Photoswitchable Temperature Nanosensors Based on the Chemical Kinetics of Photochromic Naphthopyran for Live Cell Imaging.

Microscopic temperature imaging holds significant importance in various fields, particularly in the development of nanomaterials for photothermal therapy (PTT). In this study, we present an analytical method to probe cellular temperature based on chemical kinetics and additional luminescence quenching by photoswitchable naphthopyrans. Taking advantage of the rapid ring-closing reaction of naphthopyran, temperature sensing was realized with a linear relationship between the logarithmic decay time constant (ln τ) and the reciprocal temperature (T-1). To create luminescent temperature nanosensors, we harnessed the ability of ring-opened naphthopyran to quench the luminescence of a semiconducting polymer, resulting in a diverse array of probes. Structural modifications on the naphthopyran also provided a way to fine-tune the sensitivity and response window of the nanosensors. The method allowed cellular temperature imaging on a cost-effective fluorescence microscopic setup. As an application, the temperature increase induced by gold nanorods (AuNRs) in cell lysosomes was successfully monitored, laying the foundation for a new class of photoswitchable nanosensors with promising biological applications.

Ion-selective response of visible light photoswitchable indole-hemithioindigo: toward chemical sensing of fluoride and hydroxide.

The chemical sensing of hydrophilic anions such as F- and OH- is of significant importance but also presents considerable challenges. Herein, the thermal E to Z isomerization of a visible-light-responsive photoswitch (HTI-In) is utilized to address this challenge for the first time. The isomerization of HTI-In is dependent on the concentration of F- and OH-, and exhibits excellent selectivity toward F- and OH- over other common anions and cations. Unlike irreversible chemodosimeters and other conventional fluorescent probes, the photodynamic sensing of F- and OH- (demonstrated in solvents and polyurethane hydrogels) is based on a non-equilibrium chemical kinetics and can be operated fully reversibly.

Near-Infrared Fluoride Sensing Nano-Optodes and Distance-Based Hydrogels Containing Aluminum-Phthalocyanine.

Fluoride ions are highly relevant in environmental and biological sciences, and there is a very limited number of established fluoride chemical sensors. Previous fluoride-selective optodes were demonstrated with metal-porphyrin as the ionophore and required a chromoionophore for optical signal transduction. We demonstrate here novel optical fluoride sensing with nano-optodes containing an aluminum-phthalocyanine complex (AlClPc) as the single active sensing component, simplifying the conventional ion-selective optodes approach. The fluoride nano-optodes were interrogated in the absorbance and fluorescence modes in the near-infrared region, with absorption around 725 nm and emission peaks at 720 and 800 nm, respectively. The nano-optodes exhibited a lower detection limit around 0.1 µM and good selectivity over a range of common anions including ClO4-, Cl-, Br-, I-, SO42-, NO3-, and AcO-. Furthermore, the nano-optodes were physically entrapped in agarose hydrogels to allow distance-based point-of-care testing (POCT) applications. The 3D networks of the agarose hydrogel were able to filter off large particulates in the samples without stopping fluoride ions to reach the nano-optodes. The fluoride concentrations in real samples including river water, mineral water, and groundwater were successfully determined with the distance-based sensing hydrogel, and the results agreed well with those from commercial fluoride electrodes. Therefore, the results in this work lay the groundwork for the optical detection of fluoride in environmental samples without very sophisticated sample manipulation.

Expanded single-color barcoding in microspheres with fluorescence anisotropy for multiplexed biochemical detection.

Single-color barcoding strategies could break the limits of spectral crosstalk in conventional intensity-based fluorescence barcodes. Fluorescence anisotropy (FA), a self-referencing quantity able to differentiate spectrally similar fluorophores, is highly attractive in designing fluorescent barcodes within a limited emission window. In this study, FA-based encoding of polystyrene (PS) microspheres was realized for the first time. The FA signals of fluorophores were stabilized inside PS microspheres owing to hampered rotational motion. Fluorescent labels were incorporated with similar emission but different structures, symmetries, and lifetimes. On the one hand, Förster Resonance Energy Transfer (FRET) including homo-FRET and hetero-FRET resulted in a decrease of steady-state FA with increasing dye loading, converting conventional intensity-based codes into FA-based codes. On the other hand, mixing dyes with different intrinsic FA values generated different FA values at the same fluorescence intensity level. Single color 5-plex FA-encoded microspheres were demonstrated and decoded on a homemade microscopic FA imaging platform in real time. The FA-encoded microspheres were successfully applied to detect the oligonucleotide of the foodborne bacterium, Bacillus cereus, without spectral crosstalk between the encoding and reporting dyes. Overall, FA-based encoding with an expanded coding capacity in the FA dimension holds great potential in multiplexed high-throughput chemical and biological analyses.

Proton-Coupled Photochromic Hemithioindigo: Toward Photoactivated Chemical Sensing and Imaging.

We report photoswitchable fluorescent hemithioindigos (HTIs) where the metastable E isomers were stabilized by the proton-bridged intramolecular hydrogen bond. Titration experiments and computational analysis indicated that the E isomers were much more basic than the Z isomers, which enabled photoactivated colorimetric and fluorescent pH response in solvents and polypropylene films. The HTIs exhibited reversibly switchable fluorescence with the Z isomers being the most fluorescent. Moreover, the HTIs were lysosomotropic and the kinetic fluorescence evolution during photoswitching was able to differentiate subcellular compartments with different pH. The combination of photoenhanced basicity, switchable fluorescence, and proton-coupled photochromism lay the groundwork for a broad range of chemical and biological applications.

Visible light responsive photoacids for subcellular pH and temperature correlated fluorescence sensing.

Liao's photoacids (PAs) are a well-known type of visible light-responsive photoswitches. Here, taking advantage of the temperature-dependent thermal relaxation from the ring-closed to the ring-opened forms, PAs are proposed for the first time as a fluorescent temperature sensor in cells. The logarithmic lifetime (ln τ) of the ring-closed spiro-form exhibited an excellent linear response to the reciprocal of the temperature. In addition, the fluorescent ring-opened PAs were able to highlight lysosomes and responded to lysosomal pH changes. These properties made the PAs promising fluorescent probes in the sensing of subcellular pH and temperature.

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.

Fluorescence Anisotropy as a Self-Referencing Readout for Ion-Selective Sensing and Imaging Using Homo-FRET between Chromoionophores.

Fluorescence anisotropy has been widely used in developing biosensors and immunoassays, by virtue of the self-reference and environment-sensitive properties. However, fluorescence anisotropic chemical sensors on inorganic ions are limited by the total anisotropy change. To this end, we demonstrate here fluorescence anisotropic ion-selective optodes based on the homo-FRET (Förster resonance energy transfer) of the crowded chromoionophores. The conventional fluorescence on-off mode is transformed into the anisotropic mode. Variation of the target ion concentration changes the inter-chromoionophore distance in the organic sensing phase, leading to different extents of homo-FRET and steady-state anisotropy. A theoretical model is developed by coupling homo-FRET and anisotropy. Anisotropic detections of pH, K+, and Na+ are demonstrated as examples based on the different ionophores for H+, K+, and Na+, respectively. Further, fluorescence imaging of the nano-optodes, plasticized poly(vinyl chloride) sensing films, and live cells are demonstrated using a homemade fluorescence anisotropic imaging platform. The results form the basis of an ion-selective analytical method operating in the fluorescence anisotropic mode, which could potentially be applied to other fluorescence on-off probes based on homo-FRET.

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.

Ruthenium bipyridine complexes as electrochemiluminescent transducers for ionophore-based ion-selective detection.

We report here a method to determine target ion concentrations (with Na+ as a model) based on ionophores and electrochemiluminescence (ECL). Ruthenium bipyridine complexes were released from thin polymeric films (plasticized poly(vinyl chloride) also containing a sodium ionophore) into the sample solution following an explicit ion-exchange process (between Na+ and the ruthenium complex). Two signal transducers, tris(2,2'-(pCF3)bipyridine)ruthenium(II) (Ru(p-CF3-bpy)32+) and tris(2,2'-bipyridyl)dichlororuthenium(II) (Ru(bpy)32+), were examined using the sensing film, with the latter providing a more sensitive detection range (ca. 1 to 100 µM) than that of the more hydrophobic one (0.01 to 1 mM). While the ionophore (Na+ ionophore X) offered excellent selectivity to the method, the ruthenium complexes made the measurements independent of the sample pH. Furthermore for complex biological samples such as blood serum, an indirect approach of measuring the ECL of the remaining ruthenium complexes helps avoid background matrix interference to the ECL production at the working electrode, making the ECL method more attractive for real complex samples.

Enhanced Sulfite-Selective Sensing and Cell Imaging with Fluorescent Nanoreactors Containing a Ratiometric Lipid Peroxidation Sensor.

The detection of SO2 and its derivatives is indispensable for monitoring atmospheric, water quality, and biological fluctuation of oxidative stress and metabolism of biothiols within native cellular contexts. In this article, the brush copolymer nanoreactors containing amine-terminated PDMS were used to encapsulate the fluorescent indicator C11-BDP, forming sulfite-sensitive nanoreactors (ssNRs). Surprisingly, the ssNRs were found to be highly selective to sulfite over a range of reactive oxygen/nitrogen/sulfur species and anions, which was not observed with freely dissolved indicators. The ssNRs showed a rapid response (t95 = 65 s), an excellent detection limit (0.7 µM), and a very high sensitivity (ca. 1000-fold ratiometric intensity change) to sulfite. For cellular studies, the ssNRs exhibited negligible toxicity and could be endocytosed into endosomes and lysosomes. Finally, the ssNRs allowed us to visualize the different responses of three different types of cells (pre-adipocytes, RAW264.7, and HeLa cells) to external stimuli in the culture media with sulfites and lipopolysaccharides.

Colorimetric and fluorescent turn-on detection of chloride ions with ionophore and BODIPY: Evaluation with nanospheres and cellulose paper.

Here, we present the optical detection of chloride ions with co-extraction based anion-selective optodes containing a BODIPY derivative (BDP-OH) and the ionophore In(OEP)Cl. Spectroscopic studies suggested that BDP-OH and In(OEP)Cl formed an adduct BDP-O-In(OEP), which was converted to BDP-OH and In(OEP)Cl upon increasing sample Cl- concentration, and induced signal changes in both fluorescence and absorbance modes. The method was evaluated in polystyrene-graft-poly(ethylene oxide) (PS-PEO) nanospheres (ca. 40 nm in diameter) and on cellulose paper. In contrast to Cl- probes based on fluorescence quenching, the nanospheres exhibited turn-on fluorescence and ratiometric absorbance responses to a tunable Cl- concentration range (10 µM-1 M). Through fluorescence microscopy, the nanospheres were able to respond to Cl- concentration changes in HeLa cells. Cellulose paper-based Cl- optodes with colorimetric response were successfully used to measure Cl- in artificial sweat, providing a potential analytical tool for clinical diagnosis of cystic fibrosis.

Wash-Free Detection of Nucleic Acids with Photoswitch-Mediated Fluorescence Resonance Energy Transfer against Optical Background Interference.

The optical background such as autofluorescence and light scattering poses a big challenge to quantify nucleic acids with conventional fluorescence-based methods. We report here high-contrast nucleic acid detection with photoswitch-mediated fluorescence resonance energy transfer (FRET), which strongly occurs between the open forms of the photoswitch (a naphthopyran) and the signal fluorophores brought to the surface of the nanoprobes (≲15 nm). The fluorescence change (ΔF) upon UV irradiation is highly sensitive and more robust to quantify the target DNAs than traditional intensity measurements. Therefore, the method works in samples with strong background fluorescence from the unbound fluorophores. The photoswitchable nanoprobes could be easily prepared and interrogated in capillaries for high-throughput measurements. The method was evaluated in both sandwich-like hybridization and DNA label-free detection with a nucleic stain SG. Without DNA amplification and sample pretreatment of blood serum, the photoswitchable nanoprobes provided a limit of detection of 0.5 nM, which is ∼6 to 20 times lower than conventional FRET.

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.

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.

Dual functional luminescent nanoprobes for monitoring oxygen and chloride concentration changes in cells.

A dual functional nanoprobe Pd-Q+@PDMS was proposed to simultaneously monitor Cl- and O2, leading to the determination of an average Cl- concentration of 85.7 ± 5.5 mM in lysosomes of HeLa cells. Mimicking ischemic conditions, the cells exhibited a luminescence change corresponding to a decreasing subcellular Cl- concentration.

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.