In the Laboratory
Surface-Enhanced Fluorescence on Silver Fractal-Like Structures An Experiment for Analytical or Physical Chemistry Abby Roth, Tanya Shtoyko,* Brian K. Taylor, and Arika Pravitasari Department of Chemistry, The University of Texas at Tyler, Tyler, TX 75799; *
[email protected] Zygmunt Gryczynski, Evgenia G. Matveeva, and Ignacy Gryczynski Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas, Health Science Center, Fort Worth, TX 76107 I-Fen Chang Department of Cell Biology and Genetics, University of North Texas, Health Science Center, Fort Worth, TX 76107
This laboratory experiment is designed to introduce undergraduate students in analytical or physical chemistry to the processes that occur when a molecule fluoresces near metal nanostructures. Students will gain knowledge about surfaceenhanced fluorescence and understand why certain noble metals can enhance fluorescence. The experiment also shows how
fractal-like structures
silver fractals can be electrochemically grown using an applied potential. Students will measure the enhanced fluorescence signals on fractals that are advantageous in medical diagnostics and imaging applications. In the experiment, the silver fractal structures are grown electrochemically through the reduction of silver metal (1). The setup needed to grow the fractals is shown in Figure 1. Two pieces of silver foil that serve as the anode and the cathode are “sandwiched” between two glass slides. The center is filled with distilled water and a power source is used to apply a 200 V potential to the structure. Once the potential is applied, oxidation begins occurring at the anode, specifically, at the edge of the silver:
silver cathode
deionized water
glass slides
silver anode
Figure 1. The sandwich setup for electrochemical growth of silver fractal-like structures on glass slides.
S2 S1 S0 IC
R
T2 T1 ISC1 ISC0
R
fluorescence phosphorescence
R absorbance of UV–vis light
ground state Figure 2. A Jablonski diagram of a fluorescent molecule. The dashed lines show the vibrational and rotational relaxation (R) and the dotted lines show the internal conversions (IC) and the inter-system conversions (ISC).
Ag0(s)
− Ag+(aq) + 1e
(1)
After the silver is oxidized, it travels to the cathode where it is reduced:
− Ag+(aq) + 1e
Ag0(s)
(2)
After the silver has been reduced, the fractal structure begins to form. The fractal structures grow from the cathode to the anode and are monitored visually. Once the fractal structure has reached the anode, it stops growing and an increase in current is observed. The growth process is stopped when a sufficient fractal structure is grown or once the fractal structure meets the anode. A fluorescence microscope or a confocal microscope can be used to measure the fluorescence enhancement on the silver fractal structures. However, it is recommended that a confocal microscope be used to achieve better resolution and contrast. When a molecule absorbs ultraviolet or visible light it is excited from its ground electronic state (S0) to an excited electronic state (S1), shown as a blue arrow in Figure 2. For aromatic molecules the ground state is typically a singlet state labeled S0 and the excited singlet states with increasing energy are labeled S1, S2, … . For these molecules the allowed S0 → S1 transition is typically a π → π* excitation (1). There are also lower-energy triplet exited states labeled T1 and T2. The S0 → T1 transition is not allowed owing to the change in spin. When the molecule is in the excited state S1 the molecule will quickly lose energy through vibrational and rotational relaxations (R) until it reaches the lowest level of the singlet excited state (S1). There are several possible ways for the molecule in the electronic state
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 6 June 2009 • Journal of Chemical Education
715
In the Laboratory empty conduction band
S1 to lose energy:
1. If the energy of the molecule is equal to the excited vibrational energy of its ground state S0, the molecule will go through an internal conversion (IC) to the ground state S0. Once in its ground state, the molecule will release the remaining energy nonradiatively through vibrations and rotations until it reaches the lowest level of its ground state S0. 2. The molecule can release the energy by emitting a photon in a process known as fluorescence, thus bringing the molecule back to the ground state S0. This transition can be to various vibrational ground states, which results in a broad emission band rather than a line. This process is studied in this article. 3. It can go through an inter-system crossing (ISC1) to the triplet excited state (T1). Once in the triplet state, the molecule will release energy through vibrations and rotations until it reaches the lowest level of the triplet state. Here the molecule can either release a photon through phosphorescence or undergo another inter-system crossing (ISC0) to the excited vibrational or rotational level of the ground state (S0). If phosphorescence occurs it is usually much weaker than fluorescence (2). Also, phosphorescence is a radiative process occurring at longer wavelengths than fluorescence (2). A diagram of these processes is shown in Figure 2.
Many fluorescent dyes can be used to measure the fluorescence enhancement on the silver fractal structures. Owing to its availability, Rhodamine B is chosen for this experiment. Rhodamine B is also used because its structure is favorable for fluorescence. The molecule is rather rigid, or stiff, because it has three fused rings. This hinders the molecule from vibrational and rotational transitions that make IC favorable. Thus, most of the energy absorbed will be released through fluorescence, which causes an increase in quantum yield (2) and decrease in excited-state lifetime. Quantum yield, ϕ, is the ratio of the rate of total photons emitted to the rate of total photons absorbed, per second, and can be expressed as kr ϕ = (3) knr + kr where knr represents the non-radiative decay rate due to vibrational and rotational relaxations, IC, and ISC and kr represents the radiative decay rate due to fluorescence (2). The quantum yield is always less than 1. The lifetime of the excited state, t, is the time needed for the population of the excited state to decay to 1/e of its initial value, either radiatively or non-radiatively (2): 1 t = (4) knr + kr Increasing the radiative rate kr causes the quantum yield to increase (eq 3) thus increasing the number of photons emitted. This in turn increases fluorescence signal (brightness). Some noble metals such as silver and gold can enhance fluorescence (1–3). The enhancement is seen owing to the phenomenon of localized surface plasmon resonance, or LSPR (4). A surface plasmon (SP) is an electromagnetic wave that moves along the interface boundary between a metal and an insulat716
gap
Energy
∙ e flow
filled valence band
single atom
nanostructure
bulk metal
Density of States Figure 3. Energy diagram for a single atom, a nanostructure, and bulk metal.
ing, or dielectric, common boundary (3). LSPR is the collective oscillation of conduction electrons in phase with incident light (5). For LSPR to occur, the metal must be in the form of a nanostructure such as a nanosphere or a fractal structure. The nanostructure allows for an energy gap to appear between the conduction bands and the valence bands as presented in Figure 3. This gap allows the free oscillating electrons of the metal to interact with the excited-state fluorophore. If the metal is present in bulk or in the form of a single atom, the gap between the conduction band and the valence band does not exist or is so small that quenching is seen instead of enhancement (6, 7). When looking at the scenario for a single atom, only one energy level is present, thus no band gap exists. Therefore, a single atom can not support LSPR. When looking at the situation involving nanostructures, there is a band gap present between the valence bands and conduction bands. This allows for electron (e−) flow and therefore LSPR to occur. When looking at the state of a bulk metal, the band gap is not present and LSPR does not occur. The interaction of light with metallic subwavelength particles results in a locally enhanced electromagnetic field. Molecules located in this region will be strongly excited. For example, the Raman scattering can be significantly stronger in the presence of metallic nanoparticles. In surface-enhanced Raman scattering (SERS) the signals are manifold stronger (8, 9). In the case of fluorescence, two effects should be considered. First, the locally enhanced field will provide stronger excitation. Second, the excited fluorophore (often approximated as oscillating dipole) interacts with the nanoparticles, which results in rapid radiation of the excitation energy. This can be interpreted as an increase in the radiative decay rate (10, 11). When dealing with nanostructures, the interaction between the surface plasmons and the excited-state fluorophores causes the lifetime of fluorescence, tm, to decrease and the quantum yield, ϕm, to increase,
ϕm =
tm =
kr + kr ′ knr + kr + kr ′ 1 knr + kr + kr ′
(5)
(6)
Journal of Chemical Education • Vol. 86 No. 6 June 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Figure 4. The laser scanning confocal microscope images of two samples of Rhodamine B on glass slides with fractal-like structures: (A) and (B) fluorescence images of Rhodamine B prepared on two glass slides with fractal-like structures; (C) and (D) transmission images of Rhodamine B on the glass slide with fractal-like structures; (E) and (F) superimposed fluorescence and transmittance images of Rhodamine B carried out on the glass slide with fractal-like structures.
Figure 5. Images (A) and (B) show fluorescence images of Rhodamine B prepared on the glass slide with fractal-like structures. Graphs (C) and (D) plot fluorescence intensities due to Rhodamine B measured at the cross-section of the glass slide with fractal-like structures shown as white lines in Figures 5A and 5B, respectively.
where the rate due to LSPR is designated as kr′. When no LSPR occurs kr′ = 0 and eqs 5 and 6 become eqs 3 and 4, respectively. In eqs 5 and 6, there is an additional term kr′ in the denominator that represents the radiative decay rate due to LSPR near the metal (when the free oscillating electrons located on the surface of the metal interact with the excited-state fluorophores). The contribution of the radiative decay due to LSPR near the metal in eq 5 also causes the quantum yield to increase, thus causing an increase in the total number of photons emitted (12, 13). The contribution of the radiative decay due to LSPR near the metal in eq 6 causes the lifetime to decrease.
Hazards
Experiment
Discussion
The silver fractal structures were synthesized on glass slides treated with SnCl2. The structures were produced by applying an electrical potential between two silver electrodes (13, 14). Once the structures were synthesized, they were spin coated with a solution of Rhodamine B in polyvinyl alcohol. This procedure is described in more detail in the online material. The method of synthesizing the fractal structures electrochemically yielded a non-uniform nanocomposite structure. These non-uniform nanocomposite structures form different surface plasmonic modes that can interact to produce collective surface plasmons. The formation of the collective surface plasmons yields a more productive fluorescence signal when comparing it to a more uniform structure such as nanospheres (15). This method of producing the silver fractals also produced areas of small localized plasmon activity (1, 3). These areas are referred to as “hot spots” and produced the highest enhancement in the fluorophore emission (1, 3, 15). The fractals amplify fluorescence also because there is a high metal surface area due to fractals special architecture (1, 3). Fractals have a high aspect ratio and their sharp tips act also as antennas for radiating fluorophores (3).
Two typical images obtained by students from the confocal microscope are shown in Figure 4. The fluorescence images were acquired with a laser scanning confocal microscope (Zeiss 410). The excitation wavelength was 488 nm and the emission was collected in the red Rhodamine channel. A fluorescence microscope can be also employed to obtain the images; however, lower resolution is expected. The black areas in the fluorescence images represent the low fluorescent signal in Figures 4A and 4B. The black areas are located where the dye, Rhodamine B, is placed on glass surface. The red areas in the fluorescence images (Figures 4A, 4B, 4E, 4F) represent high fluorescence signal. The red areas are located where the dye is placed on the fractal-like structures. The green areas seen in the transmittance images (Figures 4C, 4D, 4E, 4F) represent areas of high transmittance. These areas contain only the dye, Rhodamine B, on the glass surface. The gray and black areas (Figures 4C and 4D) represent areas of low transmittance and can be seen where the fractal structures along with the dye are present. The average hot spot and background signals were obtained by the students from the images in Figure 5, which shows the
Nochromix is a strong oxidizer and can cause skin, eye, and respiratory irritation and may be harmful if swallowed. Tin(II) chloride dihydrate is corrosive and can cause skin and eye burns. It can cause respiratory irritation and may be harmful if swallowed. Rhodamine B may be harmful if swallowed and can cause eye, skin, and respiratory tract irritation. Polyvinyl alcohol granules are moderately combustible and should be kept away from an open flame. During the experiment, safety goggles and gloves should be worn at all times to prevent injury.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 6 June 2009 • Journal of Chemical Education
717
In the Laboratory
fluorescence images along with cross section signals of the image. The cross section can be obtained using the software for a microscope. The students used MetaMorph 6.1 (Molecular Devices, Downingtown, PA) to obtain the signal from the corresponding cross sections shown below the fluorescence images in Figure 5. The cross section is taken such that it contains areas where there is only Rhodamine B present on the glass surface. This is done so the average background signal, or the fluorescence signal due to just the dye, can be obtained. The average background signals were 14 and 20 in Figures 5C and 5D, respectively. The average hot spot signal can be found by averaging the fluorescence signal measured on fractals. The average hot spot signals shown in Figure 5C and 5D were 164 and 142, respectively. By dividing the average hot spot signal by the average background signal, the fluorescence enhancement on the silver fractal structures was found. The average enhancement that was seen by students was 7–12. The students’ results also showed that an enhancement is present on the silver fractal structures, but it is a non-uniform signal. The non-uniform signal is seen because of the production of different surface plasmonic modes that resulted from the nonuniform nanocomposite structure.
4. Willets, K. A.; Van Duyne, R. P. Annu. Rev. Phys. Chem. 2007, 58, 267–297. 5. Wiley, B. J.; Im, S. H.; Li, Z.-Y.; McLellan, J.; Siekkinen, A.; Xia, Y. J. Phys. Chem. B 2006, 110,15666–15675. 6. Link, S.; El-Sayed, M. A. J. Phys. Chem. 1999, 103, 8410–8426. 7. Prashant, K. J. Phys. Chem. 2002, 106, 7729–7744. 8. Fleischmann, M.; Hendra, P. J.; McQuillan, A. J.; Chem. Phys. Lett. 1974, 26, 163–166. 9. Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Curr. Sci. 1999, 77, 915–924. 10. Lakowicz, J. R. Anal. Biochem. 2001, 298, 1–24. 11. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Kluwer Academic/Plenum: New York, 2006; pp 841–859. 12. Geddes, C. D.; Parfenov, A.; Roll, D.; Fang, J.; Lakowicz, J. R. Langmuir 2003, 19, 6236–6241. 13. Geddes, C. D.; Parfenov, A.; Roll, D.; Gryczynski, I.; Malicka, J.; Lakowicz, J. R. J. Fluorescence 2003, 13, 267. 14. Parfenov, A.; Gryczynski, I.; Malicka, J.; Geddes, C. D.; Lakowicz, J. R. J. Phys. Chem. B. 2003, 107, 8829–8833. 15. Karpov, S. V.; Gerasimov, V. S.; Isaev, I. L.; Markel, V. A. J. Chem. Phys. 2006, 125, 1–4.
Literature Cited
Supporting JCE Online Material
1. Shtoyko, T.; Matveeva, E.; Chang, I-Fen.; Gryczynski, Z.; Goldys, E.; Gryczynski, I, Anal. Chem. 2008, 80, 1962–1966. 2. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Kluwer Academic/Plenum: New York, 2006; p 3. 3. Goldys, E.; Xie, F. Sensors 2008, 8, 886–896.
Abstract and keywords
718
http://www.jce.divched.org/Journal/Issues/2009/Jun/abs715.html Full text (PDF) with Figures 2, 4, and 5 in color Supplement Instructor notes
Journal of Chemical Education • Vol. 86 No. 6 June 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
