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Deposition of Silver Dentritic Nanostructures on Silicon for Enhanced Fluorescence Krystyna Drozdowicz-Tomsia,*,† Fang Xie,‡ and Ewa M. Goldys† Macquarie UniVersity, Department of Physics & Engineering, Biotechnology Institute, North Ryde 2109 NSW, Sydney, Australia, and Imperial College London, Department of Materials, Exhibition Road, London, SW7 2AZ, United Kingdom ReceiVed: December 3, 2009; ReVised Manuscript ReceiVed: December 10, 2009
Silver dendritic (“fractal”) structures exhibit very high fluorescence enhancement, but their nonuniformity limits practical applications. Here, we present a novel, electroless deposition approach to produce uniform silver dendritic nanostructures on silicon for enhanced fluorescence. These metal-modified surfaces were allowed to bind a protein bovine serum albumin (BSA) labeled with the fluorescent dye Deep Purple. The effect of silver nanostructures on fluorescence intensity and lifetime in the presence of a conductive, absorbing substrate was analyzed, and high enhancement factors between 30 and 40 times were found. Such silicon-based structures with high fluorescence enhancement factors are suitable for integration with lab-on-chip and biosensing microelectronic devices. 1. Introduction Since the discovery of the Purcell effect,1 the efforts to increase the sensitivity of fluorescence detection have been focusing on control of the local electromagnetic (EM) environment of the fluorophores and taking advantage of the interaction between an emitter and its surroundings. Such optimized light emission/detection has been utilized in various situations, including in organic molecules,2 semiconductor device structures,3 photonic crystals, and more recently in life science applications.4-9 The dielectric surroundings have a profound influence on the emission of a fluorophore through its spontaneous emission rate, its angular emission pattern, and local modifications of the electromagnetic field. These effects have been explored in various scenarios by using metal nanostructures where effects such as fluorescence quenching,10-12 fluorescence enhancement,5,6,8,13-16 or both17 have been reported. The origins of such complex fluorescence effects near metal nanostructures arise from two contributions. First, the local changes of the electromagnetic field take place; these are induced by the nanostructures, by localized surface plasmon resonances (LSPR), or, in ultrathin planar metal films, by surface plasmon polaritons (SPP). Second, metal nanostructures are able to modify the radiative and nonradiative decay rates of nearby fluorophores, changing both the fluorescence lifetime and quantum yield. Additionally, these nanostructures can affect photostability, and increase the energy transfer.9 Earlier studies of surface enhanced fluorescence were carried out by using silver colloids immobilized on a glass surface, electroplated silver, roughened silver electrodes, as well as nanolithograhically produced structures.18-21 It has been shown experimentally and supported by theoretical calculations that the fluorescence enhancement factors of Au and Ag nanoparticles depend on the particle size,22,23 shape,24-26 the surrounding medium,27 as well as the particle arrangement geometry28,29 and distance between the metal and fluorophore.4,9,30 Moreover, * Corresponding author. Phone: +612 9850 7747. Fax: +612 9850 8115. E-mail:
[email protected]. † Macquarie University. ‡ Imperial College London.
surface enhanced fluorescence depends on the spectral overlap between the LSPR in metal nanostructures with spectral properties of the fluorophore. It is also affected by the locally modified EM field surrounding metal nanostructures that is strongly wavelength-dependent.31 Much of the recent work on surface enhanced fluorescence was carried out with silver islands deposited by the reduction of silver citrate.32 However, it is dendritic “fractal” silver structures that have been reported to produce the highest fluorescence amplification factors achieved so far, of up to a few hundred times.33,34 Such structures have a very complex geometry, which is expected to greatly influence their plasmonic properties. Although more complicated nanostructures with a variety of different shapes have been analyzed theoretically,35 none of these analyses can be easily extended to fractal structures. Growth of such fractal structures has been described by using the diffusion-limited aggregation model (DLA) of Witten and Sander36 and the cluster-cluster aggregation model,37 that introduce the scaling typical of fractal structures. This has implications for the understanding of surface enhanced fluorescence, because, in principle, such fractal structures can have a very high surface area and thus corrections to enhancement factors may be required, and other effects such as changes in lifetime must be considered.38 While silver fractals offer outstanding fluorescence enhancement factors, the main practical challenge in such structures is their nonuniformity of surface coverage. As growth initiation is intrinsically linked with nucleation on the surface, we hypothesize that the high quality surface of semiconducting silicon wafers offers the best opportunity for tight nucleation control. This feature should make it possible to produce both highly enhancing and relatively uniform surfaces. Moreover, it is very important to develop enhancing surfaces on conducting and semiconducting materials, as they are critically required in proteomic applications and offer easy integration in lab-on-chip for biosensors applications. 2. Experimental Section 2.1. Materials. The following materials were purchased from Sigma-Aldrich and used as received: silver nitrate (AgNO3),
10.1021/jp9114942 2010 American Chemical Society Published on Web 12/28/2009
Silver Dentritic Nanostructures 0.1% poly-L-lysine solution, acetic acid, bovine serum albumin (BSA), and phosphate buffered saline (PBS) tablets. Deep Purple (DP) total protein stain (based on epicocconone as its active ingredient) was purchased from GE Healthcare. A FluoroProfile Protein Quantification Kit also based on epicocconone was purchased from Sigma-Aldrich. Concentrated HF and methanol were obtained from J.T.Baker Inc. p-Type silicon wafers, borondoped (with a resistivity of 1-5 Ω cm), were purchased from MMRC Inc. Nanopure water (>18.2 MΩ), purified using the Millipore Mili-Q gradient system, was used in all experiments. 2.2. Formation of Silver Dendritic Nanostructures on Silicon. Silver nanostructures were produced on silicon wafers using self-assembled electroless deposition (galvanic exchange), as described in ref 39. The sample fabrication method was as follows: p-type commercial silicon wafers were cut into fragments of about 1 cm × 2 cm, which were first cleaned in acetone to degrease the Si surface, followed by etching in a diluted solution of 0.5% HF at room temperature for 10 min. The samples were rinsed in DI water and then in methanol and blow dried with compressed nitrogen. Following that, half of the area was masked with a chemical/temperature resistant 3M adhesive type and etched in a 5.0 mol/L HF solution containing 0.02 mol/L silver nitrate at 40 °C for 20 s, 30 s, 1 min, and 2 min, respectively. After the etching process, the tape was removed and silicon fragments were rinsed with deionized water and methanol alcohol and blown dry with nitrogen. 2.3. Fluorophore-Protein Conjugation Monolayer Formation. To ensure effective binding of BSA to silver fractal and silicon, the sample surface with/without silver fractal was derivatized with poly-L-lysine.40 To this aim, 50 µL of poly-Llysine solution (0.01% poly-L-lysine in 5 mM sodium phosphate buffer, pH 7.3) was added per well, incubated at room temperature for 1 h, and rinsed with water. Binding of the DPBSA monolayer to the surface was carried out by adding 50 µL of 10 µg/mL BSA solution per well and incubating overnight at 4 °C, followed by rinsing in 0.01 M PBS buffer to remove the unbound BSA molecules. Deep Purple fluorophore was then conjugated to BSA monolayers by adding 50 µL of 1:200 Deep Purple buffered solution at pH 7 to the wells and incubation of the modified substrate for 1 h at room temperature.15 Unconjugated Deep Purple was removed by rinsing the substrate with a solution of 15% methanol and 7.5% acetic acid.41 Methods. The samples partially covered with silver fractal (prepared as described elsewhere in this article) were patterned by an adhesive tape with three punched holes (5 mm diameter) to form wells on the surface. The size of the wells corresponded to the well size routinely used in 96-well plates for highthroughput screening. Their position was such that the first hole was located on silicon fully covered with a silver fractal, the second one coincided with a region covered in half by the silver fractal while the other half was unprocessed, and finally the third one was located entirely on the unprocessed silicon. In the samples used for fluorescence lifetime imaging microscopy (FLIM) measurements, only the middle well (half fractal/half silicon) was exposed to BSA and DP by incubation in the respective solutions, leading to formation of a 4 nm thick protein monolayer.15 The other two wells were incubated with BSA only to establish the fluorescence background used as a reference. For spectroscopy measurements, the samples were prepared in the same way, with the only difference that the wells were located in areas with and without silver fractal, both incubated with BSA-DP monolayers. Separate control samples with the BSA layer only were prepared for each fractal growth time to enable background subtraction. Finally, a control sample
J. Phys. Chem. C, Vol. 114, No. 3, 2010 1563 with a monolayer of BSA-DP on glass was produced, for comparison with an unprocessed silicon sample to be able to calculate the radiative rate of DP on silicon as explained further. The BSA-DP fluorescent monolayer makes it possible to quantitatively compare the fluorescence intensity of fluorophore-protein conjugates with and without various silver fractal nanostructures on silicon (after background signal subtraction and correction for differences in surface coverage). The fluorescence enhancement factor Ef is defined as Ef ) [(Efractal/DP - Efractal/BSA)/(ESi/DP - ESi/BSA)](cSi/DP/cfractal/DP), where Efractal/DP is the fluorescence intensity of BSA-DP on silver fractal, Efractal/BSA is the background fluorescence of BSA on fractal, ESi/DP is the fluorescence intensity of BSA-DP on silicon substrate, and ESi/BSA is the background fluorescence of BSA on silicon. The correction factors cfractal/DP and cSi/DP allow one to adjust for differences in the total amount of BSA deposited on various surfaces, that affects the number of conjugated fluorophore molecules and the measured fluorescence. To correct for differences in the total amount of BSA protein deposited on various surfaces, the supernatant of each well was collected by pipetting out, after overnight incubation and before the rinsing of the wells in 0.01 M PBS buffer to separate test-tubes. The same amount collected from each test tube (no less than 30 µL) was diluted to a total volume of 0.5 mL. The samples of unbounded BSA prepared in this way for all surfaces under investigation were quantified using the FluoroProfile Protein Quantification Kit from Sigma-Aldrich according to technical protocols accompanying this product. The results of this procedure and the coverage correction factors used in this study can be found in the Supporting Information. 2.4. Characterization. Scanning electron microscopy (SEM) images of the samples were collected using an environmental scanning electron microscope FEI XL 30. The fluorescence emission spectra were taken by a Fluorolog Tau 3 system from Jobin-Yvon-Horiba with 450 W Xe lamp excitation. All spectra were corrected for the spectral response, and long pass filters were used to eliminate the contribution from the scattered excitation light. The samples with DP-BSA were excited at 520 nm, and their fluorescence was measured in the range 540-650 nm using 5 nm slits by spatially averaging the fluorescence from sample regions of ∼1 mm × 2 mm. Simultaneous fluorescence intensity and lifetime measurements were taken by using a Leica SP2 MP confocal laser scanning microscopy system from Leica Microsystems equipped with a FLIM system based on a timecorrelated single photon counting (TCSPC) module with 256 × 256 channels from Becker & Hickl (SPC 830). The excitation was provided by a 405 nm pulsed laser diode modulated at 40 MHz. The fluorescence signal passed through the airy pinhole (114 µm) and was collected by a 10 × 0.7 NA air objective. After passing through a 450 nm long pass filter, the signal was detected by a PMC-100-0 detector from Becker & Hickl, that reliably measures lifetimes above 100 ps. The lifetime data were corrected for the instrument response function (IRF). The fluorescence intensity data were corrected by subtracting the number of counts measured for silicon and silver fractal surfaces with BSA monolayer only and without DP fluorophores. The lifetime analysis used the smallest number of lifetime components, which gives a good fit to the fluorescence decay data for individual pixels at the FLIM image. In all cases, two lifetime components were sufficient for a good quality fit of the fluorescence decay data. In order to calculate the emission enhancement for each sample, we used the value at the maximum of averaged lifetime distribution τm. We also measured the average lifetime of the control silicon sample covered
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Figure 1. Sample preparation steps: (a) patterning of the silicon sample with an adhesive tape, (b) silver fractal formation in HF/AgNO3 solution, (c) application of poly-L-lysine to the sample surface, (d) patterning of the substrate to create separate wells in three regions (pure silicon, half silicon/half silver fractal, silver fractal only), (e) application of 10 µg/mL BSA to all three wells and Deep Purple dye conjugation to BSA in the well over half silicon/half silver fractal. Three sets of samples were prepared for each silver fractal growth time to provide statistically valid data.
with a BSA-DB monolayer, where the value of average lifetime τ ) 1.37 ( 0.02 ns was obtained. Specular reflectance spectra were measured by using a Cary 5000 UV-visible/NIR spectrophotometer from Varian Inc., equipped with a 12.5° reflectance unit in the spectral range 200-2000 nm. 3. Results and Discussion 3.1. Fractal Formation. The formation of silver deposits via an electroless deposition can be understood on the basis of a self-assembled localized microscopic electrochemical cell model.39,42 In this model, initially, both silicon etching and silver deposition occur simultaneously at the Si surface where Si-Si bonds in the crystal lattice act as a reducing agent for the Ag+ ions in solution. The Ag+ ions in the vicinity of the silicon surface capture electrons from the valence band in silicon and form a deposit of silver nuclei that are uniformly distributed on the surface of the silicon wafer. With further deposition, SiO2 begins to be formed under these silver nanoclusters43 and it is subsequently etched away by HF. The silver nanoclusters and the surrounding Si areas act, respectively, as local cathodes and anodes, leading to electroless dendritic growth of silver deposits. The process can be expressed as two half-cell reactions 1 and 2:
Ag+ + e- f Ag(s) and
(1)
Si(s) + 6F- f SiF62- + 4e-
(2)
As the process of silver deposition continues, the surrounding silicon (anodes) is being etched, while silver nanoclusters (cathodes) are preserved and continue to grow with a fern-treelike geometry with a complex nanoscale architecture. The evolution of surface morphology is shown in the SEM images (Figure 2). The images are presented at different
magnifications to show the most frequently observed shapes at various growth times; these have different length scales as the silver fractal growth progresses. Initially, clean and smooth surfaces which have been stripped of the surface oxide in diluted HF are becoming rough with a honeycomb-like structure over which silver nuclei are formed with relatively uniform size in the range 50-100 nm (Figure 2A). Some of these nuclei remain isolated, but most start to form elongated nanoparticles at first and evolve toward small ramified clusters (Figure 2A,B). Figure 3 shows that silver nanoclusters deposited for only 20 s already show a variety of different shapes including oblate, prolate, and also some triangular prisms. As the deposition process continues (Figure 2D-F), tree-like three-dimensional (3D) silver structures are formed with clearly defined branches that continue to grow further away from the surface, and finally detach from it almost completely. Close inspection of these branches indicates that their thickness is almost constant over the entire aggregate and each section is composed from agglomerated small particles of similar size to the initial nuclei, as also observed in ref 44. At later stages of deposition (Figure 1E,F), the initial, isolated nuclei are no longer observed and the structures form a dense 3D deposit. The “fractal” character of such dendritic silver structures was confirmed by testing the fractal dimension using the “box count” definition. In this approach, the image of the structure is covered by square boxes that vary in size from 2 to 64 pixels. The number of such boxes N and their size s are used to calculate the fractal dimension D according to the formula
D ) lim(-log(Ns)/log s) sf0
The fractal dimensions D obtained for various structures are 1.74 for 20 s, 1.78 for 30 s, 1.84 for 1 min, and 1.89 for 2 min of silver deposition on silicon. It has to be noted that beyond 2 min of silicon etching/silver deposition process the silver fractal layer starts to separate from the substrate. The structures thus prepared were examined by confocal microscopy prior to the deposition of a BSA monolayer, to verify that the observed fluorescence characteristics are not attributed to fluorescent porous silicon. Fluorescent porous silicon is produced by a similar method but at much longer processing times, between 2 and 48 h.45 No visible fluorescence was observed for our very short etching/deposition times at any excitation wavelength, ruling out this possibility. Our attempts to directly measure extinction spectra to establish the position of plasmon resonances for these samples were unsuccessful, as the silicon substrate used in our experiments is absorbing. Also, the plasmon resonance was not conclusively observed by specular reflectance and for all silver fractal growth times the spectral responses were very close to those characteristic for bulk silver layers. Our theoretical calculations shown in Figure 4 for various nanoparticle sizes on glass substrate and an incident angle of 12.5° indicated that it is very difficult to observe plasmon resonance features in specular reflectance for silver nanoparticles larger than 50 nm. Such larger particles will dominate reflectance spectra for all fractal structures produced in this study. 3.2. Effect of Silver Nanostructures on Nearby Fluorophores. To establish the impact of such silver nanostructures on nearby fluorophores, we used the DP-BSA conjugate, based on earlier reports that albumin proteins spontaneously bind to glass and silver surfaces to form a complete monolayer about 4 nm thick.15 The amine groups (from poly-L-lysine) were used
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Figure 2. SEM images of the silicon wafer with silver dendrites grown at 50 °C by self-assembled electroless deposition at various times and different magnifications: (A) 20 s, (B) 30 s, (C) also 30 s but shown at low magnification, (D) 1 min, (E) 1 min but a different region and lower magnification than part D, (F) 2 min. Magnification used: 10000× for A and D (bar represents 1 µm), 5000× for E and F (bar represents 5 µm), 13000× for C (bar represents 10 µm), and 27000× for B (bar represents 500 nm).
Figure 4. Calculated specular reflection spectra for the nanoparticles of various sizes on glass and the illumination angle of 12.5°.
Figure 3. High magnification SEM image of the silicon wafer with silver dendrites deposited for 20 s.
to facilitate strong bonding of BSA to silicon, as per previously reported protocols.40 Additionally, poly-L-lysine, which forms a monolayer of about 7 nm thickness,46 acts as an additional separation layer between silver fractal and fluorophore. Such combined spacing of about 10 nm provides the optimum condition for metal plasmon mediated fluorescence enhance-
ment47,48 by minimizing nonradiative decay channels, which otherwise can lead to fluorescence quenching at small
