Multiplexed Flow Cytometric Sensing of Blood Electrolytes in Physiological Samples Using Fluorescent Bulk Optode Microspheres

Abstract

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.

Advancing Membrane Electrodes and Optical Ion Sensors

“…7 illustrates such optical sensing beads and their use in analytical flow cytometry. [56] The bead based optodes were fabricated by a sonic casting apparatus, in which an inner core stream containing all sensing ingredients is focused by a sheath flow and destabilized into droplets by vibration. [57] Flow cytometry of the cured particles may monitor the fluorescence properties of thousands of particles, hence allowing the use of statistics to narrow the confidence interval of the resulting fluorescence signal.…”

“…[57] Flow cytometry of the cured particles may monitor the fluorescence properties of thousands of particles, hence allowing the use of statistics to narrow the confidence interval of the resulting fluorescence signal. [56] This general bead based sensing approach was extended to the use of imaging arrays in which the beads were dropped into etched wells that can be individually addressed with fluorescence microscopy. [58] The volume of a 10-mm microparticle sensors is about 1 picoliter and hence drastically smaller relative to their traditional thick film counterparts.…”

“…[57] Middleimage:fluorescencemicroscope imageofionsensingmicrospheresfabricated byasonicstreampreparationinstrument. [56] Bottom:Amixtureofsensingparticlesforthe ionssodium,potassiumandcalciumcanbe reliablydetectedbyanalyticalflowcytometry, evenwhencontainingthesameindicator dye,asevidencedbythevisiblepeaksinthe histogram. [56] The variations of peak position as a function of the logarithmic ion activity correspond to theoretical expectations.…”

Microfluidic ion-sensing devices

“…Such microspheres have been utilized for silver and iodide analysis after immobilizing them on the tips of etched optical fibers [85,86]. More importantly, the particles also have been employed for the analysis of sodium, potassium, and calcium in a flow cytometry set-up [83,87,88]. These cytometric studies demonstrate that microsphere optodes are amenable to fluidic manipulation and analysis in a dynamic system much like that encountered in microfluidics.…”

Rhodamine-based ratiometric fluorescent ion-selective bulk optodes

“…The polymeric ion-selective bulk optodes can reliably measure different ions under physiological or environmental conditions without some of the drawbacks often encountered in homogeneous detection, which include dye sequestration in organelles, effects of toxicity of the dye, ion buffering, alteration of fluorescence by sample viscosity, and so forth [1,2]. The sensing membrane usually contains a pH indicator (chromoionophore), a neutral ionophore and a lipophilic anionic site immobilized in polymer matrix.…”