Fluorescence from Alexa 488 Fluorophore Immobilized on a Modified

The fluorescence intensity was measured from Alexa 488 fluorophore immobilized on a modified polycrystalline gold electrode. In the range of potential...
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Fluorescence from Alexa 488 Fluorophore Immobilized on a Modified Gold Electrode† Li Li, T. Ruzgas, and A. K. Gaigalas* Biotechnology Division, NIST, 100 Bureau Drive, Stop 8312, Gaithersburg, Maryland 20899 Received December 11, 1998. In Final Form: May 24, 1999 The fluorescence intensity was measured from Alexa 488 fluorophore immobilized on a modified polycrystalline gold electrode. In the range of potentials -0.5 to +0.2 V vs Ag(Ag/Cl), the fluorescence signal was small and of the order expected from known fluorescence quenching by metal surfaces. Immobilization of Alexa 488 with a larger separation between the fluorophore and the electrode surface resulted in an increased fluorescence signal in the potential range mentioned above. For potentials more negative than -0.5 V and more positive than +0.2 V, the fluorescence signal increased exponentially. The increase was attributed to a potential driven increase in the separation between the fluorophore and the electrode surface brought about by reorientation and dechemisorption of the fluorophore. For potentials more positive than -0.7 V vs Ag(Ag/Cl) the change in fluorescence intensity was quasi-reversible, for potentials more negative than -0.8 V the change was irreversible, and the fluorescence vanished on returning to more positive potentials. The dependence of the fluorescence intensity on potential was interpreted in terms of sequential occurrence of reorientation(E > -0.7 V) and dechemisorption (E < -0.8 V). The fluorescence signal yields significant additional information about the behavior of molecules at electrode surfaces.

Introduction The fluorescence intensity of fluorophores at or near metal surfaces is known to be a sensitive function of the distance between the fluorophore and the metal surface. The sensitivity has been attributed to the spatial dependence of the fluorescence resonant energy transfer between the fluorophore and the various energy modes in the metal. Calculations1 using a classical dipole-surface interaction have yielded excellent agreement with experiment.2,3 For smaller distances (+0.3 V vs Ag(Ag/Cl)) the fluorescence signal increased exponentially. We attribute this increase in fluorescence intensity to an increase in the net separation between the fluorophore and the gold surface where the increased separation results from potential dependent desorption of the fluorophore. The changes in fluorescence intensity were quasi-reversible if the potential did not go below -0.7 V vs Ag(Ag/Cl), otherwise the changes were irreversible. This suggests that the desorption process could be sequential consisting of a reversible reorientation step at more positive potentials and an irreversible dechemisorption step at potentials more negative than -0.8 V vs Ag(Ag/Cl). In the following we present measurements and discussion in support of the above interpretation. This model suggests that the fluorescence signal could be a sensitive probe of the initial stage of the desorption process. Experimental Technique Electrode Preparation. Gold electrode surfaces were polished initially with 5.0 µm alumina powder in water (2%, w/w) and then with 1.0 µm alumina powder and finally finished with 0.05 µm alumina powder to produce mirrorlike surfaces. The electrodes were ultrasonicated in water to remove bound alumina and electrochemically cleaned by cyclic voltammetry between -0.35 V vs Ag(Ag/Cl) and +1.7 V vs Ag(Ag/Cl) at the scan rate of 0.7 V/s (30 cycles in total) in 1 M sulfuric acid. Following a water wash, a fresh, fine gold layer was galvanostatically deposited by immersing the electrode in 0.2 M phosphate buffer solution (pH 7.4) including ∼0.2 g/mL potassium dicyanoaurate(I) while applying ∼-1.0 V vs Ag(Ag/Cl) reduction potential for 4 min. During the electrodeposition, the current density was kept constant at 0.63 mA/cm-2. After being rinsed with water, the gold electrode was once again cleaned electrochemically by cyclic voltammetry at the same conditions described above. Fluorophore Immobilization. Modification of gold electrodes by Alexa 488 was based on the method reported by Cohen et al.5 with minor revision. The freshly prepared gold electrode

10.1021/la981704d CCC: $18.00 © 1999 American Chemical Society Published on Web 06/29/1999

Fluorescence from Fluorophores on a Gold Electrode

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Figure 2. Schematic of the apparatus used to measure the fluorescence from the surface of an electrode. Four quantities were measured: F, the constant (dc) component of fluorescence intensity; ∆F, the amplitude of the modulated fluorescence intensity; ∆I, the amplitude of the electrochemical current; ∆E, the amplitude of the oscillating part of the applied potential. In the case of the latter three quantities both in-phase and out-of-phase components were measured.

Figure 1. Model configurations of the fluorophore tethered to the gold surface via “short” and “long” tethers. The formation of the S-Au bond is referred to as chemisorption in the text. The charged sulfonate, amine, and carboxyl groups can have strong electrostatic interaction with the electrode. This interaction is expected to influence the orientation of the fluorophore relative to the electrode interface. At more positive potentials it is expected that the fluorophore, designated by “X”, would be closer to the interface. was immersed in an aqueous 2 × 10-2 M cystamine solution for 14 h. Following a water wash, the cystamine-modified electrode was reacted for 2.5 h with ∼1 × 10-3 M Alexa 488 reactive fluorophore dissolved in 0.1 M phosphate buffer solution, pH 8.7.6 The resulting gold electrode was rinsed with water and used immediately for fluorescence measurements. To enlarge the distance between the gold surface and Alexa 488, the clean gold electrode was introduced into a DMSO (dimethyl sulfoxide) solution containing 1 × 10-2 M 3,3′-dithiobis(propionic acid N-hydroxysuccinimide ester) for 5 h. The modified gold electrode was washed with DMSO and then with THF (tetrahydrofuran) and further reacted for 4 h with a THF solution including both ∼2 × 10-2 M 1,12-diaminododecane and 1 × 10-4 M N,N′-diisopropylamine. The resulting electrode was rinsed with THF and subsequently with DMF (dimethylformamide). Next, the modified gold electrode was reacted for 2.5 h with a DMF solution containing ∼1 × 10-3 M Alexa 488 reactive fluorophore and 1 × 10-4 M N,N′-diisopropylamine. The resulting gold electrode was washed with DMF and then with water and finally immersed in an electrochemical cell. Figure 1 shows the assumed configuration of the fluorophore tethered to the electrode surface with “short” and “long” tethers. The fluorophore (denoted by “X” in Figure 1) could also interact with the surface directly via van der Waals and electrostatic interaction of the carboxyl, amine, and sulfonate groups. (5) Cohen, Y.; Levi, S.; Rubin, S.; Willner, I. J. Electroanal. Chem. 1996, 417, 65. (6) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, CA, 1996.

Measurements. Figure 2 shows a schematic of the apparatus used for the measurement of fluorescence intensity from fluorophores immobilized on the electrode surface. An argon ion laser operating at 488 nm was the source of illumination. A polarization rotator set the polarization of the incident beam to “s” (electric field vector perpendicular to the plane of Figure 2) or “p” (electric field vector in the plane of Figure 2), and a laser line filter blocked all other plasma lines. For some measurements a polarization analyzer was placed in front of the photomultiplier (PMT) detector. “V” polarization corresponds to electric field of the detected light in the plane of Figure 2, and “H” polarization corresponds to electric field normal to the plane of Figure 2. The laser beam was focused on a surface of an electrode to a spot approximately 1 mm in diameter. The incident power was maintained at approximately 1 mW. The electrochemical cell consisted of a quartz window imbedded in a cylindrical Teflon body which also contained openings for the electrodes and purging gas. All of the measurements were conducted under an atmosphere of Ar. The laser beam was incident at ∼55° to the normal defined by the electrode surface. The fluorescence was collected at approximately 35° relative to the normal. An f ) 1.2 lens was used to collect as much of the emitted fluorescence as possible, and a holographic notch filter tuned for 488 nm was used to block the scattered light. A long pass filter with a cutoff at 510 nm was used as an additional suppressor of stray light. The light was then incident either on the slit of a SPEX 470 monochromator or on the PMT anode. The monochromator, with a CCD detector, was used to obtain spectra of the emitted fluorescence, while the PMT was used for measuring the dependence of the fluorescence intensity on electrode potential. The constant potential applied to the electrode was controlled by the potentiostat (EG&G Princeton Applied Research 263A), while the modulating potential was derived from the reference signal of the lock-in amplifier (Stanford Research SR 830). The measured quantities were ∆V the amplitude of the applied modulating potential, ∆I the amplitude of the modulated current, ∆F, the amplitude of the modulated fluorescence signal, and F, the constant (dc) component of the fluorescence signal. All of the measured amplitudes were complex numbers consisting of a part that was in phase and a part that was out of phase with the applied modulating potential. During most of the measurements, the amplitude of the modulating potential was set to 70 mV (it was set to 10 mV when the capacitance was measured) and the constant electrode potential was changed in discrete steps of 0.01 V (not linear sweep). After each potential step, the modulated fluorescence amplitude was measured. The fluorescence amplitude ranged from several µV to several mV. To minimize the noise, the lock-in amplifier time constant was set to 3 s. The

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Figure 4. Differential capacitance measured on bare, cystamine modified, and cystamine plus Alexa 488 (“short tether”) modified gold electrodes. The frequency was set to 21 Hz, and the modulation amplitude was 10 mV. The measurements were performed on the same electrode after successive steps in the preparation sequence. The potential was measured relative to Ag(Ag/Cl).

Figure 3. (a) Measured fluorescence emission spectra of immobilized Alexa 488 at -0.6 and -0.4 V vs Ag(Ag/Cl) reference electrode. The wavelength of maximum emission is close to that observed for Alexa 488 in solution. (b) Ratio of the difference of the intensities of the two spectra to the intensity of the spectrum at -0.4 V. The approximately constant value suggests that the change in fluorescence intensity is mainly due to a change in quantum yield of Alexa 488. The band at 515 nm is a strong laser line which was not removed completely. interval between consecutive measurements(or potential increments) was approximately 9 s.

Results Figure 3a shows the fluorescence spectra taken from the immobilized Alexa 488 fluorophore at -0.6 V and -0.4 V relative to the Ag(AgCl) reference electrode. The peak at approximately 523 nm is consistent with that at 520 nm in aqueous solution. The spectra were accumulated for 8 s. The spectra taken with a bare electrode (at several potentials) are barely visible above the x axis and were not corrected for in Figure 3a. The ratio of the difference between spectra taken at -0.6 and -0.4 V and the spectra taken at -0.4 V is reasonably constant at 2.8. The small slope in the dependence of the ratio on wavelength indicates a shift toward shorter wavelengths of the spectrum at -0.6 V relative to the spectrum at -0.4 V. We estimate this shift to be of the order of several nanometers. The dominant change in the two spectra appears to be a change in the quantum yield (or absorbance) of the immobilized fluorophore. The polarization anisotropy, r, is defined by r ) (FH FV)/(FH + 2FV), where FH and FV are the detected fluorescence intensities with the polarization analyzer set in the “H” and “V” positions, respectively. The polarization anisotropy was measured at open circuit potential. The fluorescence from the immobilized fluorophores was largely depolarized in aqueous buffer; r ) 0.04 ( 0.02 for “s” incident polarization, and r ) -0.03 ( 0.02 for “p” polarization. When the electrode was immersed in a buffer containing 50% (v/v) glycerol, the anisotropy increased to

0.17 ( 0.3 for “s” polarization and -0.12 ( 0.03 for “p” polarization. This dependence on viscosity indicates that the immobilized fluorophores undergo significant rotational motion. The spectra in Figure 3 and subsequent PMT measurements were taken with “s” polarized incident light without a polarization analyzer in the detector arm. The differential capacitance was measured at 21 Hz with the modulation amplitude set to 10 mV. The differential capacitance, in units of µF, was calculated from the measured impedance Z ) ∆V/∆I using the relation C ) -106/(2πf Im(Z)). The values of the measured differential capacitance were further adjusted to approximately 20 µF by dividing by 0.15 cm2, corresponding to a product of the geometric area and an assumed electrode surface roughness factor of 1.5. The results, in units of µF/cm2, are displayed in Figure 4 for various stages of electrode modification. The bare electrode displays a classic dependence of the differential capacitance on applied constant potential where the increase in differential capacitance at positive potentials is due to the adsorption of phosphate ions.7 The electrodes with immobilized cystamine and cystamine + Alexa 488 have progressively smaller values of the differential capacitance and show a minimal variation with potential. This is consistent with the presence of increasingly thicker dielectric films on the electrode surface. Figure 5a shows the cyclic voltammograms (CV) with the negative potential limit set to -1.0 V. The first CV scan is shown by the solid line, and the second CV scan, by the dotted line. The measured current was divided by 0.15 cm2 corresponding to a product of the electrode geometric area and an assumed roughness factor of 1.5. The first CV scan displays a peak at -0.8 V vs Ag(AgCl) which most likely corresponds to the reduction of the S-Au bond.8 The second CV scan in Figure 5a has no reduction peak indicating that the cystamine has desorbed and diffused away from the interface. The difference in the current between the two scans gives an estimate of -33 ( 6 µC/cm2 for the charge involved in the reduction of the S-Au bonds. The error was estimated from the analysis of several scans with and without S-Au reduction peak. Figure 5b shows the dependence of the measured dif(7) Niaura, G.; Gaigalas, A. K.; Vilker, V. L. J. Phys. Chem. B 1997, 101, 9250. (8) Widrig, C. A.; Chung, C.; Porter, M. C. J. Electroanal. Chem. 1991, 310, 335.

Fluorescence from Fluorophores on a Gold Electrode

Figure 5. (a) First two negative linear potential scans on a freshly prepared cystamine plus Alexa 488 (“short tether”) modified electrode. The scans were initiated at -0.1 V Ag(Ag/Cl) with a scan rate of 50 mV/s. The peak at -0.8 V Ag(Ag/Cl) is associated with the reduction of the S-Au bond. (b) Dependence of the amplitude of the modulated fluorescence intensity and differential capacitance on potential. The scan was started at -0.2 V and went to -0.9 V and back to -0.2 V Ag(Ag/Cl). The fluorescence signal was greatly reduced on the return scan (-0.09 to -0.2 V Ag(Ag/Cl)) suggesting a loss of immobilized fluorophore.

ferential capacitance and the modulated fluorescence amplitude on applied potential. There is an exponential increase in the modulated fluorescence amplitude starting at about -0.6 V vs Ag(Ag/Cl). The increase is not reversible since on the return scan the modulated fluorescence amplitude is much smaller and vanishes on subsequent scans. There is an increase in the differential capacitance at -0.8 V vs Ag(Ag/Cl) which is most likely associated with the reduction of the S-Au bond. The overall value of the differential capacitance increases on the return scan indicating a thinner dielectric layer. In subsequent scans the increase in the modulated fluorescence amplitude and differential capacitance at -0.7 V vs Ag(Ag/Cl) is not observed. (The scans shown in Figure 5a,b were performed on freshly prepared electrodes.) CV scans were also taken in the direction of positive potential. A first CV taken from -0.1 to 0.5 V vs Ag(Ag/Cl) and back to -0.1 V displayed an excess of current, at potentials more positive than 0.2 V, relative to the second CV scan. Very likely this excess current is due to the oxidative desorption of cystamine.8 Figure 6a shows the same quantities as in Figure 5b for the case where the potential is kept more positive than -0.6 V vs Ag(Ag/Cl). The modulated fluorescence amplitude is approximately the same on the return scan and on subsequent scans (not shown), and the differential capacitance shows negligible variations. Figure 6b shows the “zero” frequency (DC) fluorescence response recorded during the same scan as in Figure 6a. Figure 7 shows the dependence of the DC fluorescence response on the electrode potential for different ionic strengths. At potentials more negative than -0.6 V vs

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Figure 6. (a) Dependence of the amplitude of the modulated fluorescence intensity and differential capacitance during a potential scan from -0.2 to -0.6 V and back to -0.2 V Ag(Ag/Cl). (b) DC component of fluorescence during the same potential scan as in part a. Compared to the response shown in Figure 5b the fluorescence signal is quasi-reversible. The DC component of fluorescence and the amplitude of the modulated fluorescence intensity have similar dependence on the applied potential.

Figure 7. Dependence of the DC fluorescence intensity on applied potential for different solution and immobilization conditions. The solid line is the DC fluorescence in 0.005 M phosphate buffer. The dotted line is the DC fluorescence in 0.1 M phosphate buffer. Both of the previous measurements were with the fluorophore on a “short” tether. The dashed line shows the DC fluorescence intensity for the fluorophore on a “long” tether in 0.1 M phosphate buffer.

Ag(Ag/Cl) the total fluorescence intensity increased exponentially. In the range -0.5 to 0.3 V the intensity was approximately constant for both ionic strengths with a small fluctuation at -0.1 V. Figure 7 shows the potential dependence for the case where the dye is immobilized using a tether with 18 carbons (curve titled “long”). There is significantly less variation with potential in the fluorescence intensity, and the average level of the fluorescence signal in the range -0.5 to 0.3 V is about 3-4 times higher than in the case of the short tether (4

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carbons). The comparison of absolute fluorescence intensity between the “short” and “long” is difficult because of the difficulty in estimating the absolute amount of immobilized dye; however, the qualitative conclusion that the fluorescence for the “long” tether is significantly higher is valid. Overall Figure 7 shows that the fluorescence intensity is a sensitive function of the environment of the fluorophore at the electrode surface. Discussion We will assume that the radiationless energy transfer between the immobilized fluorophore and the metal is the dominant mechanism for determining the fluorescence intensity of the immobilized fluorophore. The energy transfer depends on the third power of the separation between the fluorophore and the metal surface. Reorientation of transition moments could also lead to large changes in the fluorescence intensity if all fluorophores reoriented in the same way relative to the incident polarization. The static electric field will probably change the distribution of orientations normal to the surface of the electrode; however the distribution of transition moment orientations is not expected to change in the plane parallel to the surface. Since the incident polarization is parallel to the surface, we expect a small effect from reorientation of the transition moments. Lavrich et al.9 have shown that alkane molecules deposited on a gold crystal surface can be physisorbed via van der Waals interaction between the alkane chain and the metal and chemisorbed via the formation of the S-Au bond. We adopt a similar picture and postulate that while the fluorophore is chemisorbed via the S-Au bond of the “tether”, there is an additional electrostatic interaction between the charged groups (sulfonate, amine, and carboxyl) on the fluorophore and the electrode surface. This electrostatic interaction and the large dipole moment of the coadsorbed water molecules suggest the possibility of reorientation of the fluorophore with changes in electrode potential. Figure 5 shows that, in the case of Alexa 488 fluorophore with a “short” tether, the fluorescence intensity changes drastically at potentials more negative than -0.6 V vs Ag(Ag/Cl). We will describe this increase of the fluorescence intensity as a consequence of reorientation and dechemisorption. A similar viewpoint has been introduced previously for the case of adsorption of insoluble surfacants at electrode interfaces.10,11 Fluorescence Intensity Variation at Negative Potentials. Since the change in fluorescence intensity is quasi-reversible if the electrode potential does not go more negative than -0.6 V, it is likely that for potentials more positive than -0.6 V there is no reduction of the S-Au bond; rather the fluorophore undergoes a reorientation at the surface leading to a change in the average separation between the fluorophore and the electrode interface. This scenario would be consistent with the observed quasireversible nature of the changes of fluorescence intensity. A similar interpretation was made of the large changes in fluorescence intensity from 4-(1-pyrenyl)butyltrimethylammonium bromide (PN+Br--) adsorbed on colloidal clay upon introduction of different amounts of a (9) Lavrich, D. J.; Wetterer, S. M.; Bernasek, S. L.; Scoles, G. J. Phys. Chem. B 1998, 102, 3456. (10) Bizzotto, D.; Lipkowski, J. J. Electroanal. Chem. 1996, 409, 33. (11) Sagara, T.; Zamlynny, V.; Bizzotto, D.; McAlees, A.; McCrindle, R.; Lipkowski, J. Isr. J. Chem. 1997, 37, 197. (12) Nakamura, T.; Thomas, J. K. J. Phys. Chem. 1986, 90, 641. (13) Wheeler, J.; Thomas, J. K. Langmuir 1988, 4, 543.

Figure 8. Solid circles show the DC fluorescence intensity (data from Figure 6b) and the solid line gives the result of modeling the fluorescence dependence on applied potential using eq 1 in the text.

coadsorbate.12,13 The authors suggest that the coadsorption of another molecule leads to reorientation of the adsorbed PN+. A simple model will be used to describe the reorientation process and provide a rationalization of the quasireversible dependence of the fluorescence intensity on the applied potential. The immobilized fluorophore will be considered as a rigid body with one end tethered to the gold interface and free to rotate about the point of attachment. We assume that only two dominant orientations are prevalent and that there is a potential dependent transition between them. At more positive electrode potentials the fluorophore is attracted to the interface and on average is closer to the interface. At more negative potentials the fluorophore is repelled by the negatively charged interface and the distance from the interface increases. A first-order rate equation is used to describe the time evolution of the fraction of total immobilized fluorophores with each orientation. The rate constant depends on the applied potential E, with the explicit form of this dependence given by absolute rate theory.14 Some of the parameters which enter the description of the rate constant in this model are the frequency factor, k0, the potential, E0, at which the fluorophores are distributed equally between the two orientations, and zeff, the effective charge of the fluorophore. The applied potential, E(t), in the model varies linearly with time from -0.4 to -0.8 V and back to -0.4 V in 8 s. The reorientation process involves several molecules of water which are not considered explicitly in the simple model; their effect is hidden in the various parameters. The resulting fluorescence intensity, F(E), from the immobilized fluorophores can be written as

F(E) ) G(FFfF(E) + FC(1 - fF(E)))

(1)

where fF is the fraction of total immobilized fluorophores farther from the surface, FF and FC are the fluorescence intensities of a single fluorophore which is far from and close to the interface respectively, and G depends on instrumental settings and on the total number of immobilized molecules. The factors FF and FC will depend on the separation of the fluorophore from the electrode interface. The solid line in Figure 8 shows the correspondence between the measured fluorescence intensity and the model prediction obtained as outlined above. The parameter values were E0 ) -0.6 V, zeff ) 0.5 e, k0 ) 20 (14) Schmickler, W. Interfacial Electrochemistry; Oxford University Press: New York, 1996; p 35.

Fluorescence from Fluorophores on a Gold Electrode

s-1, G ) 0.2, and FF/FC ) 5. The first three parameters are associated with the kinetic rate constant, and FF/FC ) 5 is an estimate of the relative fluorescence intensity from fluorophores with the two orientations. Given that the potential scan is very slow, the experimental data are not sensitive to the value of k0. The reversible fluorescence intensity change at negative potentials also occurs in buffers with low ionic strength but at different values of E0. This points to the central role of electrostatic interactions in the quasi-reversible change of the fluorescence intensity. At more negative potentials than -0.7 V the S-Au bond is reduced. Since the fluorophore is very soluble, after the S-Au bond reduction the fluorophore diffuses freely and there is an irreversible loss of fluorophore at the interface.10,15 This would be consistent with the presence of a reduction peak at -0.8 V during the first CV scan and the absence of the peak during the second CV scan. The fact that the S-Au bond is reduced at -0.8 V suggests that there may be some reduction even at -0.6 V. This provides an alternate explanation for the quasireversible fluorescence variation. Terminating the potential scan at -0.6 V may still lead to the reduction of minute amounts of S-Au bonds. These amounts may be sufficient to give an observable fluorescence signal, but since the total amount of fluorophores does not change appreciably, there would be an appearance of a quasireversible fluorescence response. A model of the S-Au reduction has been described by Yang.16 Our measurement conditions would correspond to the low overpotential regime in this model where most of the reduction takes place at the periphery of “etching” sites with the diffusion of the fluorophore from the periphery of these sites as the limiting step. This model could reproduce the observed exponential increase in fluorescence signal. The observed fluorescence signals decreased by about 30% over a period of 1 h that was spent performing the measurements. Some of this decrease could be due to the accumulated reduction of the S-Au bonds at -0.6 V. However, the time spent at -0.6 V was short so that this scenario is unlikely and the signal reduction is probably due to photodegradation of the immobilized fluorophore. Figure 7 compares the fluorescence signal from a Alexa 488 fluorophore tethered via “short” and “long” tethers. In the case of the fluorophore with a long tether, the electrostatic interaction between of the fluorophore and the electrode interface may not be important since the tethering molecules form a self-organized layer and the interaction between the alkane chains dominates the energetics. As a consequence, the reduction of the S-Au bond occurs at more negative potentials17 so that the monolayer is stable over the potential range -0.8 V to +0.2 V. In addition, the longer tether results in less energy transfer between the fluorophore and the metal and hence (15) Hobara, D.; Miyake, K.; Imabayachi, S.; Niki, K.; Kakiuchi, T. Langmuir 1998, 14, 3590. (16) Yang, D.-F.; Morin, M. J. Electroanal. Chem. 1998, 441, 173. (17) Imabayashi, S.; Iida, M.; Hobara, D.; Feng, Z. Q.; Niki, K.; Kakiuchi, T. J. Electroanal. Chem. 1997, 428, 33.

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yields larger fluorescence intensity. Although there is qualitative agreement with observations, the quantitative comparison of the response with “short” and “long” tethers is difficult due to the uncertainty in the relative amount of immobilized fluorophore. However the more complex immobilization procedure in the “long” tether case makes it likely that the surface concentration of the “long” tethered molecules is smaller than that of the “short” tethered. The detailed chemical structure of the fluorophore does not appear to be important. With the use of Cy5.5 dye from Amersham Pharmacia Biotech Ltd., we have observed the same trend in fluorescence intensity at potentials more negative than -0.6 V. The Cy5.5 fluorophore is chemically very different from Alexa 488; however, they carry a charge of -3 which is comparable to the -2 charge carried by Alexa 488 fluorophore. Therefore, the sharp increase in fluorescence at negative potentials may be a general phenomenon for negatively charged fluorophores immobilized close to the gold electrode surface. Fluorescence Intensity Variation for Positive Potentials. There is a large increase in fluorescence intensity whenever the potential exceeds 0.2 V. This potential is close to the potential in the CV where excess current is observed in the first scanspresumably due to oxidative breaking of the S-Au bond. The shape of the fluorescence increase at positive potentials is similar to the shape observed at negative potentials; however, there appears to be greater sensitivity to ionic strength. We will not discuss this region further at this time. Conclusion The measured fluorescence intensity from Alexa 488 fluorophore immobilized by cystamine to a gold electrode exhibits a strong dependence on potential. This dependence has been interpreted in terms of a sequential reorientation and dechemisorption of the immobilized fluorophore. Dechemisorption is achieved via reductive breaking of the S-Au bond, while reorientation is due to changes of van der Waals and electrostatic interactions between the fluorophore and the electrode surface. The net result is the changing of the separation between the fluorophore and the electrode surface. If reorientation occurs without dechemisorption, then fluorescence intensity changes are quasi-reversible. If both processes occur, then there is complete loss of the fluorescence signal. The fluorescence intensity from Alexa 488 immobilized with a longer tether had minimal dependence on potential in the region -0.8 V to +0.5 V. Additional experimental work is in progress to ascertain the nature of the process which leads to the large and quasi-reversible changes in fluorescence signal with electrode potential. Acknowledgment. The authors are indebted to Gintaras Valincius for critical evaluation of many aspects of this work. We are grateful to Yu-zhong Zhang of Molecular Probes Inc. for providing Alexa 488. LA981704D