Photophysics and luminescence spectroelectrochemistry of [Tc(dmpe)3](+/2+) (dmpe = 1,2-bis(dimethylphosphino)ethane).

The ligand-to-metal charge transfer (LMCT) excited state luminescence of [Tc(dmpe)3](2+) (dmpe is 1,2-bis-(dimethylphosphino)ethane) has been measured in solution at room temperature and is compared to its Re analogue. Surprisingly, both [M(dmpe)3](2+)* (M = Re, Tc) species have extremely large excited-state potentials (ESPs) as oxidants, the highest for any simple coordination complex of a transition metal. Furthermore, this potential is available using a photon of visible light (calculated for M = Tc; E°'* = +2.48 V versus SCE; λmax = 585 nm). Open shell time-dependent density functional theory (TDDFT) calculations support the assignment of the lowest energy transition in both the technetium and rhenium complexes to be a doublet-doublet process that involves predominantly LMCT (dmpe-to-metal) character and is in agreement with past assignments for the Re system. As expected for highly oxidizing excited state potentials, quenching is observed for the excited states of both the rhenium and technetium complexes. Stern-Volmer analysis resulted in quenching parameters for both the rhenium and technetium complexes under identical conditions and are compared using Rehm-Weller analysis. Of particular interest is the fact that both benzene and toluene are oxidized by both the Re and Tc systems.

Spectroelectrochemical sensing of pyrene metabolites 1-hydroxypyrene and 1-hydroxypyrene-glucuronide.

Spectroelectrochemical sensing in an optically transparent thin layer electrode (OTTLE) cell was used for detecting the polycyclic aromatic hydrocarbon (PAH) biomarkers 1-hydroxypyrene (1-pyOH) and 1-hydroxypyrene-glucuronide (1-pyOglu) in phosphate buffer and artificial urine. This approach uses selective electrochemical modulation of a fluorescence signal by sequentially oxidizing the analytes in an OTTLE cell to distinguish between their overlapping fluorescence spectra. This technique allows for complete oxidation and signal modulation in approximately 15 min for each analyte; a mixture of 1-pyOH and its glucuronic acid conjugate can be analyzed in 30 min. Calibration curves consisting of the fluorescence change vs analyte concentration for 1-pyOH and 1-pyOglu yielded linear ranges from 10 nM to 1 µM and from 1 nM to 1 µM, respectively. With the use of these results, the calculated limits of detection were determined to be 1 × 10(-8) M for 1-pyOH and 9 × 10(-11) M for 1-pyOglu.

Luminescence-based spectroelectrochemical sensor for [Tc(dmpe)3]2+/+ (dmpe = 1,2-bis(dimethylphosphino)ethane) within a charge-selective polymer film.

A spectroelectrochemical sensor consisting of an indium tin oxide (ITO) optically transparent electrode (OTE) coated with a thin film of partially sulfonated polystyrene-blockpoly(ethylene-ran-butylene)-block-polystyrene (SSEBS) was developed for [Tc(dmpe)(3)](+) (dmpe = 1,2-bis(dimethylphosphino)ethane). [Tc(dmpe)(3)](+) was preconcentrated by ion-exchange into the SSEBS film after a 20 min exposure to aqueous [Tc(dmpe)(3)](+) solution, resulting in a 14-fold increase in cathodic peak current compared to a bare OTE. Colorless [Tc(dmpe)(3)](+) was reversibly oxidized to colored [Tc(dmpe)(3)](2+) by cyclic voltammetry. Detection of [Tc(dmpe)(3)](2+) was accomplished through emission spectroscopy by electrochemically oxidizing the complex from nonemissive [Tc(dmpe)(3)](+) to emissive [Tc(dmpe)(3)](2+). The working principle of the sensor consisted of electrochemically cycling between nonemissive [Tc(dmpe)(3)](+) and emissive [Tc(dmpe)(3)](2+) and monitoring the modulated emission (λ(exc) = 532 nm; λ(em) = 660 nm). The sensor gave a linear response over the concentration range of 0.16-340.0 µM of [Tc(dmpe)(3)](2+/+) in aqueous phase with a detection limit of 24 nM.

Electronic and molecular structures of trans-dioxotechnetium(V) polypyridyl complexes in the solid state.

The structures of novel Tc(V) complexes trans-[TcO(2)(py)(4)]Cl·2H(2)O (1a), trans-[TcO(2)(pic)(4)]Cl·2H(2)O (2a), and trans-[TcO(2)(pic)(4)]BPh(4) (2b) were determined by X-ray crystallography, and their spectroscopic characteristics were investigated by emission spectroscopy and atomic scale calculations. The cations adopt a tetragonally distorted octahedral geometry, with a trans orientation of the apical oxo groups. trans-[TcO(2)(pic)(4)]BPh(4) has an inversion center located on technetium; however, for trans-[TcO(2)(py)(4)]Cl·2H(2)O and trans-[TcO(2)(pic)(4)]Cl·2H(2)O, a strong H bond formed by only one of the oxo substituents introduces an asymmetry in the structure, resulting in inequivalent trans Tc-N and Tc═O distances. Upon 415 nm excitation at room temperature, the complexes exhibited broad, structureless luminescences with emission maxima at approximately 710 nm (1a) and 750 nm (2a, 2b). Like the Re(V) analogs, the Tc(V) complexes luminesce from a (3)E(g) excited state. Upon cooling the samples from 278 to 8 K, distinct vibronic features appear in the spectra of the complexes along with increases in emission intensities. The low temperature emission spectra display the characteristic progressions of the symmetric O═Tc═O and the Tc-L stretching modes. Lowest-energy, triplet excited-state distortions calculated using a time-dependent theoretical approach are in good agreement with the experimental spectra. The discovery of luminescence from the trans-dioxotechnetium(V) complexes provides the first opportunity to directly compare fundamental luminescence properties of second- and third-row d(2) metal-oxo congeners.

Simultaneous detection of two analytes using a spectroelectrochemical sensor.

Spectroelectrochemical sensors developed in our group achieve three modes of selectivity by combining electrochemistry, spectroscopy, and a chemically selective membrane in a single device. Analyte detection is based upon a change in the optical response due to the conversion of the analyte between two oxidation states that results from the cycling or stepping of the applied potential. We have demonstrated a novel approach to simultaneously detect two metals by combining optical stripping voltammetry for one metal (Pb(2+)) and the in situ ligand complexation in a film for the other metal (Fe(2+)). Using an indium tin oxide (ITO) sensor platform with a 50 nm Nafion film to preconcentrate the analytes, equimolar mixtures of Pb(2+) and Fe(2+) in 0.1 M sodium acetate buffer (pH 5) were detected. Pb(2+) was detected by optical stripping voltammetry, in which lead was deposited as metal on the ITO and detected by the optical change as it was removed by stripping. The ferrous ion was detected by the in situ ligand complexation method in which Fe(2+) was complexed with 2,2'-bipyridyl in the Nafion in the film to form an intense red complex that was detected by absorbance at 520 nm. Detection limits of 300 and 400 nM were obtained for Pb(2+) and Fe(2+), respectively. The presence of the film had no effect on the optical signal that results from the deposition and stripping of the Pb(2+). In addition, competition between the Pb(2+) and Fe(2+) for sites in the film and for the organic ligand was investigated.

Fluorescence spectroelectrochemical sensor for 1-hydroxypyrene.

A spectroelectrochemical sensor was demonstrated for an organic compound whose oxidation proceeds through an electron transfer-chemical reaction-electron transfer (ECE) mechanism to generate new chemical species that are used for detection by fluorescence. The polycyclic aromatic hydrocarbon 1-hydroxypyrene (1-PyOH) served as a representative model analyte. The spectroelectrochemical properties of 1-PyOH in solution were explored with an optically transparent thin layer electrode. Electrochemical oxidation of 1-PyOH under acidic conditions proceeds via the ECE mechanism to a diquinonepyrene, which shows reversible electrochemistry and fluoresces at 425 nm in its reduced form, dihydroxypyrene. The sensor consisted of a tin-doped indium optically transparent electrode coated with a Nafion thin-film (20 nm) that rapidly preconcentrated the analyte at the sensor surface. Fluorescence in the film was excited by the evanescent wave from attenuated total reflection spectroscopy. Electrochemical modulation of dihydroxypyrene fluorescence at 425 nm in the 500 to -200 mV (vs Ag/AgCl) potential range was used for indirect detection of 1-PyOH. The spectroelectrochemical sensor calibration curve had a range of 5 × 10(-9) to 1 × 10(-6) M with a calculated detection limit of 1 × 10(-9) M.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

Inertial microfluidics for sheath-less high-throughput flow cytometry.

Flow cytometer is a powerful single cell analysis tool that allows multi-parametric study of suspended cells. Most commercial flow cytometers available today are bulky, expensive instruments requiring high maintenance costs and specially trained personnel for operation. Hence, there is a need to develop a low cost, portable alternative that will aid in making this powerful research tool more accessible. In this paper we describe a sheath-less, on-chip flow cytometry system based on the principle of Dean coupled inertial microfluidics. The design takes advantage of the Dean drag and inertial lift forces acting on particles flowing through a spiral microchannel to focus them in 3-D at a single position across the microchannel cross-section. Unlike the previously reported micro-flow cytometers, the developed system relies entirely on the microchannel geometry for particle focusing, eliminating the need for complex microchannel designs and additional microfluidic plumbing associated with sheath-based techniques. In this work, a 10-loop spiral microchannel 100 microm wide and 50 microm high was used to focus 6 microm particles in 3-D. The focused particle stream was detected with a laser induced fluorescence (LIF) setup. The microfluidic system was shown to have a high throughput of 2,100 particles/sec. Finally, the viability of the developed technique for cell counting was demonstrated using SH-SY5Y neuroblastoma cells. The passive focusing principle and the planar nature of the described design will permit easy integration with existing lab-on-a-chip (LOC) systems.

Effect of some physico-chemical conditions on an immunoassay for viable Escherichia coli.

Viable Escherichia coli can be detected by an immunoassay in which live bacteria captured on antibody-coated paramagnetic beads are induced to synthesize the enzyme beta-galactosidase, which catalyzes the hydrolysis of the slightly fluorescent substrate 4-methyl umbelliferyl-beta-D-galactoside to the highly fluorescent product 7-hydroxy-4-methylcoumarin for detection. The effects of bacterial strain, presence of dead bacteria, and some environmental stresses on assay performance were evaluated.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 21. Selective chemical sensing using sulfonated polystyrene-block-poly(ethylene-ran-butylene)block-polystyrene thin films.

Spectroelectrochemical sensors combine three modes of selectivity in a single device (electrochemistry, spectroscopy, and selective partitioning). A thin polymer film is coated onto the sensing platform in order to facilitate chemically selective transport to the electrode. The film is an essential part of the sensor because it provides an increase in selectivity and sensitivity by selectively preconcentrating the analyte. Here, we report the next step in the characterization of partially sulfonated polystyrene-block-poly(ethylene-ran-butylene)block-polystyrene (SSEBS) films for the purpose of chemical sensing by examining the selectivity of the sensor fabricated with this novel thin film material. Binary mixtures using model analytes were used to demonstrate the sensor's ability to detect an analyte in the presence of a direct interference. The binary mixtures consisted of Ru(bpy)(3)(2+)/Fe(CN)(6)(3-), Ru(bpy)(3)(2+)/Fe(bpy)(3)(2+), and Ru(bpy)(3)(2+)/Cu(bpy)(2)(2+). Demonstration of the selective partitioning mode using the Ru(bpy)(3)(2+)/Fe(CN)(6)(3-) mixture and absorption detection showed the SSEBS film's preference for Ru(bpy)(3)(2+) over Fe(CN)(6)(3-), and therefore, Fe(CN)(6)(3-) did not interfere with the sensor's response to Ru(bpy)(3)(2+). Furthermore, the importance of the use of three modes together was demonstrated by analysis of the Ru(bpy)(3)(2+)/Fe(bpy)(3)(2+) and the Ru(bpy)(3)(2+)/Cu(bpy)(2)(2+) test mixtures, where both selection of a specific wavelength for absorption and selection of a specific potential window were required to reduce or eliminate the signal from the interference. Finally, analysis of the Ru(bpy)(3)(2+)/Fe(bpy)(3)(2+) test mixture was also demonstrated using fluorescence detection.

Characterization of partially sulfonated polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene thin films for spectroelectrochemical sensing.

The spectroelectrochemical sensor uses thin, solid polyelectrolyte films as an essential element in its operation. In this work we explored the potential of partially sulfonated polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SSEBS) thin polymer films for chemical sensing. Spectroscopic ellipsometry was used to measure optical and surface properties of the air-dried and hydrated material. SSEBS incorporates a relatively small amount of water (overall change of 25%) mainly determined by the complex dynamics of the film. The decrease in the refractive index after complete hydration of the film can be predicted based on the magnitude of swelling using effective medium approximation models. Adhesion of the material on various surfaces (glass, indium tin oxide, gold) was evaluated with the tape peel-off method. The ability of the SSEBS material to preconcentrate cations was evaluated by cyclic voltammetry, absorbance, and luminescence measurements using model analytes (Ru(bpy)(3)(2+), phenosafranine, and rhodamine 6G). The detection limits of the sensor for Ru(bpy)(3)(2+) under unoptimized conditions can be significantly improved if luminescence is used as the detection modality (DL = 5 x 10(-10) M) instead of absorbance (DL = 5 x 10(-7) M). Overall, the results demonstrate the effectiveness of the SSEBS material for spectroelectrochemical sensing.

Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection.

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-beta-D-thiogalactopyranoside, which induces formation of beta-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the beta-galactosidase. Beta-galactosidase converted 4-methylumbelliferyl-beta-D-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 x 10(4) and 1.6 x 10(7) cfu mL(-1), with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.

Simultaneous multiselective spectroelectrochemical sensing of the interaction between protein and its ligand using the redox dye Nile blue as a label.

A new binding assay for a protein and its ligand based on a spectroelectrochemical method was demonstrated using avidin-biotin and 17beta-estradiol-antiestradiol antibody. The sensor consists of a selective film coated on an optically transparent electrode (OTE) consisting of indium tin oxide (ITO). Attenuated total reflection (ATR) was used for optical detection. The binding event of the ligand to the protein was detected using the ligand labeled with the electroactive dye Nile blue (NB). The spectroelectrochemical behaviors of NB and the labeled ligand were investigated using various ion-exchange films, such as perfluorosulfonated ionomer (Nafion), Nafion-silica, poly(acrylic acid) (PAA)-silica, poly(styrenesulfonic acid) (PSSA)-silica, and heparin-silica films, which were spin-coated on the ITO electrode. The optical signal was monitored to follow the accumulation of labeled ligand in the film and its electrochemical modulation. The signal from the labeled ligand possesses three modes of selectivity based on charge-selective partitioning, the chosen electrolysis potential, and the particular wavelength for measuring absorbance. The interaction between the labeled ligand and its protein was observed by the decrease in the changes of optical response of the labeled ligand, indicating the specific binding of labeled ligand to the protein.

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis.

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.

The autofluorescence of plastic materials and chips measured under laser irradiation.

Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.

Dynamic in situ spectroscopic ellipsometry of the reaction of aqueous iron(II) with 2,2'-bipyridine in a thin Nafion film.

Dynamic in situ spectroscopic ellipsometry studies of the chemical reaction between ferrous ion and 2,2'-bipyridine (bpy) in a thin Nafion film are presented. A simple prototype system composed of a thin Nafion film on a glass substrate was used throughout the work. The reaction was detected by optically monitoring the formation of the strongly absorbing complex ion, Fe(bpy)3(2+) (epsilon 520 = 7.70 x 10(3) M(-1) cm(-1) in 0.1 M NaCl). The changes in film optical constants, n and k, and the thickness upon exposure of it to various solutions were monitored in a flow cell with the film on the backside of the substrate relative to the interrogation by light. A "step-by-step" approach was used to isolate the component parts of the system in which the film was consecutively exposed to solutions in the following order: supporting electrolyte, bpy, and, last, ferrous iron solution. The optical properties of the materials were quantitatively described before and during mass transport within the film by modeling using the appropriate multilayer optical models, i.e., the Cauchy equation for nonabsorbing media and the Urbach and Tauc-Lorentz (oscillator) functions for a film that absorbed. The experiments done allowed study of the diffusion in the film and the chemical reactions that are important in the sensing scheme for ferrous iron. Ligand (bpy) diffusion followed a two-stage diffusion mechanism described by a Berens-Hopfenberg model for incremental sorption (D25 = 7.04 x 10(-13) cm2 s(-1)). The stabilities of the appropriate systems, i.e., Nafion film with bpy, iron, and iron complex, were studied by exposing equilibrated films to circulating supporting electrolyte solutions. The measurements gave important insights into a set of film chemical reactions and, in turn, selective film dynamics. This work exemplifies the usefulness of spectroscopic ellipsometry in monitoring the kinetics of a chemical reaction in situ, as well as the changes in the film physical properties under dynamic conditions.

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection.

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.

Estimation of logP(ow) values for neutral and basic compounds by microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD).

Microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD) was used to obtain logP octanol/water (logP(ow)) values for neutral and basic compounds. Six compounds, with logP(ow) values between 0.38 and 5.03, were used to create a calibration curve relating the log of retention factors (logk) obtained from muMEEKC-IFD with the known logP(ow) values. The logP(ow) values for six additional compounds were determined using the logk values obtained by muMEEKC-IFD and the linear relationship between logP(ow) and logk established for the standard compounds. The muMEEKC-IFD buffer was composed of 50 mM 3-[cyclohexylamino]-1-propane-sulfonic acid (CAPS) buffer (pH 10.4) containing 1.2% n-heptane (v/v), 2% sodium dodecylsulfate (w/v), 8% 1-butanol (v/v) and 4 microM 5-carboxytetramethyl-rhodamine (TAMRA) as the fluorophore probe for indirect detection. The muMEEKC-IFD provided an accurate method for estimating logP(ow) values and also a means for analyzing compounds that are non-fluorescent.

Further investigations on a poly(vinyl alcohol)- polyelectrolyte chemically selective optical film.

The ionomer poly(vinylbenzyltrimethylammonium chloride) has been blended with cross-linked poly(vinyl alcohol) to form optically clear composites that can be covalently linked as thin films to oxide surfaces. Films are characterized using spectroscopic ellipsometry with refractive index (n) and extinction coefficient (k) data presented for wavelengths 300 to 1100 nm. A refractive index of 1.54 and average thickness of 709 nm are typical of an air dry film prepared by spin-coating. Dynamic in situ ellipsometry results for films exposed to 0.1 M KNO(3) and 1.0 mM Fe(CN)(6)(-3) are presented. Upon initial exposure to 0.1 M KNO(3), an air dry film expands by about 160% and stabilizes in size and refractive index at about 18 hours. Ion exchange of film cationic groups with ferricyanide is marked by slight film contraction, presumably due to electrostatic cross-linking by the multivalent anion. These films are useful in the spectroelectrochemical sensor with our newly developed fluorescence detection mode, as demonstrated by results of the reversible incorporation of the fluorescent anion fluorescein.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 20. Detection of metal ions in different oxidation states.

Spectroelectrochemical sensing a metal in two different oxidation states, both of which are weakly absorbing in the visible wavelength range, was demonstrated with ferrous and ferric ion. The sensor consisted of an indium tin oxide optically transparent electrode (ITO OTE) coated with a thin film of Nafion preloaded with the ligand 2,2'-bipyridine (bipy). Fe2+ in the sample partitioned into the film where it reacted with bipy to form Fe(bipy)3(2+), which absorbs strongly at 520 nm. The change in absorbance (DeltaA) at 520 nm associated with the accumulation of Fe(bipy)3(2+) complex in the film was measured by attenuated total reflectance spectroscopy and was proportional to the concentration of Fe2+ in the sample. Iron in the Fe3+ form can also be determined, but it has a more complex coordination chemistry involving formation of [Fe2(bipy)4O(H2O)2]4+ as well as Fe(bipy)3(3+) in the film. Fe3+ was detected indirectly by reducing the nonabsorbing Fe3+-bipy complexes that accumulated in the film to absorbing Fe(bipy)3(2+) and monitoring DeltaA at 520 nm. The effects of film thickness and ligand concentration in the film on sensor sensitivity and response time for Fe2+ were evaluated. Detection limits of 0.6 x 10(-6) M for Fe2+ and 2 x 10(-6) M for Fe3+ were obtained with 300 nm thick films after 30 min of exposure to a quiescent sample. Careful manipulation of the potential applied with simultaneous optical detection enables Fe2+ to be distinguished from Fe3+, which is the first step in developing a sensor for speciating the two oxidation states in a mixture.