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.

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.

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.

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.

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.

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.

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.

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.

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.

Chemiluminescent Ion Sensing Platform Based on Ionophores.

We report here an ionophore-based chemiluminescent platform for the selective detection of ions. This method functions on the basis of the ion-exchange between the target ion and the divalent organic cation lucigenin, which luminesces after reacting with hydrogen peroxide in alkaline conditions. Using the K+ ionophore valinomycin, chemiluminescent detection of K+ (from 10 nM to 1 M) was performed on both dichloromethane solutions and polymeric films. While the ionophores ensured excellent selectivity, the detection range could be adjusted with the ratio between the aqueous and the organic phases, and the divalent lucigenin ions also made the sensitivity for divalent target ions higher than conventional ionophore-based ion-selective optodes. The generalizability of the method was shown by changing the K+ ionophore into a Pb2+ ionophore, which successfully realized the highly selective detection of Pb2+ from 0.1 nM to 0.1 mM. Preliminary application of the method was demonstrated with the determination of K+ in diluted human blood serum (five samples), and the results agreed well with those obtained from potentiometric ion-selective electrodes.

Rapid Equilibrated Colorimetric Detection of Protamine and Heparin: Recognition at the Nanoscale Liquid-Liquid Interface.

Demand for rapid quantitation of polyions such as heparin and protamine are ever growing. Previous paper-based and polymeric optical and electrochemical sensing devices required more than several hours for signal stabilization. Therefore, signals were acquired with fixed sample exposure time modes, which was time-consuming and technically demanding. We present here for the first time the optical detection of protamine and heparin in equilibrium mode with emulsified nanospheres. The method significantly shortens the response time from hours to typically less than 10 s and offers tunable, sensitive, and colorimetric detection within the clinically relevant range (10 to 100 mg/L) for heparin. The improved characteristics are attributable to the small size of the nanospheres (ca. 50 nm in diameter) as well as the reversible recognition at the nanoscale liquid-liquid interface. Detection of the anticoagulant heparin was also successfully demonstrated in human blood serum background.

Rhodamine dye transfer from hydrogel to nanospheres for the chemical detection of potassium ions.

Smart hydrogels incorporating various functional nanomaterials are becoming popular tools for chemical sensing. Here, ion-exchange nanospheres composed of the block copolymer Pluronic F-127 played the role of a scavenger for a signal transducer dye (Rhodamine 800) in a three-phase based optical detection system for potassium ions. Rhodamine 800, a positively charged dye, was incorporated into a hydrogel together with the potassium ionophore valinomycin and an ion-exchanger (Na+R-). The concentration of Rhodamine 800 in the aqueous sample was kept low by the nanospheres containing Na+R-. Consequently, the detection limit (0.3 µM) of the three-phase based system was shifted 2 orders of magnitude lower compared with those of previously reported two-phase based sensing systems. The concept of controlling the dye transfer among the three phases provided a new train of thought for the design of ionophore-based chemical sensors.

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.

Determination of pK(a) Values of Hydrophobic Colorimetric pH Sensitive Probes in Nanospheres.

A simple and novel method is proposed here for the first time to determine pK(a) values of chromogenic hydrophobic pH sensitive probes directly in nanospheres. pK(a) values can be obtained by measuring the pH response of the nanospheres (containing the probes and ion exchanger) followed by measuring the pH and Na(+) responses of the nanospheres (containing solvatochromic dyes and ion exchanger). The pK(a) values of four chromoionophores were successfully determined. This method is in principle also applicable to characterize colorimetric probes in other water immiscible nanomaterials.

Complexometric titrations: new reagents and concepts to overcome old limitations.

Chelators and end point indicators are the most important parts of complexometric titrations. The most widely used universal chelator ethylenediamine tetraacetic acid (EDTA) and its derivatives can strongly coordinate with different metal ions. Their limited selectivity often requires the use of masking agents, and the multiple pKa values of the chelators necessitate a careful adjustment of pH during the procedure. Real world requirements for pH independent, selective and sensitive chelators and indicators call for a new design of these reagents. New concepts and structures of chelators and indicators have indeed recently emerged. We present here recent developments on chelators and indicators for complexometric titrations. Many of these advances were made possible only recently by moving the titration from a homogeneous to a heterogeneous phase using a new class of chelators and indicators based on highly selective ionophores embedded in ion-selective nanosphere emulsions. In view of achieving titrations in situ by complete instrumental control, thin layer electrochemistry has recently been shown to be an attractive concept that replaces the traditional cumbersome titration protocol with a direct reagent free sensing tool.

A near-infrared fluorescent aza-bodipy probe for dual-wavelength detection of hydrogen peroxide in living cells.

A boronic acid functionalized aza-borondipyrromethene dye (azaBDPBA) was applied to the dual-wavelength detection of hydrogen peroxide with high selectivity, which was loaded into cells to indicate the alteration of intracellular hydrogen peroxide during biological processes.

Ion-selective optode nanospheres as heterogeneous indicator reagents in complexometric titrations.

Traditionally, optical titrations of inorganic ions are based on a rapid and visible color change at the end point with water-soluble organic dyes as indicators. Adequate selectivity is required for both the indicator and the complexing agent, which is often limited. We present here alternative, heterogeneous ionophore-based ion-selective nanospheres as indicators and chelators for optical titrations. The indicating nanospheres rely on a weaker extraction of the analyte of interest by ion-exchange, owing to the additional incorporation of a lipophilic pH indicator in the nanosphere core. Ca(2+) titration was demonstrated as a proof-of-concept. Both the chelating and the indicating nanospheres showed good selectivity and a wide working pH range.

Solvatochromic Dyes as pH-Independent Indicators for Ionophore Nanosphere-Based Complexometric Titrations.

For half a century, complexometric titrations of metal ions have been performed with water-soluble chelators and indicators that typically require careful pH control. Very recently, ion-selective nanosphere emulsions were introduced that exhibit ion-exchange properties and are doped with lipophilic ionophores originally developed for chemical ion sensors. They may serve as novel, highly selective and pH independent complexometric reagents. While ion optode emulsions have been demonstrated as useful indicators for such titrations, they exhibit a pH cross-response that unfortunately complicates the identification of the end point. Here, we present pH-independent optode nanospheres as indicators for complexometric titrations, with calcium as an initial example. The nanospheres incorporate an ionic solvatochromic dye (SD), ion exchanger and ionophore. The solvatochromic dye will be only expelled from the core of the nanosphere into the aqueous solution at the end point at which point it results in an optical signal change. The titration curves are demonstrated to be pH-independent and with sharper end points than with previously reported chromoionophore-based optical nanospheres as indicator. The calcium concentration in mineral water was successfully determined using this method.