Background-Free Fluorescence-Decay-Time Sensing and Imaging of pH with Highly Photostable Diazaoxotriangulenium Dyes.

Novel fluorescent diazaoxatriangulenium (DAOTA) pH indicators for lifetime-based self-referenced pH sensing are reported. The DAOTA dyes were decorated with phenolic-receptor groups inducing fluorescence quenching via a photoinduced-electron-transfer mechanism. Electron-withdrawing chlorine substituents ensure response in the most relevant pH range (apparent p Ka' values of ∼5 and 7.5 for the p, p-dichlorophenol- and p-chlorophenol-substituted dyes, respectively). The dyes feature long fluorescence lifetimes (17-20 ns), high quantum yields (∼60%), and high photostabilities. Planar optodes are prepared upon immobilization of the dyes into polyurethane hydrogel D4. Apart from the response in the fluorescence intensity, the optodes show pH-dependent lifetime behavior, which makes them suitable for studying 2D pH distributions with the help of fluorescence-lifetime-imaging techniques. The lifetime response is particularly pronounced for the sensors with high dye concentrations (0.5-1 wt % with respect to the polymer) and is attributed to the efficient homo-FRET mechanism.

Near-infrared fluorescent aza-BODIPY dyes for sensing and imaging of pH from the neutral to highly alkaline range.

New aza-BODIPY pH indicators with spectral properties modulated solely by photoinduced electron transfer (PET) are presented. The pH sensitive hydroxyl group is located in the meta-position of a phenyl substituent with respect to the aza-BODIPY core, which eliminates the conjugation to the chromophore. The new dyes show reversible "on"-"off" fluorescence response upon deprotonation of the receptor but no changes in the absorption spectrum, which is in contrast to state-of-the-art indicators of the aza-BODIPY family. This eliminates potential changes in the efficiency of the inner filter effect and Förster resonance energy transfer (FRET) and makes the new dyes suitable acceptors in light harvesting systems used for ratiometric pH imaging. The introduction of electron-withdrawing or electron-donating groups into the receptor results in a set of indicators suitable for measurements from physiological (pH 7) to very alkaline (pH 13) conditions. The new sensors are particularly promising for monitoring of pH changes in concrete, as was recently shown elsewhere.

NIR Phosphorescent Intramolecularly Bridged Benzoporphyrins and Their Application in Oxygen-Compensated Glucose Optode.

A glucose optode measuring the internal oxygen gradient is presented. The multilayer biosensor is composed of (i) analyte-impermeable transparent support, (ii) first oxygen-sensing layer combined with an enzymatic layer, (iii) diffusion barrier, and (iv) second oxygen-sensing layer. To make this design suitable for measurement in subcutaneous tissue, a pair of NIR phosphorescent indicators with very different spectral properties is chosen. Combination of a conventional Pt(II) tetrabenzoporphyrin dye (absorption and emission maxima at 617 and 772 nm, respectively) used in the first layer and a new intramolecularly bridged Pt(II) complex (absorption and emission maxima at 673 and 872 nm, respectively) in the second layer enables efficient separation of both emission signals. This specially designed dye class is accessible via Scholl-reaction from tetraphenyltetrabenzoporphyrin complexes. For the first time, the new optode allows simultaneous glucose and oxygen measurement in a single spot and therefore accurate compensation of oxygen heterogeneities resulting from fluctuations in the tissue. The presented material covers the dynamic ranges from 0 to 150 hPa O2 and from 0 to 360 mg/dL (20 mM) glucose (at 37 °C).

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo.

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.

Ultrabright Red-Emitting Photostable Perylene Bisimide Dyes: New Indicators for Ratiometric Sensing of High pH or Carbon Dioxide.

New pH-sensitive perylene bisimide indicator dyes were synthesized and used for fabrication of optical sensors. The highly photostable dyes show absorption/emission bands in the red/near-infrared (NIR) region of the electromagnetic spectrum, high molar absorption coefficients (up to 100 000 m-1 cm-1 ), and fluorescence quantum yields close to unity. The absorption and emission spectra show strong bathochromic shifts upon deprotonation of the imidazole nitrogen atom, which makes the dyes promising as ratiometric fluorescent indicators. Physical entrapment of the indicators into a polyurethane hydrogel enables pH determination at alkaline pH values. It is also shown that a plastic carbon dioxide solid-state sensor can be manufactured by immobilization of the pH indicator in a hydrophilic polymer, along with a quaternary ammonium base. The influences of the plasticizer, different lipophilic bases, and humidity on the sensitivity of the sensor material are systematically investigated. The disubstituted perylene, particularly, features two deprotonation equilibria, enabling sensing over a very broad pCO2 range of 0.5 to 1000 hPa.

Spectral Characterization of Eight Marine Phytoplankton Phyla and Assessing a Pigment-Based Taxonomic Discriminant Analysis for the in Situ Classification of Phytoplankton Blooms.

Early stage identification of harmful algal blooms (HABs) has gained significance for marine monitoring systems over the years. Various approaches for in situ classification have been developed. Among them, pigment-based taxonomic classification is one promising technique for in situ characterization of bloom compositions, although it is yet underutilized in marine monitoring programs. To demonstrate the applicability and importance of this powerful approach for monitoring programs, we combined an ultra low-cost and miniaturized multichannel fluorometer with Fisher's linear discriminant analysis (LDA). This enables the real-time characterization of algal blooms at order level based on their spectral properties. The classification capability of the algorithm was examined with a leave-one-out cross validation of 53 different unialgal cultures conducted in terms of standard statistical measures and independent figures of merit. The separation capability of the linear discriminant analysis was further successfully examined in mixed algal suspensions. Besides this, the impact of the growing status on the classification capability was assessed. Further, we provide a comprehensive study of spectral features of eight different phytoplankton phyla including an extensive study of fluorescence excitation spectra and marker pigments analyzed via HPLC. The analyzed phytoplankton species belong to the phyla of Cyanobacteria, Dinophyta (Dinoflagellates), Bacillariophyta (Diatoms), Haptophyta, Chlorophyta, Ochrophyta, Cryptophyta, and Euglenophyta.

Compact and Low-Cost Fluorescence Based Flow-Through Analyzer for Early-Stage Classification of Potentially Toxic Algae and in Situ Semiquantification.

The occurrence and intensity of (harmful) algal blooms (HABs) have increased through the years due to rapidly changing environmental conditions. At the same time, the demand for low-cost instrumentation has increased substantially, enabling the real-time monitoring and early-stage detection of HABs. To meet this challenge, we have developed a compact multi-wavelength fluorometer for less than 400 USD. This is possible by using readily available and low-cost optical and electronic components. Its modular design results in a highly versatile and flexible monitoring tool. The algae detection module enables a continuous identification and control of relevant algal groups based on their spectral characteristics with a detection limit of 10 cells per liter. Besides its usage as a benchtop module in the laboratory, the algae module has been integrated into submersible housings and applied in coastal environments. During its first in situ application in the Port of Genoa, seawater samples of mixed algal composition were used to demonstrate the successful discrimination of cyanobacteria and dinophytes as well-known toxin producing classes. Fabrication, operation, and performance as well as its first in situ application are addressed.

Trace Ammonia Sensors Based on Fluorescent Near-Infrared-Emitting aza-BODIPY Dyes.

Highly sensitive ammonia sensors for environmental monitoring are presented. The sensing materials are based on fluorescent BF2-chelated tetraarylazadipyrromethene dyes (aza-BODIPYs) dyes physically entrapped in polyurethane hydrogels and dispersed in silicone rubber. This layer is covered by a hydrophobic porous Teflon membrane used as an additional proton barrier and light scattering layer. The dual-lifetime referenced (DLR) sensors make use of near-infrared (NIR)-emitting Egyptian blue as a reference material and in combination with optical fibers are read-out via a compact phase-fluorometer. The detectable concentration range can be tuned by the choice of aza-BODIPY dye or/and the hydrogel matrix. The most sensitive sensor has a limit of detection (LOD) of 0.11 µg/L and the upper detectable concentration of 300 µg/L. No cross-sensitivity toward pH is observed. The sensors show remarkable operational stability with no noticeable drift over a period of 2 weeks.

Sodium-Selective Fluoroionophore-Based Optodes for Seawater Salinity Measurement.

A new fluorescent sensor for Na+ is presented. The sensor relies on a Na+ selective fluoroionophore based on a bright red-emitting BODIPY chromophore. The fluorescence of the fluoroionophore is enhanced upon binding of Na+-ions to the highly selective aza-crown ether receptor due to reduction of the photoinduced electron transfer (PET) quenching. Solid state sensing materials were prepared by physically embedding the fluoroionophore into water-swellable biocompatible polymer matrices (polyurethane hydrogels), thus enabling continuous measurements of aqueous samples. Despite the simple design, the sensor showed no leaching of the indicator and featured fast and reversible response. Among different polyurethane hydrogels investigated, the hydrogel D1 featuring the highest water uptake was found to be the most suitable due to the highest dynamics between "off" and "on" states. Due to little or no cross sensitivity to other ions (e.g., Mg2+, Ca2+, K+) and its insensitivity to potential changes in pH, this sensor is promising for use in clinical diagnostics and for biological and marine applications. Fiber-optic sensors based on referenced read-out with a compact phase fluorimeter were prepared. To demonstrate their practical applicability, the sensors were used to determine the salinity in the seawater and brackish water of the Baltic Sea.

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles.

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.

Measurement of oxygen transfer from air into organic solvents.

BACKGROUND: The use of non-aqueous organic media is becoming increasingly important in many biotechnological applications in order to achieve process intensification. Such media can be used, for example, to directly extract poorly water-soluble toxic products from fermentations. Likewise many biological reactions require the supply of oxygen, most normally from air. However, reliable online measurements of oxygen concentration in organic solvents (and hence oxygen transfer rates from air to the solvent) has to date proven impossible due to limitations in the current analytical methods. RESULTS: For the first time, online oxygen measurements in non-aqueous media using a novel optical sensor are demonstrated. The sensor was used to measure oxygen concentration in various organic solvents including toluene, THF, isooctane, DMF, heptane and hexane (which have all been shown suitable for several biological applications). Subsequently, the oxygen transfer rates from air into these organic solvents were measured. CONCLUSION: The measurement of oxygen transfer rates from air into organic solvents using the dynamic method was established using the solvent resistant optical sensor. The feasibility of online oxygen measurements in organic solvents has also been demonstrated, paving the way for new opportunities in process control. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Intrinsic artefacts in optical oxygen sensors--how reliable are our measurements?

Optical oxygen sensing is of broad interest in many areas of research, such as medicine, food processing, and micro- and marine biology. The operation principle of optical oxygen sensors is well established and these sensors are routinely employed in lab and field experiments. Ultratrace oxygen sensors, which enable measurements in the sub-nanomolar region (dissolved oxygen), are becoming increasingly important. Such sensors prominently exhibit phenomena that complicate calibration and measurements. However, these phenomena are not constrained to ultratrace sensors; rather, these effects are inherent to the way optical oxygen sensors work and may influence any optical oxygen measurement when certain conditions are met. This scenario is especially true for applications that deal with high-excitation light intensities, such as microscopy and microfluidic applications. Herein, we present various effects that we could observe in our studies with ultratrace oxygen sensors and discuss the reasons for their appearance, the mechanism by which they influence measurements, and how to best reduce their impact. The phenomena discussed are oxygen photoconsumption in the sensor material; depletion of the dye ground state by high-excitation photon-flux values, which can compromise both intensity and ratiometric-based measurements; triplet-triplet annihilation; and singlet-oxygen accumulation, which affects measurements at very low oxygen concentrations.

NIR-emitting aza-BODIPY dyes--new building blocks for broad-range optical pH sensors.

New aza-BODIPY indicators which cover the pH scale from 1.5 to 13 are presented. The new indicators feature absorption/emission bands in the red/near-infrared (NIR) spectral region, exhibit high molar absorption coefficients (∼ 80,000 M(-1) cm(-1)) and show good quantum yields (∼20%). All dyes represent promising building blocks for the development of a broad-range sensor for various pH ranges. Combination of four of these pH indicators yields a pH sensor with an extended dynamic range from pH 2 to 9.

Enhancing photoinduced electron transfer efficiency of fluorescent pH-probes with halogenated phenols.

Photoinduced electron transfer (PET), which causes pH-dependent quenching of fluorescent dyes, is more effectively introduced by phenolic groups than by amino groups which have been much more commonly used so far. That is demonstrated by fluorescence measurements involving several classes of fluorophores. Electrochemical measurements show that PET in several amino-modified dyes is thermodynamically favorable, even though it was not experimentally found, underlining the importance of kinetic aspects to the process. Consequently, the attachment of phenolic groups allows for fast and simple preparation of a wide selection of fluorescent pH-probes with tailor-made spectral properties, sensitive ranges, and individual advantages, so that a large number of applications can be realized. Fluorophores carrying phenolic groups may also be used for sensing analytes other than pH or molecular switching and signaling.

Exceptional Oxygen Sensing Properties of New Blue Light-Excitable Highly Luminescent Europium(III) and Gadolinium(III) Complexes.

New europium(III) and gadolinium(III) complexes bearing 8-hydroxyphenalenone antenna combine efficient absorption in the blue part of the spectrum and strong emission in polymers at room temperature. The Eu(III) complexes show characteristic red luminescence whereas the Gd(III) dyes are strongly phosphorescent. The luminescence quantum yields are about 20% for the Eu(III) complexes and 50% for the Gd(III) dyes. In contrast to most state-of-the-art Eu(III) complexes the new dyes are quenched very efficiently by molecular oxygen. The luminescence decay times of the Gd(III) complexes exceed 1 ms which ensures exceptional sensitivity even in polymers of moderate oxygen permeability. These sensors are particularly suitable for trace oxygen sensing and may be good substitutes for Pd(II) porphyrins. The photophysical and sensing properties can be tuned by varying the nature of the fourth ligand. The narrow-band emission of the Eu(III) allows efficient elimination of the background light and autofluorescence and is also very attractive for use e.g. in multi-analyte sensors. The highly photostable indicators incorporated in nanoparticles are promising for imaging applications. Due to the straightforward preparation and low cost of starting materials the new dyes represent a promising alternative to the state-of-the-art oxygen indicators particularly for such applications as e.g. food packaging.

The effect of high light intensities on luminescence lifetime based oxygen sensing.

This study highlights possible errors in luminescence lifetime measurements when using bright optical oxygen sensors with high excitation light intensities. An analysis of the sensor with a mathematical model shows that high light intensities will cause a depopulation of the ground state of the luminophore, which results in a non-linear behaviour of the luminescence emission light with respect to the excitation light. The effect of this non-linear behaviour on different lifetime determination methods, including phase-fluorometry, is investigated and in good agreement with the output of the model. Furthermore, the consequences of increasingly high light intensities on phase fluorometric lifetime measurements are illustrated for different oxygen sensors based on benzoporphyrin indicators. For the specific case of PdTPTBPF-based sensors an error as high as 50% is possible under high light conditions (0.25 mol m(-2) s(-1) ≈ 50 mW mm(-2)). A threshold of applied excitation light intensity is derived, thus enabling the point at which errors become significant to be estimated. Strategies to further avoid such errors are presented. The model also predicts a similar depopulation of the ground state of the quencher; however, the effect of this process was not seen in lab measurements. Possible explanations for this deviation are discussed.

Diketo-pyrrolo-pyrrole dyes as new colorimetric and fluorescent pH indicators for optical carbon dioxide sensors.

New indicators for optical CO2 sensors are synthesized in two steps from the commercially available diketo-pyrrolo-pyrrole (DPP) pigments Irgazin Ruby and Irgazin Scarlet. After introduction of bis(2-ethylhexyl) sulfonamide groups via a simple two-step synthesis, the pigments are rendered highly soluble in organic solvents and in polymers and show pH-dependent absorption and emission spectra. The new indicators have molar absorption coefficients in a 20 000-50 000 M(-1) cm(-1) range, possess quantum yields close to unity, and feature good photostability. The indicators along with a quaternary ammonium base are embedded into ethyl cellulose to give optical carbon dioxide sensors. The absorption and emission spectra of the deprotonated form are bathochromically shifted by more than 100 nm compared to the neutral form (λmax absorption 496-550 nm; λmax emission 564-587 nm). This enables colorimetric read-out and self-referenced ratiometric fluorescence intensity measurements. Importantly, the dynamic range of the sensors based on the new indicators is significantly different (0-10 kPa and 1-100 kPa CO2) that enables a broad variety of applications. New DPP dyes are conveniently prepared from commercially available pigments and represent a new class of colorimetric and fluorescent pH indicators for optical carbon dioxide sensors.

New life of ancient pigments: application in high-performance optical sensing materials.

Calcium, strontium, and barium copper silicates are demonstrated to possess valuable photophysical properties which make them particularly attractive for application in optical chemosensors. Several examples of sensing materials based on these phosphors are provided. Particularly, broad excitation and near-infrared emission makes them ideal candidates for the preparation of ratiometric sensors based on absorption-based indicators. Due to their excellent chemical and photochemical stability and high brightness, these phosphors can serve as reference for fluorescent indicators to enable ratiometric intensity or dually lifetime referenced measurements. Finally, the moderate temperature dependence of the luminescence decay time enables intrinsic temperature compensation of the sensing materials at ambient temperatures. The improved sensitivity at temperatures above 100 °C makes these new materials promising candidates for high-temperature thermographic phosphors.

A new method to measure oxygen solubility in organic solvents through optical oxygen sensing.

A new method to measure oxygen concentration in air-saturated organic solvents and binary mixtures has been developed. The methodology relies on the ability of HPLC columns to retain the molecular oxygen contained in different types of solvents which are injected into the system at 298.15 K. The outlet of the HPLC is coupled with an optical oxygen sensor which continuously measures changes in oxygen partial pressure.

Synthesis and properties of new phosphorescent red light-excitable platinum(II) and palladium(II) complexes with Schiff bases for oxygen sensing and triplet-triplet annihilation-based upconversion.

New Pt(II) and Pd(II) complexes with donor-acceptor Schiff bases are conveniently prepared in only two steps. The complexes efficiently absorb in the red part of the spectrum (ε > 10(5) M(-1) cm(-1)) and show moderate to strong room-temperature phosphorescence in the near-infrared (NIR) region. Particularly, Pt(II) complexes possess phosphorescence quantum yields (Φ) of ~10%, but the emission of the respective Pd(II) complexes is less efficient (Φ ≈ 1%-2%). The complexes exhibit solvatochromic behavior, in which the absorption and emission spectra shift bathochromically in polar solvents. The Pt(II) complexes are embedded in polystyrene to produce oxygen-sensing materials. The Pd(II) and Pt(II) complexes are demonstrated to be efficient sensitizers in triplet-triplet annihilation-based upconversion systems.