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.

Insights in the determination of saxitoxin with fluorogenic crown ethers in water.

ABSTRACT: In this contribution, we present new insights and a critical discussion in the optical detection of saxitoxin using fluorophores with crown ethers. Fluorescence enhancement is caused by the reduction of photoinduced electron transfer upon complexation with the analyte. Our attempts to improve this detection method neither did yield a functioning sensor nor were the attempts to reproduce published data in this area successful. Due to the fact that only low concentrations of saxitoxin are available, multiple surrogates were investigated at high concentrations. However, no turn on response was observed. Moreover, a fluorescent decomposition product of saxitoxin that forms under UV light was discovered which was in our opinion misinterpreted as a sensor response by previous publications.

Ionophore-based optical sensors.

This review provides an overview of the key aspects of designing ionophore-based optical sensors (IBOS). Exact response functions are developed and compared with a simplified, generalized equation. We also provide a brief introduction into less established but promising working principles, namely dynamic response and exhaustive exchange. Absorbance and fluorescence are the main optical readout strategies used in the evaluation of a sensor response, but they usually require a robust referencing technique for real-world applications. Established referencing schemes using IBOS as well as those from other optical sensors are also discussed. Finally, the power of recently developed photoresponsive ion extraction/release systems is outlined and discussed in view of dynamically switchable IBOS or regenerative exhaustive exchange IBOS.

Photocurrent generation based on a light-driven proton pump in an artificial liquid membrane.

Biological light-driven proton pumps use light to move protons across a cell membrane, creating a proton gradient. Although photochromic compounds such as spiropyrans can reversibly convert between two structures with differing pKa values, spiropyrans have not been used to generate either a light-driven proton pump or an electrical current. Here, we report an artificial light-harvesting system based on a supported liquid membrane doped with a spiropyran. Irradiating the membrane with ultraviolet light induces a ring-opening reaction, converting spiropyran to merocyanine, whereas irradiation with visible light induces the reverse reaction. When the membrane is irradiated with ultraviolet and visible light on opposite sides, H(+) is taken up by merocyanine, carried through the polymeric membrane and released on the other side. We show that this system produces a light-induced proton flux, an electrical current with an efficiency of ∼0.12%, an open-circuit voltage of ∼210 mV and a membrane gradient of ∼3.6 ΔpH units. Alternating the sides illuminated with ultraviolet and visible light generates an alternating current.

Ultrasmall fluorescent ion-exchanging nanospheres containing selective ionophores.

We present a convenient precipitation procedure to fabricate ultrasmall fluorescent ion-selective nanosensors that operate on the basis of bulk ion-exchange sensing principles. The nanosphere matrix is composed of bis(2-ethylhexyl) sebacate (DOS) and a triblock copolymer Pluronic(®) F-127, which also functions as a surfactant to stabilize the nanoparticle. The particles can be prepared easily in large quantity without resorting to further complicated purification. Dynamic light scattering shows that these particles have a monodisperse size distribution with an average diameter of ∼40 nm, suggesting that the nanoparticles are among the smallest ionophore-based ion-selective nanosensors reported to date. A newly reported oxazinoindoline (Ox) as well as a Nile blue derivative (chromoionophore I) was used as a chromoionophore. Na(+)- and H(+)-selective nanospheres were characterized by absorbance and fluorescence spectroscopy. Owing to the very small size of the nanospheres, the suspension containing the particles is transparent. In the additional presence of the pH indicator HPTS, spectroscopic interrogation of pH and Na(+) in the same sample was demonstrated. As an example, the nanospheres were used to measure the Na(+) level in commercial mineral waters, and the results showed good agreement with atomic absorption spectroscopy (AAS).

Molecularly imprinted polymer microspheres containing photoswitchable spiropyran-based binding sites.

A versatile approach for the preparation of photoswitchable molecularly imprinted polymers (MIPs) is proposed where the selective recognition and the photoresponsive function are assumed by two different monomers. As a proof of concept, MIP microspheres were synthesized by precipitation polymerization for recognizing terbutylazine, a triazine-type herbicide. Formation of the selective binding sites was based upon H-bonding interactions between the template and the functional monomer methacrylic acid, whereas a polymerizable spiropyran unit was incorporated into the polymer matrix to provide light-controllable characteristics. A trifunctional monomer, trimethylolpropane trimethacrylate, was used as a cross-linker. The imprinted particles exhibited considerable morphological differences compared to their nonimprinted counterparts as observed by scanning electron microscopy. The imprinting effect was confirmed by equilibrium rebinding studies. The photoresponsiveness of the polymer particles was visualized by fluorescence microscopy and further characterized by spectroscopy. The template binding behavior could be regulated by alternating UV and visible light illumination when analyte release and uptake was observed, respectively. Binding isotherms fitted by the Freundlich model revealed the photomodulation of the number of binding sites and their average affinity. This facile synthetic approach may give an attractive starting point to endow currently existing highly selective MIPs with photoswitchable properties, thereby extending the scope of spiropyran-based photoresponsive smart materials.

Potentiometric sensors with ion-exchange Donnan exclusion membranes.

Potentiometric sensors that exhibit a non-Hofmeister selectivity sequence are normally designed by selective chemical recognition elements in the membrane. In other situations, when used as detectors in separation science, for example, membranes that respond equally to most ions are preferred. With so-called liquid membranes, a low selectivity is difficult to accomplish since these membranes are intrinsically responsive to lipophilic species. Instead, the high solubility of sample lipids in an ionophore-free sensing matrix results in a deterioration of the response. We explore here potentiometric sensors on the basis of ion-exchange membranes commonly used in fuel cell applications and electrodialysis, which have so far not found their way into the field of ion-selective electrodes. These membranes act as Donnan exclusion membranes as the ions are not stripped of their hydration shell as they interact with the membrane. Because of this, lipophilic ions are no longer preferred over hydrophilic ones, making them promising candidates for the detection of abundant ions in the presence of lipophilic ones or as detectors in separation science. Two types of cation-exchanger membranes and one anion-exchange membrane were characterized, and potentiometric measuring ranges were found to be Nernstian over a wide range down to about 10 µM concentrations. Depending on the specific membrane, lipophilic ions gave equal response to hydrophilic ones or were even somewhat discriminated. The medium and long-term stability and reproducibility of the electrode signals were found to be promising when evaluated in synthetic and whole blood samples.

Photoresponsive ion extraction/release systems: dynamic ion optodes for calcium and sodium based on photochromic spiropyran.

Photoresponsive ion extraction/release systems (PRIONERS) represent a highly interesting tool for the localized and time-controlled chemical perturbation of biological materials. We report here on our first results on phototriggered calcium and sodium exchanging materials. Such materials exist in two distinct states ("on" and "off"), depending on the wavelength of illumination. We used a combination of spectroscopic and electrochemical methods to obtain a better understanding of the dynamic processes involved in the triggered ion-exchange reaction upon activation of the photoactive compound. The driving force for the ion exchange is the light-induced acidity change of the chromoionophore. Activation with UV light generates a species in the membrane with an increased pKa. Protons are pulled into the membrane, and at the same time, ions are expelled. The selectivity of the system is determined by the employed ionophore. In contrast to photoresponsive ionophore-based systems, the concept presented here is applicable for virtually any ion of interest for which an ionophore exists.

Reversible photodynamic chloride-selective sensor based on photochromic spiropyran.

We report here for the first time on a reversible photodynamic bulk optode sensor based on the photoswitching of a spiropyran derivative (Sp). The photoswitching of Sp induces a large basicity increase in the polymeric phase, which triggers the extraction of Cl(-) and H(+). Cl(-) is stabilized by a lipophilic chloride-selective ionophore inside the membrane, while H(+) binds with the open form of Sp and induces a spectral change, hence providing the sensor signal. The system was studied with spectroscopic and electrochemical methods.

Photodynamic ion sensor systems with spiropyran: photoactivated acidity changes in plasticized poly(vinyl chloride).

We aim to introduce photoactivated optical ion sensors based on a triggered ion exchange process as novel dynamic tools. The quantification of the acidity change upon photoactivation of spiropyran within an organic membrane phase during photoexcitation is described here for the first time, suggesting a large pK(a) change of more than 6 orders of magnitude.

Electrogenerated chemiluminescence for potentiometric sensors.

We report here on a generic approach to read out potentiometric sensors with electrogenerated chemiluminescence (ECL). In a first example, a potassium ion-selective electrode acts as the reference electrode and is placed in contact with the sample solution. The working electrode of the three-electrode cell is responsible for ECL generation and placed in a detection solution containing tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] and the coreactant 2-(dibutylamino)ethanol (DBAE), physically separated from the sample by a bridge. Changes in the sample potassium concentration directly modulate the potential at the working electrode, and hence the ECL output, when a constant-potential pulse is applied between the two electrodes. A linear response of the ECL intensity to the logarithmic potassium concentration between 10 µm and 10 mM was found.

Electrogenerated chemiluminescence triggered by electroseparation of Ru(bpy)3(2+) across a supported liquid membrane.

We demonstrate here for the first time the detection of electrogenerated chemiluminescence (ECL) upon selective, electrochemically triggered tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) transport across a supported liquid membrane doped with a lipophilic cation-exchanger. This approach allows one to separate the location of ECL generation from the sample compartment, thereby avoiding the contamination of the sample with compounds necessary for ECL.

Advancing membrane electrodes and optical ion sensors.

While potentiometric sensors experienced a golden age in the 1970s that drove innovation and implementation in the clinical laboratory as sensors of choice, it has been only fairly recently that a theoretical understanding coupled with modern materials approaches transformed the area of membrane electrodes from a playful, yet empirical field to one firmly rooted in scientific understanding. This paper summarizes key progress in the field during the past two decades, emphasizing that the key impulses at the time originated from the emerging field of optical ion sensors. This simplified and transformed the underlying theory of their potentiometric membrane electrode counterparts, where subsequently substantial progress was made, including the realization of ultra-trace detection limits. The better understanding of zero-current ion fluxes and transport processes in turn allowed the development of approaches utilizing dynamic electrochemistry principles, thereby drastically expanding the field of membrane electrodes and making available a range of new methodologies that would have been difficult to predict only a few years ago. These significant developments are now starting to come back and influence the field of optical sensors, where the control and triggering of dynamic processes, away from simpler equilibrium principles, are becoming a highly promising field of research.

Magnetically remote-controlled optical sensor spheres for monitoring oxygen or pH.

Magnetic sensor macrospheres (MagSeMacs), i.e., stainless steel spheres coated with optical chemical sensors, are presented as an alternative to existing optical sensor patches and fiber-optical dip-probes. Such spheres can either be reversibly attached to the tip of an optical fiber (dip-probe) or trapped inside a vessel for read-out through the side wall. Moving the magnetic separator at the exterior enables measurements at varying positions with a single sensor. Moreover, the sensor's replacement is rapid and contactless. We measured dissolved oxygen or pH in stirred liquids, rotating flasks, and 24-well plates with a SensorDish-reader device for parallel cell culture monitoring. In these applications, MagSeMacs proved to be advantageous over conventional sensor patches and magnetic optical sensor particles because of their magnetism, spherical shape, reflectance, and size. These properties resulted in strong but reversible fixation, magnetic remote-controllability, short response times, high signal intensities, and simplified handling.

Precipitation as a simple and versatile method for preparation of optical nanochemosensors.

Optical nanosensors for such important analytes as oxygen, pH, temperature, etc. are manufactured in a simple way via precipitation. Lipophilic indicators are entrapped into nanobeads based on poly(methyl methacrylate), polystyrene, polyurethanes, ethylcellulose, and other polymers. Charged groups greatly facilitate formation of the small beads and increase their stability. Sensing properties of the beads can be tuned by choosing the appropriate indicator. Nanosensors for carbon dioxide and ammonia are found to be cross-sensitive to pH if dispersed in aqueous media. These nanobeads are successfully employed to design bulk optodes. Nanochemosensors with enhanced brightness via light-harvesting and multi-functional magnetic nanosensors also are prepared.