PittCon 2001: Paper #1338 Mark G. Hartell and W. Charles Neely STUDY OF THE CHEMICAL, PHYSICAL AND OPTICAL PROPERTIES OF A LIPOPHILIC HYDROXYCOUMARIN ANALOG IN MIXED MONOLAYERS Mark G. Hartell and W. Charles Neely Department of Chemistry Vitaly Vodyanoy Department of Anatomy, Physiology and Pharmacology Auburn University Dr. S. Pathirana Prof. S.A. de Silva FAA * USDA * NSF * DARPA * U.S. Army

I. Monolayer Studies Objectives Two different Fluorophores, A & B Both are indicators of pH Different molecular structure Different mechanism of fluorescence Surface Properties of Mixed Monolayers with Ca-Arachidate Compare and contrast properties of the two molecules Introduce a third molecule (C) which differs only in positioning of fatty alkyl chain Goals: Determine qualitative set of characteristics which differentiate each molecule Determine some quantitative criteria from which a more stable film may be constructed R = fatty alkyl chain Determine parameters to make a better sensor

The Langmuir-Blodgett Technique Molecule of interest is spread into a monolayer film Area dimensions are controlled by a movable barrier Molecular orientation well controlled in 2-dimensions Can be used to engineer highly ordered, molecularly thin layers

Langmuir-Blodgett System SYSTEM SUMMARY Self-contained system Trough is milled from solid Teflon block Computer controlled Allows for the simultaneous data collection of surface’s area, pressure and potential KSV 3000 LB System Isotherm Parameters 3-4 trials at 20  0.1 C 25 mm/min compression Subphase pH ~ 10.04 (0.3 mM CaCl2 & 0.4 mM NaHCO3) Record  and V

Surface Pressure Measurements Define net force exerted on plate downward: F = p g l w t + 2 ( t+w)cos2 - Dl g l w d 2 º 0 when plate is completely wet:  = - = -[ l g t w h/2(t + w)] h   -½ l g t w h  h when t « w

Surface Potential Determinations Yield a Measure of Dipole Moment SPECIFICATIONS Built in-house for LB system with ionizing electrode (210Po, ½ = 140 d) Simultaneous data collection inputted into native KSV software Source may be exchanged with fresh 210Po Define surface dipole moment in units of mD:  = (1/4)  (V/n) Define A as film concentration in 2/molecule:  = AV/12 210Po Ag/AgCl

Thermodynamic Definitions Isotherm Parameters  Free Energy of Compression (pure monolayer): Free Energy of Compression (mixed monolayer): A a

Arachidic Acid Baseline Data Comparison of Dipole Moment with -A Isotherm Change in dipole moment indicates rearrangement at 40 Å2/molecule before any notable rise in surface pressure Molecular orientation appears to have been assumed long before L1 and L2 state LS L2 L1 G CS state yields reduced area of approximately 20 Å2/molecule

pH Sensor Molecule: 10-C18AE Diethanolamine moiety is proton receptor Fluorescence activated by PET-based mechanism Synthesized as part of a novel class of fluorescent pH indicators: proton turns fluorescence on LUMO e- LUMO e- HOMO HOMO HOMO HOMO Symmetric Fluoroionophore Excited FL Free amine Excited FL H+-amine

Mixed Isotherms of 10-C18AE at 20oC Pure 10-C18AE yields CS state not stable at high . max = 30-35 mN/m Lower mol% mixtures indicate a phase separation in condensed states 10-C18AE may be slipping out of the plane of the film Higher surface pressure phase resembles reduced areas of pure arachidic acid Mixtures Spread in Hexane Shift towards pure arachidic acid Fluorophore only stable in mixtures when compressed below 30 mN/m

Isotherms of 10-C18AE versus C18AE Symmetrically placed alkyl chain Asymmetrically placed alkyl chain Similar two-phase isotherm yielding stable mixture below 30 mN/m

Reduced Molecular Areas of 10-C18AE Higher surface pressure states notable only in more dilute mixtures - reduced areas are identical to pure arachidic acid Lower mol% mixtures appear to be comparable to ideal mixture calculations >70 mol% indicates threat of fluorophore aggregation during analysis Average standard deviation of the mean ~ 0.3 2 Reduced Molecular Areas of Stable Lower Surface Pressure Component Reduced molecular areas indicates stability at more dilute mixtures

Reduced Molecular Areas of 10-C18AE vs. C18AE Symmetrically placed alkyl chain Asymmetrically placed alkyl chain Symmetrically placed alkyl chain yields reduced areas slightly closer to ideality

Dipole Moment Determinations at 20oC 10-C18AE has higher dipole moment than simple fatty acid Higher mol% yields larger contribution to net dipole moment of the film Concentration function is not simply linear - suggests at least one minima at 70 to 80 mol% Large data spread may be due to small variation in film due to minute impurities - molecule custom synthesized Higher concentrations resemble pure sensor molecule Lower concentrations resemble pure arachidic acid

Thermodynamic Measurements: G of Mixing for 10-C18AE Close to ideal behavior observed for mixtures less than 70 mol% Higher concentrations indicate that condensed packing assumes lower energy Concentrations relevant to sensor construction (low mol%) exhibit ideal behavior if deposited with a constant  <30 mN/m G of mixing confirms ideal behavior observed at lower mol% mixtures

G of Mixing for 10-C18AE vs. C18AE Symmetrically placed alkyl chain Asymmetrically placed alkyl chain Symmetrically placed alkyl chain yields energy curves closer to ideal mixture at concentrations <70 mol%

pH Sensor Molecule #2: a Cellular Probe Hydroxycoumarin derivative: “HC” Novel fluorescent probe: fluorescence changes as function of pH Used as cellular pH probe Used as measure of local surface potentials by measuring shift in pKa May be functional in sensor system to detect a binding event

HC-Arachidic Acid Mixed Isotherms at 20oC Pure fluorophore stable at high surface pressures max = 55-60 mN/m Can resolve condensed liquid state in higher mol% Do not see two-phase separation into pure arachidic acid at lower mol% Isotherms imply a more stable mixed monolayer versus 10-C18AE Mixtures Spread in Chloroform Shift towards pure arachidic acid Mixtures of HC appear to yield more stable isotherms at higher surface pressures compared to 10-C18AE

-A Isotherms of HC Mixtures are Reversible Two Different Reversibility Studies (1) Compression-Expansion: Isotherms are consistently reversible (2) Compression-expansion-compression-expansion: Isotherms are continuously reversible when compressed multiple times A,B,C are Study (1) and B,C are Study (2)

Determine Loss of Fluorophore During Isotherm Experiments  vs. A  A vs. t Measure loss of A at constant  Calculate loss of A vs. time

Examples of Raw Kinetic Data 25 mN/m 30 mN/m Two Studies Performed (1) 15, 20, 25 mN/m (2) 25, 30, 35 mN/m 35 mN/m Worst-Case Determination Calculate %-Loss Assuming 12 min. at Compressed State Determine Decay Function Fit 30 min. Decay to 1st Order Kinetics

Summary of Two Separate Decay Experiments Negligible Loss of Material During Experiment - Molecular Areas Not Altered Summary of Two Separate Decay Experiments const %-loss @ t=12 min. 15 mN/m 0.91 % 20 mN/m 0.69% 25 mN/m 0.74% 25 mN/m 1.5% 30 mN/m 0.38% 35 mN/m 0.38% Data assumes unrealistic worst-case scenario: 12 min. under compressed conditions Reality: Compressed state maintained for only last few minutes of experiment Conclusion: Loss is negligible for a realistic 12 min. isotherm experiment

Reduced Molecular Areas of HC Probe Avg. Standard Deviation of the mean from 3 to 4 trials is ~ 0.3 2 Molecular areas of fluorophore and arachidic acid differ by only 3 Å2/molecule Pure HC Area confirmed by literature data - rationalized by molecular modeling Lower mol% mixtures appear to be comparable to ideal mixture calculations Observed expanding effect may be artificially emphasized due to small axis scaling Reduced molecular areas indicate stability at more dilute mixtures

Fluorophore Comparison of Reduced Molecular Areas >70 mol% 10-C18AE presents unique packing arrangement yielding a more condensed area When observed on similar scale HC also indicates strong adherence to ideal areas up to 80 mol%

Dipole Moment Determinations at 20oC Higher concentrations resemble pure sensor molecule HC has higher dipole moment than simple fatty acid Higher mol% yields larger contribution to net dipole moment of the film Concentration function is quite linear Smaller data spread may be due to higher purity of this commercially available molecule vs. 10-C18AE Lower concentrations resemble pure arachidic acid

Thermodynamic Measurements: G of Mixing for HC Probe Close to ideal behavior for very dilute solutions Packing arrangements of some concentrations yield higher than ideal energies Data suggests compositions which are energetically more stable for sensor design: 0 to 20, 50 and 80 mol% G of mixing indicates optimal compositions for sensor design

II. Fluorescence Studies Objectives Two different Fluorophores, A & B Both are indicators of pH Different molecular structure Different mechanism of fluorescence Optical Properties of Solid State Films Determine fluorescence response vs pH Compare molecules in solid state with data from free solution Goals: Determine qualitative set of characteristics which differentiate each molecule and compare with solution data Determine some quantitative parameters to incorporate fluorophore B into a viable solid-state sensor system R = fatty alkyl chain Determine parameters to test a solid-state sensor

LB Film Deposition System SYSTEM SUMMARY System contained in laminar flow hood Trough is milled from solid Teflon block Computer controlled KSV 2200 LB System Deposition Parameters 20  0.1 C set = 30 mN/m 25 mm/min compression to set Subphase pH 7.41 55 mM KCl, 4 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2 & 2 mM MOPS

HC Probe Film Deposition DEPOSITION SUMMARY Quartz slides placed back-to-back: deposit material on one side only Compress 20 mol% HC to a constant 30 mN/m at 20 C ~8 layers deposited Transfer ratios indicate mixed X-Y type multi-layers Position of quartz slides during deposition 2 sets of back-to-back slides Thin layer of 20 mol% HC fluorophore in arachidic acid successfully deposited

Positioning of Sample for Fluorescence Measurements Important Quartz slide positioned in sample chamber of spectrometer Quartz slides custom made to fit diagonal of standard 1 cm cuvette Spectra collected at 90o angle through backside of slide Reflective scatter is directed away from detector Source Light Fluorophore Signal Reflective Scatter

C18AE: Solution vs. Solid-State Fluorescence Free Solution 10 mol% Solid-State Film

HC: Solution Fluorescence UN-IONIZED FORM ex = ~360 nm IONIZED FORM ex = ~385 nm Solution pH titration data consistent with literature data

HC: Solid-State Fluorescence UN-IONIZED FORM ex = ~360 nm IONIZED FORM ex = ~400 nm Shift in pKa consistent with literature data in micelles

I. Conclusions Determination of surface properties provides unique understanding of molecular orientation and monolayer stability Data provides insight towards understanding dependence of surface properties on molecular structure and packing arrangements Data provides knowledge base to make more informed choices in the construction of thin film sensors Data has been used successfully to yield optically viable sensors whose solid state optical properties mimic that of a free solution model Hartell, M.G., Pathirana, S.T., de Silva, S.A., Neely, W.C., Vodyanoy, V. “Dependence of Surface Properties of Flourophores and Calcium Arachidate on Mixed Monolayer Composition.” 13th International Symposium on Surfactants in Solution. Seminar. University of Florida, Gainesville, FL, June 11-16, 2000. Hartell, M.G., Neely, W.C., Vodyanoy, V. “Study of the Chemical, Physical and Optical Properties of a Lipophilic Hydroxycoumarin Analog in Mixed Monolayers.” 2001 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. Abstract #1338. Seminar. New Orleans, LA, March 4-9, 2001.