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Abstract
Silver nanowires with varying diameters and submillimeter lengths were obtained by changing a reducing agent used during hydrothermal synthesis. The control over the nanowire diameter turns out to play a critical role in determining their plasmonic properties, including fluorescence enhancement and surface plasmon polariton propagation. Advanced fluorescence imaging of hybrid nanostructures assembled of silver nanowires and photoactive proteins indicates longer propagation lengths for nanowires featuring larger diameters. At the same time, with increasing diameter of the nanowires, we measure a substantial reduction of fluorescence enhancement. The results point at possible ways to control the influence of plasmon excitations in silver nanowires by tuning their morphology.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Nanostructures are frequently distinguished from other materials by exhibiting a strong relation between their properties (optical, electrical, thermal, etc.) and their dimensions. One of the signature examples is the quantum confinement effect characteristic for semiconductor nanocrystals, where the maximum of the absorption shifts towards higher energies (shorter wavelengths) with decreasing of their size [1]. This dependence allows for precise tuning of the optical activity of semiconductor nanocrystals. Another example, related to metallic nanoparticles, concerns the size dependence of the plasmon resonance, attributed to collective oscillations of electrons induced by light illumination [2]. Namely, in the case of spherical nanoparticles, the maximum of the localized surface plasmon resonance (LSPR) shifts towards longer wavelengths with increasing size [2]. The ability to tune the plasmon resonance is not confined only to changing the nanoparticles’ size, as their shapes, chemical composition, or surface coating are also important factors. Nonetheless, this is the size dependence, which provides a benchmark for investigating how the properties of nanostructures vary with their dimensions.
Plasmon excitation in a metallic nanoparticle results in the emergence of a local electromagnetic field, which can influence the optical properties of fluorophores located nearby. In particular, the emission intensity of such a fluorophore might be strongly enhanced [3]. This effect often referred to as metal-enhanced fluorescence, has been widely studied for spherical nanoparticles [4–6] that provide a relatively simple configuration for both experimental and theoretical descriptions of this process. Although nanoparticles made of different metals [7], or featuring different sizes [7,8] produce distinct field distributions, strongest enhancements of fluorescence emission occur for emitters placed usually between 4-20 nm [9–11] from the metal surface.
Among several types of metallic nanostructures, with dimensions less or comparable to the wavelength of visible light, there are also elongated ones, i.e., metallic nanowires [3]. They exhibit, together with the localized plasmon resonance associated with the nanowire's diameter, also propagating surface plasmon polaritons (SPPs), which can be used for transporting energy over tens of micrometers [12]. Propagation of SPPs is a concept that has been applied for developing plasmonic nanowire-based routers [13,14], wavelength splitters [14,15], spin sorters [16], logic gates [17,18] and remote-controlled sensors [19,20]. Coupling of light in the form of SPPs has been achieved in several kinds of nanostructures: metal nanowires [21–23], metal stripes [24,25], grooves or slots, or dielectric stripes on metal surfaces [26,27]. Among these materials, silver nanowires (AgNWs) have emerged as promising, due to their nanoscale field confinement, microscale propagation lengths, and straightforward manufacturing methods [28–32]. In the case of silver nanowires LSPR can be excited over the whole visible range, making them useful for a wide range of fluorophores, although, due to poorer field confinement compared to nanoparticles, they are expected to produce lower enhancements. The nanowires, in addition to LSPR, can sustain SPPs.
Metallic nanowires can be produced by lithographic methods, yielding polycrystalline nanowires with relatively short propagation lengths, albeit with precisely controlled widths. The polycrystallinity issue has been addressed in hard template methods, where nanowires are created directly in cylindrical pores of materials such as anodic aluminum oxide [33] and carbon nanotubes [34]. While the pores provide sufficient control over the dimensions of the nanowires, the removal of the matrix often results in damage of their structure. Soft-template methods have also been developed, with crystal growth being controlled by shape-directing capping agents, such as cetyltrimethylammonium bromide (CTAB) [35] or poly(vinylpyrrolidone) (PVP) [36]. The nanowires obtained using this approach were single-crystalline with smooth surfaces, but their dimensions and uniformity were relatively poorly controlled. The polyol method, where ethylene glycol serves both as a solvent and reducing agent, is now widely used for the synthesis of silver nanowires [37–39]. This method results in AgNWs with diameters in the range of 50-200 nm and lengths of 30-80 µm. Longer AgNWs, with lengths exceeding 100 µm, can be synthesized using a hydrothermal method, where water acts as a solvent at temperatures exceeding 100°C [40–42].
In order to improve the performance of nanowires and their suitability for remotely addressed and plasmonically efficient nanostructures, two quantities have to be simultaneously taken into consideration: (i) the length of propagation of the surface plasmon polaritons (SPPs), and (ii) the fluorescence enhancement factor. It can be expected that both depend on the morphology of the nanowires, in particular on their diameter. In this work, we describe a wet-chemistry synthesis approach for fabricating silver nanowires with relatively uniform diameters, which depend on the reducing agent. Such nanowires can be synthesized with the hydrothermal method, which at the same time enables better control of the lengths of silver nanowires. Comprehensive experiments carried out using fluorescence imaging allowed to associate variation of both fluorescence enhancement and SPP propagation length with the diameter of the nanowires. Enhancement of the fluorescence intensity of photoactive proteins was determined using wide-field fluorescence imaging of a sample, where the emitters were evenly distributed over AgNWs placed randomly on the surface. For assessing the dependence of SPP propagation on the nanowire morphology, a two-objective fluorescence microscope with independent control of excitation and detection was used. The experimental results lead to the conclusion that thicker AgNWs, obtained with aniline and H2O2 as reducing agents, for which longer SPP propagation lengths were observed, produce weaker fluorescence enhancement. Therefore, through the choice of reducing agent, it is possible to obtain AgNWs with reasonably well-controlled diameters, which then determines the plasmonic properties - fluorescence enhancement and SPP propagation – of these highly intriguing metallic nanostructures.
2. Materials and methods
2.1 Chemicals
Silver nitrate (AgNO3, 99.9999%), polyvinylpyrrolidone (PVP, Mw ≈ 40 000), aniline (99.5%), sodium chloride (NaCl, 99.99%), and poly(vinyl alcohol) ((PVA), Moviol 20-98) were purchased from Sigma-Aldrich. Hydrogen peroxide (H2O2, 30%) was purchased from Stanlab. Peridinin-chlorophyll-protein complex conjugated with streptavidin (PCP) was purchased from BD Biosciences. All reagents were used as received without any further modifications or purifications, and their solutions were prepared in deionized (DI) water (Sartorius, Arium Comfort II, >15 MΩ/cm).
2.2 AgNWs synthesis
AgNWs were synthesized using a hydrothermal method, similar to previously reported [43]. First, aqueous solutions of silver nitrate (0.02M, 7.5 mL), PVP (0.5 g, 2.5 ml), and sodium chloride (0.03 M, 7.5 mL) were mixed and afterwards a “reducing solution” (2.5ml) of either
- (1) 20 µl aniline in the case of AgNWs aniline;
- (2) 18 µl aniline + 2 µl hydrogen peroxide solution in the case of AgNWs aniline + H2O2; or
- (3) only water in the case of AgNWs.
Next, each of the mixtures was transferred to a 50 ml Teflon-lined stainless-steel autoclave reactor, as presented in Fig. 1. The reactors were heated to 100°C (4°C/min) in an oven, kept in this temperature for 1 h, and then heated up to 165°C (4°C/min) and left at this temperature for 24 h. This step was followed by slow cooling of the autoclave to reach room temperature. The final product in the form of a grey-white precipitate was collected by centrifugation (1000 rpm, 20 min) and was washed three to five times with DI.
To purify the AgNWs and remove any nanoparticles, the precipitate was dispersed in 10 ml of 0.5% PVP solution. Afterwards, 30 ml of acetone was slowly added, while gently mixing. After 10 minutes, the precipitate at the bottom was collected, and the procedure was repeated 2 times. Finally, AgNWs were dispersed in 3 ml of water and stored at 4°C.
2.3 Peridinin–chlorophyll-protein (PCP)
As an emitter suitable for studying the influence of nanowire morphology on fluorescence enhancement and SPP propagation, a water-soluble photoactive protein, peridinin-chlorophyll-protein (PCP) was applied. The structure of this protein contains eight peridinins, two chlorophylls, and lipid molecules embedded in a protein matrix [44]. Importantly, PCP has been previously used for several studies concerning interactions in hybrid systems involving metallic nanostructures [44,45].
2.4 Structural characterization
Lengths of AgNWs were measured using a dark-field mode of LV150 optical microscope (Nikon, 10x objective), with a Fi-color CCD camera (Nikon) as a detector. Samples were prepared by depositing 2 µl of 100-fold diluted AgNWs on an ITO substrate. Diameters of AgNWs were measured from SEM images collected using a Nova NanoSEM 450 microscope at 10 kV in immersion mode from a sample prepared from 10-fold diluted AgNWs.
2.5 Optical characterization
Extinction spectra of AgNWs (50-fold diluted dispersions) and PCP (40 µg/ml) in solution were measured using Evolution 300 dual-beam spectrophotometer (Thermo Electron Corporation). The suspensions were measured in the cuvettes made from polymethyl methacrylate with a 1-cm optical path. The fluorescence spectrum of PCP in solution was collected using Fluorolog 3 (Jobin Yvon) spectrofluorometer using an excitation wavelength of 480 nm.
2.6 Fluorescence microscopy measurements
Samples for fluorescence imaging experiments were prepared by spin coating (1000 rpm) a mixture of 50-fold diluted AgNWs, 0.04% PVA, and 4 µg/ml PCP on a glass coverslip. Three samples were prepared, each containing silver nanowires synthesized with different reducing agents. Wide-field fluorescence microscopy experiments were performed using Nikon Eclipse Ti-U inverted microscope. Excitation was provided by an LED illuminator (Prizmatix) at the wavelength of 480 nm and 100 µW of power. For excitation and detection of fluorescence a Plan Apo, 100x/1.4 NA oil immersion objective (Nikon) was used. The signal was filtered using a dichroic mirror T650LPXR (Chroma) combined with FEL650 and FB670-10 (Thorlabs) optical filters and detected with an iXon Du-888 EMCCD (Andor) camera. The electromagnetic gain was set to 5, binning was 2 × 2, and acquisition time was 1 s.
Measurements of the propagation length were carried out with a two-objective fluorescence microscope setup (Nikon Ti-U2) where 635 nm diode laser (LS635-150, Spectra Laser) coupled with single-mode optical fiber was used for sample excitation. The light was focused at the end of a selected nanowire using an Apo TIRF 1.49NA/60x oil immersion objective (Nikon). Fluorescence intensity maps were acquired using a second objective at the bottom: 0.7NA/60x S Plan Fluor (Nikon). Fluorescence was extracted with optical filters FELH650 (Thorlabs) and ET675-20m (Chroma) and imaged using Zyla 5.5 sCMOS (Andor Technology) camera. Collection time was set to 100 ms and binning to 2 × 2.
3. Results and discussion
In order to study the effect of AgNW morphology on the SPP propagation and the MEF effect, nanowires with varied diameters should be synthesized. To this end, we have chosen three combinations of reducing agents in the hydrothermal method [41,43]. In all three protocols PVP was used, which in addition to being a growth modifier, surface stabilizer, and nanoparticle dispersant, also played the role of a sole reducing agent in the first synthesis [46]. The second synthesis involved aniline, classified as a modest reducing agent, previously applied to obtain silver nanoparticles [47]. Finally, the third approach involved a combination of aniline and hydrogen peroxide, as described in our previous work [43].
In Fig. 2 we compare representative scanning electron microscopy (SEM) pictures of individual AgNWs obtained using three different synthesis routes. The images are accompanied by corresponding distributions of diameters determined from sets of SEM collected across the samples. All three approaches yield silver nanowires, and the procedures were tested to be reproducible. The nanowires obtained with PVP only are relatively thin, with a narrow diameter distribution. The average value was equal to 76 nm, while their lengths were on average 81 µm. The introduction of aniline to the solution influenced both the diameter and length of the obtained AgNWs. The distribution of diameters, which turned out to be rather broad, for the AgNWs obtained during this synthesis ranged from 50 nm to 250 nm, with an average of 153 nm. The average length of these AgNWs was higher than in the previous case, amounting to 186 µm. The ability to grow longer nanowires was facilitated presumably by either accelerated diffusion of surface atoms [48] or enhanced generation rate of silver atoms in the vicinity of the AgNW tip due to catalytic process in the presence of aniline. The final batch of AgNWs, resulting from the protocol, which involved aniline in combination with hydrogen peroxide, was characterized by the average diameter and length of 131 nm and 277 µm, respectively with some of the nanowires featured lengths exceeding 1 mm. The role of hydrogen peroxide in this synthesis is not fully understood. We assume that it may help to eliminate non-twinned nanoparticles at the onset of crystal growth [49] and later may also act as a catalyst in the growth of (111) facets of Ag [50,51]. SEM imaging results indicate that the choice of the reducing agent substantially influences the morphology of AgNWs, including their diameters. At the same time, the results of SEM imaging indicate (Fig. 2 insets) that the thickness of the stabilizing layers are almost the same for all three types of AgNWs, and equal to approximately 6 nm.
Fig. 2. SEM images of AgNWs (A), AgNWs aniline (B), AgNWs aniline + H2O2 (C) and histograms of respective diameter distribution (D,E,F).
Extinction spectra of aqueous suspensions of AgNWs obtained using the three synthesis protocols are displayed in Fig. 3. All samples exhibit a very broad band at around 390 nm, attributed mainly to the localized surface plasmon resonance. This band stretches from the UV to the near-infrared spectral range. The spectrum measured for AgNWs reduced only with PVP, in addition to the main peak at around 388 nm, also features a shoulder at around 348 nm, ascribed to the transverse quadrupole plasmon modes in AgNWs [52]. For the AgNWs reduced in the presence of aniline, the extinction spectrum is shifted toward longer wavelengths, and both the shoulder and the main peak are broadened. Addition of hydrogen peroxide to the aniline results in the shoulder being better pronounced with simultaneous narrowing of the main peak. We attribute these changes in the extinction spectra to the differences in the AgNWs diameter, induced by the type of the reducing agent.
Fig. 3. Extinction spectra of AgNWs (red), AgNWs Aniline (blue), AgNWs aniline + H2O2 (green), absorption (black) and emission (pink) spectra of PCP.
The absorption spectrum of PCP complexes, which were used as emitters in the fluorescence imaging experiments, exhibits a broad band between 400 and 550 nm, and the second at around 670 nm. The emission appears at 673 nm, in agreement with previous results [53]. Overlapping between the extinction spectra of silver nanowires and the fluorescence spectrum of the PCP protein should facilitate efficient coupling with plasmon excitations.
The coupling between the fluorescent protein PCP and surface plasmons in silver nanowires was studied with wide-field fluorescence imaging microscopy. First, the positions of AgNWs were verified using transmission mode. Next, for the selected location on the sample, fluorescence images were acquired. In Fig. 4, we compare representative fluorescence images of PCP deposited on silver nanowires synthesized with three approaches.
Fig. 4. Wide field fluorescence maps of PCP with AgNWs (A), AgNWs aniline (B), AgNWs aniline + H2O2 (C) and fluorescence intensity profiles extracted along the nanowire (D).
Similarly to previous research focused on imaging emitters coupled with silver nanowires [54], the images displayed in Fig. 4 feature bright lines, whose positions correlate with the positions determined using transmission mode. This implies that the fluorescence emission of the PCP complexes located in the vicinity of the nanowires is enhanced due to plasmonic excitations. The enhancement is observed for all three types of silver nanowires. To analyze the results of fluorescence imaging, cross-sections of intensity were extracted along the nanowires, as shown in Fig. 4(d), where the profiles along the nanowires are compared. In addition to the profiles extracted along the nanowires, reference profiles were also determined. The results, summarized in Fig. 4(d), indicate that fluorescence intensity is the highest for the nanowires synthesized with PVP only (average intensity along the chosen nanowire is 3400 cps). In contrast, emission of PCP in the structures, where the chosen AgNWs obtained with aniline (Fig. 4(b)) or aniline with hydrogen peroxide (Fig. 4(c)) were used, is considerably less, in this case amounting to 1000 cps and 900 cps, respectively. We note, however, that these are selected examples, not necessarily the typical ones. Therefore, in order to obtain statistically relevant results for comparison, many such nanowires of each kind should be analyzed.
The fluorescence enhancement factors were calculated by dividing average intensities along the wire (line 1) and beside the wire (line 2) for a particular image. Such a procedure was applied to 30 individual nanowires from each synthesis batch. The distributions of the fluorescence enhancement factors are shown in Fig. 5, and the average values thereof are given in Table 1.
Table 1. Comparison of average diameter, length of propagation, and intensity ratio for about 30 AgNWs for each synthesis.
It turns out that for the AgNWs synthesized with PVP only, the enhancement is very strong, approximately 19-fold. This value is considerably higher than reported in previous experiments [45], where thick layers of PCP were studied. For similarly prepared samples [55] comparable enhancements of fluorescence intensity have been measured. For the other two samples, we observe around a 6-fold increase in fluorescence intensity, with the AgNWs obtained with aniline exhibiting slightly lower values compared to that for AgNWs synthesized with aniline + H2O2 (Fig. 5). We conclude, that the nanowires with the smallest diameter yield the strongest enhancement of fluorescence emission of the PCP complexes. This result is expected despite the increase of the scattering cross-section with diameter, as the leading mechanism in this case is the variation of electric field distribution with the size of the nanowires. Indeed, structures of smaller diameter, e.g. nanoparticles [8], can provide stronger field enhancements in their vicinity, causing more pronounced modifications of the fluorophore optical properties. Thus, the larger fluorescence enhancement observed for thinner nanowires can be associated with stronger electric field confinement.
The second characteristics, in addition to the metal-enhanced fluorescence, of elongated metallic nanostructures, concerns propagation of surface plasmon polaritons [56]. The propagation in the three types of nanowires with different diameters was studied using a two-objective fluorescence microscope [55]. The SPPs in a single AgNW were excited at one of its ends via tightly focused laser beam with the fluorescence detected in a wide-field imaging mode using a second objective. Representative images obtained in such a configuration are shown in Fig. 6. When one end of the selected nanowire coupled with PCP is illuminated, we observe bright elongated shapes corresponding to the fluorescence emission of PCP complexes along the nanowire. The intensity of this emission is reduced with increasing distance from the illuminated end of the nanowire. The emergence of such bright lines, which correlate with the positions of nanowires determined in a dark-field microscopy mode, indicates that plasmons in AgNWs can efficiently excite the emission of PCP complexes in their closest vicinity. Indeed, the observed fluorescence originates from the coupling between PCP and surface plasmons propagating in a silver nanowire [43,56].
Fig. 6. Fluorescence intensity maps obtained from two-objective microscopy of AgNWs (A), AgNWs aniline (B), AgNWs aniline + H2O2 (C), fluorescence intensity profiles along the nanowires (D, E, F,) and distribution of length of propagation for each of the syntheses (G, H, I).
In order to compare the propagation in AgNWs obtained with the three synthesis routes, we extracted intensity profiles along the nanowires (displayed in Fig. 6(d)-(f)). The profiles can be approximated with a monoexponential decay function:
where x stands for position along the AgNW and LSPP is a parameter describing the SPP propagation. The LSPP values extracted for about 20 single nanowires for each synthesis are presented in the form of histograms in Fig. 6(g)-(i). Taking into account the average values, the shortest propagation length 4.1 µm was obtained for AgNWs prepared with PVP only. In the case of silver nanowires obtained with aniline+ H2O2 the average propagation of 5.1 µm was measured, which increases further to 6.2 µm for the aniline AgNWs. When correlating these results with average diameters of AgNWs (Table 1) we can conclude that nanowires with the smallest diameter exhibit the shortest propagation lengths. At the same time, they feature the strongest fluorescence enhancement. This is direct evidence that by tuning the morphology of elongated metallic nanostructures that facilitate propagation of SPPs, it is very feasible to control both the length of surface plasmon propagation and the enhancement of fluorescence. In this case this is achieved by synthesizing silver nanowires with various combinations of reducing agents. These results open the possibility of designing and fabricating nanowires with defined properties for applications as a plasmonic waveguides for remote sensing.The fluorescence intensity measured using wide-field fluorescence microscopy and the intensity observed in the experiment aimed at determining the propagation length both concern the physical mechanisms of plasmon generation and the properties thereof. In the first case, localized surface plasmons are mainly excited in the silver nanowires, but all the PCP complexes within the excitation spot (both close and far away from the nanowires) also contribute to the measured signal. In contrast, in the propagation experiments predominantly surface plasmon polaritons are excited and the emission occurs only from the PCP complexes located in the close vicinity to the nanowires. Therefore, the intensities measured in the two experiments are very difficult to compare directly.
4. Conclusions
Three combinations of reducing agents used in hydrothermal synthesis of silver nanowires are shown to yield nanowires with different diameters and, accordingly, with distinct optical properties. In particular, by imaging the fluorescence of photoactive proteins deposited over the nanowires, we find longer propagation lengths of SPP for nanowires with larger diameters. In contrast, such nanowires exhibit considerably lower values of fluorescence enhancements. Indeed, a two-fold change of the diameter (from 75 nm to 150 nm), translates into a four-fold reduction of emission intensity of the PCP protein. We conclude that through the choice of reducing agent it is possible to obtain AgNWs with relatively well-controlled diameters, that determine their plasmonic properties - fluorescence enhancement and SPP propagation – of these highly intriguing metallic nanostructures.
Funding
Narodowe Centrum Nauki (2016/22/E/ST5/00531, 2017/27/B/ST3/02457); Narodowe Centrum Badań i Rozwoju (POWR.03.05.00-00-Z302/17).
Disclosures
The authors declare no conflicts of interest.
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