Patil et al., Phys. Chem. Chem. Phys. 2000, 2, Methylene blue undergoes a concentration and ionic strength-dependent dimerization: λ max (monomer) ~ 660 nm λ max (dimer) ~ 610 nm ABSTRACT Ionotophoresis is a technique for the transdermal administration of drugs that is commonly used in various clinical and laboratory settings. In neuropharmacology, the related technique of microionotophoresis utilizes very small implanted pipets to allow for precise ejection of drugs and related compounds into specific regions of the brain. Despite its many applications, microionotophoresis is a semi-quantitative approach to drug delivery since there is presently no means of directly measuring the amount of compound ejected. Research in our group aims to develop a calibration protocol suitable for in vivo applications of microionotophoresis using popular multi- barrel carbon fiber probes. In our approach, absolute ejection rates will be determined in vitro by photometric measurements and are correlated with simultaneous voltammetric measurements. This protocol will allow accurate quantification of molecules ejected from the probes during in vivo use when photometric measurements are not possible. We have used methylene blue to provide proof of concept for the photometric measurements, confirming the ejection rate follows a linear trend with time for a given ionotophoresis current. Present work is directed towards characterizing the electrochemical traits of methylene blue at carbon fiber electrodes, and future work will combine and correlate electrochemical and photometric measurements to construct an accurate and effective quantifiable model of microionotophoretic ejection. EXPERIMENTAL SETUP Development of a calibration scheme for in vivo microiontophoresis 1 Marsalis Smith and 2 Paul A. Flowers 1 Department of Biology, University of North Carolina at Pembroke 2 Department of Chemistry and Physics, University of North Carolina at Pembroke Pembroke, North Carolina ACKNOWLEDGMENTS This work was supported in part by grant #5R25GM from the NIGMS (National Institute of General Medical Sciences) supporting the UNCP RISE Program and the Pembroke Undergraduate Research and Creativity Center. a standard compound microscope was configured to permit simultaneous videography and spectrometry an optical cuvet containing an integral Ag counter electrode was fabricated from 1-mm glass capillary tubing micropipets with tip diameters of ~2 – 4 µm were prepared using a programmable puller methylene blue (MB) was chosen as a model compound due to its strong absorbance in the visible region of the spectrum and its electroactivity (per planned use in future studies with carbon fiber iontophoresis probes) EJECTION IMAGING PHOTOMETRIC CALIBRATIONELECTROCHEMICAL ANALYSIS successful experiments yield visibly distinct ejections of MB ejection rates vary with control parameters (MB concentration, applied current) and uncontrolled phenomena (for example, clogging of pipet tip) image analysis can be used to convert pixel intensities to nominal concentrations concentration profiles derived from images at typical high- and low-ejection rates show reasonable agreement with expectations the MB dimerization equilibrium complicates photometric calibration optimal sensitivity and acceptable precision are obtained using the sum of peak absorbances for both species or the integrated area beneath the spectral envelope between 500 – 750 nm using DC potential amperometry (DCPA) we proved concept by obtaining a calibration curve using a model redox couple, ferri/ferrocyanide (see above) – procedure: sequentially spike 10 mL 0.1M KNO 3 solution with 10 µL of 0.1M K 3 [Fe(CN) 6 ] with continuous stirring cyclic voltammetry (CV) of MB was performed to confirm the favorable electrochemical characteristics of MB (at right) FUTURE WORK repeat DCPA calibration using MB replace carbon disk working electrode with multi-barrel carbon fiber iontophoresis probes (see below); and correlate photometric and electrochemical signals measured during in-vitro ejections to yield calibration factors for use during typical in-vivo applications