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Abstract
To achieve a surface-enhanced Raman spectroscopy (SERS) sensor that is easy to make and use, we propose periodic gold nanotrench arrays, which can be fabricated without surface contamination and intricate sensor alignment. Deep and narrow plasmonic nanotrenches for amplifying local electromagnetic fields were reliably generated on a wafer-scale substrate by nanoimprint lithography and two successive oblique-angle depositions. Electromagnetic simulations and Raman measurements show that the proposed plasmonic nanostructures function as SERS sensors, enabling nanomolar sensitivity. Furthermore, we successfully confirmed the microRNA detection capability of the proposed nanostructures to demonstrate their promising potential and feasibility for use in biomedical diagnostic sensors.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
The single-molecule detection capability and molecule-specific signal of surface-enhanced Raman spectroscopy (SERS) make it a promising sensing platform in a variety of fields, such as monitoring of environmental toxins, food safety, and biomedical markers [1–10]. Because the high sensitivity of SERS is mainly based on the enhancement of electromagnetic fields that amplify the cross section of Raman scattering, numerous studies have reported on plasmonic structures with nanoscale tips or gaps [11–17]. As part of this effort, for the SERS detection of microRNA (miRNA), which is a biomarker for human cancers and the target analyte of this study, specially-designed nanostructures such as Ag-coated Au nanostars, Ag nanobundles in Au nanobowls, and hollow Au/Ag nanospheres have been proposed [18–6]. However, plasmonic nanostructures with sophisticated shapes are difficult to fabricate reliably and often suffer from chemical contamination due to surfactant molecules used in chemical synthesis or chemical solvents used in physical nanofabrication [20–23]. Furthermore, the plasmonic nanostructures should be aligned with the incident light to maximize and, more importantly, to homogenize the electromagnetic field enhancement. For example, the length direction of nanotips and the width direction of nanogaps should be parallel and perpendicular to the light polarization, respectively. A small perturbation in the alignment between a plasmonic nanostructure and light causes a significant change in the SERS intensity that is proportional to the fourth power of the electromagnetic field enhancement [3]. Therefore, essential prerequisites for the practical use of SERS are to fabricate reliable plasmonic nanostructures easily and to align them correctly with the incident light. In this report, we propose a periodic gold nanotrench (GNT) array as an easy-to-make-and-use SERS sensor based the following results: easy-to-make SERS sensor with hot spots generated spontaneously on a wafer-scale substrate, easy-to-use SERS sensor without concerns about intricate alignment between hot spots and incident light, and successful demonstration of nanomolar sensitivity to miRNA.
2. Experimental (fabrication of the periodic gold nanotrench array)
Figure 1(a) shows the sample fabrication procedure. Using UV-nanoimprint lithography, one-dimensional polymer nanogratings (200 nm period, 100 nm linewidth, 100 nm height) were generated on a flexible polyurethane acrylate (PUA)-coated polyethylene terephthalate (PET) substrate. Then, a 50 nm thick Au film was deposited on the patterned polymer substrate with an oblique evaporation angle of 35° from the surface normal, followed by a second Au deposition with the same thickness and angle from the opposite direction. As illustrated in Fig. 1(a), the two Au film depositions covered the entire surface of the polymer nanogratings and reduced the space between adjacent gratings. Deep and narrow trench structures were fabricated spontaneously. The scanning electron microscopy (SEM) images in Fig. 1(b) show the proposed SERS substrate, namely, the GNT array in which the width of the GNTs was approximately 40 nm. The atomic force microscopy (AFM) image in Fig. 1(c) shows a three-dimensional view of the GNT array. Although the sharpness of the AFM tip was not sufficient to clearly detect the bottom of the GNTs, the cross-sectional profile in Fig. 1(c) shows that the depth and aspect ratio of the nanotrenches were at least approximately 65 nm and 1.6, respectively. Therefore, the proposed nanofabrication produced plasmonically active GNTs on a wafer-scale substrate without sophisticated wet chemical processes (such as resist development or metal etching), which can cause surface contamination.
Fig. 1. (a) Schematic of the sample fabrication procedure based on nanoimprint lithography and oblique-angle deposition. PUA and PET represent polyurethane acrylate and polyethylene terephthalate, respectively. The dimensions of the polymer grating are expressed in nanometers. (b) Plane-view SEM images of the GNT array; the image on the right is a magnified view of the one on the left. Inset photograph shows the GNT array fabricated on a wafer-scale substrate. (c) AFM image and cross-sectional profile obtained from the sample shown in (b).
3. Results and discussion
To demonstrate the potential of the GNT array as a SERS sensor, the enhancement of local electric fields near the nanostructures was calculated using a three-dimensional finite-difference time-domain simulation software (FDTD Solutions, Lumerical Inc.), as shown in Fig. 2(a). Simulation mesh was set to a 1 nm cubic grid except for the uppermost plane of the GNT array where a 0.5 nm cubic grid was applied. Refractive index of the polymer grating was assumed as 1.5 and bulk dielectric properties of Au from Palik’s handbook were used for the simulation [24]. The cross-sectional dimensions of the modeled GNT array were based on the SEM and AFM images shown in Fig. 1(b, c). Although the deposited Au film has the surface roughness thought to be developed during the evaporation step, the modeled nanostructure was assumed to have a flat surface to reduce the simulation time and to understand the electromagnetic contribution of the trench structure. Even with a 10-fold increase in the surface roughness, it is known that SERS activity only increases by about 2–3 times [25]. Perfectly matched layers (PML) and periodic boundaries were used as boundary conditions in the vertical and lateral directions, respectively. The direction of incident light (532 nm wavelength) was from top to bottom, and the electric field was polarized in the lateral x-direction. The calculated contours of |E/Eo|2 in Fig. 2(b, c), where Eo and E denote the amplitudes of the incident and enhanced electric fields, respectively, were monitored in the uppermost and cross-sectional planes of the GNT array. The red regions in the field contours clearly demonstrate that the local electric fields were highly enhanced inside and along the nanotrenches. Because the SERS enhancement factor is known to be comparable with the |E/Eo|4 value [3], the Raman signal of target analytes sitting on the upper edge of the nanotrench is expected to be enhanced by approximately 104 or more.
Fig. 2. (a) Schematic of the cross-sectional view of the FDTD simulation geometry. (b, c) Squared amplitudes of the local electric fields monitored in the (b) uppermost and (c) cross-sectional planes of the GNT array. Light direction and polarization angle are indicated by black and gray arrows, respectively. The interfaces between air, gold, and polymer are marked by dotted lines. The color scales all correspond with the color bar in (c).
For the experimental assessment of Raman signal enhancement, the GNT array was treated with R6G by immersing the samples in an aqueous R6G solution for 20 min. Then, the samples were rinsed twice with distilled water for 20 s and dried with nitrogen gas. Although this method for treating R6G molecules cannot provide the exact number of molecules for SERS analysis, the molecules are more uniformly delivered to the sample surface rather than the drop-and-dry method that induces a coffee-stain effect [7]. It is known that R6G molecules strongly adsorb on a negatively charged gold surface [26], and bare gold has a zeta potential of approximately −30 mV at neutral pH [27]. After the R6G treatment, SERS measurements were carried out using a commercial confocal Raman microscope (alpha300, WITec). The details of SERS measurements are described in the Supplement 1. Figure 3(a) shows the Raman spectra measured from the GNT arrays treated with various R6G concentrations from 2 μM to 2 nM; their peak positions correspond well with the reported vibrational bands of R6G molecules [28]. This nanomolar-level sensitivity is attributed to the enhanced electromagnetic fields in the nanotrenches (as shown in Fig. 2), which was experimentally demonstrated by changing the polarization angle of the incident light (as shown in Fig. 3(b)). The Raman intensities measured with different polarization angles clearly show that the Raman signal was maximized when the light polarization was perpendicular to the longitudinal direction of the trench, which is consistent with the simulation results (Fig. 2 and Fig. S2 in Supplement 1). Because the GNT arrays had the same orientation over the entire 4 inch wafer, which was defined by nanoimprint lithography and simply perceived by a visible marker, accurate alignment of the trench direction and light polarization was routinely possible. Furthermore, the nanotrenches can be useful for accumulating target analytes by capillary forces [29].
Fig. 3. (a) Raman intensities measured from GNT arrays treated with various R6G concentrations (2 μM (black), 20 nM (blue), and 2 nM (red)). The inset shows the R6G treatment process. The four major peaks marked by black dots originates from C–C–C ring in-plane bending (615 cm−1), C–H out of plane bending (780 cm−1), and aromatic C–C stretching (1365 cm−1, 1650 cm−1) [28]. (b) Raman intensities measured from the GNT array treated with 2 μM R6G using different polarization angles (90° (black), 45° (blue), and 0° (red) to the longitudinal direction of the trench). The inset illustrates the polarization angles applied for the measurements.
Finally, the GNT array was used to detect miRNA-125b. Because the expression or downregulation of miRNA-125b is known to be involved in many human cancers (such as colon, bladder, and mammary tumors) through the regulation of multiple cell processes [30,31], accurate monitoring of the expression level of miRNA-125b can provide tangible benefits to patients. However, miRNA families with four common nucleotides in a short length (19-22 nucleotides) exhibit similar SERS spectra inherently and require statistical methods, such as partial least squares discriminate analysis (PLS-DA), to differentiate them from one another [32]. To detect the miRNA-125b specific spectrum without additional statistical aids, the sensing method shown in Fig. 4(a) was used. In the presence of miRNA-125b, the probe DNAs on the GNT surface changed their conformation from a hairpin structure to a double-stranded structure and consequently bound with the enzyme (alkaline phosphatase). This enzyme can convert aqueous soluble substrates (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) into insoluble precipitates (nitro blue tetrazolium diformazan) on the GNT surface, and the amount of precipitate is controlled by the concentration of the target miRNA. The experimental details of the miRNA measurements are described in the Supplement 1. The amplification of the precipitates combined with the enhanced electromagnetic field on the GNT surface enabled the detection of the Raman signal of the target miRNA at the nanomolar level. Figure 4(b) shows representative SERS spectra measured from the GNT arrays with and without miRNA. Primary peaks, indicated by black dots in Fig. 4(b), originated from the breathing mode of aromatic carbon rings (∼1000 cm−1), vibration of carbon double bonds (∼1350 cm−1), and symmetric stretching of nitrogen dioxide (∼1600 cm−1) [33,34]. These characteristic Raman peaks are consistent with the chemical structure of the precipitate, that is, nitro blue tetrazolium diformazan. Therefore, the target miRNA can be detected by monitoring the Raman peaks of the abundant precipitates instead of the trace amount of miRNA. Although nonspecific binding of the enzyme on the sample surface produced a background signal, as presented by the blue curve in Fig. 4(b), the SERS spectra from the GNT arrays treated with 1 nM (black curve) and 0.1 nM (red curve) miRNA-125b showed approximately 2 and 1.3 fold larger intensities than that of the background signal, respectively. The two-dimensional maps of Raman intensities in Fig. 4(c) show more distinct visible evidence of the nanomolar sensitivity for the target miRNA, as well as the uniform spatial distribution of the Raman signal.
Fig. 4. (a) Schematic of miRNA detection based on a target-specific enzymatic reaction that converts soluble substrate into insoluble SERS analytes. (b) Raman intensities of precipitated insoluble analytes in the presence of 1 nM (black), 0.1 nM (red), and 0 nM (blue) miRNA. (c) Two-dimensional intensity maps of the Raman signal between 1580 and 1630 cm−1 from GNT arrays with (upper panel) and without (lower panel) the target miRNA. The scale bars for Raman intensity and length are the same for both maps.
4. Conclusion
In summary, we developed an easy-to-make-and-use SERS sensor, namely, GNT arrays. Deep and narrow plasmonic nanotrenches were spontaneously generated by nanoimprint lithography and two successive Au depositions, and the trenches can be easily aligned with the excitation laser to maximize the SERS performance. The proposed GNT arrays induce a large enhancement of local electric fields, as demonstrated by an electromagnetic simulation, and successfully function as a SERS sensor with nanomolar sensitivity, as confirmed by miRNA-125b detection. Because large-scale fabrication and simple use of the highly-sensitive GNT arrays are possible, it is thought that the practical application of SERS sensors would become more feasible.
Funding
National Research Foundation of Korea (2017M3A7B4041754, 2021R1I1A3048262).
Disclosures
The authors declare no conflicts of interest.
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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