Surface PEGylation of ionophore-based microspheres enables determination of serum sodium and potassium ion concentration under flow cytometry.

We present here an ionophore-based ion-selective optode (ISO) platform to detect potassium and sodium concentrations in serum through flow cytometry. The ion-selective microsensors were based on polyethylene glycol (PEG)-modified polystyrene (PS) microspheres (PEG-PS). Ratiometric response curves were observed using peak channel fluorescence intensities for K+ (10-6 M to 0.1 M) and Na+ (10-4 M to 0.2 M) with sufficient selectivity for clinical diagnosis. Due to the matrix effect, proteins such as albumin and immunoglobulin caused an obvious increase in response for serum sample determination. To solve this problem, 4-arm PEG chains were covalently attached onto the surface of PS microspheres through a two-step reaction, which improved the stability and combated pollution of microspheres. As a preliminary application, potassium and sodium concentrations in human serums were successfully determined by the PEG-PS microsensors through flow cytometry.

Perspective on fluorescence cell imaging with ionophore-based ion-selective nano-optodes.

Inorganic ions are ubiquitous in all kinds of cells with highly dynamic spatial and temporal distribution. Taking advantage of different types of fluorescent probes, fluorescence microscopic imaging and quantitative analysis of ion concentrations in cells have rapidly advanced. A family of fluorescent nanoprobes based on ionophores has emerged in recent years with the potential to establish a unique platform for the analysis of common biological ions including Na+, K+, Ca2+, Cl-, and so on. This article aims at providing a retrospect and outlook of ionophore-based ion-selective nanoprobes and the applications in cell imaging.

Phase transfer of fatty acids into ultrasmall nanospheres for colorimetric detection of lipase and albumin.

Colorimetric detection of fatty acids during biological interactions is extremely difficult since they are optically silent. Here, fatty acids are found to function as ion-exchangers in ultrasmall polymeric nanospheres to facilitate the protonation of chromoionophores, causing a vivid color change between red and blue. With an excellent detection limit of 1.8 µg mL-1 for oleic acid, colorimetric assays for lipase and albumin are developed with quick response, high sensitivity, and low cost.

Photoswitch-Based Fluorescence Encoding of Microspheres in a Limited Spectral Window for Multiplexed Detection.

Fluorescence barcoding with multicolor fluorophores is limited by spectral crowding. Herein, we propose a fluorescence encoding method in a single-color channel with photoswitches. The photochromic naphthopyran was used to mediate the fluorescence of polystyrene microspheres through resonance energy transfer. The initial fluorescence intensity (F0) and the fluorescence after UV light activation (F/F0) were combined to generate hundreds of 2-dimensional barcodes. The coding capacity was further expanded with the different chemical kinetics of the photoswitches. The photoswitch-based fluorescence barcodes were applied to simultaneously and selectively detect the DNA sequences of COVID-19 (with related mutations) as a proof-of-concept for real applications. The compatibility with the state-of-the-art fluorescence microscopes and simple encoding and decoding make the method very attractive for multiplexed and high-throughput analyses.

One-pot synthesized organosilica nanospheres for multiplexed fluorescent nanobarcoding and subcellular tracking.

Multicolor microbeads are widely used in flow cytometry for various cellular and immunoassays. However, they are limited by their large size of around one to tens of micrometers. Nanomaterials for multiplexed analysis are emerging as valuable tools in high-throughput assays and fluorescence cell barcoding. We present barcoding and related cellular studies based on mass-produced organosilane-derived multifunctional nanospheres with a uniform size. Functional groups including thiols, amines, and azides were integrated in one step from various organosilanes without additional orthosilicates. Fluorescent nanobarcodes (NBs) were achieved through flexible physical adsorption and chemical ligation of spectrally separated fluorescent dyes. Live cells labeled with the NBs were readily distinguished by flow cytometry. The NBs have a small and uniform size (ca. 27 nm in diameter), excellent biocompatibility, rapid cellular uptake, and low dye leakage. The fluorescent nanospheres were applied for long-term cell tracking during multiple rounds of cell division and monitored over 48 hours. While most nanospheres were endolysosome-targeting, modification with fluorescein isothiocyanate (FITC) surprisingly lighted up the cell nucleus. This work lays the foundation of a unique family of functional nanomaterials promising for multiplex detection and other chemical and biological applications.

UNet retinal blood vessel segmentation algorithm based on improved pyramid pooling method and attention mechanism.

The segmentation results of retinal vessels have a significant impact on the automatic diagnosis of retinal diabetes, hypertension, cardiovascular and cerebrovascular diseases and other ophthalmic diseases. In order to improve the performance of blood vessels segmentation, a pyramid scene parseing U-Net segmentation algorithm based on attention mechanism was proposed. The modified PSP-Net pyramid pooling module is introduced on the basis of U-Net network, which aggregates the context information of different regions so as to improve the ability of obtaining global information. At the same time, attention mechanism was introduced in the skip connection part of U-Net network, which makes the integration of low-level features and high-level semantic features more efficient and reduces the loss of feature information through nonlinear connection mode. The sensitivity, specificity, accuracy and AUC of DRIVE and CHASE_DB1 data sets are 0.7814, 0.9810, 0.9556, 0.9780; 0.8195, 0.9727, 0.9590, 0.9784. Experimental results show that the PSP-UNet segmentation algorithm based on the attention mechanism enhances the detection ability of blood vessel pixels, suppresses the interference of irrelevant information and improves the network segmentation performance, which is superior to U-Net algorithm and some mainstream retinal vascular segmentation algorithms at present.

Hydrogel-Based Optical Ion Sensors: Principles and Challenges for Point-of-Care Testing and Environmental Monitoring.

Hydrogel is a unique family of biocompatible materials with growing applications in chemical and biological sensors. During the past few decades, various hydrogel-based optical ion sensors have been developed aiming at point-of-care testing and environmental monitoring. In this Perspective, we provide an overview of the research field including topics such as photonic crystals, DNAzyme cross-linked hydrogels, ionophore-based ion sensing hydrogels, and fluoroionophore-based optodes. As the different sensing principles are summarized, each strategy offers its advantages and limitations. In a nutshell, developing optical ion sensing hydrogels is still in the early stage with many opportunities lying ahead, especially with challenges in selectivity, assay time, detection limit, and usability.

Densely connected U-Net retinal vessel segmentation algorithm based on multi-scale feature convolution extraction.

PURPOSE: The segmentation results of retinal blood vessels have a significant impact on the automatic diagnosis of various ophthalmic diseases. In order to further improve the segmentation accuracy of retinal vessels, we propose an improved algorithm based on multiscale vessel detection, which extracts features through densely connected networks and reuses features. METHODS: A parallel fusion and serial embedding multiscale feature dense connection U-Net structure are designed. In the parallel fusion method, features of the input images are extracted for Inception multiscale convolution and dense block convolution, respectively, and then the features are fused and input into the subsequent network. In serial embedding mode, the Inception multiscale convolution structure is embedded in the dense connection network module, and then the dense connection structure is used to replace the classical convolution block in the U-Net network encoder part, so as to achieve multiscale feature extraction and efficient utilization of complex structure vessels and thereby improve the network segmentation performance. RESULTS: The experimental analysis on the standard DRIVE and CHASE_DB1 databases shows that the sensitivity, specificity, accuracy, and AUC of the parallel fusion and serial embedding methods reach 0.7854, 0.9813, 0.9563, 0.9794; 0.7876, 0.9811, 0.9565, 0.9793 and 0.8110, 0.9737, 0.9547, 0.9667; 0.8113, 0.9717, 0.9574, 0.9750, respectively. CONCLUSIONS: The experimental results show that multiscale feature detection and feature dense connection can effectively enhance the network model's ability to detect blood vessels and improve the network segmentation performance, which is superior to U-Net algorithm and some mainstream retinal blood vessel segmentation algorithms at present.

Ionophore-Based Ion-Selective Nanospheres Based on Monomer-Dimer Conversion in the Near-Infrared Region.

Here, we report ion-selective nanospheres with readout in the near-infrared (NIR) region in both fluorescence and absorbance modes. The nanospheres rely on an ionophore-mediated monomer-dimer conversion of an NIR transducer, DTTC. The DTTC monomer in the nanospheres emits fluorescence around 820 nm, while the dimer in the aqueous environment generates strong blue-shifted emission around 660 nm. With a lead ionophore, an unprecedented lower detection limit of 3 pM for Pb2+ was achieved, allowing us to determine Pb2+ levels in river water without diluting the sample. Also, the Cu2+-selective nanospheres showed a detection limit of 5 nM. Taking advantage of the biologically desired NIR window, blood potassium concentrations were also determined without a complicated sample pretreatment. The sensing process was explained with a theoretical model. The detection range was found finely adjustable by the amount of nanospheres used. Therefore, the nanospheres formed a highly selective, sensitive, versatile, and rapid analytical platform for metal-ion sensing.

Ionophore-based pH independent detection of ions utilizing aggregation-induced effects.

Ionophores have been integrated into various electrochemical and optical sensing platforms for the selective detection of ions. Previous ionophore-based optical sensors rely on a H+ chromoionophore as the signal transducer and consequently, suffered from a pH cross-response. pH independent methods were proposed very recently by utilizing the solvatochromic dyes or the exhaustive mode. Here, we report a pH independent sensing principle based on nanospheres containing ionophores. As the ion-exchange occurs, the signal transducer undergoes aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ), leading to a dramatic change in fluorescence intensity. The principle was evaluated on different ionophores including those selective for K+, Na+, Ca2+, and Pb2+. The nanospheres were also introduced into microfluidic chips and successfully applied for the determination of sodium and potassium ion concentrations in diluted blood serum and urine samples.

Distance and Color Change Based Hydrogel Sensor for Visual Quantitative Determination of Buffer Concentrations.

We present here an innovative platform for the determination of pH buffer capacity based on FITC-dextran loaded hydrogels. Optical signals from the pH-sensitive hydrogels were analyzed by simple parameters including distance and color change. The methodology was validated on five different buffer systems and exhibited wide linearity (0.1 to 100 mM), good batch-to-batch reproducibility, high versatility, and resistance to background ionic strength changes. Experimental results also fit well with a theoretical model based on numerical simulation. Preliminary application in carbonate alkalinity determination of seawater proved very successful. This hydrogel buffer concentration sensor is fundamentally different from conventional acid-base titrations, brings minimum perturbation to samples, and shows great potential in real applications.

A rapid point-of-care optical ion sensing platform based on target-induced dye release from smart hydrogels.

We report here a rapid and versatile metal ion analytical platform based on the dye release from hydrogels entrapping ion-selective microdroplets. Using Ca2+, Pb2+, and Na+ as model ions, the ion-selective hydrogels were developed in combination with four positively charged dyes, making the hydrogels highly promising for point-of-care diagnostics as well as environmental monitoring.

Graphene Quantum Dots Integrated in Ionophore-Based Fluorescent Nanosensors for Na+ and K.

To enrich the recipes of ion-selective nanosensors, graphene quantum dots (GQDs) were integrated into ionophore-based fluorescent nanosensors with exquisite selectivity and high sensitivity for Na+ and K+. The unique property of GQDs gave the nanosensors ultrasmall size (ca. 10 nm), high brightness, good biocompatibility, and potential pH sensing possibility. At pH 7.4, the sensors exhibited a detection range from 0.1 mM to 1 M for Na+ and from 3 µM to 1 mM for K+. The nanosensors were successfully applied to blood serum and urine samples. Chemically induced intracellular sodium concentration change in HeLa cells was also qualitatively monitored.

Electrochemical-to-Optical Signal Transduction for Ion-Selective Electrodes with Light-Emitting Diodes.

Optical ion sensors normally have a relative narrow sensitive detection window. Here, based on multicolor light-emitting diodes (LEDs), we report on an electrochemical-to-optical signal transduction scheme under chronoamperometry control to convert the potentiometric response of ion-selective electrodes (ISEs) to optical output with tunable sensitivity and much wider response range. The sensing principle was demonstrated on K+, Ca2+, and Pb2+. LED light intensity was found to depend linearly on the concentration of monovalent ions. Optical signals could be captured with photomultiplier tubes or digital cameras, and a visual alarming system to monitor abnormal ion concentration was also developed from super-Nernstian electrodes.

A Plasticizer-Free Miniaturized Optical Ion Sensing Platform with Ionophores and Silicon-Based Particles.

Nanoscale ionophore-based ion-selective optodes (nano-ISOs) are effective sensing tools for in situ and real time measurements of ion concentrations in biological and environmental samples. While searching for novel sensing materials, nano-ISOs free of plasticizers are particularly important for biological and environmental applications. This work described plasticizer-free nano-ISOs based on Si-containing particles including PEGylated organosilica nanoparticles, PDMS nanospheres, and SiO2 microspheres, with diameters around 50 nm, 100 nm, and 5 µm, respectively. The platform enabled the use of highly selective ionophores, where the nanomatrices played important roles in tuning the ion-carrier complex formation constants and led to better selectivity for the PEGylated organosilica nano-ISOs than those based on PDMS. With use of the versatile silica chemistry, pH and ion dual sensing was achieved on SiO2 microspheres. In addition, increasing the cross-linking degree of the PDMS nano-ISOs extended the linear response range, and cellular uptake experiments showed that the nano-ISOs could readily enter HeLa cells with very low cytotoxicity.

Non-Equilibrium Diffusion Controlled Ion-Selective Optical Sensor for Blood Potassium Determination.

Blood electrolyte measurements play important roles in clinical diagnostics. Optical ion sensors as simple and elegant as a mercury thermometer are in high demand. We present here an analytical method to quantify potassium ions in undiluted human blood and plasma by measuring the distance or the rate of the color propagation. The sensor was composed of K+-selective nanospheres embedded in an agarose hydrogel where mass transport was diffusion controlled. The sensor's color-changing rate and the distance of color propagation depended linearly on the logarithm of K+ activity. A theoretical model was established and fully supported the experimental findings. This work lays the foundation of a new family of optical ion sensors for direct determination of common blood electrolytes.

Thermochromic Ion-Exchange Micelles Containing H+ Chromoionophores.

Thermochromic composites constitute a classical subfamily of stimuli responsive materials. We report here the thermochromic effect in Pluronic F-127 (F127) micelles containing hydrophobic ion-exchanger and H+ chromoionophores. The highly versatile and reversible thermochromism is attributed to the temperature-induced hydration-dehydration of the peripheral layer of the micelles, which in turn controls the ion-exchange process between the core and the periphery of the micelles. The color typically changes abruptly within 3-5 °C, and the color transition temperature can be tuned within 5-25 °C upon varying the F127 concentrations. This work lays the foundation of a new variety of thermochromic materials involving ion-exchange.

Osteopontin knockdown inhibits αv,ß3 integrin-induced cell migration and invasion and promotes apoptosis of breast cancer cells by inducing autophagy and inactivating the PI3K/Akt/mTOR pathway.

BACKGROUND: Osteopontin (OPN) is associated with tumor formation, progression and metastasis, and increased OPN levels have been associated with poor survival in breast cancer. We investigated the mechanisms responsible for OPN activity, and the relationships between OPN expression and clinical parameters in breast cancer. METHODS: OPN mRNA and protein levels were compared in malignant and benign breast tumors by polymerase chain reaction (PCR) and immunohistochemistry, respectively, and levels in breast cancer cells were determined by PCR and western blotting. The effects of lentiviral-mediated knockdown of OPN on OPN and αv,ß3 integrin expression, cell invasion and migration, autophagy and apoptosis were analyzed in MDA-MB-231 cells. RESULTS: OPN expression increased with aggressiveness of breast cancer phenotype. OPN knockdown inhibited αv,ß3 integrin expression in MDA-MB-231 cells, with subsequent inhibition of cell migration and invasion. Knockdown also inhibited the PI3K/Akt/mTOR pathway, promoted expression of the autophagy-related gene products LC3 and Beclin 1, and increased apoptosis. OPN expression was positively associated with tumor grade and lymph node metastasis. CONCLUSION: These results suggest that knockdown of OPN may inhibit breast cancer metastasis by regulating αv,ß3 integrin expression and inducing autophagy and subsequent inhibition of PI3K/Akt/mTOR signaling, thus providing further insights into the complex mechanisms regulating tumor growth and metastasis.