Single-Molecule Detection Using Surface-Enhanced Resonance

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Anal. Chem. 2001, 73, 3674-3678

Single-Molecule Detection Using Surface-Enhanced Resonance Raman Scattering and Langmuir-Blodgett Monolayers Carlos J. L. Constantino, Tibebe Lemma, Patricia A. Antunes, and Ricardo Aroca*

Materials and Surface Science Group, School of Physical Sciences, University of Windsor, Windsor, Ontario, N9B 3P4 Canada

The Langmuir-Blodgett (LB) technique has been used to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra of single dye molecules dispersed in the matrix of a fatty acid. The experimental results presented here mimic the original electrochemical surface-enhanced Raman scattering (SERS) work where the background bulk water did not interfere with the detection of the SERS signal of molecules adsorbed onto the rough silver electrode. LB monolayers of the dye in fatty acid have been fabricated on silver island films with a concentration, in average, of one probe molecule per micrometer square. The properties of single-molecule spectroscopy were investigated using micro-Raman including mapping and global images. Blinking of the SERRS signal was also observed. The concept of spatially resolved surface-enhanced Raman scattering (SERS) was first introduced by Van Duyne et al.1 showing that attomole mass sensitivity could be achieved using micrometer-sized sampling areas. The spatial resolution in the farfield experiments, although limited by the optical wavelength in use, is a powerful tool for spatially resolved analytical applications. In Raman microscopy experiments, 1-µm2 spatial resolution is commonly achieved. Recently, and taking advantage of the giant local field enhancement observed in SERS, which permits the observation of effective cross section for Raman scattering up to 10-16 cm2/molecule,2-6 spatially resolved single-molecule detection in Raman scattering has now been reported on silver aggregates3,4 and silver particles.5,6 Raman scattering is now part of the family of single molecular spectroscopies (SMS). Most of the SMS work has been done using fluorescence signals thanks to the large cross sections of the emission process.7,8 A typical fluorescence cross section will be 10-19 cm2, * Corresponding author: (e-mail) [email protected]. (1) Van Duyne, R. P.; Haller, K. L.; Altkorn, R. I. Chem. Phys. Lett. 1986, 126, 190. (2) Markel, V. A.; Shalaev, V. M.; Zhang, P.; Huynh, L.; Tay, T. L.; Haslett, T. L.; Moskovits, M. Phys. Rev. B 1999, 16, 8080. (3) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1996, 76, 2444. (4) Kneipp, K.; Wang, Y.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667. (5) Nie, S.; Emory, S. R. Science 1997, 275, 1102. (6) Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932.

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while a Raman scattering cross section would be ∼10-29 cm2. SMS has been realized in the near field,7 and it has also been demonstrated that it operates in the optical far-field providing a noninvasive technique for single-molecule studies.8 The present report is the first application of the spatially resolved surface-enhanced resonance Raman scattering (SERRS) approach to achieve single molecular detection (SMD) using Langmuir-Blodgett (LB) monolayers on silver island films. The large effective resonant Raman cross sections are exploited to achieve SMD of dye molecules dispersed in a fatty acid, a twodimensional host matrix fabricated using the LB technique. The fatty acid Langmuir monolayer containing the guest dye is transferred to silver island films to study the SERRS signal from the dye. The multiplicative effect of resonance Raman enhancement and surface enhancement, in addition to the high sensitivity of the new dispersive Raman instruments, makes the SMS a viable analytical technique. In the SMS of fluorescence probes in solids, the molecule of interest is in an optically transparent matrix and the only signal would be that of the guest molecule. In our experiments, the SERRS spectrum of the single dye molecule would have to be observed together with the SERS of the fatty acid matrix in the LB monolayer and with the common inelastic background that is observed in SERS spectra.9 The results presented here show that the SERRS signal from single molecules is detectable and that SMS work can be carried out using mixed LB monolayers. Widefield or global imaging of the most intense SERRS band in the spectrum was recorded. The silver island surfaces coated with a LB monolayer were mapped with spatial steps of 3 µm. Surfaceenhanced fluorescence (SEF) was recorded as a complementary result to support the SERRS SMD. The 514.5-nm laser line used in the work allowed us to record the SERRS and the SEF from LB monolayers in the same spectrum. EXPERIMENTAL SECTION Spectroscopy. The Raman scattering and Raman imaging (mapping and global imaging) were obtained with a Renishaw Research Raman microscope system RM2000 equipped with a (7) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422. (8) Single-Molecule Detection, Optical Detection, Imaging and Spectroscopy; Basche, T., Moerner, W. E., Orrit, M., Wild, U. P., Eds.; VCH: Weinhem, 1997; p 10. (9) Gao, Y.; Lopez-Rios, T. Surf. Sci. 1985, 162, 976. 10.1021/ac0101961 CCC: $20.00

© 2001 American Chemical Society Published on Web 06/30/2001

computer-controlled three-axis encoded (XYZ) motorized stage with a minimum step of 0.1 µm. The RM2000 uses a Leica microscope (DMLM series) and is equipped with a Peltier cooled CCD array (-70 °C). The high throughput of the instrument permits the use of very low powered lasers. The spectrograph was equipped with a 1200 g/mm grating with additional angletuned band-pass filter optics. Raman spectra were recorded with ∼4 cm-1 resolution using the 514.5-nm laser line and 0.32 mW on the sample, 50× (NA 0.75) microscope objective to focus the laser beam onto a spot of ∼1 µm2. The Raman spectra were also recorded at different spots using 0.13 mW power, high gain, 20 accumulations, and 20 s as collection time to better resolve the background signal. SERRS spectra were recorded in a range of temperature from -190 °C to +200 °C with a step of 20 °C. The area mappings were recorded within a 42 by 42 µm area with a 3-µm step, using ∼0.13 mW power at the sample, high gain, 3 accumulations, and 5 s of collection time. Global images were recorded using angled tuned dielectric filters to select the scattering light at 1370 cm-1, which is the strongest SERRS band in single-molecule LB monolayer spectrum. The 514.5-nm laser line spot size was defocused to an area of ∼40 µm by 40 µm and the global images were collected using 0.32 mW power (∼20 W cm-2), high gain, low speed, and 10 s as collection time for a large number of sites on the surface of the single-molecule LB monolayer. The Raman imaging (mapping and global imaging) was recorded not only at room temperature but also at -100 and +100 °C. The SEF on silver islands was recorded using a 514.5nm laser line, 50× microscope objective, with ∼0.06 mW power. The LINKAN THMS 600 heating-cooling stage was used for SEF and SERRS temperature dependence. Data acquisition and analysis were carried out using the WIRE software for Windows and Galactic Industries GRAMS/32 C software including the 3D package. UV-visible spectra of the silver films were recorded in Cary 50 scan UV-visible spectrophotometer before and after the LB deposition to check the presence of the LB film by changes in the absorption spectra due to changes in the dielectric function of the coated silver plasmons. Solution. The concentration of the spreading solution was calculated to achieve, on average, 1 molecule of bis(benzimidazo)perylene (AzoPTCD) and 4 × 106 molecules of eicosanoic or arachidic acid C19H39COOH (AA) per micrometer square of trough area. According to the π-A area isotherm, the molecular area of AA is ∼25 Å2, or 4 × 106 molecules of AA/µm2 (108 Å2). During the Langmuir experiment on the Lauda trough, 8.640 × 1016 molecules are commonly spread on the subphase of which 2.16 × 10-10 molecules should be AzoPTCD, needed to keep the proportion of 1/µm2 of surface area. The total volume of solution chosen to spread 8.640 × 1016 molecules on the subphase was 250 µL. Therefore, solutions of AzoPTCD and AA with 1.44 × 10-10 and 5.7 × 10-4 mol L-1, respectively, were required. The AA (312.55 g mol-1) solution was prepared directly by weighting 17.97 mg and diluting it in a 100-mL flask. The AzoPTCD (536.58 g mol-1) solution was prepared by two dilutions from a more concentrated solution, whose initial concentration was 3.10 × 10-4 mol L-1 (18.24 mg in 100 mL), and the second dilution was done in the same flask as AA. The AzoPTCD solutions were prepared using 10% trifluoroacetic acid (TFA) and 90% dichloromethane

spectroscopic grade solvents. The TFA is necessary since the AzoPTCD is not soluble in conventional organic solvents. All the flasks and microsyringe used here were previously cleaned with neutral detergent, chromic acid, and acetone. After each step, distilled and deionized water was used. Thin Films. Langmuir film of AzoPTCD in AA was fabricated in a Lauda Langmuir film balance, which was had been cleaned and rinsed with acetone and ultrapure water (18.2 MΩ cm) to remove any residual particles. Then 800 mL of ultrapure water containing cadmium chloride (2.5 × 10-4 mol L-1) at 15 °C was used as subphase. After spreading the solution containing AzoPTCD in AA, 20 min were allowed for evaporation of the solvent. The film was compressed at 17.5 mm min-1 by a single barrier and an area of 150 mm (trough width) × 188 mm (distance between the barrier and the sensor pressure) ) 2.82 × 104 mm2 was occupied by the Langmuir film for a surface pressure of 32 mN m-1, which correspond to the solid phase of the Langmuir film. The average number of AzoPTCD molecules confined per micrometer square on the subphase can be estimated by the product of the AzoPTCD solution (1.44 × 10-10 mol L-1) × Avogadro’s number × volume of the solution spread divided by the area occupied by the Langmuir film in its condensed phase in micrometers square. The last step before starting Z-deposition of the LB was to allow time (∼15 min) to achieve stability of the Langmuir film in its solid phase. The film is considered to be stable if the barrier moves less than 1 mm in 5 min keeping the pressure constant at 32 mN m-1. The LB monolayer was transferred to Corning 7059 glass slides half coated with silver islands using an electronically controlled dipping device, Lauda Film Lift FL-1 and keeping a constant pressure at 32 mN m-1. The LB monolayer was deposited previously by immersing the substrate into the water subphase, then spreading the solution, waiting for the solid-phase stabilized Langmuir film to be reached, and finally lifting the substrate at 3 mm min-1 (Z-deposition). The transfer ratio is defined as the area due to the displacement of the barrier to keep the surface pressure constant during the deposition divided by the area of the substrate covered during the deposition was calculated. The area due to the barrier displacement was found to be 150 mm (barrier width) × 18 mm (barrier displacement) ) 2.7 × 103 mm2 while the area of the covered substrate is 2[(50 × 25) + (50 × 1)] ) 2.6 × 103 mm2. The transfer ratio found from the relation between 2.7 × 103 and 2.6 × 103 mm2 was 1.0. Neat LB monolayers of AzoPTCD and AA were also deposited onto glass substrates half coated with silver islands. Resonance Raman scattering (RRS) spectra were recorded using the LB monolayer on glass and SERRS spectra using the LB monolayer on silver islands. The substrates half coated with silver island films were prepared by vacuum evaporation (10-6 Torr) onto a heated substrate at +200 °C. Films of 4-, 6-, and 10-nm mass thickness as determined by a quartz crystal balance were fabricated. The glass substrates were carefully cleaned before depositing the silver islands. They were kept for 30 min in the neutral detergent and then exhaustively washed with distilled and deionized water. The substrates were kept in a chromic acid solution for 30 min and then exhaustively washed with distilled and deionized water again and with acetone as the last step. Just before immersing the substrates into the subphase for LB preparation, they were washed with ultrapure water. Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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Table 1. Values of Wavenumber and fwhm for Neat AzoPTCD LB Monolayer on Glass (Neat LB/glass), Neat AzoPTCD LB Monolayer on Silver Islands (Neat LB/Ag), and Single-Molecule LB Monolayer (SM LB) temp/ °C

wavenumber/ cm-1

fwhm/ cm-1

neat LB/glass RRS

room temp

neat LB/Ag SERRS

+190

1291 1370 1290 1367 1291 1371 1293 1374 1295 1372

12 13 14 16 12 13 11 13 19 19

+10 -190 SM LB/Ag SERRS

room temp

Figure 1. SERRS spectra of a neat AzoPTCD LB monolayer on a 6-nm silver island film at +190, +10, and -190 °C.

RESULTS AND DISCUSSION The dye molecule selected for the SMD studies was AzoPTCD, which has a trans and a cis isomer.10 The cis isomer of AzoPTCD is shown in Figure 1. A detailed study of the absorption, emission, and SERRS imaging of neat AzoPTCD LB monolayers has been published.10 Although AzoPTCD is a large molecule with 168 fundamental vibrational modes, its Raman spectrum is quite simple and dominated by less than 10 vibrations of the perylene moiety. In particular, the two distinct and strongest bands at 1292 and 1371 cm-1 10 are the signature used in the SMD studies. The band at 1566 cm-1 is also a fingerprint for AzoPTCD; however, in the single-molecule LB monolayer, which contains the AzoPTCD probe molecule dispersed in AA matrix, the strongest signals in the SERS spectrum of the AA are also in this spectral region. The SERRS spectrum of the neat AzoPTCD LB monolayer is the reference for the SMD studies, and the published vibrational spectra10 was complemented here with a temperature dependence SERRS and SEF study of the neat LB monolayer within a range of temperature from liquid nitrogen to a maximum of +200 °C. The SERRS results are illustrated with three spectra recorded in these experiments as can be seen in Figure 1, for the spectral region that includes all the main Raman bands observed. From the simple peak-to-peak intensity of the Raman bands it is observed that the absolute intensity of the SERRS bands gains a 2-fold increase going from +190 to -190 °C. A small, consistent shift of band center to a higher wavenumber is also observed as shown in Figure 1. A slight increase in the full width at half-maximum (fwhm) with temperature in the SERRS spectra of neat LB films is expected,11 as seen in Table 1. The PTCD materials are characterized for strong excimer fluorescence12 that is also enhanced on metal island films, producing SEF.10,13 The spectra for neat AzoPTCD LB monolayer are presented in Figure 2 where SEF spectra at -100, +100, and +200 °C are shown. The inset in Figure 2 is a blowup of the section containing the SERRS spectra within the emission spectra. The absolute SEF intensity also (10) Constantino, C. J. L.; Aroca, R. J. Raman Spectrosc. 2000, 31, 887. (11) Sherwood, P. M. A. Vibrational Spectroscopy of Solids; Cambridge University Press: Cambridge, U.K., 1972. (12) Hochstrasser, R. M.; Nyi, C. A. J. Chem. Phys. 1980, 72, 2591. (13) Aroca, R.; Constantino, C. J. L. Langmuir 2000, 16, 5425.

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Figure 2. SERRS and SEF spectra of the neat AzoPTCD LB monolayer at +200, +100, and -100 °C. The inset shows the blowup for the SERRS.

Figure 3. SERRS spectrum of the single-molecule LB monolayer on a 6-nm silver island film and RRS spectrum of the neat AzoPTCD LB monolayer on glass.

increases with decreasing temperature; however, the rate is higher than that of SERRS. Using again the peak-to-peak intensities, the SEF signal increases by a factor of 4 going from +200 to -100 °C. Therefore, the SEF/SERRS intensity ratio increases with decreasing temperature from 47 at +200 °C, to 62 at +100 °C, and reaching 77 at -100 °C. An apparent linear increase in SEF and SERRS intensity with decreasing temperature is observed. The results presented in Figures 1 and 2 can be explained in terms of some softening in the molecular packing due to increasing temperature. Table 1 shows that the wavenumber of the most characteristic SERRS vibrational modes is comparable with the corresponding RRS values at the same temperature. It can also be concluded that the characteristic vibrations of the perylene ring are not strongly affected by the metal surface.

Figure 4. SERRS spectra of the single-molecule LB monolayer extracted from the mapping experimental results recorded at -100 °C.

Figure 5. SERRS line mapping of the neat AzoPTCD LB monolayer (top) and single-molecule LB monolayer (bottom).

Figure 3 presents a spatially resolved SERRS spectrum for a single molecule in an LB monolayer. There are significant differences between the single-molecule LB monolayer spectrum and the neat AzoPTCD LB monolayer on silver islands or glass spectra for all three band parameters: relative intensities, fwhm, and wavenumber. These differences are illustrated in Table 1. The 1372-cm-1 band is seen with a higher relative intensity than the 1295 cm-1 in the single-molecule LB monolayer spectrum. The

opposite is observed in all the RRS and SERRS spectra for a neat AzoPTCD LB monolayer. Notably, the calculated Raman intensities for perylene14 and our own computation of Raman intensities for AzoPTCD (HF/6-31G) give a larger absolute intensity for the band assigned to the 1372 cm-1 in comparison with 1295-cm-1 band. The fwhm of the single-molecule LB monolayer spectrum is larger than the fwhm of the neat LB monolayer SERRS or RRS spectra. The two characteristic SERRS bands of the singlemolecule LB monolayer are always observed at a higher wavenumber than the corresponding SERRS or RRS bands of the neat AzoPTCD LB monolayer at the same temperature. Figure 4 contains five SERRS spectra of the single-molecule LB monolayer extracted from the area mapping experiments carried out at -100 °C. It can be seen that for a single molecule the observed wavenumbers are slightly higher and show minor variations from spot to spot. The fwhm are found to be wider for the two characteristic bands of the single molecule in comparison with the corresponding bands seen in SERRS of the neat AzoPTCD LB monolayer at -100 °C. The changes in the wavenumbers observed on different spots of the sample as shown in Figure 4 are also indirect evidence of a single-molecule spectrum. Similar results were obtained with R6G in time-resolved single-molecule SERS spectra.5 Therefore, the higher wavenumber for characteristic bands may be attributed to a single molecule in the AA matrix, while the lower values of the Raman bands correspond to AzoPTCD aggregates. The increase in the fwhm in the singlemolecule spectrum is another expression of single-molecule SERRS against SERRS of aggregates. It is well known that the SERRS spectra of neat AzoPTCD contain a strong excimer emission signal due to SEF10 and that excimer emission (and SEF) can only be observed in the presence of aggregates.12 Fluorescence spectra were recorded exciting a neat AzoPTCD LB monolayer on silver islands with the 514.5-nm laser line and extending the scanning in the Stoke’s region to 9000 cm-1, where excimer emission (or SEF) is clearly observed. In the lowwavenumber section of the spectrum (100-1800 cm-1) the SERRS bands are also observed. The SEF mapping is shown in Figure 5

Figure 6. One global image for neat AA LB monolayer (top) and two global images for single-molecule LB monolayer, all recorded at 1370 cm-1.

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Figure 7. SERRS spectra, in time sequence, recorded on a single spatially resolved spot of the LB monolayer on silver containing 1 AzoPTCD molecule/µm2.

(top) where spectra were recorded with 2-µm step. Although the SEF signal is much stronger than the SERRS bands, the SERRS is evident in the spectrum. The mapping results of the singlemolecule LB monolayer on silver are at the bottom in Figure 5, where a weak SERRS is seen; but the fluorescence is not observed. Since excimer emission (and SEF) can be observed solely in the presence of aggregates, it may be concluded that the absence of any excimer emission in the single-molecule LB monolayer spectrum is experimental evidence for the lack of AzoPTCD aggregates in the single-molecule LB monolayer. Area mapping and global images were obtained for a singlemolecule LB monolayer containing ∼1 AzoPTCD molecule/µm2 of surface area, fabricated on silver islands films with mass thicknesses of 4, 6, and 10 nm used as SERS substrates. Some of the results are illustrated in Figure 6 for images collected at 1370 cm-1. Two selected global images are overlaid with one global image from a reference sample of the neat AA LB monolayer on 6-nm silver island film. All the global images collected from the neat AA LB monolayer on silver islands provide a flat background (top image). The single-molecule LB monolayer gave global images with sporadic high light intensity at 1370 cm-1. The high illumination center at 1370 cm-1 is attributed to the presence of the AzoPTCD molecule. The on and off behavior of the vibrational signal or large variations of their relative intensities have been dubbed “blinking”. Series of spectra were collected on a single spot of the single-molecule LB monolayer with a total collection time of 1 s and time interval ranging from 1 to 30 s. In all cases, blinking was observed. Spectra from these experiments are shown in Figure 7. The statistic of the experimental results for 30-s time intervals is as follows: (a) at -100 °C, ∼20% of the spectra result (14) Ong, K. K.; Jensen, J. O.; Hameka, H. F. J. Mol. Structure (THEOCHEM) 1999, 459, 131.

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in a well-defined signal of the two characteristic bands; (b) at room temperature also, ∼20% of the recorded spectra contain a “good signal”; (c) finally, at +100 °C, only ∼13% of the total spectra contain the single-molecule vibrational fingerprint. Single-molecule studies using dyes and the analytical technique presented here are ongoing in our laboratory to gain further insight into the properties of the single-molecule spectral behavior. CONCLUSIONS The single-molecule LB monolayer technique has been employed to achieve single-molecule detection of AzoPTCD using SERRS. The spectra attributed to a single molecule in the matrix of the fatty acid present the characteristic signature of singlemolecule detection: small variation of the wavenumbers for molecules on different sites; increase in the fwhm also differs from the SERRS spectra of AzoPTCD aggregates. Blinking has also been observed. It is demonstrated that single-molecule detection is attainable using spatially resolved surface-enhanced resonance Raman scattering and common SERS substrates such as silver island films thanks to signal enhancement produce by the resonance Raman effect, the surface enhancement, and high sensitivity of the new dispersive Raman instruments. ACKNOWLEDGMENT Financial assistance from the Natural Science and Engineering Research Council of Canada is gratefully acknowledged. C.J.L.C. acknowledges a fellowship from the Fundac¸ ˜ao de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) Brazil. Received for review February 15, 2001. Accepted May 24, 2001. AC0101961