Controlling FRET Enhancement Using Plasmon Modes on Gold

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Controlling FRET Enhancement Using Plasmon Modes on Gold Nanogratings Jennifer M Steele, Chae M Ramnarace, and William R Farner J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07317 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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The Journal of Physical Chemistry

Controlling FRET Enhancement using Plasmon Modes on Gold Nanogratings Jennifer M. Steele,* Chae M. Ramnarace, and William R. Farner Department of Physics and Astronomy, Trinity University, One Trinity Place, San Antonio, Texas, 78212.

ACS Paragon Plus Environment

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ABSTRACT The optical properties of structured metal surfaces and nanoparticles can be engineered to influence the fluorescence properties of nearby quantum emitters through the manipulation of the local density of optical states (LDOS). Applying these techniques to Förster resonance energy transfer (FRET) is appealing, but has proven to be a complicated and debated issue. In this paper, surface plasmons modes for a gold nanograting are found to enhance the FRET efficiency between nearby donor and acceptor molecules. Nanogratings support traveling surface plasmon waves with a broad range of wavelengths that follow a dispersion relationship allowing for increases in the LDOS at targeted portions of the spectra of FRET paired molecules. Nearby excited fluorescent molecules may decay by launching a surface plasmon wave that couples into free-space light, which can be recovered. With this system, we measured the FRET efficiency for different plasmon wavelengths spanning both the donor absorption and acceptor emission spectra. The increase in efficiency was found to be greatest when the surface plasmon modes, and therefore the increase in LDOS, overlapped the acceptor emission spectrum.


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Introduction Förster resonance energy transfer (FRET) describes the nonradiant, dipole-dipole transfer of energy between an excited fluorescent molecule, termed the donor, and an acceptor fluorescent molecule when the distance between donor and acceptor molecules is much less than the wavelengths of light used. The transfer of energy depends on the spectral overlap between donor emission and acceptor absorption, the relative orientation of the two dipole moments of the fluorescent molecules, and the distance between the donor and acceptor molecules.1

The

distance dependence scales as r-6, making FRET useful in many biological applications that involve changes in molecular conformations,2,3 photosynthesis,4 as well as other applications such as photovoltaics.5 It has been well documented that the emission of a fluorescence molecule located near a metal nanoparticle or metal surface can be modified by surface plasmon modes. Metal enhanced fluorescence (MEF) is achieved through two pathways – an excitation enhancement and an emission modification.6-11 If surface plasmon modes overlap the absorption spectrum of the fluorophore, the enhanced electromagnetic field induced by the surface plasmon will increase the excitation of the fluorophore. Surface plasmon modes that overlap the emission spectrum of the fluorophore increase the local density of optical states (LDOS).12 A fluorophore in the excited state may then decay by either emitting a photon or exciting a surface plasmon. Fermi’s Golden Rule states that this increase in decay channels will cause an increase in the emission rate (the inverse of the lifetime) of the excited state of the fluorophore. This has the effect of increasing the emission. These two mechanisms have been well documented experimentally for nanoparticles8,13 and structured metal surfaces.14,15 Applying MEF to FRET has many potential advantages, chief of which is the ability to enhance the energy transfer rate The number of systems that FRET can be applied to is limited by the Förster radius, R0, defined as the distance between the donor and acceptor molecule where the energy transfer probability is 50%.1 For typical FRET pairs, this distance is between 1-10 nm. Extending this range by increasing the FRET transfer rate will expand the usefulness of FRET. Both experimental and theoretical work, however, have so far shown a less than clear picture of how the LDOS effects the FRET rate and efficiency. Förster theory expresses the FRET rate as


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Γ = Γ , where Γ is the decay rate of the donor absent the acceptor.12 Because the decay rate of the donor depends on the LDOS at the donor emission, this predicts a linear dependence of rate on LDOS. This straightforward relationship has not been borne out by experiment or theory, as studies have found that the LDOS can increase both the FRET rate and efficiency,16-18 that the LDOS only increases the FRET efficiency,19,20 and that the LDOS does not affect the FRET rate or efficiency.21,22 That the photonic environment has a complex relationship to the FRET and efficiency is not too surprising if one considers change in the LDOS of the donor molecules from the acceptor molecules. The presence of the acceptor molecules increases the decay rate of the donor molecule even in the absence of an engineered increase in the LDOS from a plasmonic structure. Introducing plasmon modes at wavelengths overlapping the donor emission can either compete with the FRET process if donors preferentially decay to free space light, or contribute to it if the modes increase the correlation between donor and acceptor. Metal nanogratings provide a unique plasmonic substrate to study the effect of the LDOS on FRET efficiencies as a function of wavelength. Unlike nanoparticles, antennae, and apertures which support single,22,23 often broad16 localized plasmon resonances, surface plasmon modes on gratings are traveling waves that follow a dispersion relationship. The dispersion relationship allows for a broad range of surface plasmon wavelengths that can be accessed individually. In this work, we measure the FRET efficiency for a range of surface plasmon wavelengths spanning both the donor and acceptor emission spectra. This will allow us to compare FRET efficiencies by increasing the LDOS at donor emission wavelengths, acceptor emission wavelengths, as well as between those two spectral regions in contrast to previous studies that focus only on plasmon modes that overlap the donor emission. We are able to do this on a single substrate, reducing uncertainty that comes from ensemble measurements or sample to sample variations in fluorophore concentrations. FRET between a donor and acceptor molecule can be characterized by the FRET rate, Γ , and the efficiency, EFRET. The decay rate for an isolated donor molecule, Γ , is comprised of both radiative and non-radiative rates, the latter being Ohmic losses to the environment. In the presence of acceptor molecules, energy transfer to the acceptors provide an additional decay channel, increasing the donor decay rate: Γ = Γ + Γ .

The FRET rate is therefore


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Γ = Γ − Γ . The FRET rate may also be thought of in terms of rate equations. If the probability of a donor molecule is in its excited state is given by D1, the probability that an acceptor molecule is in its ground state is A0, with kT as a measure of the dipole-dipole coupling strength, then the transfer rate is simple k .20 The efficiency is defined as the probability energy is transferred to the acceptor molecule: =

=

(1)

The efficiency will only increase if the FRET rate is increased more than the donor decay rate. Unlike nanoparticles, surface plasmon excitations on gratings are traveling waves of electrons confined to the surface of the metal. The surface plasmons may be excited by TM-polarized light, with the wavelength of the traveling wave for a particular excitation energy determined by the dispersion relationship.24,25 For a smooth metal film, the dispersion relationship is: =

!" !#

!" !#

/&

$

(2)

where = 2(⁄) is the wavevector of the surface plasmon along the grating, + is the frequency dependent dielectric function of the metal, +& is the dielectric function of the medium surrounding the metal, ω is the frequency of the incident light, and c is the speed of light. Both corrugated films and slotted gratings can be thought of as a small permutation of smooth metal films.24,25 Although the surface plasmon dispersion lies beyond the light cone for smooth films, the presence of a periodic structure allows for the direct excitation by TM-polarized light through momentum matching.14,24,25 For light incident at an angle θ to the surface normal, only the component of light parallel to the surface may excite a surface plasmon. The periodicity of the grating allows for an integer number of reciprocal lattice vectors to be added to the wavevector along the grating surface: ,-./0 = 1234 ± 67 where =

&8 9

(3)

and λ the wavelength of the incident light in vacuum. The reciprocal lattice

vector is given by 7 =

&8 :

, where d is the period of the grating and m is the diffraction order.

When ksurface equals ksp from Equation 1, a surface plasmon will be excited on the grating surface


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with a wavelength equal to ) = 2(⁄ . The dispersion relationship therefore defines sets of wavelengths and angles required to excite surface plasmons for a particular grating. The dispersion of the surface plasmons launched on gratings is what allows for the separation of the two mechanisms of MEF. The first mechanism relies on a plasmon being excited by the excitation laser, which then creates an enhanced electromagnetic field. Plasmons will only be excited when the laser is incident on the grating at the angle determined by the dispersion relationship. Experiments have shown that this enhancement can be turned on an off by simply changing the incident angle of the excitation laser.14

For the second mechanism, excited

fluorophores may channel their energy into a surface plasmon excitation. This energy can couple back out of the nanograting as photons, and the light will beam out at an angle determined by the wavelength of the plasmon.14,15,26 By sweeping a detector at various angles around the grating, fluorescence from the second mechanism can be isolated.

By measuring the

fluorescence emission as a function of detector angle we can effectively track the effect on the emission and FRET efficiency as the surface plasmon wavelength shifts through both the donor and acceptor emission spectra, changing the LDOS. This can be done on a single grating using the same coating of fluorescent molecules, eliminating variations between individual nanoparticles or any uncertainty introduced from measurements of ensembles.

Experimental Methods Gold nanowire gratings with a period of 500 nm and a height of 50 nm were produced using a template stripping method. Silicon master patterns with a period of 500 nm and a 220 nm wire width were purchased from LightSmyth Technologies. Stamps were created from 184 polydimethylsiloxane (PDMS) using a typical mix of monomer plus 10% by weight curing agent, then baked at 70° C for about one hour. The PDMS stamp is a relief pattern, so the expected transferred wire width is approximately 300 nm. 50 nm of gold was sputtered onto the PDMS stamps at a low deposition rate. A thin layer of five-minute epoxy was spread onto clean glass microscope slides. The stamps were placed gold side down onto the epoxy. After 10 minutes of curing, the PDMS stamps were peeled off the glass substrate, leaving the gold wires behind. Sample quality was measured with atomic force microscopy (AFM), as seen in Figure 1.


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The width of the transferred wires was measured to be 330 ± 40 nm. An exact measurement of the height was complicated by the underlying epoxy.

Figure 1. AFM micrograph of a gold nanograting. 50 nm of gold was deposited onto a PDMS stamp formed from a silicon master grating with a period of 500 nm. AFM cross-sections show a width of 330 ± 40 nm for the wires. The surface plasmon dispersion relationship was measured with white light transmission. Polarized, collimated white light from a 100 W tungsten halogen bulb was used. The sample was mounted on a rotating sample holder. Data was taken for light incident from 0° to 50° from the surface normal in one degree increments. Light was collected with an optical fiber and recorded using a linear silicon CCD array detector from Ocean Optics (USB4000). The fiber was placed directly behind the sample to collect the zeroth order transmission. The dispersion shown in Figure 2 shows two plasmon modes, excited on both sides of the grating. From Equation 2, when the incident angle is zero, the plasmon waves are excited only by the reciprocal lattice vector of the grating (7 = 2(⁄< ). The feature starting at ~500 nm can therefore be associated with the air side first order (m = 1) plasmon. The feature starting at 750 nm then splitting into a blue and red moving feature can be associated with the first order (m = 1) glass side plasmon. This feature is red shifted from the grating period by the index of refraction of the glass substrate (n = 1.5). The white light absorption was taken with a bare grating.


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Figure 2. White light percent transmission spectrum. Transmission was recorded as a function of wavelength for a range of incident angles. Plasmons appear as a peak followed by a dip in transmission.

Lines showing the first order air (solid) and first order glass (dashed) slide

plasmons are shown to guide the eye. For FRET measurements, Atto-532 and Atto-633 (Sigma-Aldrich) were chosen as the donor and acceptor molecules respectively.

The fluorescent molecules were loaded into a 5% PVA

aqueous solution and spin cast on to the gratings at a speed of 3000 RPM for 30 seconds. The gratings were then heated at 70° C on a hotplate for about 10 minutes to fix the coating, shown schematically in Figure 3(b). To remove the PVA coating, a 10% aqueous solution by weight of PVA was dropped onto the sample, and heated at 50° C until fixed. This layer could be easily peeled off, stripping the fluorophore loaded coating with it, making it possible to reuse the same grating for different donor and acceptor concentrations.

In this paper, all fluorescence

measurements were taken with the same grating.


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Figure 3. (a) Schematic showing the optical set up to measure the fluorescence emission at a variety of angles relative to the surface normal. (b) Schematic showing the grating with a PVA coating loaded with the donor and acceptor molecules. (c) Spectra of the FRET pair on a blank microscope slide. The maximum emission from the donor is 555 nm and the maximum emission from the acceptor is 650 nm. The small peak at 532 nm is the laser leaking through the high pass filter. Fluorescence measurements were made on a custom-built setup that allowed for varying both the incident angle of the laser as well as the angle of fluorescence collection, shown in Figure 3(a). A 532 nm diode laser provided the excitation of the donor. The incident laser was fixed at an angle of φ = 40° with respect to the sample normal, avoiding any plasmon excitation from the grating at this angle and wavelength combination, verified by the dispersion relationship shown in Figure 2. The same detector was used to collect fluorescence as the white light experiment. The fiber was mounted on a swing arm and rotated from θ = -5° to 20° to the sample normal in 1° increments. A 550 nm high pass filter was used to filter out the excitation light. Clean, blank


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microscope slides were coated with the same fluorophore loaded PVA layer to provide the baseline measurements for enhancement calculations. An example of the emission spectra of the FRET paired molecules on a glass slide is shown in Figure 3(c). The maximum emission for the donor and acceptor molecules is 555 nm and 651 nm respectively. The sharp drop-off is from the long pass filter, and the small peak at 532 nm is a portion of the laser light leaking through the filter.

A slight variance in fluorescence was measured at different detector angles,

normalization scans were therefore taken for the same range of angles as the grating fluorescence measurements.

Results and Discussion A grating was prepared as described above, with concentrations of 75µM and 70µM for the donor and acceptor molecules respectively loaded in the PVA film. The excitation enhancement mode or MEF is turned off by the choice of angle for the excitation laser. Any enhanced fluorescence therefore comes solely from the increase in LDOS provided to the excited fluorophores by plasmon modes on the grating. These plasmons may decay into free-space light, but only by beaming out of the sample at the wavelength dependent angle defined by the dispersion relationship. The effect of this enhancement on the emission spectra can be seen in Figure 4 which shows the emission spectra for a range of detector angles. When the detector is located at the surface normal, the surface plasmon available at this angle has a wavelength of 616 nm. This is redshifted from the expected 500 nm because of the thin PVA coating resulting in the effective index of refraction above the grating to be greater than one. The blue arrow in Figure 4 shows the location of this plasmon. As the detector is rotated from the surface normal, the plasmons split into red-shifting and blue-shifting features as seen in Equation 2 and Figure 2. The colored arrows show the progression of surface plasmon peaks as the detector angle is changed; the asymmetry in the movement between the red and blue shifting peaks is due to the spectra being plotted as a function of wavelength and not energy. The individual spectrum can therefore be thought of as the typical emission of the system punctuated with an enhancement at a particular wavelength. Similar results have been obtained with spherical nanoparticle dimers.27 However, unlike a previous study on different sized nanorods22 that only observed spectral modification for plasmons with energy lower than the intrinsic emission peak, we observe


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spectral modification for plasmons both lower and higher in energy than the intrinsic emission peak for the acceptor. We suspect the same is true for the donor, but the high-pass filter put in place to block the laser light also blocks the higher energy portion of the donor emission.

Figure 4. Normalized fluorescence spectra recorded at different detector angles for a grating with a PVA layer containing 75 µM and 70 µM concentrations of donor and acceptor molecules respectively. The arrows locate the surface plasmon wavelength for each angle obtained from the enhancement plots. The spectra were normalized to the number of counts at the donor maximum emission wavelength, 555 nm, for clarity. In order to isolate and measure the enhancement from the gratings, fluorescence was measured for samples with an identical PVA coating on a blank glass slide for the same detector angles. Dividing the enhanced fluorescence by the background yield spectra like the ones shown in Figure 5(a). Here the MEF is plotted for a detector angle of 0°, 4°, and 8°. The surface plasmons are easily identified as 616 nm, 638 nm and 599 nm, and 665 nm and 575 nm for each of the detector angles respectively. This grating shows a modest enhancement of 2X. Additional samples have shown enhancements from 2-10X depending on sample and fluorophore concentration. The enhancement data is plotted in Figure 5(b) as a surface plot, clearly showing the two plasmon branches. These plots are used to determine the surface plasmon wavelengths at each detector angle. Because they are taken with the PVA layer on the grating, any redshifting from the effective index of refraction is accounted for.


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Figure 5. Enhancement data for a grating with a PVA layer loaded with 75 µM and 70 µM of donor and acceptor molecules. Enhancement is measured by dividing the emission spectrum from a grating sample by the emission spectrum from the same concentration of donor and acceptor fluorescent molecules spin cast on a blank glass microscope slide. (a) Enhanced fluorescence for a selection of detector angles. (b) Fluorescence enhancement plot for all detector angles measured between 0° and 12°. The ability to measure the fluorescence as the plasmon wavelength moves through the emission spectra of the donor and acceptor molecules allows for a detailed investigation of both the MEF of the donor and acceptor fluorophores and any change in efficiency in the FRET process between the two fluorophores as a function of plasmon wavelength. It is clear from Figure 4 that the overall fluorescence of the donor and acceptor is enhanced when a plasmon mode is available


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near the emission peak. To determine if there is any change in the efficiency in the FRET process, the efficiency, E, was calculated using: =

= >=?@

(3)

= >=?@ A

where FD and FA are the integrated fluorescence of the donor and acceptor respectively, and B = CD /C accounts for the difference in quantum yields. The quantum efficiencies reported for these molecules are QD = 90% and QA = 64%. Any direct excitation of acceptor molecules is accounted for in the term ED:0 . A small amount of fluorescence was measured, typically less than 5% of the FRET pair acceptor emission. In order to separate the donor and acceptor emissions, measurements were taking with 75 µM of donor molecules loaded into the PVA film with no acceptor molecules on the same grating. Fluorescence emission was measured for the same range of detector angles as the FRET system. To account for slight variations in counts for each emission spectrum, the spectra were normalized to the maximum counts of the donor at 555 nm. The donor only spectrum was then subtracted from the FRET pair spectrum at each detector angle. An example of the spectrum is shown in Figure 6 for a detector angle of 8°. To obtain FD, the donor only spectrum was numerically integrated, and to obtain FA, the difference spectrum was numerically integrated starting from 600 nm. Atto 633 has no emission below 600 nm, and omitting these wavelengths will ensure the slight variances in the difference spectrum for the donor emission will not affect the calculation of FA.

Figure 6. Corrected and normalized spectra for the same grating with a PVA layer containing both the donor and acceptor molecules, just the donor molecules, and then the difference


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between the two. The detector angle for this plot was 8°. For 8°, the red-shifting plasmon is located at 575 nm, where an additional peak is clearly seen. The blue-shifting plasmon is located at 664 nm, and therefore is not as visible as a separate peak, but as a taller and wider single peak. Table 1 summarized the results of this analysis. For each angle, the wavelength of the available plasmon modes are given along with the ratio of donor to acceptor fluorescence and the efficiency of FRET transfer. For comparison, an identical analysis was performed for the same FRET pair concentrations loaded PVA film spun cast on a clean glass slide. For the blank sample, the difference in fluorescence and efficiency did not vary appreciably with angle, as expected. The average ratio was measured to be 0.92 with E = 60%. Angle (degrees)

Blue Plasmon (nm)

Red Plasmon (nm)

FD/FA

E

0

616

616

0.80

64%

2

607

630

0.80

65%

4

599

638

0.71

66%

6

586

651

0.68

68%

8

575

664

0.75

65%

10

564

677

0.84

63%

11

558

683

0.85

62%

12

553

688

0.75

65%

Table 1. FRET efficiency and fluorescence ratio a grating coated in a PVA layer containing 75 µM and 70 µM concentrations of donor and acceptor molecules respectively. The increased LDOS provided by the grating has a clear effect on the efficiency of FRET between the donor and acceptor. Looking at Equation 3, in order to increase the efficiency of the FRET pair, it is necessary to increase Γ more than Γ . When the detector is at 0° to the surface normal, the plasmon wavelength is 616 nm, which is far off the maximum emission wavelength for both the donor (555 nm) and the acceptor (651 nm) molecules. However, the increase in fluorescence is greater for the acceptor molecule, driving down the ratio FD/FA and increasing the efficiency slightly. As the plasmon splits into a blue and red shifting modes, the plasmon wavelengths start to significantly overlap the emission peaks. The greatest increase in


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efficiency occurs when the detector angle is 6° from normal.

The red-shifting plasmon

wavelength is located at the acceptor maximum emission, and the blue-shifting wavelength overlaps the donor emission. The effect is to increase the fluorescence from the acceptor, driving down the ratio FD/FA and achieving an efficiency of 68%, 13% greater than what was measured without the gold grating. As the plasmon wavelengths shift further, the blue-shifting nears the maximum donor emission as the red shifting plasmon moves away from the maximum emission of the acceptor. This causes the efficiency to decrease. As the blue-shifting plasmon moves close to the high pass filter cut-off, the increase in fluorescence for the donor will be filtered out as well. The increase in efficiency measured from a detector angle of 12° is likely to be artificially high because the filter blocks some of the increase of FD, artificially decreasing the ratio FD/FA. Typical fluorescent measurements are not made at discrete angles. To simulate measuring the fluorescence with a microscope objective with a modest numerical aperture, the spectrum from all scans with a detector angle between 0° and 14° were added together for grating samples with both the donor and acceptor and just the donor molecules. The normalized results plus the difference spectrum are plotted in Figure 7. Remarkably, the modulations of the spectra at individual angles are smoothed out, and the FRET emission spectrum does not appear significantly different than Figure 3(c) except that the relative peak heights and widths of the donor and acceptor emission are altered. Calculating the integrated fluorescence as described above yields FD/FA = 0.79 and a FRET efficiency of 64%. The overall effect of the grating therefore causes an increase in FRET efficiency.


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Figure 7. Sum of all spectra resulting from detector angles between 0° and 14° taken from the same grating with a PVA layer loaded with both the donor and acceptor molecules, just the donor molecules, and the difference between those two spectra. The above data indicates that the FRET efficiency is enhanced more when the LDOS is increased at wavelengths overlapping the acceptor emission. To test this in a less idea case, a PVA film loaded with 75µM and 40µM concentrations of the donor and acceptor molecules was spin cast onto the same grating as described above, cutting the number of acceptor molecules in about half.

The fluorescence enhancement for this sample is shown in in Figure 8(a).

Normalized spectra from a range of detector angles is shown in Figure 8(b), with arrows indicating the plasmon wavelengths. The overall effect is the same, with the native emission spectra punctuated with enhancements at particular wavelengths for each detector angle. The same analysis was performed on the spectra, and the results can be found in Table 2. Analysis was also performed for an identical PVA layer loaded with 75µM and 40µM concentrations of the donor and acceptor molecules on a blank glass microscope slide. For this sample, FD/FA = 1.6 and E = 46%.


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Figure 8. Fluorescence data for a PVA layer loaded with 75µM and 40µM of donor and fluorescent molecules. (a) Fluorescence enhancement plot for all detector angles measured between 0° and 12°. (b) Normalized fluorescence spectra recorded at different detector angles for a grating with a PVA layer containing 75µM and 40µM concentrations of donor and acceptor molecules. The arrows locate the surface plasmon wavelength for each angle. The plasmon mode available at 0° is 633nm, which is slightly redshifted from the 616nm in the last data set. This difference is due to a slightly thicker layer of PVA, causing an increase in the effective index of refraction above the gratings. The difference is only about 3% - from n = 1.23 to n = 1.27.

This is a well-known aspect of surface plasmon excitations on films and

nanoparticles – that small changes in the local index of refraction result in large changes in the surface plasmon wavelength is why these metal structures are often used as sensors.28-30 In this case, the shift is fortuitous in that at 0° the plasmon wavelength is just shy of the maximum emission of the acceptor, and does not overlap the donor emission. As the angle is increased to


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2°, the red-shifting plasmon matches the maximum emission of the acceptor, while the blueshifting plasmon still mostly overlaps the emission of the acceptor. This leads to the largest increase in FRET efficiency, a 23% increase over the efficiency of the blank sample. As the blue-shifting plasmon moves into the emission of the donor, the FRET efficiency decreases as both the fluorescence from the donor and acceptor are increased. For 10° and 12°, the blueshifting plasmon significantly overlaps the emission of the donor, and the red-shifting plasmon has moved to the far red side of the acceptor emission.

This yields the lowest FRET

enhancement. This data clearly shows that increasing the LDOS at wavelengths overlapping the acceptor emission causes an increase in the FRET efficiency, while increasing the LDOS at wavelengths overlapping donor emission can decrease the FRET efficiency. Adding the spectrum from 0° to 12° as before, FD/FA = 1.2 and E = 52%. As before, the grating overall increases the FRET efficiency.

Angle (degrees)

Blue Plasmon (nm)

Red Plasmon (nm)

FD/FA

E

0

633

633

1.2

55%

2

622

653

1.1

57%

4

609

667

1.2

53%

6

598

679

1.4

50%

8

584

692

1.7

45%

10

572

703

1.6

46%

12

561

716

1.5

46%

Table 2. FRET efficiency and fluorescence ratio a grating coated in a PVA layer containing 75 µM and 40 µM concentrations of donor and acceptor molecules respectively.

Conclusions


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The FRET efficiency was measured for Atto 532 and Atto 633 molecules located near a gold nanograting. By collecting the fluorescence as a function of detector angle, the FRET efficiency was calculated as a function of plasmon wavelength. The dispersion of the plasmons excited on the gratings allowed for an increase in the LDOS at the donor emission wavelengths, the acceptor wavelengths, and a combination of the two. The overall effect of the gratings was found to increase the FRET efficiency. The largest increases in efficiency occurred when plasmon wavelengths strongly overlapped the acceptor emission. When the LDOS is increased at the acceptor emission, the lifetime of the acceptor excited state is decreased, leading to a greater number of acceptor molecules in the ground state, making FRET more likely to occur. Plasmons overlapping just the donor emissions showed the lowest enhancement of the FRET efficiency. When the LDOS is increased at the donor emission, it competes with the FRET channel. We also found that the increase in efficiency was larger for samples containing fewer acceptor molecules. This seems to support the results found for nanoapertures and antennae that found a greater increase in both the FRET rate and efficiency for molecules that were spaced further apart,16,17 although more work is necessary to explore this trend.


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AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. Funding Sources This work was supported by an award from the Research Corporation for Science Advancement and from the W.M. Keck Foundation Undergraduate Education Program. REFERENCES (1) (2) (3) (4)

(5)

(6) (7) (8) (9)

(10) (11) (12)

Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, NY, 2006. Weiss, S. Measuring Conformational Dynamics of Biomolecules by Single Molecule Fluorescence Spectroscopy. Nat. Struct. Biol 2000, 7 (9), 724–729. Jares-Erijman, E. A.; Jovin, T. M. FRET Imaging. Nat. Biotechnol. 2003, 21 (11), 1387– 1395. Hildner, R.; Brinks, D.; Nieder, J. B.; Cogdell, R. J.; Van Hulst, N. F. Quantum Coherent Energy Transfer Over Varying Pathways in Single Light-Harvesting Complexes. Science 2013, 340 (6139), 1448–1451. Hardin, B. E.; Hoke, E. T.; Armstrong, P. B.; Yum, J.-H.; Comte, P.; Torres, T.; Fréchet, J. M. J.; Nazeeruddin, M. K.; Grätzel, M.; McGehee, M. D. Increased Light Harvesting in Dye-Sensitized Solar Cells with Energy Relay Dyes. Nat. Photon. 2009, 3 (7), 1–7. Ming, T.; Chen, H.; Jiang, R.; Li, Q.; Wang, J. Plasmon-Controlled Fluorescence: Beyond the Intensity Enhancement. J. Phys. Chem. Lett. 2012, 3 (2), 191–202. Aslan, K.; Malyn, S. N.; Geddes, C. D. Metal-Enhanced Fluorescence From Gold Surfaces: Angular Dependent Emission. J. Fluoresc. 2007, 17 (1), 7–13. Zhang, Y.; Dragan, A.; Geddes, C. D. Wavelength Dependence of Metal-Enhanced Fluorescence. J. Phys. Chem. C 2009, 113 (28), 12095–12100. Lakowicz, J. R.; Ray, K.; Chowdhury, M.; Szmacinski, H.; Fu, Y.; Zhang, J.; Nowaczyk, K. Plasmon-Controlled Fluorescence: A New Paradigm in Fluorescence Spectroscopy. Analyst 2008, 133 (10), 1308–1346. Lakowicz, J. R. Radiative Decay Engineering 3. Surface Plasmon-Coupled Directional Emission. Anal. Biochem. 2004, 324 (2), 153–169. Lakowicz, J. R. Radiative Decay Engineering 5: Metal-Enhanced Fluorescence and Plasmon Emission. Anal. Biochem. 2005, 337 (2), 171–194. Novotny, L.; Hecht, B. Principles of Nano-Optics, 2nd ed.; Cambridge University Press: Cambridge, U.K 2012.


20

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22) (23)

(24) (25)

(26)

(27)

(28) (29)

Aslan, K.; Malyn, S. N.; Zhang, Y.; Geddes, C. D. Conversion of Just-Continuous Metallic Films to Large Particulate Substrates for Metal-Enhanced Fluorescence. J. Appl. Phys. 2008, 103, 084307. Steele, J. M.; Gagnidze, I.; Wiele, S. M. Efficient Extraction of Fluorescence Emission Utilizing Multiple Surface Plasmon Modes From Gold Wire Gratings. Plasmonics 2010, 5, 319–324. Hure, R.; Simoneau, S.; Chandler, B. D.; Steele, J. M. Two-Color Metal-Enhanced Fluorescence Using Gold Slotted Nanogratings. Plasmonics 2016. DOI 10.1007/s11468016-0426-8 Ghenuche, P.; Mivelle, M.; de Torres, J.; Moparthi, S. B.; Rigneault, H.; Van Hulst, N. F.; García-Parajó, M. F.; Wenger, J. Matching Nanoantenna Field Confinement to FRET Distances Enhances Förster Energy Transfer Rates. Nano Lett. 2015, 15 (9), 6193–6201. Ghenuche, P.; de Torres, J.; Moparthi, S. B.; Grigoriev, V.; Wenger, J. R. M. Nanophotonic Enhancement of the Förster Resonance Energy-Transfer Rate with Single Nanoapertures. Nano Lett. 2014, 14 (8), 4707–4714. Ren, J.; Wu, T.; Yang, B.; Zhang, X. Simultaneously Giant Enhancement of Förster Resonance Energy Transfer Rate and Efficiency Based on Plasmonic Excitations. Phys. Rev. B 2016, 94 (12), 125416–125418. Blum, C.; Zijlstra, N.; Lagendijk, A.; Wubs, M.; Mosk, A. P.; Subramaniam, V.; Vos, W. L. Nanophotonic Control of the Förster Resonance Energy Transfer Efficiency. Phys. Rev. Lett. 2012, 109 (20) 203601-1-5. Schleifenbaum, F.; Kern, A. M.; Konrad, A.; Meixner, A. J. Dynamic Control of Förster Energy Transfer in a Photonic Environment. Phys. Chem. Chem. Phys. 2014, 16 (25), 12812–12817. Konrad, A.; Metzger, M.; Kern, A. M.; Brecht, M.; Meixner, A. J. Controlling the Dynamics of Förster Resonance Energy Transfer Inside a Tunable Sub-Wavelength Fabry-Pérot-Resonator. Nanoscale 2015, 7 (22), 10204–10209. Zhao, L.; Ming, T.; Shao, L.; Chen, H.; Wang, J. Plasmon-Controlled Förster Resonance Energy Transfer. J. Phys. Chem. C 2012, 116 (14), 8287–8296. Bidault, S.; Devilez, A.; Ghenuche, P.; Stout, B.; Bonod, N.; Wenger, J. Competition Between Förster Resonance Energy Transfer and Donor Photodynamics in Plasmonic Dimer Nanoantennas. ACS Photonics 2016, 3 (5), 895–903. Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag, Berlin, Germany, 1988. Steele, J. M.; Moran, C. E.; Lee, A.; Aguirre, C. M.; Halas, N. J. Metallodielectric Gratings with Subwavelength Slots: Optical Properties. Phys. Rev. B 2003, 68 (20), 2051031–2051037. Jiang, Y.; Wang, H. Y.; Weng, H.; Gao, B. R.; Hao, Y. W.; Jin, Y.; Chen, Q. D.; Sun, H. B. Surface Plasmon Enhanced Fluorescence of Dye Molecules on Metal Grating Films. J. Phys. Chem. C 2011, 115 (25), 12636–12642. Ringler, M.; Schwemer, A.; Wunderlich, M.; Nichtl, A.; Kürzinger, K.; Klar, T. A.; Feldmann, J. Shaping Emission Spectra of Fluorescent Molecules with Single Plasmonic Nanoresonators. Phys. Rev. Lett. 2008, 100 (20), 681–684. Mayer, K. M.; Hafner, J. H. Localized Surface Plasmon Resonance Sensors. Chem. Rev. 2011, 111 (6), 3828–3857. McFarland, A. D.; Van Duyne, R. P. Single Silver Nanoparticles as Real-Time Optical


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(30)

Page 22 of 22

Sensors with Zeptomole Sensitivity. Nano Lett. 2003, 3 (8), 1057–1062. West, J. L.; Halas, N. J. Engineered Nanomaterials for Biophotonics Applications: Improving Sensing, Imaging, and Therapeutics. Annu. Rev. Biomed. Eng. 2003, 5, 285– 292.

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Controlling FRET Enhancement Using Plasmon Modes on Gold

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