SERS Detection of Biomolecules by Highly Sensitive and Reproducible Raman-Enhancing Nanoparticle Array
© The Author(s). 2017
Received: 5 November 2016
Accepted: 2 May 2017
Published: 10 May 2017
This paper describes the preparation of nanoarrays composed of silver nanoparticles (AgNPs: 20–50 nm) for use as surface-enhanced Raman scattering (SERS) substrates. The AgNPs were grown on porous anodic aluminum oxide (AAO) templates by electrochemical plating, and the inter-channel gap of AAO channels is between 10 and 20 nm. The size and interparticle gap of silver particles were adjusted in order to achieve optimal SERS signals and characterized by scanning electron microscopy, atomic force microscopy, and Raman spectroscopy. The fluctuation of SERS intensity is about 10–20% when measuring adenine solutions, showing a great reproducible SERS sensing. The nanoparticle arrays offer a large potential for practical applications as shown by the SERS-based quantitative detection and differentiation of adenine (A), thymine (T), cytosine (C), guanine (G), β-carotene, and malachite green. The respective detection limits are <1 ppb for adenine and <0.63 ppm for β-carotene and malachite green, respectively.
Uniform and reproducible Raman enhancement enabled by Ag nanoparticle array embedded in anodic aluminum oxide differentiates and helps quantify DNA canonical nucleobases (adenine, thymine, cytosine, and guanine).
KeywordsSurface-enhanced Raman scattering (SERS) Nanoparticle arrays Biomolecules detection
Surface-enhanced Raman spectroscopy (SERS) nanotechnology is an interesting platform for rapid and precise identification of small biomolecules and becomes a potentially fingerprinting and bio-detecting technology, due to enhance Raman signals by 6–13 orders of magnitude in the SERS-active surface [1–11]. The SERS-active surface was fabricated by the arrangement of silver or gold nanoparticle arrays, which generated the localized surface plasmon resonances and the laser exposure in the analytic biomolecules.
The key point of SERS technology is focused on controlling the interparticle gap and the diameter of the metal nanoparticles. The report demonstrated that “hot-spot” effect can be generated when the interparticle gap is lower than 10 nm, which will enormously increase SERS signals when the analytic biomolecules are close to the SERS-active surface. However, the hot-spot effect of the SERS-active substrate is immensely enhanced with decreased interparticle gap-to-particle diameter ratio, which can affect the stability of SERS activity if the variation of the interparticle gap and the particle diameter cannot be brought into control. The detection of this SERS-active substrate has achieved monolayer sensitivity, which is useful to detect a small number of biomolecules observed in a cellular compartment [12, 13], such as sensing in immunoassays, DNA, cancer cells, and microbes [4, 14–20].
In our previous study of Ag nanoparticles embedded in ordered array of anodic aluminum oxide (AgNP/AAO substrate) , scattering spectra-rather than transmission or reflection spectra owing to their optical inference-of such array with different interparticle gaps were acquired to reveal their electromagnetic resonance properties. Analytic formulae were derived based on electrostatic dipole approximation to describe the resonance wavelength and width as a function of dimensional factors of the Ag nanoparticle array (particle diameter and interparticle spacing). The experimental results are in good agreement with the derived formulate. For SERS applications, since the localized surface plasmon resonance (LSPR) wavelength in the present work (interparticle spacing: 10–30 nm) is in the range of 500–700 nm (peaked at 620 nm), He–Ne laser (632.8 nm) was used as the excitation light source to boost the local excitation field and the ensuing emission propensity. Furthermore, for simulation study, we performed high precision electrodynamic simulation based on pseudo-spectral time-domain (PSTD) method in our previous study . The far-field scattering spectra thus obtained from calculation also showed good agreement with experimental findings. The surface electric and magnetic fields of the Ag nanoparticle array under two polarization excitation schemes (along x- and y-axis) were calculated. The enhanced electric local field was manifested at the gap between adjacent nanoparticles.
The AgNP/AAO substrate was investigated with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Its SERS sensitivity and reproducibility are presented in this report. Finally, the SERS detection of small biomolecules (adenine (A), thymine (T), cytosine (C), guanine (G) from DNA, and β-carotene) and water pollutants (malachite green) is also demonstrated here.
Fabrication of AgNP/AAO Substrate
A glass slide with 150 nm of aluminum (Al) thin film deposited by sputtering was anodized in the sulfuric acid (0.3 M) using a voltage of 16 V to form porous AAO substrates with arrays of self-organized nanochannels with the specific pore diameters and interparticle gaps. The AAO nanochannels were then chemically etched in phosphoric acid and chromic acid at 35 °C to achieve optimal pore size and interparticle gap for this study. To grow Ag nanoparticles in the AAO nanochannels by electrochemical plating, an alternating voltage of 9 V was applied to the AAO substrate in a solution of silver nitrate (0.006 M) and magnesium sulfate (0.165 M) (molarity ratio: 1: 27.5) for different time duration. After depositing Ag nanoparticles to the desired length, the substrate was washed by hydrochloric acid (0.2 M) and formed the final AgNP/AAO SERS-active substrate .
SERS Measurements of Biomolecules and Water Pollutant
DNA canonical nucleobases (adenine, thymine, cytosine, and guanine), β-carotene, and malachite green, used as model pollutants in water, were purchased from Sigma-Aldrich. They were all used without further purification. Sample solutions were prepared by dissolving in water at designated concentrations. Five microliters of each sample solution was dropped on the surface of the AgNP/AAO substrate and dried for 20 min before the Raman measurements . The morphology of AgNP/AAO substrates was performed on a field-emission scanning electronic (SEM) microscope (FESEM, JSM-6700F, JEOL) and atomic force microscope (AFM) (Dimension 3100, Bruker) in tapping mode.
Raman measurements were carried out in a commercial Raman microscope (HR800, Horiba) with a He–Ne laser (632.8 nm) as the excitation light source. The laser beam, after passing through a laser-line filter to remove residual plasma lines, was focused by an objective lens to the substrate surface. In this experiment, ×20 objective lens (spot size is about 20–30 μm) was used to evaluate the uniformity and reproducibility of SERS signal. The scattering radiation was collected by the same objective lens and sent to an 80-cm spectrometer (1800 gr/mm) plus liquid-nitrogen-cooled charge-coupled device for spectral recording. The resultant spectral resolution and accuracy are 3 and 0.1 cm−1, respectively. The irradiated laser power was adjusted to prevent any laser-induced damage such that the portrayed Raman signal is linearly related to the laser power. The signal acquisition time was 60 s. Each acquired SERS spectrum was processed with a home-made software to remove high-frequency noise and continuum background .
Results and Discussion
Characterization of AgNP/AAO Substrate
Performances of SERS Substrate: Reproducibility and Enhancement
where N bulk is the number of analyte molecules (adenine) sampled in the bulk, and N SERS is the number of adenine adsorbed on the SERS substrates. I SERS and I Raman denote the integrated intensities at specific peaks (732.8 cm−1) in the SERS and Raman spectra. With the same spot size of the laser and the same content of adenine, the ratio of N SERS to N bulk could be deemed as the ratio of two concentrations of adenine. The EF value of SERS substrate with and without the AAO template are 1.9 × 108 and 1.1 × 107, respectively. The result shows that the particle size and interparticle gap of Ag nanoparticles can be effectively manipulated by AAO templets to enhance the SERS sensitivity. From the literatures [25–29], the EF of the Ag-based SERS substrate is in the broad range of 102~109, which depends on the density and layers of Ag nanoparticles, molecule adsorbability on the SERS substrate, and the parameter setting of Raman spectroscopy (e.g., different wavelengths of the laser). In other words, the higher EF would induce the poorer reproducibility. For the one layer of Ag nanoparticles system, our SERS substrate exhibits enough enhancement factor (>108) with great reproducibility (~10%) from substrate to substrate.
By the way, although it is common to compare the enhancement factor among different SERS enhancers, it is not the only performance factor that is relevant to the applications of SERS technology. The following factors are equally important, if not more: reliability, uniformity, ease of operation, large size, speed, etc. Our Ag/AAO SERS substrate in this work exhibits these advantages, compared with that of other Ag systems.
SERS Detection of Canonical Nucleobases of DNA
SERS Detection of Water Pollutant (Malachite Green)
SERS Detection of β-Carotene
In our knowledge, compared with other Ag systems, Ag/AAO SERS substrate could detect a variety of small biomolecules (adenine (A), thymine (T), cytosine (C), guanine (G), β-carotene) and water pollutants (malachite green), which can be used extensively in different fields without further labelling or modification.
This paper demonstrates that Ag–AAO nanoparticle arrays can be reproducibly fabricated and exploited for SERS-based detection of small biomolecules such as canonical nucleobases of DNA, β-carotene, and water pollutants. The uniformity of SERS signal of adenine varies about 10–20% at seven different spots on AgNP/AAO substrate. The reproducibility result shows that SERS signal of adenine is varied by about 10% at seven different AgNP/AAO substrates. Furthermore, the durability result shows that the signal strength of adenine varied by 8% even after 28 days. The high sensitivity, uniformity, and reproducibility of this SERS-active substrates based on AgNP/AAO can in principle be used for the quantitative determination of the label-free health diagnostics, bio-sensing, and water detection, such as sensing antioxidant capability (β-carotene), detecting DNA canonical nucleobases (adenine, thymine, cytosine, guanine), and in situ monitoring water pollutants (malachite green), respectively.
This work was financially supported by the Ministry of Science and Technology of Taiwan (MOST 105-2623-E-016-001-D and MOST 105-2628-M-001-001) and partially supported by Academia Sinica.
The authors declare that they have no competing interests.
TYC, TYL, JKW, and YLW had conceived and designed the experiments. TYC, KSW, ZXC, YQT, and CHW performed the experiments. KSW, KTT, ZXC, and YCC contributed the ideas and material analyses. TYC, TYL, JKW, and YLW wrote the manuscript. All authors read and approved the final manuscript.
TYC, KSW, YQT, and CHW are undergraduate students at Ming Chi University of Technology. TYL holds an assistant professor position at Ming Chi University of Technology. KTT and YCC are postdoctoral fellows at Academia Sinica. ZXC is a research assistant at Academia Sinica. JKW is a research fellow at Center for Condensed Matter Sciences, National Taiwan University and Academia Sinica, Taiwan. YLW is a distinguished research fellow at Academia Sinica, Taiwan, and an adjunct professor at National Taiwan University.
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