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).
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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- Nie S, Emory SR (1997) Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 275:1102View ArticleGoogle Scholar
- Tripp RA, Dluhy RA, Zhao Y (2008) Novel nanostructures for SERS biosensing. Nanotoday 3:31View ArticleGoogle Scholar
- Fang Y, Seong NH, Dlott DD (2008) Measurement of the distribution of site enhancements in surface-enhanced Raman scattering. Science 321:388View ArticleGoogle Scholar
- Demirel MC, Kao P, Malvadkar N, Wang H, Gong X, Poss M, Allara DL (2009) Bio-organism sensing via surface enhanced Raman spectroscopy on controlled metal/polymer nanostructured substrates. Biointerphases 4:35View ArticleGoogle Scholar
- Kao P, Malvadkar NA, Cetinkaya M, Wang H, Allara DL, Demirel MC (2008) Surface‐enhanced Raman detection on metalized nanostructured poly (p‐xylylene) films. Adv Mater 20:3562View ArticleGoogle Scholar
- Li JF, Huang YF, Ding Y, Yang ZL, Li SB, Zhou XS, Fan FR, Zhang W, Zhou ZY, Wu DY, Ren B, Wang ZL, Tian ZQ (2010) Shell-isolated nanoparticle-enhanced Raman spectroscopy. Nature 464:392View ArticleGoogle Scholar
- Chen Z, Liu R, Wang Y, Zhu H, Sun Z, Zuo T, Chang X, Zhao F, Xing G, Yuan H, Xiang J, Gao X (2010) Ag nanopaticles coated SWCNT with surface enhanced Raman scattering (SERS) signals. J Nanosci Nanotechnol 10:8538View ArticleGoogle Scholar
- Jarvis RM, Brooker A, Goodacre R (2006) Surface-enhanced Raman scattering for the rapid discrimination of bacteria. Faraday Discuss 132:281View ArticleGoogle Scholar
- Sajanlal PR, Pradeep T (2012) Functional hybrid nickel nanostructures as recyclable SERS substrates: detection of explosives and biowarfare agents. Nanoscale 4:3427View ArticleGoogle Scholar
- Lee HO, Chae WS, Kim JW, Yu H (2011) Surface enhanced Raman scattering from porous gold nanofibers of different diameters. J Nanosci Nanotechnol 11:566View ArticleGoogle Scholar
- Li DW, Zhai WL, Li YT, Long YT (2014) Recent progress in surface enhanced Raman spectroscopy for the detection of environmental pollutants. Microsc Acta 181:23Google Scholar
- Wang HH, Liu CY, Wu SB, Liu NW, Peng CY, Chan TH, Hsu CF, Wang JK, Wang YL (2006) Highly raman‐enhancing substrates based on silver nanoparticle arrays with tunable sub‐10 nm gaps. Adv Mater 18:491View ArticleGoogle Scholar
- Liu TT, Lin YH, Hung CS, Liu TJ, Chen Y, Huang YC, Tsai TH, Wang HH, Wang DW, Wang JK, Wang YL, Lin CH (2009) A high speed detection platform based on surface-enhanced Raman scattering for monitoring antibiotic-induced chemical changes in bacteria cell wall. PLoS One 4:e5470View ArticleGoogle Scholar
- Liu TY, Tsai KT, Wang HH, Chen Y, Chen YH, Chao YC, Chang HH, Lin CH, Wang JK, Wang YL (2011) Functionalized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood. Nature Commun 2:538View ArticleGoogle Scholar
- Hardiansyah A, Chen AY, Liao HL, Yang MC, Liu TY, Chan TY, Tzou HM, Kuo CY, Wang JK, Wang YL (2015) Core-shell of FePt@ SiO2-Au magnetic nanoparticles for rapid SERS detection. Nanoscale Res Lett 10:412View ArticleGoogle Scholar
- Mevold AHH, Hsu WW, Hardiansyah A, Huang LY, Yang MC, Liu TY, Chan TY, Wang KS, Su YA, Jeng RJ, Wang JK, Wang YL (2015) Fabrication of gold nanoparticles/graphene-PDDA nanohybrids for bio-detection by SERS nanotechnology. Nanoscale Res Lett 10:397View ArticleGoogle Scholar
- Mevold AHH, Liu JY, Huang LY, Liao HL, Yang MC, Chan TY, Wang KS, Wang JK, Wang YL, Liu TY (2015) Core-shell structure of gold nanoparticles with inositol hexaphosphate nanohybrids for label-free and rapid detection by SERS nanotechnology. J Nanomater 2015:Article ID 857154. https://www.hindawi.com/journals/jnm/2015/857154/.
- Ho JY, Liu TY, Wei JC, Wang JK, Wang YL, Lin JJ (2014) Selective SERS detecting of hydrophobic microorganisms by tricomponent nanohybrids of silver-silicate-platelet-surfactant. ACS Appl Mater Interfaces 6:1541View ArticleGoogle Scholar
- Liu TY, Ho JY, Wei JC, Cheng WC, Chen IH, Shiue J, Wang HH, Wang JK, Wang YL, Lin JJ (2014) Label-free and culture-free microbe detection by three dimensional hot-junctions of flexible Raman enhancing nanohybrid platelets. J Mater Chem B 2:1136View ArticleGoogle Scholar
- Liu TY, Chen Y, Wang HH, Huang YL, Chao YC, Tsai KT, Cheng WC, Chuang CY, Tsai YH, Huang CY, Wang DW, Lin CH, Wang JK, Wang YL (2012) Differentiation of bacteria cell wall using Raman scattering enhanced by nanoparticle array. J Nanosci Nanotechnol 12:5004View ArticleGoogle Scholar
- Biring S, Wang HH, Wang JK, Wang YL (2008) Light scattering from 2D arrays of monodispersed Ag-nanoparticles separated by tunable nano-gaps: spectral evolution and analytical analysis of plasmonic coupling. Opt Express 16:5312View ArticleGoogle Scholar
- Lin BY, Hsu HC, Teng CH, Chang HC, Wang JK, Wang YL (2009) Unraveling near-field origin of electromagnetic waves scattered from silver nanorod arrays using pseudo-spectral time-domain calculation. Opt Express 17:4222Google Scholar
- Chen YW, Liu TY, Chen PJ, Chang PH, Chen SY (2016) A high-sensitivity and low-power theranostic nanosystem for cell SERS imaging and selectively photothermal therapy using anti-EGFR-conjugated reduced graphene oxide/mesoporous silica/AuNPs nanosheets. Small 12:1458View ArticleGoogle Scholar
- Chiang CY, Liu TY, Su YA, Wu CH, Cheng YW, Cheng HW, Jeng RJ (2017) Au nanoparticles immobilized on honeycomb-like polymeric films for surface-enhanced raman scattering (SERS) detection. Polymers 9:93View ArticleGoogle Scholar
- Wang X, Wen H, He T, Zuo J, Xu C, Liu FC (1997) Enhancement mechanism of SERS from cyanine dyes adsorbed on Ag2O colloids. Spectrochim Acta A 53:2495View ArticleGoogle Scholar
- Lin WC, Liao LS, Chen YH, Chang HC, Tsai DP, Chiang HP (2011) Size dependence of nanoparticle-SERS enhancement from silver film over nanosphere (AgFON) substrate. Plasmonics 6:201View ArticleGoogle Scholar
- Yi Z, Niu G, Luo J, Kang X, Yao W, Zhang W, Yi Y, Yi Y, Ye X, Duan T, Tang Y (2016) Ordered array of Ag semishells on different diameter monolayer polystyrene colloidal crystals: an ultrasensitive and reproducible SERS substrate. Sci Rep 6:32314View ArticleGoogle Scholar
- Liu H, Zhang L, Lang X, Yamaguchi Y, Iwasaki H, Inouye Y, Xue Q, Chen M (2011) Single molecule detection from a large-scale SERS-active Au79Ag21 substrate. Sci Rep 1:112View ArticleGoogle Scholar
- Huang Q, Wen S, Zhu X (2014) Synthesis and characterization of an AgI/Ag hybrid nanocomposite with surface-enhanced Raman scattering performance and photocatalytic activity. RSC Adv 4:37187View ArticleGoogle Scholar
- Srivastava S, Sinha R, Roy D (2004) Toxicological effects of malachite green. Aquat Toxicol 66:319View ArticleGoogle Scholar
- Sudova E, Machova J, Svobodova Z, Vesely T (2007) Negative effects of malachite green and possibilities of its replacement in the treatment of fish eggs and fish: a review. Vet Med 52:527Google Scholar
- Wang HH, Cheng TY, Sharma P, Chiang FY, Chiu SW, Wang JK, Wang YL (2011) Transparent Raman-enhancing substrates for microbiological monitoring and in situ pollutant detection. Nanotechnology 22:385702View ArticleGoogle Scholar
- Ermakov IV, Ermakova MR, Gellermann W, Lademann J (2004) Noninvasive selective detection of lycopene and β-carotene in human skin using Raman spectroscopy. J Biomed Opt 9:332View ArticleGoogle Scholar
- Olives Barba AI, Camara Hurtado M, Sanchez Mata MC, Fernandez Ruiz V, Lopez Saenz de, Tejada M (2006) Application of a UV–vis detection-HPLC method for a rapid determination of lycopene and b-carotene in vegetables. Food Chem 95:328View ArticleGoogle Scholar