Soft UV nanoimprint lithography-designed highly sensitive substrates for SERS detection
© Cottat et al.; licensee Springer. 2014
Received: 28 July 2014
Accepted: 15 November 2014
Published: 21 November 2014
We report on the use of soft UV nanoimprint lithography (UV-NIL) for the development of reproducible, millimeter-sized, and sensitive substrates for SERS detection. The used geometry for plasmonic nanostructures is the cylinder. Gold nanocylinders (GNCs) showed to be very sensitive and specific sensing surfaces. Indeed, we demonstrated that less than 4 ×106 avidin molecules were detected and contributed to the surface-enhanced Raman scattering (SERS) signal. Thus, the soft UV-NIL technique allows to obtain quickly very sensitive substrates for SERS biosensing on surfaces of 1 mm 2.
Surface-enhanced Raman scattering (SERS) technique was shown to be a very effective analytical tool for the detection and identification of molecules, thanks to its high sensitivity [1, 2]. It has been widely used for ultrasensitive chemical analysis down to the single molecule sensitivity. Its field of applications is as varied as to include chemical-biochemical analysis, nanostructure characterization as well as biomedical applications [3–7]. Especially in chemistry, SERS is applied for the detection of conformational changes and structural differences regarding preferred orientations of molecules with respect to a metal surface . The facts that SERS gives a specific fingerprint of a molecule and is sensitive to very small molecules make it a good candidate for application in the fields of chemical and biological sensors. The SERS enhancement is due to the localized surface plasmon resonance (LSPR) of the metallic nanostructure. The nanostructured LSPR properties need to be designed and strongly controlled in order to produce highly reproducible active SERS substrates [9, 10]. In previous studies, we have discussed the necessity to optimize the size of gold nanocylinders (GNCs) in order to achieve the highest possible SERS enhancement. In addition, we have demonstrated the necessity to optimize the LSPR in the case of each studied molecules .
For industrial applications, nanostructured surfaces of at least 1 mm 2 have to be produced. In the past, electron beam lithography (EBL) has helped us to demonstrate that by optimizing the nanostructure assembly parameter, enhancement factors estimated at 10 5 to 10 7 could be obtained for such proteins as bovine serum albumin (BSA) or RNase-A . However, EBL is expensive and time-consuming. Techniques to produce large and organized nanostructured assemblies on transparent substrates have been developed since several years in order to maximize Raman scattering enhancement [12, 13]. The most popular is probably the nanosphere lithography (NSL) . The advantage of this technique is to obtain large areas (several mm 2) of nanostructures on a substrate. However, the shape and arrangement of the nanostructures are more hardly tuned.
In this article, we propose to use another technique called soft UV-nanoimprint lithography (UV-NIL)  in order to fabricate SERS substrates. UV-NIL is biocompatible, since it can be implemented on any flat surface. Another essential advantage is that the samples produced with the same mold are all identical. This is important to guarantee the reproducibility of the results. We have already demonstrated the use of UV-NIL for the detection of biomolecules using a LSPR shift  and for the realization of nanoholes for AFM studies of membrane proteins . Large arrays of reproducible nanostructures are more and more implemented for SERS [18, 19]; nevertheless, they are rarely used for biosensing. Galarreta et al. have demonstrated from functionalized nanotriangles obtained by NSL the detection of avidin . We can explain this phenomenon by making several assumptions. First of all, the technologies available to produce large gratings of nanostructures on transparent substrates are quite recent and still the domain of physicists. The second point is that for biodetection, the nanoparticles must be functionalized, firstly, in order to preserve the biomolecules from surface interactions and, secondly, to guarantee specific biosensing. Eventually, biomolecules are difficult to handle and have low Raman cross section. In order to determine the properties of our UV-NIL substrates as a SERS sensor and determine its sensing performances, we have chosen to study the biotin/avidin system.
The fabrication of gold nanocylinders by UV-NIL
Raman spectrum acquisition
Raman spectra were recorded using a Labram spectrophotometer from Horiba Scientific (Kyoto, Japan) for all experiments. The acquisition parameter was fixed to 500 s for avidin/biotin system. A 633-nm laser was used for all experiments with a power of 100 μ W. The laser excitation was focused on the substrate using a microscope objective (×80, N.A. = 0.75). The same objective was used to collect the Raman signal from SERS substrates in a backscattering configuration. The Raman spectra were recorded with a spectral resolution of 1 cm -1 and a spatial resolution about 1 μ m. For classical Raman measurements in solution, a macro-objective with a focal length of 40 mm (N.A. = 0.18) and a 633-nm laser were used. All obtained spectra have been corrected for acquisition time and laser power so they can be compared.
The SPR measurements were recorded using a BIAcore 1990 GE Healthcare system (Pewaukee, WI, USA) with bare gold chips. It was mainly used to test the biotin functionalization procedure and the specificity of this biotin - DDT layer. For this experiment, increasing concentrations of avidin (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM) were used to test the surface sensitivity. Furthermore, the functionalized surface was also exposed to a flow of concentrated BSA (1.5 μ M) in order to determine the surface specificity for avidin detection.
Results and discussion
Gold nanocylinder fabrication
SERS results obtained on biotin and avidin with UV-NIL GNCs are shown in Figure 4b. Since SERS is highly sensitive to the first layer deposited on the gold surface, the red Raman spectrum should then be relevant of the whole functionalization layer (i.e., cysteamine, DDT, and biotin). Yet, cysteamine and DDT have a low Raman cross section; thus, we assume that they would be barely visible and that the red spectrum is mainly due to the biotin molecule in interaction with cysteamine. It is difficult to compare this spectrum with the biotin-NHS powder spectrum. The main reason for this is that the NHS group has been removed during the reaction between biotin and the cysteamine. The main visible peaks in the red spectrum are located at 1145, 1202, 1276, 1493, 1537, 1572, and 1591 cm -1. In a second step, avidin was added onto the sample, giving rise to the green spectrum. A new set of peaks is seen revealing the interaction between avidin and biotin. The spectrum of avidin on biotin is quite different from the one of the avidin solution. Two explanations are given: the interaction between avidin and biotin (this behavior has already been observed in references [22, 23]) and the fact that the biotin-avidin spectrum has been acquired in dry conditions giving rise to probable conformational changes.
where ISERS and IRaman are the SERS and Raman intensities, respectively. NSERS and NRaman are the number of excited molecules in SERS and Raman experiments, respectively. Thus, we found an EF value of approximately 3.7 ×107 for avidin/biotin system. This EF value is comparable or slightly higher than that obtained by other fabrication techniques such as EBL. Moreover, the advantages of the UV-NIL technique are the fabrication speed, the low cost, reproducibility, and homogeneity of nanostructures on large pattern areas. These advantages are very important parameters for the SERS application. In addition, giving this EF value and considering the signal/noise ratio of the SERS spectrum, it would be possible to dilute the concentration of avidin at least 100 times and still be able to detect it. Thus, we assume that we could detect avidin at concentrations in the micromolar range, which is in agreement with the detection limit already calculated for other proteins but using nanocylinders produced by EBL .
In summary, we have fabricated by the UV-NIL technique a large area of ordered GNCs, which was used as SERS substrate. The use of UV-NIL enabled to reach comparable results with EBL in terms of enhancement factor. Moreover, this UV-NIL technique is faster and less expensive than EBL. The size and shape of nanostructures are homogenous and reproducible on large pattern areas, which are important points for industrial applications of SERS. We detected a weak number of avidin molecules (3.8 ×106) by SERS measurements. A great advantage of SERS is the fingerprint provided by each molecule in order to identify the detected molecules. Finally, the SERS substrates fabricated by UV-NIL have thus showed their ability in biosensing with a good sensitivity.
This research was partially funded by the French National Agency ANR-11-ECOT-010-Remantas project and the French National Agency ANR-12-NANO-0016 Piranex project. The publication is supported under the Campus for Research Excellence And Technological Enterprise program.
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