferation, and promote apoptosis by activating the DNA damage response. Importantly, both Lip-(L1)2Zn and Lip-(L2)2Zn decreased the size of breast cancer cell-based spheroids, indicating they may be capable of suppressing tumor growth. Our work represents an important proof-of-concept exercise demonstrating that successful liposomal formation of phenanthro[9,10-d]imidazole-based Zn(II) complexes with inherent optical properties have great promise for the development of imaging probes and efficient anticancer drugs.

Multifunctional IrOx Neural Probe for In Situ Dynamic Brain Hypoxia Evaluation.

Perioperative cerebral hypoxia and neonatal hypoxia-ischemic encephalopathy are the main triggers that lead to temporary or permanent brain dysfunction. The pathogenesis is intimately correlated to neural activities and the pH of the microenvironment, which calls for a high demand for in situ multitype physiological signal acquisition in the brain. However, conventional pH sensing neural interfaces cannot obtain the characteristics of multimodes, multichannels, and high spatial resolution of physiological signals simultaneously. Here, we report a multifunctional implantable iridium oxide (IrOx) neural probe (MIIONP) combined with electrophysiology recording, in situ pH sensing, and neural stimulation for real-time dynamic brain hypoxia evaluation. The neural probe modified with IrOx films exhibits outstanding electrophysiology recording and neural stimulation performance and long-term stable high spatial pH sensing resolution of about 100 µm, and the cytotoxicity of IrOx microelectrodes was investigated as well. In addition, 4 weeks' tracking of the same neuron firing and instantaneous population spike captured during electrical stimulation was achieved by MIIONP. Finally, in a mouse brain hypoxia model, the MIIONP has demonstrated the capability of synchronous in situ recording of the pH and neural firing changes in the brain, which has a valuable application in dynamic brain disease evaluation through real-time acquisition of multiple physiological signals.

The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells.

The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 µm using PLIM and showed a gradient in the intracellular O2 level towards their center.

Mechanisms of Chemosensory Transduction in the Carotid Body.

The mammalian carotid body (CB) is a polymodal chemoreceptor, which is activated by blood-borne stimuli, most notably hypoxia, hypercapnia and acidosis, thus ensuring an appropriate cellular response to changes in physical and chemical parameters of the blood. The glomus cells are considered the CB chemosensory cells and the initial site of chemoreceptor transduction. However, the molecular mechanisms by which they detect changes in blood chemical levels and how these changes lead to transmitter release are not yet well understood. Chemotransduction mechanisms are by far best described for oxygen and acid/carbon dioxide sensing. A few testable hypotheses have been postulated including a direct interaction of oxygen with ion channels in the glomus cells (membrane hypothesis), an indirect interface by a reversible ligand like a heme (metabolic hypothesis), or even a functional interaction between putative oxygen sensors (chemosome hypothesis) or the interaction of lactate with a highly expressed in the CB atypical olfactory receptor, Olfr78, (endocrine model). It is also suggested that sensory transduction in the CB is uniquely dependent on the actions and interactions of gaseous transmitters. Apparently, oxygen sensing does not utilize a single mechanism, and later observations have given strong support to a unified membrane model of chemotransduction.