Silica nanoparticles as a tool for fluorescence collection efficiency enhancement
© Krajnik et al.; licensee Springer. 1900
Received: 23 September 2012
Accepted: 8 March 2013
Published: 28 March 2013
In this work we demonstrate enhancement of the fluorescence collection efficiency for chlorophyll-containing photosynthetic complexes deposited on SiO2 spherical nanoparticles. Microscopic images of fluorescence emission reveal ring-like emission patterns associated with chlorophyll-containing complexes coupled to electromagnetic modes within the silica nanoparticles. The interaction leaves no effect upon the emission spectra of the complexes, and the transient behavior of the fluorescence also remains unchanged, which indicates no influence of the silica nanoparticles on the radiative properties of the fluorophores. We interpret this enhancement as a result of efficient scattering of electromagnetic field by the dielectric nanoparticles that increases collection efficiency of fluorescence emission.
KeywordsSilica nanoparticles Fluorescence collection efficiency Fluorophores Light-harvesting complex 87.64.K- 87.85.jf 77.22.-d
One of the most commonly used approaches to tune the fluorescence properties of fluorophores is to couple them to plasmonic excitations in metallic nanoparticles . Large variations of shapes and sizes of metallic nanostructures provide almost infinite space for spectral engineering of optical properties of emitters, ranging from control of the fluorescence intensity, fluorescence decay dynamics, as well as the emission spectrum itself. Remarkable effects of plasmon coupling have been demonstrated on a single-molecule level, where a fluorophore was approached in a controllable way by a spherical metallic nanoparticle . For large distances, the emission remained unaffected; however, as the separation decreased, a strong enhancement of the fluorescence emission has been measured. Upon further reduction of the separation between the fluorophore and metallic nanoparticle, the intensity of the fluorescence emission decreased rapidly. This result demonstrates allimportant effects of plasmon coupling in such experimental configuration, and they are associated with modifications of fluorescence quantum yield of the fluorophore, enhancement of its excitation rate, and quenching due to nonradiative energy transfer to the metallic nanoparticle. As these processes compete against each other, in order to achieve strong enhancement of the fluorescence intensity, it is crucial to put attention to the geometry of the hybrid plasmonic nanostructure, in particular to the control of the separation between fluorophores and metallic nanoparticles.
The concept of coupling fluorescent molecules with metallic nanoparticles has been then extended to other nanostructures, such as multichromophoric photosynthetic pigment-protein complexes . In this case the distance between metallic nanoparticles and proteins was controlled via silica layers with defined thickness. It has been shown that depending upon actual arrangement of the hybrid nanostructure, it is possible to obtain strong enhancement of the absorption rate  or increase of the fluorescence rate  in such a system. Importantly, in order to determine which of the two processes is responsible for the observed enhancement of the fluorescence, it is necessary to combine standard steady-state experiment with time-resolved fluorescence spectroscopy .
Another method applied to increase the fluorescence of molecules is based on applying dielectric nanospheres . Such structures feature strong magnetic resonances, thus can be used for changing emission of molecules that feature not only magnetic but also electric dipole moment . On the other hand, such nanoparticles are characterized with high refractive index; therefore, placing them between collection optics and emitters results in improvement of optical resolution and collection efficiency [9–14]. One of the examples is a solid immersion lens , frequently a hemispherical macroscopic lens made of high-refractive-index glass (n = 1.84 and n = 1.69 in ), using of which can yield a significant (factor of n) increase of the optical resolution. It has also been shown that solid immersion lenses can be applied for high-resolution imaging of semiconductor structures at cryogenic temperatures . On the other hand, application of dielectric nanoparticles has been discussed in the context of enhancing optical response in the infrared as well as in the visible spectral range. It has been shown that for the emission of a single molecule placed onto a surface of a dielectric microsphere, it is possible to observe up to fivefold enhancement of the fluorescence intensity when such a structure is illuminated with a Gaussian beam . This effect was attributed to strong confinement of the electromagnetic field near the particle. Importantly, dielectric nanostructures have been also suggested as an efficient source of absorption enhancement in solar cell architectures due to creation of whispering gallery modes by properly chosen illumination . All these findings point towards a broad range of possibilities of introducing spherical dielectric nanoparticles for controlling the optical properties in many applications. In addition, it has been shown that such nanoparticles can be coated with metallic islands for enhanced Raman scattering [15, 16].
In this work we focus on hybrid nanostructures composed of photosynthetic complexes and spherical silica nano(micro)spheres. By fluorescence microscopy and spectroscopy, we demonstrate almost a threefold enhancement of the fluorescence emission of the photosynthetic complexes located near the dielectric spheres. The results suggest that the enhancement factor depends upon the size of nanoparticles. The spectral shape as well as dynamic behavior of the emission remains unchanged upon coupling with the nanospheres; therefore, we attribute the observed enhancement as being due to enhanced efficiency of light collection from molecules in the vicinity of the silica nanoparticles.
Peridinin-chlorophyll-protein (PCP) photosynthetic molecules were obtained according to the protocol by Miller et al. . Briefly, PCP apoprotein in 50 mM Tris-HCl (pH 8.0) solution was added to 25 mM tricine and 10 mM KCl (pH 7.6), mixed with a stoichiometric amount of PCP pigments dissolved in ethanol. The sample was held in 4°C for 72 h. Reconstituted samples were equilibrated to 5 mM tricine with 2 mM KCl (pH 7.6) by passage through a PD-10 column and bound to a column of DEAE Trisacryl (Sigma-Aldrich, St. Louis, MO, USA). Reconstituted PCP was then removed with 5 mM tricine with 2 mM KCl (pH 7.6) containing 0.06 M NaCl. The protein solution was characterized by absorption and fluorescence spectroscopy.
All reagents for silica nanoparticle synthesis were purchased and used as received from the indicated suppliers: nitric acid, hydrochloric acid, ammonium hydroxide (25%), and glucose from Chempur (Karlsruhe, Germany); potassium hydroxide and ethanol from POCh (Gliwice, Poland); tetraethylorthosilicate from Sigma-Aldrich (St. Louis, MO, USA); and silver nitrate from Lach-ner (Neratovice, Czech Republic). Deionized water was purified to a resistance of 18.2 MΩ (HLP 5UV System, Hydrolab, Hach Company, Loveland, CO, USA) and filtered using a 0.2-μm membrane filter to remove any impurities. All glassware and equipment were first cleaned in an aqua regia solution (3:1, HCl/HNO3) and rinsed with ultrapure water prior to use. All solutions were prepared under stirring and/or sonication, using 18.2 MΩ cm of ultrapure water. Silica particles with diameters of 250 nm to 1.1 μm and low dispersities were prepared using a variation of the method developed by Stöber et al. . The obtained nanoparticles were characterized by scanning electron microscopy and absorption spectroscopy.
The samples for fluorescence measurements were prepared by spin-coating the solution of silica nanoparticles onto a clean microscope cover slip. For that purpose, equal volumes of nanoparticle solution were mixed with PCP solution at a concentration of 2 μg/mL. After that, a solution of the PCP complexes was deposited on the nanoparticles. Alternative approach of mixing both samples prior to spin-coating was used, and the results were qualitatively identical.
Absorption spectra were recorded on a Varian-Cary 50 UV-visible spectrophotometer (Palo Alto, CA, USA). Steady-state fluorescence measurements were performed using a FluoroLog 3 spectrofluorometer (Jobin Yvon) equipped with a double grating monochromator. A xenon lamp source was used for excitation, and the signal was detected with a thermoelectrically cooled photomultiplier tube with a dark current less than 100 cps.
Fluorescence intensity maps were measured with a Nikon Eclipse Ti inverted wide-field microscope (Tokyo, Japan) equipped with Andor iXon Du-888 EMCCD (Belfast, UK) with a dark current 0.001 e-/pix/s at −75°C. The excitation was provided by a LED illuminator with a central wavelength of 480 nm. In order to narrow down the excitation beam spectrally, we used in addition a band-pass filter, FB480-10. The beam was reflected with a dichroic beam splitter (Chroma 505DCXR, Rockingham, VT, USA) to the microscope objective (Plan Apo, ×100, oil immersion, Nikon). The excitation power of illumination was about 60 μW. Fluorescence intensity maps of the PCP complexes were obtained by filtering the spectral response of the sample with a band-pass filter (Chroma HQ675-20).
Measurements of fluorescence spectra and decays were carried out using our home-built fluorescence microscope based on the Olympus long working distance microscope objective LMPlan ×50, NA 0.5 . First of all, silica nanoparticles were localized on the sample surface using the scanning mode of the microscope, and then from selected points corresponding to the emission of the PCP complexes placed close to the silica nanoparticles, spectra and decays were measured. For the reference, we also measured a similar set of data from areas away from the nanoparticles. The excitation was provided by a picosecond pulsed laser at 485 nm with an excitation power of 60 μW at a repetition rate of 50 MHz. The fluorescence spectra were measured by dispersing the emission using an Amici prism and detecting the spectrum with a CCD detector (Andor iDus DV 420A-BV). Fluorescence decays were obtained using a time-correlated single-photon counting approach, with a fast avalanche photodiode as the detector. The emission of the PCP complexes was extracted using a band-pass filter, HQ675-20.
Results and discussion
We find that coupling of photosynthetic, chlorophyll-containing complexes with dielectric silica nanoparticles leads to an enhancement of the fluorescence emission. The interaction leaves no measurable effect on the shape of the emission as well as on the transient behavior of the fluorescence. We conclude that the effect of fluorescence enhancement originates from high scattering of electromagnetic field by dielectric nanoparticles that leads to improvement of the collection efficiency. Although several aspects of the results described in this work are still to be understood completely, the experiment is a vital step towards assembling functional nanostructures that exploit enhancement effects associated with dielectric nanoparticles.
electron-multiplying charge-coupled device
The research was supported by the WELCOME project ‘Hybrid Nanostructures as a Stepping Stone Towards Efficient Artificial Photosynthesis’ funded by the Foundation for Polish Science and the EUROCORES project ‘BOLDCATS’ funded by the European Science Foundation. MG-R, PN, and BJ acknowledge the partial support by the Polish Ministry of Science and Higher Education (Poland) under grant no. OR00 005408 (2009–2011).
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