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.

1,3,5-2,4,6-Functionalized Benzene Molecular Cage: An Environmentally Responsive Scaffold that Supports Hierarchical Superstructures.

New stimulus-responsive scaffolds are of interest as constituents of hierarchical supramolecular ensembles. 1,3,5-2,4,6-Functionalized, facially segregated benzene moieties have a time-honored role as building blocks for host molecules. However, their user as switchable motifs in the construction of multi-component supramolecular structures remains poorly explored. Here, we report a molecular cage 1, which consists of a bent anthracene dimer 3 paired with 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene 2. As the result of the pH-induced ababab↔bababa isomerization of the constituent-functionalized benzene units derived from 2, this cage can reversibly convert between an open state and a closed form, both in solution and in the solid state. Cage 1 was used to create stimuli-responsive hierarchical superstructures, namely Russian doll-like complexes with [K⊂18-crown-6⊂1]+ and [K⊂cryptand-222⊂1]+. The reversible assembly and disassembly of these superstructures could be induced by switching cage 1 from its open to closed form. The present study thus provides an unusual example where pH-triggered conformation motion within a cage-like scaffold is used to control the formation and disassociation of hierarchical ensembles.

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.

Recent advance in dual-functional luminescent probes for reactive species and common biological ions.

The chemical microenvironment in cells is extremely complex involving numerous species with drastically different concentration as well as temporal and spatial distribution. Especially, reactive species including ROS, RNS, RSS, and many inorganic ions are recognized to play very important roles. The concentrations of these species are constantly regulated and often depend on each other. While fluorescent probes have been widely used to study various cell biological processes, those simultaneously probing two species just emerged recently. In this review, we highlight dual-functional luminescent probes for the detection and fluorescence imaging of two and more analytes in cells. The content will cover small-molecule synthetic fluorescent probes, DNA nanoassemblies, and nanoparticle-based nanoprobes. The target analytes include reactive species such as ROS, RNS, and RSS and other ions and molecules such as H+, Cl-, Ca2+, Cu2+, and O2.

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.

Chrysophanol Protects Against Acute Heart Failure by Inhibiting JNK1/2 Pathway in Rats.

BACKGROUND Acute heart failure (AHF) usually requires urgent therapy. Myocardial damage, oxidative stress, and inflammation are major components in the pathology of AHF. This study was designed to investigate the effects of chrysophanol on AHF. MATERIAL AND METHODS Sprague-Dawley rats were injected with isoprenaline hydrochloride to construct AHF rat models. AHF rats were treated with normal saline (negative control), chrysophanol, the combination of chrysophanol and SP600125, or benazepril (positive control) using sham rats as blank controls. Echocardiography, histological staining, and enzyme activity analysis were performed to assess the heart functions and myocardial damage. Effects on apoptosis, oxidative stress (OS), and inflammation were evaluated by biochemical analysis, TUNEL staining, and ELISA. RESULTS Chrysophanol improved the parameters of cardiac functions and alleviated the myocardial damage accompanied by the reduction of creatine kinase and lactate dehydrogenase activity. Meanwhile, chrysophanol inhibited the myocardial apoptosis along with the upregulation of Bcl-2 and downregulation of Bax and cleaved caspase-3. AHF-induced abnormal changes of OS parameters (MDA, GPx, CAT, SOD) and inflammatory markers (IL-6, IL-1ß, TNF-alpha, IFN-γ) were alleviated by chrysophanol. Benazepril treatment showed similar results with chrysophanol, while the addition of SP600125 enhanced the chrysophanol-mediated protection effects in AHF rats. Western blot analysis demonstrated that chrysophanol inhibited the phosphorylation of JNK1/2 and its upstream/downstream factors. CONCLUSIONS Chrysophanol improved cardiac functions and protected against myocardial damage, apoptosis, OS, and inflammation by inhibiting activation of the JNK1/2 pathway in AHF rat models. These finding indicate that chrysophanol may be a promising approach for treatment of AHF.

A Solid-State Reference Electrode Based on a Self-Referencing Pulstrode.

The design of solid-state reference electrodes without a liquid junction is important to allow miniature and cost-effective electrochemical sensors. To address this, a pulse control is proposed using an Ag/AgI element as reliable solid-state reference electrode. It involves the local release of iodide by a cathodic current that is immediately followed by an electromotive force (EMF) measurement that serves as the reference potential. The recapture of iodide ions is achieved by potentiostatic control. This results in intermittent potential values that are reproducible to less than one millivolt (SD=0.27 mV, n=50). The ionic strength is shown to influence the activity coefficient of released iodide in accordance with the extended Debye-Hückel equation, resulting in a predictable change of the potential reading. The principle is applied to potentiometric potassium detection with a valinomycin-based ion-selective electrode (ISE), demonstrating a completely solid-state sensor configuration.

Naphthocage: A Flexible yet Extremely Strong Binder for Singly Charged Organic Cations.

We report a quite flexible naphthol-based cage (so-called "naphthocage") which adopts a self-inclusion conformation in its free state and is able to bind singly charged organic cations extremely strongly ( Ka > 107 M-1). Ion-selective electrodes prepared with this naphthocage show a super-Nernstian response to acetylcholine. In addition, the highly stable complex (1010 M-1) between ferrocenium and the naphthocage can be switched electrochemically, which lays a basis for its application in stimuli-responsive materials.

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.

Electrogenerated Chemiluminescence for Chronopotentiometric Sensors.

We introduce here a general strategy to read out chronopotentiometric sensors by electrogenerated chemiluminescence (ECL). The potentials generated in chronopotentiometry in a sample compartment are used to control the ECL in a separate detection compartment. A three-electrode cell is used to monitor the concentration changes of the analyte, while the luminol-H2O2 system is responsible for ECL. The principle was shown to be feasible by theoretical simulations, indicating that the sampled times at a chosen potential, rather than traditional transition times, similarly give linear behavior between concentration and the square root of sampled time. With the help of a voltage adapter, the experimental combination between chronopotentiometry and ECL was successfully implemented. As an initial proof of concept, the ferro/ferricyanide redox couple was investigated. The square root of time giving maximum light output changed linearly with ferrocyanide concentration in the range from 0.70 to 4.81 mM. The method was successfully applied to the visual detection of carbonate alkalinity from 0.06 to 0.62 mM using chronopotentiometry at an ionophore-based hydrogen ion-selective membrane electrode. The measurements of carbonate in real samples including river water and commercial mineral water were successfully demonstrated.

Simplified Fabrication for Ion-Selective Optical Emulsion Sensor with Hydrophobic Solvatochromic Dye Transducer: A Cautionary Tale.

It has recently been reported that polystyrene microbeads may be modified to realize plasticizer-free ion-selective optical sensors (optodes) on the basis of solvatochromic dye transducers. We show here that the functionalized microbeads, individually isolated by flow cytometry, exhibit unexpectedly poor fluorescent properties and that the sensor response is instead attributed to the supernatant. A more thorough study reveals that such optical microemulsion sensors can be made operationally functional and chemically selective, seemingly in the absence of any solvent matrix or added surfactant. Instead, it is shown that residual THF used in the fabrication of the emulsified sensors may solubilize the sensing components and give a functional optode response. To evaluate this further, the number of sensing components was stepwise simplified to assess their need. Variation of residual THF levels has no effect on the ion optode response when plasticizer is present, in support of established results. Lipophilic solvatochromic dye transducers are also shown not to require an added surfactant as their nature already endows the emulsified sensors with a stabilizing ionic surface charge. The ionophores are shown to exhibit much larger stability constants in the surfactant-free formulations than surfactant-based ones (valionomycin, log ß > 9.2 compared to 6.1; Na+-ionophore X, 6.7 vs 4.7), which is attributed to a less polar solvent environment for the ionophore. Potassium-, sodium-, and calcium-selective sensors were used as model systems in this study.

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.