Anal. Chem. 199466, 2433-2440
Phase-Resolved Evanescent Wave Induced Fluorescence. An in Situ Tool for Studying Heterogeneous Interfaces Jeffrey S. Lundgren, Evan J. Bekos, Run Wang, and Frank V. Bright' Department of Chemistry, Acheson Hall, State Universiw of New York at Buffalo, Buffalo, New York 14214
A new technique that we call phase-resolved evanescent wave induced fluorescence (PREWIF) is introduced and is shown to be useful for studying heterogeneousinterfaces under ambient conditions. PREWIF is based on the traditional interfacial selectivity offered by total internal reflection (i.e., the evanescent wave) combined with the picosecond resolving power of phaseresolved fluorescence spectroscopy. Using this new technique we demonstrate the following: (1) the recovery of weak, interfacialfluorescence from a sol-gel-derived thin film doped with a low concentration of fluorophore in the presence of strong Rayleigh scatter; (2) resolution of the individual componentsin synthetic binary mixtures of fluorophores doped within thin sol-gel-derived films that are coated onto fused silica substrates; (3) elimination of bulk fluorescence and selective monitoring of the interfacial (- 100 di) species in a model immunosurface; (4) extraction of the individual spectra that contribute to the fluorescence of a fluorescently labeled protein physisorbed to a silica surface.
There are many instrumental approaches designed to study interfaces.' For example, ultra-high-vacuum techniques like X-ray photoelectron spectroscopy (XPS or ESCA), secondary ion mass spectrometry (SIMS), and ion scattering spectroscopy (ISS) exhibit excellent surface selectivity (down to 10 A) and superb detection limits and allow one to determine the atomic and molecular composition of a complex interfa~e.29~Unfortunately, one cannot easily uses these techniques with most liquids or under ambient conditions. Infrared (IR) and Raman spectroscopy4 have been more successful at probing the composition and structure of interfaces under ambient conditions. However, water can sometimes complicate these measurements, and unenhanced Raman sometimes lacks the requisite detection limits. Surface plasmon resonance techniques also work well in certain situations, and atomic force microscopy (AFM) can provide insights on an atomic scale.5 Hirschfeld first suggested fluorescence as a tool for studying the liquid/solid interfaces6 Over the years this lead to a host of in situ surface analytical techniques for probing interfacial phenomena under ambient conditions. Fluorescence spectroscopy offers excellent detection limits and high information ~~~
~
[email protected] (bitnet). (1) Tingey, K. G.; Andrade, J. D. Lcrngmuir 1991, 7, 2471. (2) McGuire, G. E.; Ray, M. A.; Simko, S. J.; Perkins, F. K.; Brandow, S. L.; Dobisz, E. A.; Nemanich, R. J.; Chourasia,A. R.; Chopra, D. R. Anal. Chem. 1993, 65, 311R. (3) Turner, N. H.; Schreifels, J. A. Anal. Chem. 1992, 64, 302R. (4) Gcrrard, D. L.; Birnie, J. Anal. Chem. 1992, 64, 502R. (5) Snyder, S. R.; White, H. S. Anal. Chem. 1992, 64, 117R. (6) Hirschfeld, T. Can. Spectrosc. 1965, IO, 128. 0003-2700/94/03662433$04.50/0
0 1994 American Chemical Society
content7v8but is not (unlike ESCA, ISS, AFM) inherently surface selective. In order to use fluorescence to probe interfaces, one must ensure that the observed luminescence arises solely from the interfacial species.g This is generally achieved by using an evanescent field scheme to excite selectively the interfacial fluorophore(s) or by ensuring that only the species at the interface can/will fluoresce. During the past decade, there has been increased attention directed toward fluorescence as a means to study interfacial phenomena.lG15 Fields ranging from polymer chemistry to bioadhesion and studies on "biocompatability" have used total internal reflection fluorescence (TIRF) and its variants as a means to better understand interfacial chemistry, adsorption kinetics, and interfacial dynamics. The bulk of this work has used static fluorescence to follow the aforementioned processes. Thus, even though this approach has been extremely successful, it is inherently limited because even a "simple" interface is not necessarily composed of a single type of fluorescent center. As examples, one might envision two forms of a single protein (e.g., strongly or weakly sorbed), native and denatured conformations of a particular antibody, or subpopulations of fluorescent centers distributed randomly within a thin polymer film. In these situations it is clearly inappropriate to use the static fluorescence spectrum as an accurate measure of the true interfacial environment. To obtain a more clear picture of the interface, additional selectivity is required. Time resolution is an ideal means to this end,7J and several research groups have already used surface selectivity (Le., TIRF) in concert with various levels of time resolution.1G55 To date, this approach has been used to study interfacial photo physic^,'^^^ accessibility of silica-immobilized lumi(7) Warner, I. M.; Patonay. G.; Thomas, M. P. Anal. Chem. 1985, 57, 463A. ( 8 ) Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Plenum Press: New York, 1991; Vols. 1-3. (9) Harrick, N . J. Internal Reflection Spectroscopy; Harrick Scientific Corp.: New York, 1967. (IO) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.;Plenum Press: New York, 1983; Vol. 1. (1 1) Axelrod, D.; Burghardt, T. P.; Thompson, N . L. Annu. Rev. Biophys. Biaeng. 1984, 13, 247.
(12) Andrade, J. D.; Hlady, V. Ann. N. Y.Acad. Sci. 1987,516, 158. (13) Hellen, E. H.; Fulbright, R. M.; Axelrod, D. In Spectroscopic Membrane Probes; CRC Press: Boca Raton, FL, 1988; Vol. 11. (14) Reichert, W. M. CRC Crit. Rev. Biocompa?. 1989, 5, 173. (IS) Andrade, J. D.; Hlady, V.; Wei, A. P. Pure Appl. Chem. 1992, 64, 1777. (16) Yanagimachi, M.; Tamai, N.; Masuhara, H. Chem. Phys. L.et?. 1993,201, 115. (17) Rumbles, G.; Brown, A. J.; Philip, D. J. ChemSoc., Faraday Trans. 1991, 87,
825.
Wong, A. L.; Harris, J. M. J. Phys. Chem. 1991, 95, 5895. Pankasem, S.;Thomas, J. K. J. Phys. Chem. 1991, 95, 7385. Liu, Y. S.; Ware, W. R. J . Phys. Chem. 1993, 97, 5980. Liu, Y. S.; De Mayo, P.; Ware, W. R. J. Phys. Chem. 1993, 97, 5987. Liu, Y. S.; De Mayo, P.; Ware, W. R. J. Phys. Chem. 1993, 97, 5995. Aussencgg, F. R.; Leitner, A.; Lippitsch, M. E.; Reinisch, H.; Riegler, M. Sur$ Sci. 1987, 189/190, 935. (24) Lieherherr, M.; Fattinger, Ch.;Lukosz, W. Sur$ Sci. 1987, 189/190, 954. (18) (19) (20) (21) (22) (23)
Analytical Chemistty, Vol. 88, No. 15, August 1, 1994 2433
nescent centers to quenchers,18 sorbate distributions,20-22 biochemical interface^,'^^ distribution of chemical recognition elements at biosensor interface^?^^^^ aspects of diffusion at model biointerfa~es,4~-~'*5~ and picosecond rotational reorientation of fluorescent solutes at model liquid/liquid and liquid/solid interface^.^^.^^^^^ Surprisingly, the most powerful time-resolved techniques (i-e., those with the highest time resolution) have not yet been used in concert with wavelength selectivity to resolve the interfacial fluorescence from a spatially and spectrally heterogenous system into the actual contributions from the individual fluorescent species.56 That is, although it is now routine to use nanosecond or picosecond fluorescence techniques (time or frequency domain) to recover the associated underlying spectra that contribute to complex emission processes in the b ~ l k ,the ~ . same ~ scheme has yet to be fully demonstrated56 for interfaces. This is unfortunate sinceone would gain a wealth of information on the composition and distribution of the interfacial species if one could resolve the static surface fluorescence into contributions from the individual fluorescent specie^.^^^ (25) Levitz, P.; El Miri, A.; Keravis; D.; Van Damme, H. J . Colloid InterjuceSci. 1984, 99, 484. (26) Murayama, A.; Oka, Y.; Fijisaki, H. Surf. Sci. 1985, 158, 222. (27) Wirth, M. J. Appl. Specfrosc. 1993, 47, 651. (28) Cnossen, G.; Drabe, K. E.; Wiersma, D. A. J. Chem. Phys. 1993. 98, 5276. (29) Arias, J.; Aravind, P. K.; Metiu, H. Chem. Phys. Left. 1982, 85, 404. (30) Ohshima,S.; Kajiwara,T.; Hiramoto, M.; Hashimoto, K.; Sakata, T. J . Phys. Chem. 1986, 90,4474. (31) Itaya, A.; Kurahashi, A.; Masuhara, H.; Tamai, N.; Yamazaki, I. Chem. Len. 1987, 1079. Fattinger, Ch.; Lukosz, W. J. Luminescence 1984, 31, 32, 933. Toriumi, M.; Masuhara, H. Specfrochim. Acto Rev. 1991, 14, 353. Sasaki, K.; Koshioka, M.; Masuhara, H. Appl. Spectrosc. 1991, 45, 1041. Yanagimachi, M.; Toriumi, M.; Masuhara, H. Mureriuls 1991, 3, 413. Toriumi, M.; Masuhara, H. In Irrudiution of Polymeric Mureriuls;Reichmanis, E.,Frank,C. W.,ODonnell,J. H.,Eds.;ACSSympiumSeris 527;American Chemical Society: Washington, 1993. Masuhara, H.J. Colloid Taniguchi,Y.; Mitsuya, M.;Tamai,N.;Yamazaki,I.; Inferfuce Sci. 1985, 104, 341. Rainbow, M. R.; Atherton, S.;Eberhart, R. C. J. Biomed. Murer. Res. 1987, 21, 539. Crystall, B.; Rumbles, 0.;Smith, T. A.; Philip, D. J . Colloid Interface Sci. 1993. 155. 247. Kruli, U. J.; Brown, R. S.;Vandenberg, E. T.; Heckl, M. M. J . Electron Microsc. Tech. 1991, 18, 212. Brennen, J. D.; Brown, R. S.;McClintock, C. P.; Krull, U. J. A n d . Chim. Acra 1990, 237, 253. K ~ l lU. , J.; Brown, R. S.;Hougham, B. D.; McGibbon, G.;Vandenberg, E. T. Tulunro 1990, 37, 561. Brennan, J. D.; Brown, R. S.;Foster, D.; Kallury, R. K.; Krull, U.J. Anal. Chim. Acro 1991, 255, 73. Brennan, J. D.; Brown, R. S.;Manna, A. D.; Kallury, K. M. R.; Piunno, P. A.; Krull, U. J. Sens. Acruurors B 1993, 11, 109. Suci, P.; Hlady, V. Colloids Surf. 1990, 51, 89. Bright, F. V. Appl. Specrrosc. 1993, 47, 1152. Bright, F. V.; Wang, R.; Li, M.; Dunbar, R. A. Immunomerhods 1993,3,104. Burghardt, T. P.; Ajtai, K. Proc. Nurl. Acud. Sci. U.S.A. 1985, 82, 8478. Burghardt, T. P.; Ajtai, K. Biochemistry 1986, 25, 3469. Burghardt, T. P.; Thompson, N. L. Biochemisfry 1985, 24, 3731. Thompson, N. L.; Poglitsch, C. A.; Timbs, M. M.; Pisarchick. M. L. Acc. Chem. Res. 1993, 26, 567. Wirth, M. J.; Burbage, J. D. J . Phys. Chem. 1992, 96, 9022. Piasecki, D. A.; Wirth, M. J. J . Phys. Chem. 1993, 97, 7700. Fukumura, H.; Hayashi, K. J . Colloid Inferfuce Sci. 1990, 135, 435. Wirth, M. J.; Burbage, J. D. Anal. Chem. 1991, 63, 1311. A report (Masuhara, H.; Mataga, N.; Tazuke, S.;Murao, T.; Yamazaki, I. Chem. Phys. Leu. 1983, 100, 415) using time-resolved spectroscopy has appeared demonstrating time and spectral rcsolution at interfaces. However, the fluorescence centers (POPOP and N-ethylcarbazole) were prepared as discrete, spatially separated films. We recently reported on using our multifrequency phase and modulation total internal reflection fluorescence (MPM-TIRF) technique (refs46 and 47) torwaver theactualdecay-associated spectra (Bright, F. V.; Wang, R.; Narang, U.; Bekos, E. J.; Li, M.; Dunbar, R. A.; Lundgren, J. S.;Jordan, J. D. 20th FACSS Meeting, Detriot, MI, October 17-22, 1993; paper 122) for antifluorcscein/fluoresceinat a fused silica substrate. However, the total aquistion time for this single experiment was nearly 23 h.
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Ana&ticalChemlstry, Vol. 86, No. 15, August 1, 1994
Phaseresolved fluorescence spectroscopy574 is a frequencydomain technique that exploits differences between excitedstate fluorescence lifetimes to resolve directly the total fluorescence spectrum into the contributions from the individual fluorophores. In this paper, we describe a new hybrid technique that wecall phase-resolved evanescent wave induced fluorescence (PREWIF) which combines the interfacial selectivity produced by TIRF with the temporal and spectral resolution of phase-resolved fluorescence. To illustrate the power and capabilities of PREWIF, we present results on thin sol-gel-derived films doped with fluorescent solutes, a model immunosurface, and a fluorescently labeled protein sorbed at a silica substrate.
THEORY Total Internal Reflection Fluorescence. If electromagnetic radiation passes from a more optically dense medium (nl) and impinges upon the interface between nl and n2 at an angle greater than the critical angle (I', = sin-*(nz/nl), the radiation is totally internally reflected.'~~-~~ At the nl/n2 interface, analysis of Maxwell's wave equations9shows that an evanescent field is produced which extends some fraction of the wavelength of light into n2. The penetration depth (d,) of the evanescent wave into n2 is given by
where I'i is the angle of incidence, Xi is the wavelength of the incident electromagnetic radiation, and the other terms are defined above. In a traditional TIRF experiment, the evanescent wave is used to excites fluorescent species within dp of the n1/n2 interface. Phase Resolution. Phase-resolved is performed in the frequency domain by exciting the sample with high-frequency (MHz to GHz) sinusoidally modulated radiation of the form Ex(t) = Zcx( 1 + mcxsin at)
(2)
where I,, is the average intensity of the modulated excitation, mcxis the excitation depth of modulation (acex/dcex),w is the angular modulation frequency (w = 27rf, f is the linear modulation frequency) and t is time. The resulting fluorescence (Fl(t)) is also sinusoidally modulated and is written F1 (t) = Zem( 1
+ m,,M sin(wt - 6))
(3)
In this expression, Zcm is the wavelength-dependent, timeaveraged fluorescence, M is the demodulation factor, and 6 is phase angle or phase shift. The excited-state lifetime ( T ) is related to 6 and M by (57) Vcselova, T. V.; Cherkasov, A. S.;Shirokov, V. I. Opr. Specrrosc. 1970, 29, 617. (58) Lakowicz, J. R.; Cherek, H. J . Biochem. Biophys. Merhods 1981, 5, 19. ( 5 9 ) McGown, L. B.; Bright, F. V. Anal. Chem. 1984, 56, 1400A. (60) Bright, F. V.; Bctts, T. A.;Litwiler, K. S.CRCCrir. Reo. Anal. Chem. 1990, 21. 389.
1
7=-tan8
(4)
w
By measuringthe time-dependent fluorescence (eq 3) with a phase-sensitivedetector (Le., a lock-in amplifier) one obtains a time-independent phase-resolved signals7“O
that depends on the relative difference between B and the detector phase angle (OD) (Le., 7 ) . The result of eq 6 is that one can control the relative phase-resolved signal by adjusting OD. Further, if OD = 8 90°, PR(OD) goes to zero. To better illustrate the ramifications of eq 6, consider the case of two different fluorophores (A and B) distributed randomly within dpof the nl/n2 interface that are excited by a modulated evanescent field. The total evanescent wave induced fluorescence will combine to yield the static fluorescence spectrum. In this case, the observed phase-resolved fluorescence can be written57d0
+
PR(8,) = C, cos(@,- 8,)
+ C, cos(8, - )e,
(7)
where Cl represents the terms Zem,~me,Mt (i is component A or B). Inspection of eq 7 shows that one can eliminate selectiuely the entire contributionfrom a particular interfacial fluorescent species (A or B) by judicious choice of 8D. For example, by adjusting 6~ to 8A f goo, one “nulls” the contribution from component A and can in principle acquire the fluorescence spectrum for component B directly (in the presence of A’s emission). Similarly, one can adjust OD to Be f 900, thereby nulling component B, and acquire the fluorescence spectrum for component A. In both cases, the resulting phase-resolved intensity scales as sin (18, - OBI); therefore, ones ability to resolve A from B depends directly on the excited-state lifetimes of A and B, the modulation frequency (eq 4), and the degree of spectral overlap. Phase resolution has been used previously on bulk mixtures of f l u o r ~ p h o r e to s ~resolve ~ ~ species with strongly overlapping spectra with differences in excited-state lifetimes of only 100 PS.
PREWIFcombines the interfacial selectivity of TIRF with the spectral and temporal resolution of phase-resolved fluorescence and provides a means to record in situ the individual fluorescence spectra that combine to make up the fluorescence from a heterogeneous interface.
EXPERIMENTAL SECTION Materials. The following chemicals were used: tetramethylorthosilicate (TMOS), (glycidoxypropy1)trimethoxysilane (GOPS), CHsCN, and eosin Y (Aldrich); HC1, NaCl, Na2HP04, and NaH2P04.2H20 (Fisher); ethanol (200proof; Quantum); rhodamine 6G, (R6G; Exciton); bovine serum albumin (BSA), proteinG (PG) and fluorescein (F) (Sigma); and 6-acryloyl-2(dimethylamino)naphthalene (acrylodan), 6-propionyl-2(dimethylamino)naphthalene(PRODAN), 2-ptoluidinyl-6-naphthalenesulfonicacid (TNS), and antifluo-
rescein (AF; Molecular Probes). All reagents were used as received and aqueous solutions were prepared in double distilleddeionized water. Sol-Gel Film Preparation. In a small glass vial are mixed 2 mL of TMOS, 1 mL of 0.1 N HCl, and 4 mL of ethanol. The ethanol is added slowly at first, and the entire mixture is stirred for 5 h. A sol-gel solution containing the dopant(s) is prepared by mixing 2 mL of the stock sol-gel solution (vide supra) with 2 mL of an aqueous solution containing the dopant(s). This mixture is stirred for 1 h. Sol-gel-derived thin films are prepared using two different schemes. In the spin casting method, fused silica plates (25 X SO X 2 mm; ESCO) are initially washed with methanol and copious amounts of water and dried in an oven at 125 OC for 3 4 h. Sol-gel-derived films (blanks or those containing dopants) are then prepared in a simple three-step process. First, a cleaned plate is wetted with 2-propanol for 30 s on a spin coating apparatus (2000 rpm). The spinning is then stopped and 100 pL of sol-gel solution is pipetted directly onto the center of .the plate and the spinning continued for an additional 30 s. Finally, the plates are placed in Petri dish and allowed to air-dry for 1 week to 10 days. A dip coating method is also used whereby a 4-6-cm-long segment of 600-pm core diameter silica optical fiber (General Fiber Optic) serves simultaneouslyas the optical coupler and the substrate. In this scheme, a l-cm section of the cladding is removed from the distal end of the fiber optic; the ”bare” silica portion is immersed in warm concentrated nitric acid for 20 min and rinsed with water. This protocol removes all the polymer cladding from the distal end of the optical fiber and leaves a clean silica surface. A sol-gel-derived thin film is produced on the clean fused silica surface by dipping the fiber into the sol-gel solution (vide supra) and slowly (0.1 cm/s) extricating the fiber from the solution. The coated fiber is then placed in Petri dish and allowed to air-dry for at least 1 week. Preparationof Acrylodan-Labeled BSA.61 A stocksolution containing 50 pM BSA is prepared in 0.10 M phosphate buffer (pH 7.0).A 40-mL aliquot of this solution is taken and enough acrylodan (in the minimum amount of CH3CN) is added such that the molar ratio of BSA to acrylodan is 1: 1. This mixture is stirred gently and maintained at room temperature for 10 h. The reaction mixture is then loaded into a 12 000 MW cutoff cellulose dialysis bag and dialyzed at 4 OC against 250 mL of 1 :20(v/v) acetonitrile and phosphate buffer (0.1 M, pH 7.0). The 250-mL solution is replaced every 12 h for 4 days. Dialysis is complete when there is no detectable acrylodan fluorescence in the dialysate solution. The BSA/ acrylodan solution is stored at 4 OC. The molar ratio of acrylodan to BSA is 0.80 & 0.05.61 Preparation of a Model Immunosurface. Antibody immobilization involves two main steps: (1) preparation of silicaimmobilized protein G and (2) reaction of the protein G with antifluorescein antibodies. The specific protocol used in the current work is adapted from Sportsman and Wilson62and Alarie et al.63 (61) Garrison, M. D.; Iuliano, D. J.;Saavedra,S. S.;Truskcy,G. A.; Reichert, W. M.J. Colloid Interface Sci. 1992, 148, 415. (62) Sportsman, J. R.; Wilson, G. S. Anal. Chem. 1980,52,2013. (63) Alarie, J. P.; Scpaniak, M. J.; Vo-Dinh, T. Anal. Chlm. Acta 1990,229,169.
Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
2495
Briefly, the fused silica substrates are first cleaned with H F for 10 min, soaked in chromic acid for 1 h, and immersed in warm HNO3 for 24 h. The substrates are rinsed with copious amounts of water and stored at room temperature. Three to five of the cleaned substrates are then immersed in 50 mL of 10% (v/v) aqueous GOPS. This solution is then sonicated for 20 min, the pH is adjusted to 3 (with 1 M HCl), and the system is heated to 90 OC for 4 h. These substrates are slowly cooled to room temperature, and each substrate is removed and rinsed with 250 mL of water. The GOPS-treated substrates are immediately activated by placing them individually into 50 mL of periodic acid (4mg/mL in 75% (v/v) acetic acid). The solutions are gently shaken for 1 h and then rinsed with five washings (10 mL each) of pH 7.5 phosphate buffer (0.01 M, 1% NaCl; PBS). The activated substrates are then placed in a special Teflon base, which allows liquid to contact only one face of the substrate, and covered with 1.5 mL of PBS containing 0.5 mg/mL protein G. This mixture is allowed to incubate at 4 O C . After 48 h the substrates are washed (10 X 100 mL) with PBS. The Schiff base is subsequently reduced by immersing the individual substrates in 10 mL of 30 mg/mL sodium cyanoborohydride for 1 h at 4 "C. Following reduction, the protein-G-coated substrates are stored in PBS at 4 OC. In order to immobilize the antifluorescein (AF), the individual protein-G-coated substrates are immersed in 5 mL of PBS containing 0.01 mg/mL antiflu~rescein.~~ After 48 h, the substrates are rinsed with 100 mL of cold PBS and stored in PBS at 4 "C. Instrumentation. All absorbance spectra are acquired using a UV/visible/near-IR scanning spectrophotometer (UV2101/3101 PC Shimadzu). Film thicknesses are measured using a profilometer (Alpha-step 100, Tencor Instruments). Measurement precision is on the order of 10.0 nm. Typical film thicknesses are 0.5-0.6 bm. A spin coating apparatus (Headway Research, Inc.) is used to prepare all the platebased sol-gel-derived thin films. The general capabilities and basic hardware associated with our frequency-domain fluorometer and its modification for TIRF experiments have been described p r e v i ~ u s l y . ~For ~ . ~the ~ current PREWIF experiments, the instrument is simply converted from our more typical multiharmonic Fourier s ~ h e m e to ~ ~a , discrete ~~ frequency mode.60 In the experiments involving the optical fiber configuration, the modulated excitation radiation is focused into the proximal end of the fiber and excites the sorbates at the distal end of the fiber. This type of scheme has been used by others.65 The distal end of the optical fiber is positioned within the collection zone of the emission/ detection optics. The current configuration allows us to perform PREWIF experiments in the UV and visible with modulation frequencies between 1 and 275 MHz.
RESULTS AND DISCUSSION Minimization of Rayleigh Scatter. One of the more troublesome aspects of TIRF is associated with light scattered into the detection volume. Judicious control over alignment (64) Wang, R.; Narang, U.;Prasad, P. N.; Bright, F. V. Anal. Chem.1993,65, 267 I . (65) Newby, K.;Andradc, J. D.; Bcnncr, R. E.;Reichert, W. M.J.Colloidlnrerjace Sci. 1986, 11I , 280.
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Analytical Chemistry, Vol. 66,No. 15, August 7, 1994
3 , Static
- Null
-1
-2
11
Rayleigh S c a t t e r
0
v)
0.05
. \
1 /'
Null TNS
\ \
\
V'
0 c
'
v,
-0.10 I 400
I
420
-
I
I
I
I
I
1
1
440
460
480
500
520
540
560
Emission Wavelength (nm)
Flgure4. Raw statk (- -) and "allzed PREWIF spectra (- or for a sd-gel derived thin film doped wfth TNS and PRODAN. (-) Nulling the TNS contribution minus PRODAN emission. Nulling the PROOAN contribution minus TNS emission. Emission wavelength resolution, 8 nm; >bx = 363.8 nm; modulation frequency, 100 MHz. -0)
(-0)
not until one acquires the PREWIF spectra that one becomes convinced there are two discrete fluorescent species contributing. These results and all others shown in this paper were obtained using a single film/substrate. Single-component films were not used to aid in adjusting the null-phase angles. Thus, these results represent a fair demonstration of how PREWIF would function on a truly "unknown" system. A more complicated situation arises for a binary mixture composed of rhodamine 6G and eosin Y. In this particular system, the spectral maxima of the individual components differ by only 10 nm, the excited-state lifetimes are 2.5 ns apart, and the red edge of the fluorescence spectra overlap strongly. Figure 5 presentsthe staticand normalized PREWIF spectra for this sample. Here, unlike the PRODAN/TNS results described above, one is hard pressed to comment on the system heterogeneity from the static spectra alone. The PREWIF spectra, acquired by selectively nulling at the blue A M ! y t l ~ a I ~ b b Vd. y , 66, No. 15, Atgust 1, 1994
2457
100 MHz
/
i
-
\ \
I
I
.--
100 M H z
.. ..... N u l l R6G
\
i
I
1
Static
\
1
N u l l dimer I - .
/
Null Eosin Y
-
1
Static
- Null
.
\
monomer
\ :,.
. ._.
,
W"
al
+ v,
-1.5 510
530
550
570
590
61 0
~~~
500
520
540
560
580
600
€
Emission W a v e l e n g t h ( n m )
-
-
Emission Wavelength (nm)
Flgurr 5. Raw static (- -)and normalizedPREWIF spectra (- or for a sol-gelderived thin film doped with rhodamine 60 and eosin Y. (-)Nulling the eosin Y contribution minus rhodamine 6 0 emission. Nullingthe rhodamine 6 0 contributionminus eosin Y emission. Emission wavelength resolution, 8 nm; A,, = 488.0 nm; modulation frequency, 100 MHz. a,.)
(.e.)
(-) and red (-) edges of the fluorescence, demonstrate that the static spectrum (- - -) is indeed composed of two discrete
components. A still morecomplicated example is a binary mixture where one of the components is a minor contributor to the observed fluorescence. To illustrate this situation we investigated the rhodamine 6G dimer and monomer system studied previously by Narang et a1.66 In this experiment a rhodamine 6G-doped thin sol-gel film is prepared66such that 10-1 5% of the emission arises from the rhodamine 6G dimer. The excited-state lifetimes for the dimer and monomer differ by -3 ns, and the spectral maxima are separated by -25 nm. The dimeric species is also moderately photolabile.66 Figure 6 presents the static and normalized PREWIF spectra for this system. One can clearly see that PREWIF is able to easily recover the dimer and monomer spectra that combine to make up the total interfacial fluorescence even when one of the components is a minor contributor and photolabile. Discrimination of Bulk and Antibody-Bound Fluorescein. Oneof the difficulties associated with using TIRF for surfaceselective measurements is the possibility of background fluorescence from species in the bulk. This arises because the evanescent field propagates well into the bulk (up to 2500 A under our conditions) and the specific interfacial region of interest may be substantially less than the zone probed by the evanescent field. For example, in the protein G-antifluorescein (PG-AF) model system studied here, the actual immunosurface extends only 100 A from the silica/liquid interface (Figure 7). Thus, if there are fluorescing species in the bulk solvent (e.g., residual fluorescein), such could make a substantial contribution to the observed fluorescence. Several research groups have developed elegant multistep calibration methods to address this type of pr~blem.~~-'O
-
(66) Narang, U.; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993,47, 229. ( 6 7 ) Lok, B. K.; Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91, 8 7 .
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Anatytical Chemistty, Vol. 66,No. 15, August I, 1994
Figure 8. Raw static (- -)and normalized PREWIF spectra (-or - e ) for a sol-gelderived thin film doped wRh a moderately high level of rhodamine 60. (-) Nullingthe rhodamine 6 0 monomer minus rhodamine 6Gdimer emission.(.-)Nulling the rhodamine 6 0 dimer minus rhodamine 6G monomer emission. Emission wavelength resolution, 8 nm; = 488.0 nm; modulation frequency, 100 MHz.
B
A
k C
E;(((
D
antifluorescein
3
surface-immobilized protein G
4
fluorescein
Figure 7. Simplified schematic of the model immunointerfaceprobed by PREWIF.
However, these methods require either radioactive labels or assumptions regarding the quantum yield of the fluorophore in the bulk and at the interface. This latter issue is especially troublesome here because the excited-state fluorescence lifetimes for free and antifluorescein-bound fluorescein are 4000 and 500 ps, re~pectively.~~ Thus, the species of interest in our biosurfaces (antifluorescein-bound fluorescein) has a quantum yield significantly lower than the same species in the bulk. Further, if one is interested in the spectroscopy of only the interfacial species, one would have tremendous difficulty distinguishing surface and bulk fluorescence using static TIRF alone. (68) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J . Colloid Interface Sci. 1986, 1 1 1 , 555. (69) Rockhold, S. A.; Quinn,R. D.; Van Wagenen, R. A.; Andrade, J . D.; Reichert, W.M. J . Electroanal. Chem. 1983, 150, 261. (70) Reichert, W. M.; Ives, J. T.; Suci, P. A,; Hlady, V. Appl. Specrrosc. 1987, 41, 636.
.
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Flgure 8. (Panel A) Raw statlc (- -)and normalized PREWIF spectra (- or -.) for the protein Gantifluoresceln-fluorescein system when an excess of fluorescein Is present such that there is a significant population of unbound fluorescein within the evanescent field probe volume. (-) Nulllng the unbound fluorescein mlnus antlfluorescelnfluorescein emission. (.-)Nulling the antibody boundfluorescein mlnus unbound fluorescein emission. (Panel B) Relative PREWIF spectra illustrating the relative slgnal level from the interfacial (- -) and bulk = (-)fluorescein species. Emission wavelength resolution, 8 nm; bX 457.9 nm; modulation frequency, 100 MHz.
-
Fortunately, because the interfacial (antibody associated) and bulk (free) fluorescein species exhibit different excitedstate fluorescent lifetimes, PREWIF may allow one to discriminate between interfacial and bulk species. This concept can be better illustrated by looking back to Figure 7, which shows the silica-immobilized PG-AF system as one titrates the surface-immobilized antifluorescein with fluorescein (panels A-D). Initially there is no fluorescein present (panel A) and no fluorescence. As we increase the concentration of fluorescein (panels B-D) we begin to fill the available A F binding sites (panels B and C). In these cases all the fluorescence is from antibody-bound fluorescein. However, once all the antibody binding sites are occupied, any additional fluorescein will reside in the bulk (panel D). This latter situation presents a serious problem for any static TIRF method and is exacerbated when the quantum yield for the interfacial species is less than the same species in the bulk. Figure 8 presents a series of raw static and PREWIFspectra for the silica-immobilized PG-AF system in the presence of excess fluorescein (F). In these particular experiments, the molar ratio of free to antibody-bound fluorescein is on the order of 3: 1. This results in a 20-30-fold differencein intensity between surface (PG-AF-F) and bulk (F) species. In panel A we present the normalized static fluorescence spectrum (- -) and the recovered normalized PREWIF spectra nulling the free (-) and antibody-bound (.-) forms of fluorescein. These PREWIF spectra are nearly identical to the spectra recovered in the bulk for fluorescein alone and fluorescein in the presence of excess antiflu~rescein.~~ Panel B presents the recovered relatiue PREWIF spectra for this same system when the detector phase angle is adjusted to null free (- - -) and
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Flgure 8, Raw statlc (- -)and normalizedPREWIF spectra (- 01 e-) for acrykdaklabeied BSA physlsobd to elllca. (-) Nulllng the red edge and recoveringthe locally excitedacrykdan emission. (-) Nulling the blue edge and recovering the dipolar relaxed acrylodan emission. Emlsslon wavelength resolution, 8 nm. = 351.1 nm; modulation frequency, 50 MHz.
antibody-bound (-) fluorescein. These results serve to demonstrate the remarkable power of PREWIF and underscores the viability of this technique for probing selectively interfacial species even when fluorescing species in the bulk are inherently more fluorescent. These results bode well for the continued use of PREWIF to study immunosurfaces. Heterogeneity of BSA-Acrylodan Sorbed to S ilica. Reichert and co-workers61 reported previously on using the fluorescent probe a ~ r y l o d a n ~to l -label ~ ~ BSA and study BSA adsorption to several silica-based surfaces. Acrylodan and BSA were chosen because the former reacts only with free thiol groups and the latter possesses a single free thiol at Cys34 in loop one of domain one. Thus, this system represents a simplification over other probe-labeled protein systems in which the location and position of the fluorescent reporter group is often ill-defined. If the acrylodan-BSA (Ac-BSA) system were truly so simple, one would anticipate the emission to arise from a single species. However, Reichert and his associates61used static TIRF to show that the emission from acrylodan in BSA-Ac was complex. Figure 9 shows the raw static (- -) and PREWIF spectra (- or -.) for Ac-BSA physisorbed to a fused silica optical fiber. The static spectrum shows no obvious evidence of any complexity; however, the PREWIF spectra clearly demonstrate that theobserved static fluorescence results from at least two very different emissive forms of a~rylodan.~* These results again demonstrate the power of PREWIF and
-
~~
(71) Reid, S. W.; Koepf, E. K.;Burtnick, L. D. Arch. Biochem. Biophys. 1993. 302. 31. (72) Yem, A. W.; Epp, D. E.; Mathews, W. R.; Guido, D.M.; Richard, K. A,; Staite, N.D.;Deibcl, M. R., Jr. J. Biol. Chem. 1992, 267, 3122. (73) Epp, D. E.; Ycm,A. W.; Fisher, J. F.; McGee, J. E.; Paslay, J. W.; Dcibcl, M. R., Jr. J. Biol. Chem. 1992, 267, 3122. (74) Marriott, G.; Zechcl, K.;Jovin, T. M. Biochemistry 1988, 27,6214. (75) Lehrer, S. S.; Ishii, Y. Biochemistry 1988, 27. 5899. (76) Saavcdra, S.S.;Grobin, A. W.; Lochmiiller, C. H. AMI. Chem. 1988, 60.
2158.
( 7 7 ) F'rendcrgast, F. 0.;Mcyers, M.; Carlson, G. L.; Iida, S.; Potter, J. D. J. Biol. Chem. 1983.258, 7541.
AnalyHcalChemkby, Vol. 66, No. 15, August 1, 1994
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suggest how it might be used to more accurately describe the chemistry of proteins sorbed to interfaces. In fact, by using PREWIF in concert with MPM-TIRF,46,47one can in principle obtain a much improved picture of biointerfaces and even speciate between the same molecular species in different forms (e.g., native and denatured, neutral and ionized).
CONCLUSIONS A new fluorescence-based technique for probing interfaces, surface-immobilized bioreceptors, and biointerfaces is reported. Phase-resolved evanescent wave induced fluorescence is used to (1) recovery the weak, interfacial fluorescence from a sol-gel-derived thin film doped with a low level of rhodamine 6G in the presence of strong Rayleigh scatter; (2) resolve the individual components in dilute TNS/PRODAN, rhodamine 6G/eosin Y, and rhodamine 6G dimer/monomer binary mixtures formed within 0.5-pm-thick sol-gel-derived films (78) Additional work from our group (Bekos, E. J.; Wang, R.; Lundgren, J. S.; Gardella, J. A,, J.; Bright, F. V. paper No. 270 and Wang, R.; Lundgren, J. S.;Bekos, E. J.; Bright, F. V. paper No. 27 1at the 45th Pittsburgh Conference, Chicago, IL, February 27-March 4,1994) shows that thereis clear picosecond and nanosecond dipolar relaxation of the local microdomain about the Ac residue in native and surface-sorbed Ac-BSA.
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Analytical Chemistry, Vol. 88, No. 15, August 1, 1994
overcoated on fused silica substrates; (3) eliminate the contribution from bulk fluorescein and selectively monitor antibody-bound fluorescein at a model immunosurface; and (4) yield evidence on the inherent emission heterogeneity for acrylodan-labeled bovine serum albumin physisorbed to silica surfaces. In summary, PREWIF offers significant advantages over static TIRF techniques. It will help to more accurately describe the fluorescence arising from complex interfaces, and it provides a convenient tool for studying heterogeneous interfaces under ambient conditions.
ACKNOWLEDGMENT This work was generously supported by the National Science Foundation (CHE-9300694). A preliminary account of this work was presented at the 45th Pittsburgh Conference, Chicago, IL, February 27-March 4, 1994, paper 117. Received for review March 11, 1994. Accepted May 23, 1994.' *Abstract published in Advance ACS Abstracts. July 1, 1994.
