Gobind Das*a,
Salma Alrasheeda,
Maria Laura Coluccio
b,
Francesco Gentilebc,
Annalisa Nicastrid,
Patrizio Candeloro
b,
Giovanni Cudad,
Gerardo Perozziellob and
Enzo Di Fabrizioa
aPhysical Sciences and Engineering (PSE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia. E-mail: gobind.das@kaust.edu.sa
bBio-Nanotechnology and Engineering for Medicine (BIONEM), Department of Experimental and Clinical Medicine, University of Magna Graecia Viale Europa, Germaneto, Catanzaro 88100, Italy
cDepartment of Electrical Engineering and Information Technology, University of Napoli, Federico II – Corso Umberto I 40, 80138 Napoli, Italy
dAdvanced Research Center on Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, University of Magna Graecia Viale Europa, Germaneto, Catanzaro 88100, Italy
First published on 27th October 2016
Advancements in nanotechnology fabrication techniques allow the possibility to design and fabricate a device with a minimum gap (<10 nm) between the composing nanostructures in order to obtain better control over the creation and spatial definition of plasmonic hot-spots. The present study is intended to show the fabrication of nanolens and their application to single/few molecules detection. Theoretical simulations were performed on different designs of real structures, including comparison of rough and smooth surfaces. Various molecules (rhodamine 6G, benzenethiol and BRCA1/BRCT peptides) were examined in this regard. Single molecule detection was possible for synthetic peptides, with a possible application in early detection of diseases.
There are several fabrication techniques to produce SERS substrates, including metal island films,9 electrochemically modified electrodes,10,11 nanosphere lithography,12 nano-imprinter,13 and electron beam lithography.14,15 Various types of SERS substrates have been reported such as ordered,14,16–18 random,19 functionalized,20 urchin like,21 superstructures,22,23 metal supercrystals,24,25 and graphene based SERS26 devices. However, the most conventional technique is to produce Ag colloids and to adsorb biomolecules to them.27,28
In this study, we fabricated self-similar chains (SSCs) of nanospheres (NSs) following the design proposed by Li et al.29 and Dai et al.30 A self-similar chain, also called a nanolens, is a sequence of three metal NSs in a plane, in which the gap between the spheres and the diameter of the spheres is progressively reduced and maintained in the few nanometer range.31 The term nanolens is employed because of the predominant hot-spot found at a specific location, i.e., at the smallest gap. This represents a nano-optics apparatus, i.e., a hierarchical structure which triggers resonance effects and collective oscillations of electrons over the surface of the device. Due to these effects, the electric field is well confined in the gap between the two smaller NSs; in this hot-spot, Raman signal is largely enhanced. A tight control of size, shape, and positioning of the fabricated nano-structures is required; the smallest gap in the nanolens is normally below 10 nm. To attain a similar control, we used a combination of top-down and bottom-up fabrication techniques; more precisely, E-beam lithography (EBL) as a top-down process and metal electroless deposition as a bottom-up one. First, EBL allowed defining the nanolithographic structures, giving the possibility to fabricate a controllable and reproducible device. Then, metal deposition was carried out by means of a site-selective electroless technique to satisfy the strict nanostructure size and shape requirements in order to attain the hot-spot at the anticipated position. SERS substrates, such as bow-tie, nanostars, nanoAntennas, PLLA nanofibres, provide more than one hot-spot, which means Raman interpretation will be confusing or impossible when it comes to the analysis of a mixture of different types of molecules because it will be composed of an overlap of all molecules present in several hot-spots. However, this problem can be resolved by using an SSCs device such as ours wherein a single dominant hot-spot is well precisely localized at the nanogap.31
We used numerical Finite Difference Time Domain (FDTD) simulations to verify the ability of the device to enhance the electro-magnetic (e. m.) field. In the simulations, we maintained the true characteristic parameters of the nanolens, including dielectric constant of the polymer resist, quality factor Q of the silver-constituted lens, and experimentally observed size, gap, shape and roughness of the nano-spheres. We found a theoretical enhancement factor of 106 to 107, which was very close to the experimentally observed values as measured for different probing molecules.
We demonstrated the device proficiency using rhodamine 6G (R6G, a fluorescent dye molecule), benzenethiol, and control/mutated synthetic BRCA peptides. For all the configurations, molecular species were detected in very low abundance ranges.
The approach to perform a highly precise nanochemistry over the lithographic surface was an important step to carry out a site-selective metal deposition.32 The electroless deposition process33 is found to be the right choice in this regard to deposit metallic Ag on the predefined area, after which reduction of the oxidized metallic species leads to the formation of the neutral metal atom (neutral Ag surface). Ag nanometal deposition is carried out using a solution of AgNO3 and HF.33,34 Thereafter, the sample is rinsed with double-distilled water in order to wash out the metal salt from the nanostructure surface. After the Ag-electroless deposition over the exposed area, the final device is achieved with two gaps between the lenses around 23 nm and 9 nm, whereas the diameter of smallest NS is found to be 26 nm. The schematic of the fabrication process is shown in Fig. 1a–d. In addition, the SEM images of the device after electron beam writing and after site-selective electroless metal deposition are shown in Fig. 1b′ and c′.
Various nanolens structures with single, double, triple and quadruple lenses were fabricated for assessment and to compare their capability to localize the Plasmon Polaritons (PPs) close to the smallest NS (see ESI,† Section: 1). Au-based nano-optics devices were also fabricated using the same techniques.
Various molecules (fluorescent dye, thiol and synthetic peptides) were deposited on the device to verify its SERS activity. In micro-Raman mapping measurements of the samples, the step size in the X and Y direction was maintained below 150 nm (typical step size is 100 nm).
We first present the results regarding the single nanolens device. A monolayer of an organic fluorescent molecule R6G was deposited on the device using an immersion technique. An optical image of the device is shown in Fig. 2a. Micro-Raman measurements were performed in the region of interest (ROI), which contains a single nanolens.
Raman spectra, acquired in the 1050–1925 cm−1 range on the devices with (red arrow) and without (black arrow) a nanolens structure, are shown in Fig. 2b for direct comparison. The spectra show the effect of the nanolens structure for enhancing the Raman signal of R6G. Well known R6G SERS peaks were observed at around 1360 cm−1, attributed to COO− vibration and at 1506, 1569, 1649 cm−1 related to the C–C ring stretching vibration.18,36,37 In addition to these bands, a few more bands were evident, centered at 1126 and 1188 cm−1, which can be attributed to C–Hx in-plane bending, and 1310 cm−1, which can be attributed to C–OH stretching vibrations. No spectral feature (i.e. only background) was observed outside the nanolens, indicating that the rinsing procedure was effective. The fluorescence of the dye molecule was efficiently quenched in the vicinity of the metal surface,38,39 and therefore the Raman spectrum of R6G was clearly visible. The 2D-contour image for the Raman band centered at 1650 cm−1 is shown overlapping with the optical image of the mapping area in Fig. 2c. In the figure, the maximum Raman intensity was found to correspond with the location of the nanolens, and this revealed consistency of the measurements. In this and other similar experiments, we observed SERS activity solely over the nanolenses, whereas the signal was vanishingly small in the remaining regions of the measurement area. We remark here that the direction of polarization of the incident radiation should be collinear to the plane of the nanostructure to attain the maximum SERS signal. 3D-mapping analysis for the same reference band at 1650 cm−1 is shown in Fig. 2d. Highly sensitive and selective SERS signals were observed for R6G using this device. The observed broadness of the area of the map with high Raman signal may be ascribed to the convolution of the laser spot-size with the nanometric gap of the nanolens structure. Considering that the major SERS contribution originated from the distance between the two smallest metal spheres, the SERS enhancement factor was estimated to be 1.75 × 107 (see ESI,† Section: 2 for SERS enhancement factor calculation). The surface area of a R6G molecule was assumed to be around 0.9 nm2. The Raman signal originating from the nanolens was due to a maximum of 100–140 closely packed molecules.
Next, we discuss the response of the devices when the number of nanolenses varies from 2 to 4 (see ESI,† Section: 1). Fig. 3 reports the measured Raman intensity as a function of the number (N) of nanolenses in the device. The enhanced signal varies linearly with N, ranging from 700 counts for N = 1 to nearly 2000 counts for N = 4. Raman mapping measurements on the devices are reported in the ESI,† Section: 3. Asymmetry was observed in the hot-spot of 2D mapping analysis (see Fig. S4(b and c)†), which was due to the convolution of instrument aberration and the plasmonic hot-spot. However, we verified that this instrumental asymmetry does not give low reliability in Raman because the photon count was stable on each lens when we used mono-disperse samples.
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Fig. 3 SERS intensity with standard deviation of R6G reference band centered at around 1650 cm−1 for the device with varying number of nanolens. |
Finite Difference Time Domain (FDTD) simulation was performed for the nanolens devices. The design of the nanolens device was chosen according to the experimental parameters of the fabricated ones. The designs for the near-field simulations are reported in the upper panel of Fig. 4. A linearly polarized plane wave, parallel to the nanolens major axis, was used to excite the plasmonic surface. The propagation direction of the plane wave was set in such a manner that the wave vector k was orthogonal to the nanolens' major axis. The subgriding method was retained to achieve a mesh size down to 2 × 2 × 2 nm3. The 2D electrical field map was calculated by placing the monitor at half height of the thinnest nanosphere (14 nm from the bulk silicon). Three silver NSs were integrated in a PMMA polymer (i.e., the resist), which was deposited over the silicon substrate. The dispersion relationship proposed by Palik et al. was employed for silicon and silver materials during all the FDTD calculations.40 Theoretical simulation for the self-similar device with smoothed surface was carried out. Devices were excited with a plane wave with a wavelength of 514.5 nm. For all simulations, the polarization of incident light was maintained parallel to the SSCs' axis. The distribution of the electric field in the near-field approximation is shown in Fig. 4 (lower panel). The localized electric field in between two smaller NSs was found to be around 16 V m−1 when the metal nanosphere surface was smooth (see lower-left panel of Fig. 4). Thereafter, a roughness of 2 nm was artificially included to reproduce the imperfections of the fabricated device. Simulations were performed for devices with a nominal smooth surface for comparison. Previous reports suggested that the electric field was augmented over random irregular surfaces.31,41 We observed that the maximum electric field enhancement factor was around 44 V m−1 (see lower-right panel of Fig. 4); thus, the theoretical Raman signal enhancement was (44)4 = 3.7 × 106. The present result was consistent with the previously reported results.42,43 The experimental SERS enhancement factor and the calculated signal enhancement lie very close to each other, thanks to introduction of disorder over the NSs (resembling the FDTD simulation designed to the experimental device). Numerical simulations for one to four nanolenses were also reported in Fig. S5 (see ESI,† Section: 4). The numerical study of these 4 cases showed that both the intensity and the electrical field distribution changed from monomer to tetramer nanolenses. Considering that the molecules are distributed in the same manner covering the entire surface, we found an agreement with the experimental trend reported in Fig. 2. This led to an increase in Raman signal with the number of nanolenses, as observed in the experimental findings.
Repeated experiments and numerical simulations demonstrated the increased efficiency of the 3-NSs device over the one with only 2 spheres. Previously reported theoretical FDTD calculations showed that the intensity for a 3-NSs device is significantly larger than that of the 2 spheres design;44 herein, we present experimental measurements of benzenethiol molecules to reinforce these findings. Raman intensity profiles of benzenethiol for the 2- and 3-spheres device (Fig. S6†) show the increased sensitivity of a design with 3-NSs with respect to a device with 2-NSs. The peak was centered at 1584 cm−1 (ref. 45 and 46) (see ESI,† Section: 5). In the inset of Fig. S6,† near-field electric field distribution is shown. The electric field simulation also demonstrated that the field enhancement in 3-NSs is greater than that of 2-NSs.
Finally, we challenged the devices with synthetic peptides to assess its sensitivity. BRCA1 gene was employed to test the device sensitivity. BRCA1 gene is a tumor suppressor localized in the long arm of chromosome 17 (17q21), which is associated with breast and other types of malignant neoplasia. There are commonly 7 types of mutations in the BRCA1 gene within the BRCT domain in which only one peptide is replaced by the other peptide, causing neoplasia. Herein, we employed W1837R wherein tryptophan is substituted by arginine, to test the functionality of the device. We analysed wild type W1837 (Fig. 5a–c) and mutated W1837R (Fig. 5a′–c′) BRCA1 peptides with a sequence of 16 amino acids. The mutation occurs in a single point of the sequence. Detailed information related to the peptides is reported in the ESI,† Section: 6. Peptides were deposited over the nanolens devices using the chemisorption technique, resulting in a monolayer on the nanostructure. Micro Raman mapping measurements were performed over the device. The peptides' Raman activity was observed over a limited number of points over the entire grid (Fig. 5). Optical images of the area over which wild type (Fig. 5a) and mutated (Fig. 5a′) peptides were analysed are shown in Fig. 5b–b′. Examples of the acquired Raman spectra for wild type and mutated peptides are reported in Fig. 5c and c′. The variation in Raman spectra at different grid points showed the excitation of individual molecules lying at different angles.47 This demonstrates the device sensitivity at a single molecule detection level. Raman measurements of bulk synthetic peptides are shown in Fig. 5d for comparison. These results support the prospect of using similar nanolens devices as biosensors to distinguish wild type from mutated peptides with high sensitivity, selectivity and reliability.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23301e |
This journal is © The Royal Society of Chemistry 2016 |