Octadecylamine was derivatized with 8-anilino-1-naphthalene sulfonic acid in order to simplify and understand the Langmuir-Blogdett (LB) Films films of fluorescent probe labeling proteins. Its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were carried out to elucidate the microenvironment of molecules in the LB films.

Keywords: LB film, Fluorescent probe labeling, Fluorescent spectra and lifetime

Introduction

Fluorescent probe labeling ^{1, 2}, microchipand LB films of DNA and proteins³ are important in molecular electronic and biotechnological applications such as information processing and molecular recognition. As an imitated and simplified model of above complex cases, here we prepare the Langmuir-Blogdett (LB) films of 8-anilino-1-naphthalene sulfonic acid (ANS) labeled octadecylamine (AM). A molecule based naphthalene sulfonate is well known fluorescent probes which react with primary and secondary amino groups to form highly fluorescent derivatives ^{4, 5}. It ionically binds to amines. The LB films of ANS-derivatized octadecylamine were deposited on solid substrates. Their steady-state fluorescent spectra and fluorescent lifetime were studied.

Experimental Section

ANS was purchased from Aldrich Chem. Co. It was used as received. Other reagents were of analytical pure grade. Equal molar amounts of ANS and octadecylamine were dissolved in a small amount of ethanol, then the solution was diluted with chloroform to a concentration of 3 ґ 10^-4 M for ANS-derivatization. Spreading solution are ANS labeled amine (ANS-AM) and ANS labelled amine in stearic acid (ANS-AM/SA) (1:5). The trough used was a domestic product. An aliquot of the sample solution taken up by a micro syringe was spread on the aqua subphase in the trough. ANS-AM monolayer was transferred at 25 mN/m, while ANS-AM/SA was transferred at 40 mN/m. Fluorescence emission spectra was taken on a Shimadzu model RF-5000 spectrofluorimeter. The excitation was at ~360 nm and the emission was monitored at 400~600 nm. The lifetimes were measured with an Applied Photophysics model SP-70 nanosecond spectrofluorimeter by the method of time-correlated single photon counting. The excitation wavelength was 337 nm and the monitoring wavelength was ~ 480 nm.

Results and Discussion

The surface pressure-molecular area curves (p –A) for ANS labelled octadecylamine in presence and absence of stearic acid (SA) show that solid condensed film is formed. The collapse pressure for ANS-AM and ANS-AM/SA (1:5) is 35 and 58 mN/m respectively. As the consequence of labelling, the amphiphilicity of the resultant octadecylamine derivatives is lowered in some extent, so the collapse pressure for pure ANS-AM is lower than that for corresponding mixed films. However, the values of 35 mN/m are not low comparing to 18 mN/m of DLA⁶. The images of LB monolayers of the labeled molecules alone are still even under electron microscopy. The p-A isotherms of ANS-AM and ANS-Am/SA have a classical shape without inflections or plateau as is show in Fig.1. The surface area per molecule is ~0.25 nm² for ANS-AM/SA (1:5) and is 0.55 nm² for ANS-AM. In the former case the stearic acid contributes the main part in the molecular area of ANS-AM/SA system, and in the latter case the area represents ANS-AM itself. Estimated by using bond lengths⁷, the molecular area of the based naphthalene sulfonyl group in the flat configuration is about 0.75 nm² and the cross-sectional area of group standing vertically on the surface is assumed to be on the order of 0.5 nm². So it means that in our deposited ANS-AM monolayer the head group of labelled molecules is in a tilted orientation.

No significant difference was found between solution spectra in the absence or presence of stearic acid for ANS labeled octadecylamine (Fig.2). It means that the diluent stearic acid has no substantial perturbation to the spectral properties of fluorescent derivatives of AM. It is reported that a fluorescent probe ANS is virtually no fluorescent in aqueous solution. However, when the dye is present in a nonpolar medium or when they interact with proteins, their quantum yield increases and the fluorescence emission maximum shifts to shorter wavelengths. The emission bands of films fabricated have a small blue shift with respect to correspondent solution bands. This likely implies that the fluorescent probe experience more hydrophobic microenvironment in ordered LB films than in the random state in solution.

The lifetime of ANS has been reported to be 16 ns⁸, but no detail information was cited. Our lifetime results for solutions and films are collected in Table 1. The data for ANS-AM show that both solution and film values are approximately 8 ns. The mono-exponential analysis is consistent with the bi-exponential analysis. The results of monolayers films coincide with that of multilayers film on quartz. The lifetimes of multilayers have a little shorter value than that of 3 layers. This led us envisage that interlayer aggregates have formed. H. Toshiharu et al.⁹ in measuring the fluorescence lifetime of ANS in the human erythrocyte ghost membrane suspension obtained two types of lifetimes: t1=15 ns at low concentration of ANS which bonded in protein and  t2=8.4 ns at high ANS concentration. The ANS-AM is consistent with high ANS concentration case.

_{Table 1 Fluorescence lifetimes of labeled octadecylamine}

ANS-AM film                                     3 layers                             8.01                   1.22

ANS-AM film                                     21 layers                           8.16                   1.27

ANS-AM/SA solution                         CHCl₃                                       7.60                     1.30

ANS-AM/SA film                                 3 layers                             7.82                   1.28

ANS-AM/SA film                                 13 layers                          7.95                   1.30

The results of the present work show that the introduction of fluorescent probe ANS into primary amines can make them accessible to spectral study. The ease of derivitization, successful deposition of monolayers and sensitive fluorimetric characterization promise a great potential utility of this labeling approach in structural and photophysical studies of LB films of molecules without suitable chromophores. It also seems that ANS is a convenient fluorescent probe to label amphiphilic amines, because the derivatization procedure needs no special preparation and the p –A isotherm and transfer ratio of the LB monolayer are normal.

Acknowledgments

• This work was supported by the National Natural Science Foundation of China and The Minister Science Foundation of the Chinese Academy of Sciences.

Cunningham Robert E. Fluorescent Labeling of DNA, Humana Press Inc. (Washington DC, USA), 1999, 115.

Farinas Javier; Verkman A. S., J. Biol. Chem. 1999, 274(12), 7603-7606.

Gabrielli G.; Rustichelli, F.(Ed.); The Seventh International Conference on Organized Molecular Films, Thin Solid Films 284-285 (1996)1-23.

Williams, A. T. Rhys and Winfield, S. A.; Analyst 107, (1982) 1092

Baumann, C. G.; Bloomfield, V. A.; Lovrien, R. E., Biopolymers, 49(6) , (1999)451-458

Kano K; Ishimura T; Hashimoto S.; J. Phys. Chem., 95, (1991) 7839.

Grieser F.; Thistlethwaite, P.; Urquhart, R.; Patterson, L. K.; J. Phys. Chem., 91, 5286 (1987)

Diamandis, E.P.; ; Clin. Biochem., 21, 139 (1988).

Horie, T.; Sugiyama, Y.; Awazu, S.; Hanano, M.; Pharmacobio-Dyn. 5(2), 73 (1982).