Polymeric bulk optode microsphere ion sensors in combination with suspension array technologies such as analytical flow cytometry may become a power tool for measuring electrolytes in physiological samples. In this work, the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors was explored. The simultaneous determination of Na(+), K(+), and Ca(2+) in diluted sheep blood plasma was demonstrated for the first time, using a random suspension array containing three types of mixed microsphere bulk optodes of similar size, fabricated from the same chromoionophore without additional labeling. Sodium ionophore X, potassium ionophore III, and grafted AU-1 in poly(butyl acrylate) were the ionophores used in the bulk optode microsphere ion sensors for Na(+), K(+), and Ca(2+), respectively, in combination with the cation-exchanger NaTFPB (sodium tetrakis-[3,5-bis(trifluoromethyl)phenyl]borate) and the same concentration of the chromoionophore ETH 5294 (9-(di-ethylamino)-5-octadecanoylimino-5H-benzo[a]phen-oxazine) in plasticized poly(vinyl chloride). Excellent reproducibility was achieved for the sensing of potassium ions. The effect of sample pH was relatively small at near-physiological pH and followed theoretical predictions, yet the sample temperature was found to influence the sensor response to a larger extent. Multiplexed ion sensing was achieved by taking advantage of the chemical tunability of the sensor response, adjusting the sensor compositions so that the three types of ion sensors responded with distinct levels of protonation of the chromoionophore. Consequently, three well-resolved peaks were simultaneously observed in the single-channel histogram during the multiplexed calibration as well as in the subsequent measurement of the three cations in 10-fold-diluted sheep plasma. The assigned peak positions corresponded very well to the physiological range of the measured ions.

A chemically nonreactive method for the fabrication of uniform core/shell fluorescent polymeric microspheres (see figure) is demonstrated with a flow apparatus. Various dyes and labeled proteins are doped into the appropriate particle phases, and an early example of chemical sensing is demonstrated.

A simple method for doping ion-sensing ingredients into silanized silica particles (∼3.5μm) was developed to fabricate carrier-based ion-selective microsphere optical ion sensors. These ion sensors operate according to established bulk optode principles. Na+-selective microsphere optical sensors were prepared with the ionophore tert-butylcalix[4]arene tetraacetic acid tetraethylester, the H+-chromoionophore 9-(diethylamino)-5-octadecanoylimino-5H-benzo[a]phenoxazine (ETH 5294), the ion exchanger sodium tetrakis[3,5-bis(tri-fluoromethyl)phenyl]borate (NaTFPB), PVC and the plasticizer bis(2-ethylhexyl)sebacate (DOS). For Ca2+ sensing, the grafted Ca2+ ionophore AU-1 in poly(n-BA) was applied with ETH 5294, NaTFPB and DOS. Fluorescence microscopy and flow cytometry were used for characterization. The resulting microsphere optical ion sensors were found to obey bulk optode theory with reliable response, high selectivities, dynamic ranges suitable for physiological measurements and a shelf time of over 6 months. The measuring ranges (logarithmic molar concentrations) at pH 7.4 were −3.5 to −1.5 for the fabricated Na+-selective optical-sensing microspheres, and −4.5 to −1.5 for Ca2+-selective optical-sensing microspheres. Corresponding thin optode films and regular PVC/DOS-based microspheres were prepared for comparison and showed similar response characteristics. The fabricated microsphere optical ion sensors were also deposited on an optical fiber bundle, and reproducible fluorescence signals were observed.

The use of flow cytometry as a rapid, high-throughput diagnostic tool for the analysis of ions is described. Monodisperse, uniform microspheres, which obey bulk optode theory and are governed by bulk extraction processes rather than surface phenomena, were prepared under mild, nonreactive conditions using a sonic stream particle casting apparatus. As an initial example demonstrating the utility of this approach, microspheres that contained a H+-selective fluorescent chromoionophore (ETH 5294), a cation-exchanger (NaTFPB), and either a highly sodium-selective (sodium ionophore X) or a potassium-selective ionophore (BME-44) were prepared. Separate solution analysis of sodium- and potassium-selective microspheres resulted in the generation of functional response curves using peak channel fluorescence intensities. The selectivity observed for both types of particles is sufficient for the clinical determination of Na+ and K+. Furthermore, sodium- and potassium-selective microspheres were analyzed in parallel using sodium sample solutions, resulting in the successful determination of sodium ion concentrations and providing important information about the selectivity of the potassium-selective sensors over sodium. This work demonstrates the potential applicability of flow cytometry as a means for developing multiplexed, rapid, high-throughput analyses for clinically relevant ions.

[structure: see text] A modular fluorescence intramolecular energy transfer saccharide sensor 2 has been prepared with phenanthrene as the donor and pyrene as the acceptor.

A convenient method for the preparation of monodisperse, plasticized poly(vinyl chloride) particles based on an automated particle casting technique is described. The particles are made highly selective for a number of ions by doping them with ionophores and other active components, in complete analogy to thin-film or fiber-optic chemical sensors. The approach used here produces spheres of high monodispersity at a rate of approximately 20000 particles/s. The casting process is based on a reproducible polymer drop formation and precipitation process, and the particles are formed under very mild, nonreactive conditions. This allows one to conveniently incorporate known amounts of different active components into the polymers. As an initial example, the particles are doped with three optical sensing components, the sodium ionophore tert-butylcalix[4]arene tetraethyl ester, the H+-chromoionophore ETH 5294, and the anionic additive sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate. The particles are found to be of spherical shape with a diameter of approximately 10 microm. They respond individually and selectively to sodium according to classical optode theory, as determined by fluorescence microscopy. With a RSD of 1.6%, sensing reproducibility from particle to particle is excellent. This technique may allow the development of mass-produced chemically selective microspheres on the basis of bulk extraction processes.

A diethanolamine pendant polystyrene polymer was prepared by the copolymerization of diethanolaminomethyl styrene, styrene, and divinylbenzene. The composition of the polymer resins produced agreed with copolymerization starting ratios. The polymer resins readily immobilized phenylboronic acid in THF and released pure phenylboronic acid quantitatively in THF/water/acetic acid. (C) 2000 Elsevier Science Ltd. All rights reserved.