A fluorescent ratiometric potassium sensor based on IPG4-silica microparticles for selective detection and fluorescence imaging of potassium cations.

Potassium cations play many important roles in living organisms, especially in electro-physiology, since they are involved in neurotransmission and muscle contractions. We report the synthesis of a ratiometric fluorescent microsensor for potassium (K+) detection, based on the fluorescent probe ION potassium green 4. Potassium-sensitive fluorescent microparticles were obtained by using silica as the core material. We obtained silica-based microsensors with sizes in the micrometer range, spherical shapes, good monodispersity, optimal selectivity and a sensitivity range of 0 to 40 mM. The microsensors also proved to be non-toxic in cell cultures and suitable for fluorescence imaging, offering new possibilities for non-invasive optical detection, quantification and in situ monitoring of K+ variations in cell culture systems.

Facile One Pot Synthesis of Hybrid Core-Shell Silica-Based Sensors for Live Imaging of Dissolved Oxygen and Hypoxia Mapping in 3D Cell Models.

Fluorescence imaging allows for noninvasively visualizing and measuring key physiological parameters like pH and dissolved oxygen. In our work, we created two ratiometric fluorescent microsensors designed for accurately tracking dissolved oxygen levels in 3D cell cultures. We developed a simple and cost-effective method to produce hybrid core-shell silica microparticles that are biocompatible and versatile. These sensors incorporate oxygen-sensitive probes (Ru(dpp) or PtOEP) and reference dyes (RBITC or A647 NHS-Ester). SEM analysis confirmed the efficient loading and distribution of the sensing dye on the outer shell. Fluorimetric and CLSM tests demonstrated the sensors' reversibility and high sensitivity to oxygen, even when integrated into 3D scaffolds. Aging and bleaching experiments validated the stability of our hybrid core-shell silica microsensors for 3D monitoring. The Ru(dpp)-RBITC microparticles showed the most promising performance, especially in a pancreatic cancer model using alginate microgels. By employing computational segmentation, we generated 3D oxygen maps during live cell imaging, revealing oxygen gradients in the extracellular matrix and indicating a significant decrease in oxygen level characteristics of solid tumors. Notably, after 12 h, the oxygen concentration dropped to a hypoxic level of PO2 2.7 ± 0.1%.

A 3D Pancreatic Cancer Model with Integrated Optical Sensors for Noninvasive Metabolism Monitoring and Drug Screening.

A distinct feature of pancreatic ductal adenocarcinoma (PDAC) is a prominent tumor microenvironment (TME) with remarkable cellular and spatial heterogeneity that meaningfully impacts disease biology and treatment resistance. The dynamic crosstalk between cancer cells and the dense stromal compartment leads to spatially and temporally heterogeneous metabolic alterations, such as acidic pH that contributes to drug resistance in PDAC. Thus, monitoring the extracellular pH metabolic fluctuations within the TME is crucial to predict and to quantify anticancer drug efficacy. Here, a simple and reliable alginate-based 3D PDAC model embedding ratiometric optical pH sensors and cocultures of tumor (AsPC-1) and stromal cells for simultaneously monitoring metabolic pH variations and quantify drug response is presented. By means of time-lapse confocal laser scanning microscopy (CLSM) coupled with a fully automated computational analysis, the extracellular pH metabolic variations are monitored and quantified over time during drug testing with gemcitabine, folfirinox, and paclitaxel, commonly used in PDAC therapy. In particular, the extracellular acidification is more pronounced after drugs treatment, resulting in increased antitumor effect correlated with apoptotic cell death. These findings highlight the importance of studying the influence of cellular metabolic mechanisms on tumor response to therapy in 3D tumor models, this being crucial for the development of personalized medicine approaches.

Fluorescent nano- and microparticles for sensing cellular microenvironment: past, present and future applications.

The tumor microenvironment (TME) demonstrates distinct hallmarks, including acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion fluxes, which are crucial targets for early cancer biomarker detection, tumor diagnosis, and therapeutic strategies. Various imaging and sensing techniques have been developed and employed in both research and clinical settings to visualize and monitor cellular and TME dynamics. Among these, ratiometric fluorescence-based sensors have emerged as powerful analytical tools, providing precise and sensitive insights into TME and enabling real-time detection and tracking of dynamic changes. In this comprehensive review, we discuss the latest advancements in ratiometric fluorescent probes designed for the optical mapping of pH, oxygen, ROS, ions, and biomarkers within the TME. We elucidate their structural designs and sensing mechanisms as well as their applications in in vitro and in vivo detection. Furthermore, we explore integrated sensing platforms that reveal the spatiotemporal behavior of complex tumor cultures, highlighting the potential of high-resolution imaging techniques combined with computational methods. This review aims to provide a solid foundation for understanding the current state of the art and the future potential of fluorescent nano- and microparticles in the field of cellular microenvironment sensing.

pH-sensing hybrid hydrogels for non-invasive metabolism monitoring in tumor spheroids.

The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.

A pH-sensor scaffold for mapping spatiotemporal gradients in three-dimensional in vitro tumour models.

The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.

Novel hyphenation of DGT in-situ passive sampling with YES assay to ascertain the potency of emerging endocrine disruptors in water systems in New Zealand.

This study represents the first attempt to investigate selected estrogenic compounds that include 17α-ethynylestradiol (EE2), 17ß-estradiol (E2) bisphenol A (BPA), and bisphenol AF (BPAF) along the drinkable water, from river-to-tap, and wastewater, from effluent-to-treated wastewater, treatment processes of the Hamilton City Council and the monitoring of the freshwater, from source-to-outfall, of the Waikato River in New Zealand. This was accomplished by the adoption of a novel combination of diffusive gradients in thin films (DGTs) in-situ passive sampling coupled with high-performance liquid chromatography/mass spectrometry analysis (HPLC/MS) and the Yeast Estrogen Screen (YES). Estradiol equivalency quantities, integrated in time, were evaluated theoretically (cEEQ) by DGT-HPLC/MS and experimentally (EEQ) by DGT-YES assay. cEEQ and EEQ highlighted that primary treatments are not suitable for estrogens and bisphenolic plastics removal both at drinkable and wastewater treatment plants in Hamilton where they worsen the water quality in terms of estrogenicity making these pollutants more available in the water phase. All downstream sites monitored along the Waikato River showed higher cEEQ and EEQ, moreover the Waikato River water quality showed a moderate worsening moving from Taupo (source) to Tuakau (outfall). The most polluted sites were downstream of Hamilton city and Huntly township wastewater treatment plants that serve the main conurbations in the area. cEEQ and EEQ generally showed good agreement at low concentrations but differed substantially at more polluted sites where cEEQ consistently underestimated estrogenic potency, possibly due to DGT accumulation of estrogenic compounds not quantified by HPLC/MS.

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Invited for the cover of this issue are Anil Chandra, Loretta L. del Mercato and co-workers at the Institute of Nanotechnology of National Research Council and the University of Salento. The image depicts how negatively charged pH-sensitive pyranine (HPTS) molecules were successfully immobilized on silica microparticles (SMPs) without compromising the molecules' pH sensitivity. These resulting sensors can be used to measure pH in vitro and in vivo due to the cytocompatibility of HPTS molecules and SMPs. Read the full text of the article at 10.1002/chem.202101568.

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Pyranine (HPTS) is a remarkably interesting pH-sensitive dye that has been used for plenty of applications. Its high quantum yield and extremely sensitive ratiometric fluorescence against pH change makes it a very favorable for pH-sensing applications and the development of pH nano-/microsensors. However, its strong negative charge and lack of easily modifiable functional groups makes it difficult to use with charged substrates such as silica. This study reports a methodology for noncovalent HPTS immobilization on silica microparticles that considers the retention of pH sensitivity as well as the long-term stability of the pH microsensors. The study emphasizes the importance of surface charge for governing the sensitivity of the immobilized HPTS dye molecules on silica microparticles. The importance of the immobilization methodology, which preserves the sensitivity and stability of the microsensors, is also assessed.