Biosensors and Bioelectronics 72 (2015) 61–70
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Intense Raman scattering on hybrid Au/Ag nanoplatforms for the distinction of MMP-9-digested collagen type-I fiber detection Kundan Sivashanmugan a, Jiunn-Der Liao a,b,n, Pei-Lin Shao a, Bernard Haochih Liu a, Te-Yu Tseng a, Chih-Yu Chang a a b
Department of Materials Science and Engineering, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan Center for Micro/Nano Science and Technology, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 28 March 2015 Accepted 27 April 2015 Available online 29 April 2015
Well-ordered Au-nanorod arrays were fabricated using the focused ion beam method (denoted as fibAu_NR). Au or Ag nanoclusters (NCs) of various sizes and dimensions were then deposited on the fibAu_NR arrays using electron beam deposition to improve the surface-enhanced Raman scattering (SERS) effect, which was verified using a low concentration of crystal violet (10–5 M) as the probe molecule. An enhancement factor of 6.92 108 was obtained for NCsfibAu_NR, which is attributed to the combination of intra-NC and NR localized surface plasmon resonance. When 4-aminobenzenethiol (4ABT)-coated Au or Ag nanoparticles (NPs) were attached to NCsfibAu_NR, the small gaps between 4-ABTcoated NPs and intra-NCs allowed detection at the single-molecule level. Hotspots formed at the interfaces of NCs/NRs and NPs/NCs at a high density, producing a strong local electromagnetic effect. Raman spectra from as-prepared type I collagen (Col-I) and Ag-NP-coated Col-I fibers on NCsfibAu_NR were compared to determine the quantity of amino acids in their triple helix structure. Various concentrations of matrix-metalloproteinase-9-digested Col-I fibers on NCsfibAu_NR were qualitatively examined at a Raman laser wavelength of 785 nm to determine the changes of amino acids in the Col-I fiber structure. The results can be used to monitor the growth of healing Col-I fibers in a micro-environment. & 2015 Elsevier B.V. All rights reserved.
Keywords: Au nanorods Nanoclusters Surface-enhanced Raman scattering 4-aminobenzenethiol Digested type I collagen
1. Introduction The growth process during wound healing is of great interest. Skin tissue reconnection with active scaffolds or stem cells is often used to study cell differentiation and proliferation on the wound bed (Jain et al., 2014; Ngo et al., 2014; Ngo Thi et al., 2014). Biodegradable and nonbiodegradable proteins and peptides are promising supports for tissue reconnection owing to their useful properties, such as chemical structural diversity, large number of architectures, mechanical strength, and biocompatibility (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Shoulders and Raines, 2009). A variety of natural proteins and peptides, such as laminin, fibronectin, and collagen (Col) fibers, are often used to improve the efficacy of tissue reconstruction (Silver et al., 2003; Stamov et al., 2008). Col fibers are most commonly used in tissue regeneration due to their excellent rates of biointegration, biodegradability, and biocompatibility (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Silver et al., 2003; Stamov et al., 2008). The n Corresponding author at: Department of Materials Science and Engineering, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan. Fax: þ886 62346290. E-mail address: [email protected] (J.-D. Liao).
http://dx.doi.org/10.1016/j.bios.2015.04.091 0956-5663/& 2015 Elsevier B.V. All rights reserved.
triple helix structure of Col fibers may improve tissue growth via several cross-linking procedures (Kadler et al., 1996; Shoulders and Raines, 2009). However, the degradation of Col fibers is also an important process in wound healing, which generally occurs during the growth and development of adult connective tissues within pericellular and extracellular environments. In mature tissues, existing Col fibers are replaced by new Col fibers (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Silver et al., 2003; Stamov et al., 2008). Complex tissue reconstruction requires type I Col (Col-I) fiber protein, which is an important structural component in blood vessels, skin, tendons, ligaments, and bone (Bozec et al., 2007; Fang et al., 2013). The degradation of Col-I, still unclear in tissue reconstruction, can occur through either the extracellular or intracellular pathway (Fligiel et al., 2003; Orza et al., 2011; Sun et al., 2008b). The growth and degradation of Col-I fibers is tightly regulated. From in vitro studies, matrix metalloproteinases (MMPs), including MMP-1, -9, -13, and -14, can effectively degrade native Col-I fibers surrounding a cell surface through the glycine in Col-I fibers (Aimes and Quigley, 1995; Bigg et al., 2007; Fini et al., 1992; Knauper et al., 1997). In addition, the degradation of Col-I, -II, and -III fibers using various temperature-dependent treatments has been investigated (Cárcamo et al., 2012; Jain et al., 2014;
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Su et al., 2014). Methods for analyzing the degradation of Col-I fibers include the enzyme-linked immunosorbent assay and highperformance liquid chromatography (Akhtar et al., 1999; Hudson et al., 2012), which have excellent sensitivity for the detection of specific degraded enzymes in Col-I fibers. However, labeling techniques usually require sophisticated procedures for sample preparation and subsequent data analysis, as well as repetitive tests, making them high-cost. Therefore, a simple, quick, cost-effective, and sensitive detection method for monitoring Col-I fibers is highly required. Raman spectra provide structural and chemical information about target species in a chemical or biological sample (Luo et al., 2014; Xie and Schlucker, 2013). However, the very low efficiency of Raman data collection leads to long acquisition time for detecting normal analyte molecules. In addition, biological samples, e.g., cells, proteins, and viruses, require a method with high resolution or enhancement and sensitivity or selectivity. A strong Raman signal is generated when a Raman-active analyte interacts with a noble metal, e.g., Ag, Au, or Cu, surface. A surface-enhanced Raman scattering (SERS)-active substrate allows detection down to a single-molecule level (Betz et al., 2014; Harper et al., 2013; Kleinman et al., 2013). The effect of SERS is mainly attributed to two primary mechanisms: the chemical effect and the electromagnetic (EM) effect (Betz et al., 2014; Harper et al., 2013; Kleinman et al., 2013). Very few studies have investigated Col-I fiber structure using SERS, e.g., by detecting signals from an Au or Ag nanoparticle (NP)-coated Col-I fiber surface (Orza et al., 2011; Sun et al., 2008b). Ag-NP-coated Col-I fiber produces strong Raman signals, but with low reproducibility. Alternatively, nanolithography techniques such as electron beam lithography (Luo et al., 2014), focused ion beam (FIB) (Sivashanmugan et al., 2013a, 2013b), and nanomechanical indentation (Chang et al., 2011; Yao et al., 2012) have been employed to fabricate various types of nanostructure (NS), e.g., nanorods (NRs), nanowires, nanocavities. Nanofabrication methods create highly precise and regular patterns that have a high enhancement factor (EF). SERS-active substrates have been used to detect nucleoproteins, oligonucleotides, and viruses due to the strong light scattering in the vicinity of nanogaps and NSs (Shao et al., 2014; Sivashanmugan et al., 2015, 2013a). In our previous work, SERS-active substrates with well-ordered
Au or Au/Ag multilayered NR arrays were fabricated using FIB technology (Sivashanmugan et al., 2015, 2013a, 2013b). It is hypothesized that a high EM effect due to the availability of multiple edges and a small curvature induced in the substrate (Costa et al., 2009; Shao et al., 2014; Sivashanmugan et al., 2014, 2015, 2013a). In addition, the localized surface plasmon resonance (LSPR) effect strongly depends on NR shape and the spacing between NRs. The LSPR effect is known to increase SERS sensitivity up to the singlemolecule level. Notably, the SERS effect highly varies with the size of the target species due to laser interaction strength. Large biotarget species, e.g., cells and proteins, usually require a substrate with strong and highly reproducible LSPR. Therefore, a substrate with a high SERS effect is required for practical applications (Que et al., 2011, 2012). In the present work, FIB and electron beam deposition were combined for the fabrication of Au and Ag nanoclusters (NCs) on a FIB-made Au NR arrays (NCsfibAu_NR) as a SERS substrate, as illustrated in Fig. 1(a). The Au or Ag NCs on fibAu_NR influence the detection of Raman-active species. This influence was examined using a low concentration (10–5 M) of crystal violet (CV) as the probe molecule. As shown in Fig. 1(b), 4-aminobenzenethiol (4ABT)-coated Au or Ag NPs were deposited on an optimized NCsfibAu_NR substrate to create a large plasmonic effect via a large number of hotspots. As shown in Fig. 1(c), Col-I, Ag-NPcoated Col-I fibers, and MMP-9-digested Col-I fibers were deposited on an optimized NCsfibAu_NR substrate to determine changes in Col-I fiber structure. The former application utilizes a target chemical species bonded with NPs and attached to NCs®bAu_NR, while the latter utilizes a target biological sample with NPs and attached to NCsfibAu_NR. These applications were used to confirm whether the hybrid Au/Ag nanostructure can intensify the Raman scattering effect on the target species simply with NPs.
2. Experimental section 2.1. Fabrication of Au or Ag NCs on fibAu_NR Au thin film was deposited onto polished single-crystal silicon (100) wafers primed with a 5- nm-thick adhesion layer of Ti using an electron beam evaporator (VT1-10CE, ULVAC, Taiwan) at a
Fig. 1. Schematic illustrations of experimental route for (a) as-fabricated NCsfibAu_NR, (b) 4-ABT-coated Au or Ag NPs on NCsfibAu_NR, and (c) Col-I, NP-coated Col-I, and MMP-9-digested Col-I fibers on NCsfibAu_NR.
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controlled deposition rate. The optimal layer thickness for Au film (E 420 nm) was maintained. Patterns were designed using CorelDRAW software. The NR designs were implemented by applying an FIB (SMI 3050, SII Nanotechnology, Japan). The pattern size was about 120 mm 120 μm. The following beam conditions were used: 30-kV acceleration voltage, 0.07-μm depth, 10-pA aperture, 70-μs dwell time, þ0.56 optical lens, and 8-image scale (Sivashanmugan et al., 2013a, 2013b). The spacing between adjacent NRs, diameter, and shape of Au NR arrays (fibAu_NR) were maintained by adjusting the beam current. Au or Ag NCs were then deposited on the fibAu_NR arrays to increase the effect of SERS, as illustrated in Fig. 1(a). Au or Ag NCs on fibAu_NR were prepared using electron beam evaporation deposition with estimated diameters of 2, 5, and 8 nm; the corresponding samples are denoted as G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR for Au NCsfibAu_NR and S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR for Ag NCsfibAu_NR. The size of the as-prepared NCs was based on the spacing between NRs (D-spacing, i.e., E 35 nm). Information about all NCs and NCsfibAu_NR are given in Tables S1 and S2 (in Supporting Information), respectively. Field-emission scanning electron microscopy (FE-SEM, JSM-7001, JEOL, Japan), atomic force microscopy (AFM, Bruker, ICON SPM, USA) and high-resolution field-emission transmission electron microscopy (HR-FETEM, JEM-2100 F, JEOL, Japan) with energy-dispersive X-ray spectroscopy (EDS) were employed to analyze the morphologies of the as-prepared NCs, fibAu_NR, and NCsfibAu_NR.
2.2. EF determination using molecular probe The molecular probe CV was diluted in aqueous solution to a concentration in the range of 10–5–10 15 M. To verify EF, fibAu_NR or NCsfibAu_NR with a molecular-probe-containing solution was covered with a glass slide and then immediately measured using Raman spectroscopy. Raman spectra were acquired using a confocal microscopy Raman spectrometer (inVia Raman microscope, Renishaw, United Kingdom) with diode lasers at an excitation wavelength of 785 nm, and then scanned with an integration time of 10 s over an area of 1 μm 1 μm (the size of the laser spot was 1 μm) using a 50 objective. Before each batch, the Raman shift was calibrated using the absolute intensity of signal at 520 cm 1 from a standard silicon wafer. The EF measurement was estimated according to the standard equation (Sivashanmugan et al., 2013a, 2013b)
I N EF = sers × bulk Ibulk Nsers
(1)
where Isers and Ibulk are the SERS and normal Raman scattering intensities, respectively, and Nsers and Nbulk are the numbers of molecules contributing to the inelastic scattering intensity respectively evaluated by SERS and normal Raman scattering measurements. The Raman intensity was averaged from 10 consecutive measurements. The highest peak intensity for all samples was recorded as the specific band and treated as the Isers value in Eq. (1). The ideal average Nsers value (E6.17 107 molecules) was obtained from the volume of the uniform monolayer of CV ideally adsorbed on NCsfibAu_NR. The Nsers value is correlated with the concentration of 10 5 M of CV-containing solution. The spot size of the laser and the average value of Nbluk were obtained as E1 μm2 and E9.23 108 molecules, respectively. The Ibulk values were measured as E 280 (arbitrary units). The above values were substituted into Eq. (1) to calculate the SERS EF values for samples.
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2.3. Fabrication of 4-ABT-coated Au or Ag NPs on NCsfibAu_NR Gold (III) chloride trihydrate (HAuCl4 3H2O), silver nitrate (AgNO3), sodium citrate (HOC(COONa)(CH2COONa)2 2H2O), and 4-ABT (H2NC6H4SH) were purchased from Sigma-Aldrich. Ultrapure water (E10 MΩ cm) was used as a solvent in the experiments. All glassware used in the experiments was cleaned with nitric acid before use. The NPs to be coated with 4-ABT for deposition on NCs®bAu_NR were prepared based on a well-established method (Sivashanmugan et al., 2013b). Firstly, an Au sol was prepared by dissolving 1 mM HOC(COONa)(CH2COONa)2 2H2O and 1 mL of 0.01 mM HAuCl4 3H2O in 20 mL of aqueous solution and then boiled for 30 min. In this mixture solution, Au seed turned reddish after 4 min. After continuous stirring for an additional 5 min, the mixture was allowed to stand at room temperature, and then washed five times using water with some specific parameters as follows: the radius and rotating speed were 5 cm and 12,000 rpm, respectively, to remove impurities and stored for further studies. For the growth of Ag sol, 100 mL of aqueous solution containing 1 mM AgNO3 was brought near boiling temperature. 3 mL of Ag seed solution and HOC(COONa)(CH2COONa)2 2H2O (with a final concentration of 1 mM in solution) was then added to the boiling solution. The mixture was heated until the color of the solution turned greenish yellow. The solution was removed and brought to room temperature, washed five times using water with some specific parameters as follows: the radius and rotating speed were 5 cm and 12,000 rpm, respectively, to remove impurities, and then stored for further studies. The morphology and structure of the NPs were characterized using HR-FETEM. The optical properties of the NPs were monitored as a function of time in 10- mm-opticalpath-length quartz cuvettes with an ultraviolet-visible (UV–vis) spectrophotometer (UV–3600, Shimadzu). The as-prepared Au or Ag NPs were then mixed with an optimized concentration of 5 mL of 4–ABT (10 mM ethanolic solution) and stabilized for 10 h. The solution was then washed with water and ethanol in sequence. Au or Ag NCsfibAu_NR was then covered with a droplet containing 4-ABT-coated Au or Ag NPs and dried in a vacuum for 2 h, washed with ethanol, and then dried in air. The 4-ABT-coated Au or Ag NPs on Au, fibAu_NR and NCsfibAu_NR substrates were examined at a Raman laser wavelength of 785 nm. The morphology and structure of 4-ABT-coated Au or Ag NPs on NCsfibAu_NR were respectively characterized using HR-FETEM and AFM. 2.4. Raman studies of Col-I fibers Rat tail Col-I was purchased from Merck Millipore and then diluted to 0.1 mg/mL in a 10-mL solution of phosphate-buffered saline (PBS) before use. The as-prepared samples were incubated at 37 °C for 30 min to grow Col-I fibers. Col-I was diluted to 0.1 mg/mL in a 5-mL solution of PBS with 5 mL (an optimized concentration) of Ag NPs and then incubated at 37 °C for 1 h to create NP-coated Col-I fibers. Col-I and Ag-NP-coated Col-I fibers were then placed on an optimized NCsfibAu_NR. After 15 min, the samples were dried with nitrogen gas and subjected to Raman spectroscopic studies. The morphology and structure of Col-I and Ag-NP-coated Col-I fibers on an optimized NCsfibAu_NR was characterized using AFM. MMP-9 was purchased from Merck Millipore. To activate MMP-9, MMP-9 with a concentration of 0.005 or 0.01 mg/mL was diluted in a 10-mL solution of PBS. To digest Col-I fibers, the fibers were mixed with various concentrations of MMP-9, and then incubated at 27 °C for 30 min. The MMP9-digested Col-I fibers were then placed on an optimized NCs®bAu_NR and subjected to Raman analysis. The morphology and structure of the digested Col-I fibers on an optimized NCsfibAu_NR
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were characterized using AFM.
3. Results and discussion 3.1. Characterization of as-fabricated Au or Ag NCsfibAu_NR As shown in Fig. S1, the morphology of fibAu_NR was analyzed using FE-SEM, AFM, and HR-FETEM. The specified spacing between adjacent NRs was maintained by adjusting the working current and etching time during FIB fabrication. Fig. S1(a) and (b) show top- and side-view images of fibAu_NR, respectively. The length, diameter, and spacing between NRs for fibAu_NR were E400, E 170, and 35 nm, respectively. The AFM topography of fibAu_NR is shown in Fig. S1(c). The as-formed NRs were uniformly fabricated within the patterned area, as indicated by the AFM line profiles shown in the inset in Fig. S1(c). The Au or Ag NCs were anticipated to slightly reduce the spacing between adjacent NRs and the tip ring diameter of NRs (Sivashanmugan et al., 2013b). To confirm the size, dimension, and distribution of Au or Ag NCs, the NCs were deposited on an Si (100) substrate and the resulting values were used as the reference. Fig. S2(a–f) show two-dimensional top-view AFM and FE-SEM images of the as-prepared Au or Ag NCs, evenly distributed on an Si (100) substrate. The gap between NCs significantly increased with
increasing size and dimension of the deposited Au or Ag NCs. In addition, the average roughness (Ra) of Ag NC sample S_3 (Ra E4.33 nm) was higher than that of Au NC sample G_3 (Ra E2.68) due to the shape and physical properties of Au or Ag NCs (Rycenga et al., 2009; Sivashanmugan et al., 2013b; Yamamoto et al., 2013; Yang et al., 2014), as listed in Table S1. The formation and distribution of Au or Ag NCs on various substrates were respectively examined using FE-SEM and HR-FETEM. Fig. 2(a–c) and S3(a–c) show side and cross-sectional (insets) FE-SEM images of Au or Ag NCsfibAu_NR. Both Au and Ag NCs were uniformly distributed on fibAu_NR. Furthermore, FE-SEM EDS spectra and elemental mapping of Au or Ag NCsfibAu_NR confirm the formation (as shown in Fig. S4) and distribution of NCs on fibAu_NR. Ag NCs on fibAu_NR were highly aggregated compared to Au NCs on fibAu_NR due to their varied deposition rates, e.g., by measuring the decrease of Au element from fibAu_NR, as listed in Table S2. In addition, Ra values, measured from AFM, for Ag NCsfibAu_NR ranged from 18 to 33 nm and followed the sequence S_1fibAu_NR 4S_2fibAu_NR 4S_3fibAu_NR. These values were higher than the corresponding values for Au NCsfibAu_NR samples G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR (13– 21 nm). Notably, the EM effect, and thus the SERS effect, is significantly increased by surfaces with a relatively high Ra (Costa et al., 2009; Kleinman et al., 2013; Rycenga et al., 2009; Shao et al., 2014; Sivashanmugan et al., 2014, 2015, 2013a, 2013b; Yamamoto
Fig. 2. FE-SEM top- and lateral-view (insets) images of Ag NCs on fibAu_NR: (a) S_1fibAu_NR, (b) S_2fibAu_NR, and (c) S_3fibAu_NR (inset image scale bar: 50 nm). HRFETEM images of Ag NCs on fibAu_NR: (a-i) S_1fibAu_NR, (b-i) S_2fibAu_NR, and (c-i) S_3fibAu_NR. (a c-ii) HR-FETEM images of Ag NC distribution on fibAu_NR. Marked regions in (a c-ii) indicate thickness of Ag NCs on fibAu_NR surface in (a c-iii).
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et al. 2013; Yang et al. 2014). Fig. 2(a c-i) and S3(a c-i) show HR-FETEM images of Ag or Au NCsfibAu_NR. A relatively high density of NCs on NRs was obtained by varying the deposition angle (e.g., the Au or Ag target was placed perpendicular to fibAu_NR). The spacing between adjacent NRs was not fully enclosed by Ag or Au NCs, as shown in Fig. 2(a–c-ii) and S3(a–c-ii). As shown in Fig. 2(a–c-iii) and S3(a–ciii), a small gap (e.g., o2 nm) formed between NCs on the side surface of NRs, which may greatly enhance Raman scattering around NCs. The geometry of NCs on NRs is also an important factor: increasing the size and dimension of Ag or Au NCs on Au NRs tended to decrease the D-spacing of NRs, as summarized in Table S2. The optimized sample S_3fibAu_NR had lattice spacings of 0.26 and 0.24 nm for Au and Ag, respectively, which match the lattice spacing of (111) face-centered cubic planes (Fig. S5) (Rycenga et al., 2009; Yang et al., 2014). Moreover, a tiny gap (e.g., o1 nm) formed at the interface between NCs (intra-NCs) and NRs (NCs on NRs), greatly increasing the effect of LSPR around NCs and NCs on NRs and therefore increasing the sensitivity of SERS (Rycenga et al., 2009; Yamamoto et al., 2013; Yang et al., 2014; Yilmaz et al., 2014). 3.2. SERS effect on fibAu_NR and Au or Ag NCsfibAu_NR The sensitivity and enhancement effect of CV were determined at a Raman laser wavelength of 785 nm. The insets in Fig. 3(a) and (b) show 10 5 M CV solution dispersed on fibAu_NR, Au NCs®bAu_NR (G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR), and Ag NCsfibAu_NR (S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR). For comparison, Raman spectra of 10 5 M CV solution dispersed over Au or Ag NCs on an Si substrate are shown in Fig. S6(a) and (b). The Raman enhancement effect was intensified by increasing the size of NCs. In addition, Ag NCs were adsorbed and resulted in a higher Raman scattering effect as compared to Au NCs, presumably attributable to the adjustments of surface roughness and distribution of NCs. The strongest CV Raman scattering was observed for Ag NCsfibAu_NR. The most intense Raman shift from the characteristic peaks of CV usually appears at 1618 cm 1 (CVpeak), which is assigned to the ring C–C stretching modes. The other Raman band shifts are listed in Table S3 (Sivashanmugan et al. 2015, 2013a, 2013b). Based on Eq. (1), the estimated EF values for the samples are shown in Fig. 3(a) and (b). Ag NCsfibAu_NR sample S_3fibAu_NR exhibited the highest Isers and EF (6.92 108). Since the intra-Ag NC and Au NR plasmon energies are most probably matched that increase the electron oscillation at Raman-active sites, the intensity of Raman signals and the value of EF thus increased (Lee et al., 2013; Rycenga et al., 2009; Sivashanmugan et al., 2015, 2013a, 2013b; Yamamoto et al. 2013; Yang et al. 2014; Yilmaz et al. 2014). In Fig. S7, strong Raman intensities at 1618 cm 1 for low concentrations of 10 7, 10 9, 10 13, and 10 15 M of CV on S_3fibAu_NR can be seen. Presumably, the high Ra value of NCs on the surfaces of NRs, along with the SPR effect around NCs and the NC/NR interface and the LSPR effect around NRs, contributes to the improved low-concentration single-molecule detection. 3.3. Quality of 4-ABT-coated Au or Ag NPs on S_3fibAu_NR The optimized Ag NCsfibAu_NR sample S_3fibAu_NR was utilized as the substrate for attachment of 4-ABT-coated Au or Ag NPs. The shape-controlled Au or Ag NPs were analyzed using UV– vis absorption spectroscopy and HR-FETEM. Fig. S8(a) and (b) shows the UV–vis spectra peak position for Au or Ag NP solution, which appeared at 525 nm for Au and 425 nm for Ag. The results correspond well to characteristic nanometer-scale sizes of Au or Ag NPs. HR-FETEM images of Au or Ag NPs are shown in the
Fig. 3. Raman-active peaks of 10 5 M CV molecules on (a) Au NCs on fibAu_NR (G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR) and (b) Ag NCs on fibAu_NR (S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR) examined at Raman laser wavelength of 785 nm (insets show corresponding Raman spectra). Enhancement factors and relative Raman intensities for Au or Ag NCs on fibAu_NR were compared. CV peak at 1618 cm 1 was used as index for relation of relative Raman intensities with respect to Au or Ag NCs on fibAu_NR.
insets of Figs. 4(a) and (b) and S8. Based on the HR-FETEM images, Au or Ag NPs were spherical with an average diameter of 20 nm. In Fig. 4(a) and (b), the likely interaction (or slight aggregation, as shown in the insets) of 4-ABT-coated Au or Ag NPs is shown in the HR-FETEM images. The 10 7 M 4-ABT mixed with Au or Ag NP solution after 10 h is shown in the insets of Fig. 4(a-i) and (b-i). The interactions of thiol and NH2 groups of 4-ABT with Au or Ag NPs are illustrated in Fig. 4(a-i) and (b-i) (Abdelsalam, 2009; Kim et al., 2012). In general, the newly formed bonds between sulfur/ Au and NH2/Ag are very strong; however, the bond strength differs from that of metals. Notably, 4-ABT-coated Ag NPs exhibit more aggregation than that of 4-ABT-coated Au NPs, since a relatively strong covalent bond is formed between Ag and NH2. On the other hand, few layer of surfactant citrate may significantly influence the aggregations of sample with 4-ABT. Fig. 4(c) and (d) show HR-FETEM images of the top surface of 4-ABT-coated Au and Ag NPs, respectively, on S_3fibAu_NR. The area within the dotted lines has small gaps between 4-ABT-coated Au or Ag NPs and Ag NCs/Au NR (o2 nm). The 4-ABT-coated Ag NPs exhibited a relatively large aggregation on S_3fibAu_NR as compared in Figs. 4(c-i) and (d-i) owing to the increased surface
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Fig. 4. HR-FETEM images of as-fabricated 4-ABT-coated (a) Au and (b) Ag NPs. Insets show HR-FETEM images of Au and Ag NPs. Schematic diagrams of 4-ABT-coated (a-i) Au and (b-i) Ag NPs. Insets show 4-ABT-coated Au or Ag NPs in solution. HR-FETEM images of 4-ABT-coated (c) Au and (d) Ag NPs on S_3fibAu_NR. High-density NPs distributed on top surfaces of NR (c d-i) are shown. The two inter-junction gaps between (d-ii) Ag NCs on Au NR and (d-iii) Ag NPs on Ag NCs attached on Au NR surface are shown. (e) Two- and (e-i) three-dimensional AFM images of optimized 4-ABT-coated Ag NPs on S_3fibAu_NR (scale bar: 170 nm).
chemistry of Ag (relatively dark) caused by 4-ABT (Kim et al., 2011a, 2011b). In Fig. 4(d-ii) and (d-iii), the inter-junction gap between Ag NCs and Au NR ( o3 nm) and that between 4-ABTcoated Ag NPs and Ag NCs ( o3 nm) can be seen. In Figs. 4(e) and (e-i) and S9, two- and three-dimensional AFM and HRTEM images of 4-ABT-coated Ag NPs on S_3fibAu_NR are shown, respectively; the distribution of 4-ABT-coated Ag NPs was suggested to the resulted HR-FETEM image in Fig. 4(d). In the 4-ABT-coated Au or Ag NPs on Ag NCs (or S_3) fibAu_NR system, the effect of SERS with low-concentration (10 7 M) 4-ABT was evaluated at a Raman laser wavelength of 785 nm. In Fig. 5 (a) and (b), strong Raman bands appear, which are attributed to the b2-type bands of 4-ABT (as listed in Table S4), in particular for sample N_6 (i.e, 4-ABT-coated Ag NPs on S_3fibAu_NR). The most intense Raman shift appeared at 1586 cm 1, which is assigned to the ring C–C stretching modes (Abdelsalam, 2009; Cao and Che, 2014; Kim et al., 2012, 2011a, 2011b; Shao et al., 2014; Uetsuki et al., 2010), most probably due to a strong electronic charge transfer between 4-ABT-coated Ag NPs and Ag NCsfibAu_NR at the small gaps, as shown in Fig. 4(d-ii) and (d-iii) (Abdelsalam, 2009; Cao and Che, 2014; Kim et al., 2012, 2011a, 2011b; Shao et al., 2014; Uetsuki et al., 2010). In Fig. 5(c) and (d), Isers at 1586 cm 1 and they integrated surface areas are compared. The results
indicate that the increase in the SERS effect is most likely due to the excitation of electrons oscillation at the nanosized gaps of NPs/ NCs and NCs/NR (Abdelsalam, 2009; Dendisová et al., 2013; Kim et al., 2012; Kim and Lee, 2005; Shao et al., 2014; Sivashanmugan et al., 2015, 2013b). 3.4. Quality assessment of Col-I fibers As illustrated in Fig. 6(a), Col-I fibers form a triple helical structure that consists of three polypeptide chains (R) that repeat a triplet amino acid sequence (Jalan et al., 2014; Kadler et al., 1996; Shoulders and Raines, 2009). The three R chains twist together into a unique triple helical structure, whose length and diameter range from 100 to 500 nm and 30 to 100 nm, respectively. The length and diameter of Col-I fibers strongly depend on the incubation time (Capaldi and Chapman, 1984; Jalan et al., 2014). To verify the sensitivity of SERS on Ag NCsfibAu_NR (S_3), Ag NP-coated Col-I fibers were placed on the substrate and measured. Since the peptides are easily incorporated through their biochemical immobilization at the Ag surface without additional modification of the Col-I fiber structure (Orza et al., 2011; Sun et al., 2008a, 2008b), Ag NPs and the Col-I fiber surface exhibit cross-linking, as shown in Fig. S10(a) and (b). The incorporation of
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Fig. 5. Raman-active peaks of 10 7 M 4-ABT and that coated on (a) Au and (b) Ag NPs on S_3fibAu_NR examined at Raman laser wavelength of 785 nm. (c, d) Peak position at 1586 cm 1 (4-ABT) was used as index for comparison between integrated surface area of Raman peak position and relative Raman intensities of samples. T_1 and N_1: 4-ABT on Au; T_2 and N_2: 4-ABT-coated Au and Ag NPs on Au; T_3 and N_3: 4-ABT on fibAu_NR; T_4 and N_4: 4-ABT-coated Au and Ag NPs on fibAu_NR; T_5 and N_5: 4-ABT on S_3fibAu_NR; T_6 and N_6: 4-ABT-coated Au and Ag NPs on S_3fibAu_NR.
Ag NPs leads to an increase of structural disorder on Col-I fibers. For comparison, Fig. S11(a) shows Rama spectra for Col-I fibers, Ag NP-coated Col-I fibers on S_3fibAu_NR, Col-I fibers on a glass substrate, Ag NP-coated Col-I fibers on a glass substrate, Col-I fibers on fibAu_NR, and Ag NP-coated Col-I fibers on fibAu_NR. Among them, Ag NP-coated Col-I fibers on S_3fibAu_NR exhibited the highest Raman signals, i.e., those for amides I and II, Pro or Hyp, and (Gly-X-Y)n (Gullekson et al., 2011; Lee et al., 2010), and the highest integrated area of the Raman peak at 810 cm 1 (Fig. S11(b)). The characteristic Raman peak at 810 cm–1 is assigned to the ʋ(C–C) in Pro and Hyp rings (other Raman bands are listed in Table S5) (Gullekson et al., 2011; Lee et al., 2010). The results suggest that the coupling of Ag NP-coated Col-I fibers attached to NCsfibAu_NR enhances the LSPR near the NPs/NCs-NR system. The sensitivity of SERS for the detection of digested Col-I fibers was verified. In general, the proteolysis of the MMP family can cleave Col and native Col- I, II, and III fibers at a specific site in all three chains of the triple helix structure, with the cleavage starting at approximately the end of C- and N-terminus (Knauper et al., 1997; Kridel et al., 2001; Tam et al., 2002; Verma and Hansch, 2007). Among the MMP family, MMP-9 is most important because it is generated on diseased cell surfaces (Kridel et al., 2001). MMP9 has three common domains, namely propeptide, catalytic, and hemopexin-like domains, the latter of which is linked to the catalytic domain by a flexible hinge region, as illustrated in Fig. 6(b). In this study, Col-I fibers were digested by MMP-9 at concentrations of 0.005 and 0.01 mg/mL. Fig. 6(c) and Fig. S12 shows the two-dimensional AFM topography of Col-I fibers (i and ii), 0.005 mg/mL MMP-9-digested Col-I
fibers (iii and iv), and 0.01 mg/mL MMP-9-digested Col-I fibers (v and vi). The inset of Fig. 6(c-i and ii) shows an AFM image of the as-formed twisted triple helical structure of a Col-I fiber. Since MMP-9 contains catalytic and hemopexin C domains, the ability to bind with Col-I fibers and slowly start to cleave Col-I fibers are thought to be mediated (Aimes and Quigley, 1995; Knauper et al., 1997; Sun et al., 2008a; Verma and Hansch, 2007), as shown in Fig. 6(c-v and vi). Fig. 6(d) shows Raman spectra of Col-I fibers, 0.005 mg/mL MMP-9-digested Col-I fibers, and 0.01 mg/mL MMP-9-digested Col-I fibers on S_3fibAu_NR. Strong Raman signals from amide I (1664 cm 1), Pro and Hyp (1562 cm 1), amide III (1230 cm 1), and ʋ(C–C) in Pro and Hyp rings (810 cm 1) for Col-I fibers on S_3fibAu_NR can be seen in Fig. 6(d-i) and are listed in Table S5 (Cárcamo et al., 2012; Gullekson et al., 2011; Lee et al., 2010). This confirms that S_3fibAu_NR is the most SERS-active substrate, which likely creates a strong local EM effect between intra-NCs and at the NCs/NR interface. In Fig. 6 (d-ii and iii), Raman signals of amides I and III and Pro and Hyp from Col-I fibers slightly decreased with increasing concentration of MMP-9. When MMP-9 was added to Col-I fibers, they gradually lost their integrated structure due to the breaking of hydrogen bonds between Gly peptide chains, as indicated in Fig. 6 (c) and (d) (Aimes and Quigley, 1995; Fini et al., 1992; Knauper et al., 1997; Verma and Hansch, 2007). Raman spectra for 0.005 mg/mL MMP-9 interacting with Col-I fibers (Fig. 6 (d-ii)) show strong aromatic ring breathing at 1004 cm–1 and reduced amides I and II and Gly-X-Y Raman bands. Furthermore, the ends of C- and N-terminal peptides in Col-I fibers were cleaved when
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Fig. 6. Schematic diagrams of structures of (a) Col-I fiber and (b) MMP-9. (c) Two-dimensional and color quantitative AFM topography images of (i and ii) Col-I fibers, (iii and iv) Col-I digested by 0.005 mg/mL MMP-9, and (v and vi) Col-I digested by 0.01 mg/mL MMP-9 on S_3fibAu_NR. Triple helix structure of Col-I fiber and its possibly denatured structure are respectively shown in insets of AFM images (i) and (v) (scale bar: 60 nm). (d) Raman-active peaks of (i) Col-I fibers, (ii) Col-I fibers digested by 0.005 mg/mL MMP-9, and (iii) Col-I digested by 0.01 mg/mL MMP-9 on S_3fibAu_NR.
the MMP-9 concentration was increased to 0.01 mg/mL, as shown in Fig. 6 (d-iii). It is known that an increased interaction of the MMP-9 hemopexin domain with Col-I fibers leads to highly expressed Col-I fiber end terminals as hydrogen bonds between the three chains are thoroughly broken (Aimes and Quigley, 1995; Fini et al., 1992; Knauper et al., 1997; Verma and Hansch, 2007), which is confirmed by the disappearing Raman band, shown in Fig. 6 (diii), i.e., amide bands. Temperature-denatured Col-I fibers on S_3fibAu_NR were studied. The Col-I fibers were rapidly denatured by temperature due to lowered strength and fusion. Their morphology and Raman spectra are shown in Fig. S13. It is difficult to distinguish temperature-denatured Col-I fibers from MMP-9-digested Col-I fibers from Raman spectra. Further study is required. The proposed EM mechanism of Ag NP-coated Col-I fibers on NCsfibAu_NR is illustrated in Fig. 7. In Fig. 7(a), a relativity strong LSPR effect is obtained on the fibAu_NR surface, and the spacing between adjacent NRs and the length of NRs contribute to LSPR. The high SERS may be induced by the combined NC SPR and NR LSPR effects from NCsfibAu_NR, which occurred at the interface of NCs and NRs, as shown in Fig. 7(b). In addition, the intra-NC effect increased the local field effect around NCs and NRs. Interestingly, multi-polar
excitations are mostly generated at the surfaces of Ag NP-coated 4-ABT or Col-I fibers on NCsfibAu_NR, as shown in Fig. 7(c) and (d). The generations of large plasmonic fields around nanogaps in NSs systems, contributing to the detection of single molecules, were also theoretically and experimentally evidenced and thereafter suggested that around nanogaps, the EM effect is significantly enhanced (Qian et al., 2010; Chen et al., 2013). A similar effect was obtained for 4-ABT-coated Ag NPs on NCsfibAu_NR. Notably, Ag NP-coated 4-ABT on NCsfibAu_NR SERS effect systematically enhances the LSPR effect as compared to the Ag NP-coated Col-I fibers on NCsfibAu_NR due to the size effect of target species.
4. Conclusion Highly SERS-active substrates, Au or Ag NCs on fibAu_NR, are fabricated by combining FIB and electron beam deposition methods. The diameter of clusters and the formation of NCs on NR affect the SERS effect, which was evaluated using CV as a molecular probe at low concentration. In this work, the EF of S_3 (Ag NCs) fibAu_NR significantly increased with an Ag cluster size of 8 nm (to 6.92 108). It is very likely that the intra-NC LSPR effect on the
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Fig. 7. Proposed schematic diagrams of SERS effect on (a) Au NR, (b) Ag NCs on Au NR, (c) 4-ABT-coated Au NPs on Ag-NC-coated Au NR, and (d) Ag-NP-coated Col-I fibers on Au NR.
surface of NRs contributes to the detection of single molecules. In addition, a double inter-juction hotspot effect resulted from 4-ABT-coated Au or Ag NPs on S_3fibAu_NR, which provides more hotspot areas at the interface of NPs/NCs and NCs/NRs and significantly increases the number of SERS-active sites. The optimized SERS-active substrate S_3fibAu_NR was applied to detect MMP-9digested Col-I fibers under various MMP-9 concentrations. The results indicate that the Raman band signals of Col-I fibers decrease with increasing MMP-9 concentration. Presumably, MMP9-digested Col-I fibers may gradually degrade the Col-I fiber structure through the scission of hydrogen bonds between Gly peptide chains. Au or Ag NCsfibAu_NR thus has potential for investigating tissue growth in various bio-environments.
Acknowledgment This work was financially supported by the Ministry of Science and Technology, Taiwan under grant 103-2221-E-006-067-MY3.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.091.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Intense Raman scattering on hybrid Au/Ag nanoplatforms for the distinction of MMP-9-digested collagen type-I fiber detection Kundan Sivashanmugan a, Jiunn-Der Liao a,b,n, Pei-Lin Shao a, Bernard Haochih Liu a, Te-Yu Tseng a, Chih-Yu Chang a a b
Department of Materials Science and Engineering, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan Center for Micro/Nano Science and Technology, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan
art ic l e i nf o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 28 March 2015 Accepted 27 April 2015 Available online 29 April 2015
Well-ordered Au-nanorod arrays were fabricated using the focused ion beam method (denoted as fibAu_NR). Au or Ag nanoclusters (NCs) of various sizes and dimensions were then deposited on the fibAu_NR arrays using electron beam deposition to improve the surface-enhanced Raman scattering (SERS) effect, which was verified using a low concentration of crystal violet (10–5 M) as the probe molecule. An enhancement factor of 6.92 108 was obtained for NCsfibAu_NR, which is attributed to the combination of intra-NC and NR localized surface plasmon resonance. When 4-aminobenzenethiol (4ABT)-coated Au or Ag nanoparticles (NPs) were attached to NCsfibAu_NR, the small gaps between 4-ABTcoated NPs and intra-NCs allowed detection at the single-molecule level. Hotspots formed at the interfaces of NCs/NRs and NPs/NCs at a high density, producing a strong local electromagnetic effect. Raman spectra from as-prepared type I collagen (Col-I) and Ag-NP-coated Col-I fibers on NCsfibAu_NR were compared to determine the quantity of amino acids in their triple helix structure. Various concentrations of matrix-metalloproteinase-9-digested Col-I fibers on NCsfibAu_NR were qualitatively examined at a Raman laser wavelength of 785 nm to determine the changes of amino acids in the Col-I fiber structure. The results can be used to monitor the growth of healing Col-I fibers in a micro-environment. & 2015 Elsevier B.V. All rights reserved.
Keywords: Au nanorods Nanoclusters Surface-enhanced Raman scattering 4-aminobenzenethiol Digested type I collagen
1. Introduction The growth process during wound healing is of great interest. Skin tissue reconnection with active scaffolds or stem cells is often used to study cell differentiation and proliferation on the wound bed (Jain et al., 2014; Ngo et al., 2014; Ngo Thi et al., 2014). Biodegradable and nonbiodegradable proteins and peptides are promising supports for tissue reconnection owing to their useful properties, such as chemical structural diversity, large number of architectures, mechanical strength, and biocompatibility (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Shoulders and Raines, 2009). A variety of natural proteins and peptides, such as laminin, fibronectin, and collagen (Col) fibers, are often used to improve the efficacy of tissue reconstruction (Silver et al., 2003; Stamov et al., 2008). Col fibers are most commonly used in tissue regeneration due to their excellent rates of biointegration, biodegradability, and biocompatibility (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Silver et al., 2003; Stamov et al., 2008). The n Corresponding author at: Department of Materials Science and Engineering, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan. Fax: þ886 62346290. E-mail address: [email protected] (J.-D. Liao).
http://dx.doi.org/10.1016/j.bios.2015.04.091 0956-5663/& 2015 Elsevier B.V. All rights reserved.
triple helix structure of Col fibers may improve tissue growth via several cross-linking procedures (Kadler et al., 1996; Shoulders and Raines, 2009). However, the degradation of Col fibers is also an important process in wound healing, which generally occurs during the growth and development of adult connective tissues within pericellular and extracellular environments. In mature tissues, existing Col fibers are replaced by new Col fibers (Desimone et al., 2011; Lin et al., 2012; Lv et al., 2014; Silver et al., 2003; Stamov et al., 2008). Complex tissue reconstruction requires type I Col (Col-I) fiber protein, which is an important structural component in blood vessels, skin, tendons, ligaments, and bone (Bozec et al., 2007; Fang et al., 2013). The degradation of Col-I, still unclear in tissue reconstruction, can occur through either the extracellular or intracellular pathway (Fligiel et al., 2003; Orza et al., 2011; Sun et al., 2008b). The growth and degradation of Col-I fibers is tightly regulated. From in vitro studies, matrix metalloproteinases (MMPs), including MMP-1, -9, -13, and -14, can effectively degrade native Col-I fibers surrounding a cell surface through the glycine in Col-I fibers (Aimes and Quigley, 1995; Bigg et al., 2007; Fini et al., 1992; Knauper et al., 1997). In addition, the degradation of Col-I, -II, and -III fibers using various temperature-dependent treatments has been investigated (Cárcamo et al., 2012; Jain et al., 2014;
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Su et al., 2014). Methods for analyzing the degradation of Col-I fibers include the enzyme-linked immunosorbent assay and highperformance liquid chromatography (Akhtar et al., 1999; Hudson et al., 2012), which have excellent sensitivity for the detection of specific degraded enzymes in Col-I fibers. However, labeling techniques usually require sophisticated procedures for sample preparation and subsequent data analysis, as well as repetitive tests, making them high-cost. Therefore, a simple, quick, cost-effective, and sensitive detection method for monitoring Col-I fibers is highly required. Raman spectra provide structural and chemical information about target species in a chemical or biological sample (Luo et al., 2014; Xie and Schlucker, 2013). However, the very low efficiency of Raman data collection leads to long acquisition time for detecting normal analyte molecules. In addition, biological samples, e.g., cells, proteins, and viruses, require a method with high resolution or enhancement and sensitivity or selectivity. A strong Raman signal is generated when a Raman-active analyte interacts with a noble metal, e.g., Ag, Au, or Cu, surface. A surface-enhanced Raman scattering (SERS)-active substrate allows detection down to a single-molecule level (Betz et al., 2014; Harper et al., 2013; Kleinman et al., 2013). The effect of SERS is mainly attributed to two primary mechanisms: the chemical effect and the electromagnetic (EM) effect (Betz et al., 2014; Harper et al., 2013; Kleinman et al., 2013). Very few studies have investigated Col-I fiber structure using SERS, e.g., by detecting signals from an Au or Ag nanoparticle (NP)-coated Col-I fiber surface (Orza et al., 2011; Sun et al., 2008b). Ag-NP-coated Col-I fiber produces strong Raman signals, but with low reproducibility. Alternatively, nanolithography techniques such as electron beam lithography (Luo et al., 2014), focused ion beam (FIB) (Sivashanmugan et al., 2013a, 2013b), and nanomechanical indentation (Chang et al., 2011; Yao et al., 2012) have been employed to fabricate various types of nanostructure (NS), e.g., nanorods (NRs), nanowires, nanocavities. Nanofabrication methods create highly precise and regular patterns that have a high enhancement factor (EF). SERS-active substrates have been used to detect nucleoproteins, oligonucleotides, and viruses due to the strong light scattering in the vicinity of nanogaps and NSs (Shao et al., 2014; Sivashanmugan et al., 2015, 2013a). In our previous work, SERS-active substrates with well-ordered
Au or Au/Ag multilayered NR arrays were fabricated using FIB technology (Sivashanmugan et al., 2015, 2013a, 2013b). It is hypothesized that a high EM effect due to the availability of multiple edges and a small curvature induced in the substrate (Costa et al., 2009; Shao et al., 2014; Sivashanmugan et al., 2014, 2015, 2013a). In addition, the localized surface plasmon resonance (LSPR) effect strongly depends on NR shape and the spacing between NRs. The LSPR effect is known to increase SERS sensitivity up to the singlemolecule level. Notably, the SERS effect highly varies with the size of the target species due to laser interaction strength. Large biotarget species, e.g., cells and proteins, usually require a substrate with strong and highly reproducible LSPR. Therefore, a substrate with a high SERS effect is required for practical applications (Que et al., 2011, 2012). In the present work, FIB and electron beam deposition were combined for the fabrication of Au and Ag nanoclusters (NCs) on a FIB-made Au NR arrays (NCsfibAu_NR) as a SERS substrate, as illustrated in Fig. 1(a). The Au or Ag NCs on fibAu_NR influence the detection of Raman-active species. This influence was examined using a low concentration (10–5 M) of crystal violet (CV) as the probe molecule. As shown in Fig. 1(b), 4-aminobenzenethiol (4ABT)-coated Au or Ag NPs were deposited on an optimized NCsfibAu_NR substrate to create a large plasmonic effect via a large number of hotspots. As shown in Fig. 1(c), Col-I, Ag-NPcoated Col-I fibers, and MMP-9-digested Col-I fibers were deposited on an optimized NCsfibAu_NR substrate to determine changes in Col-I fiber structure. The former application utilizes a target chemical species bonded with NPs and attached to NCs®bAu_NR, while the latter utilizes a target biological sample with NPs and attached to NCsfibAu_NR. These applications were used to confirm whether the hybrid Au/Ag nanostructure can intensify the Raman scattering effect on the target species simply with NPs.
2. Experimental section 2.1. Fabrication of Au or Ag NCs on fibAu_NR Au thin film was deposited onto polished single-crystal silicon (100) wafers primed with a 5- nm-thick adhesion layer of Ti using an electron beam evaporator (VT1-10CE, ULVAC, Taiwan) at a
Fig. 1. Schematic illustrations of experimental route for (a) as-fabricated NCsfibAu_NR, (b) 4-ABT-coated Au or Ag NPs on NCsfibAu_NR, and (c) Col-I, NP-coated Col-I, and MMP-9-digested Col-I fibers on NCsfibAu_NR.
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controlled deposition rate. The optimal layer thickness for Au film (E 420 nm) was maintained. Patterns were designed using CorelDRAW software. The NR designs were implemented by applying an FIB (SMI 3050, SII Nanotechnology, Japan). The pattern size was about 120 mm 120 μm. The following beam conditions were used: 30-kV acceleration voltage, 0.07-μm depth, 10-pA aperture, 70-μs dwell time, þ0.56 optical lens, and 8-image scale (Sivashanmugan et al., 2013a, 2013b). The spacing between adjacent NRs, diameter, and shape of Au NR arrays (fibAu_NR) were maintained by adjusting the beam current. Au or Ag NCs were then deposited on the fibAu_NR arrays to increase the effect of SERS, as illustrated in Fig. 1(a). Au or Ag NCs on fibAu_NR were prepared using electron beam evaporation deposition with estimated diameters of 2, 5, and 8 nm; the corresponding samples are denoted as G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR for Au NCsfibAu_NR and S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR for Ag NCsfibAu_NR. The size of the as-prepared NCs was based on the spacing between NRs (D-spacing, i.e., E 35 nm). Information about all NCs and NCsfibAu_NR are given in Tables S1 and S2 (in Supporting Information), respectively. Field-emission scanning electron microscopy (FE-SEM, JSM-7001, JEOL, Japan), atomic force microscopy (AFM, Bruker, ICON SPM, USA) and high-resolution field-emission transmission electron microscopy (HR-FETEM, JEM-2100 F, JEOL, Japan) with energy-dispersive X-ray spectroscopy (EDS) were employed to analyze the morphologies of the as-prepared NCs, fibAu_NR, and NCsfibAu_NR.
2.2. EF determination using molecular probe The molecular probe CV was diluted in aqueous solution to a concentration in the range of 10–5–10 15 M. To verify EF, fibAu_NR or NCsfibAu_NR with a molecular-probe-containing solution was covered with a glass slide and then immediately measured using Raman spectroscopy. Raman spectra were acquired using a confocal microscopy Raman spectrometer (inVia Raman microscope, Renishaw, United Kingdom) with diode lasers at an excitation wavelength of 785 nm, and then scanned with an integration time of 10 s over an area of 1 μm 1 μm (the size of the laser spot was 1 μm) using a 50 objective. Before each batch, the Raman shift was calibrated using the absolute intensity of signal at 520 cm 1 from a standard silicon wafer. The EF measurement was estimated according to the standard equation (Sivashanmugan et al., 2013a, 2013b)
I N EF = sers × bulk Ibulk Nsers
(1)
where Isers and Ibulk are the SERS and normal Raman scattering intensities, respectively, and Nsers and Nbulk are the numbers of molecules contributing to the inelastic scattering intensity respectively evaluated by SERS and normal Raman scattering measurements. The Raman intensity was averaged from 10 consecutive measurements. The highest peak intensity for all samples was recorded as the specific band and treated as the Isers value in Eq. (1). The ideal average Nsers value (E6.17 107 molecules) was obtained from the volume of the uniform monolayer of CV ideally adsorbed on NCsfibAu_NR. The Nsers value is correlated with the concentration of 10 5 M of CV-containing solution. The spot size of the laser and the average value of Nbluk were obtained as E1 μm2 and E9.23 108 molecules, respectively. The Ibulk values were measured as E 280 (arbitrary units). The above values were substituted into Eq. (1) to calculate the SERS EF values for samples.
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2.3. Fabrication of 4-ABT-coated Au or Ag NPs on NCsfibAu_NR Gold (III) chloride trihydrate (HAuCl4 3H2O), silver nitrate (AgNO3), sodium citrate (HOC(COONa)(CH2COONa)2 2H2O), and 4-ABT (H2NC6H4SH) were purchased from Sigma-Aldrich. Ultrapure water (E10 MΩ cm) was used as a solvent in the experiments. All glassware used in the experiments was cleaned with nitric acid before use. The NPs to be coated with 4-ABT for deposition on NCs®bAu_NR were prepared based on a well-established method (Sivashanmugan et al., 2013b). Firstly, an Au sol was prepared by dissolving 1 mM HOC(COONa)(CH2COONa)2 2H2O and 1 mL of 0.01 mM HAuCl4 3H2O in 20 mL of aqueous solution and then boiled for 30 min. In this mixture solution, Au seed turned reddish after 4 min. After continuous stirring for an additional 5 min, the mixture was allowed to stand at room temperature, and then washed five times using water with some specific parameters as follows: the radius and rotating speed were 5 cm and 12,000 rpm, respectively, to remove impurities and stored for further studies. For the growth of Ag sol, 100 mL of aqueous solution containing 1 mM AgNO3 was brought near boiling temperature. 3 mL of Ag seed solution and HOC(COONa)(CH2COONa)2 2H2O (with a final concentration of 1 mM in solution) was then added to the boiling solution. The mixture was heated until the color of the solution turned greenish yellow. The solution was removed and brought to room temperature, washed five times using water with some specific parameters as follows: the radius and rotating speed were 5 cm and 12,000 rpm, respectively, to remove impurities, and then stored for further studies. The morphology and structure of the NPs were characterized using HR-FETEM. The optical properties of the NPs were monitored as a function of time in 10- mm-opticalpath-length quartz cuvettes with an ultraviolet-visible (UV–vis) spectrophotometer (UV–3600, Shimadzu). The as-prepared Au or Ag NPs were then mixed with an optimized concentration of 5 mL of 4–ABT (10 mM ethanolic solution) and stabilized for 10 h. The solution was then washed with water and ethanol in sequence. Au or Ag NCsfibAu_NR was then covered with a droplet containing 4-ABT-coated Au or Ag NPs and dried in a vacuum for 2 h, washed with ethanol, and then dried in air. The 4-ABT-coated Au or Ag NPs on Au, fibAu_NR and NCsfibAu_NR substrates were examined at a Raman laser wavelength of 785 nm. The morphology and structure of 4-ABT-coated Au or Ag NPs on NCsfibAu_NR were respectively characterized using HR-FETEM and AFM. 2.4. Raman studies of Col-I fibers Rat tail Col-I was purchased from Merck Millipore and then diluted to 0.1 mg/mL in a 10-mL solution of phosphate-buffered saline (PBS) before use. The as-prepared samples were incubated at 37 °C for 30 min to grow Col-I fibers. Col-I was diluted to 0.1 mg/mL in a 5-mL solution of PBS with 5 mL (an optimized concentration) of Ag NPs and then incubated at 37 °C for 1 h to create NP-coated Col-I fibers. Col-I and Ag-NP-coated Col-I fibers were then placed on an optimized NCsfibAu_NR. After 15 min, the samples were dried with nitrogen gas and subjected to Raman spectroscopic studies. The morphology and structure of Col-I and Ag-NP-coated Col-I fibers on an optimized NCsfibAu_NR was characterized using AFM. MMP-9 was purchased from Merck Millipore. To activate MMP-9, MMP-9 with a concentration of 0.005 or 0.01 mg/mL was diluted in a 10-mL solution of PBS. To digest Col-I fibers, the fibers were mixed with various concentrations of MMP-9, and then incubated at 27 °C for 30 min. The MMP9-digested Col-I fibers were then placed on an optimized NCs®bAu_NR and subjected to Raman analysis. The morphology and structure of the digested Col-I fibers on an optimized NCsfibAu_NR
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were characterized using AFM.
3. Results and discussion 3.1. Characterization of as-fabricated Au or Ag NCsfibAu_NR As shown in Fig. S1, the morphology of fibAu_NR was analyzed using FE-SEM, AFM, and HR-FETEM. The specified spacing between adjacent NRs was maintained by adjusting the working current and etching time during FIB fabrication. Fig. S1(a) and (b) show top- and side-view images of fibAu_NR, respectively. The length, diameter, and spacing between NRs for fibAu_NR were E400, E 170, and 35 nm, respectively. The AFM topography of fibAu_NR is shown in Fig. S1(c). The as-formed NRs were uniformly fabricated within the patterned area, as indicated by the AFM line profiles shown in the inset in Fig. S1(c). The Au or Ag NCs were anticipated to slightly reduce the spacing between adjacent NRs and the tip ring diameter of NRs (Sivashanmugan et al., 2013b). To confirm the size, dimension, and distribution of Au or Ag NCs, the NCs were deposited on an Si (100) substrate and the resulting values were used as the reference. Fig. S2(a–f) show two-dimensional top-view AFM and FE-SEM images of the as-prepared Au or Ag NCs, evenly distributed on an Si (100) substrate. The gap between NCs significantly increased with
increasing size and dimension of the deposited Au or Ag NCs. In addition, the average roughness (Ra) of Ag NC sample S_3 (Ra E4.33 nm) was higher than that of Au NC sample G_3 (Ra E2.68) due to the shape and physical properties of Au or Ag NCs (Rycenga et al., 2009; Sivashanmugan et al., 2013b; Yamamoto et al., 2013; Yang et al., 2014), as listed in Table S1. The formation and distribution of Au or Ag NCs on various substrates were respectively examined using FE-SEM and HR-FETEM. Fig. 2(a–c) and S3(a–c) show side and cross-sectional (insets) FE-SEM images of Au or Ag NCsfibAu_NR. Both Au and Ag NCs were uniformly distributed on fibAu_NR. Furthermore, FE-SEM EDS spectra and elemental mapping of Au or Ag NCsfibAu_NR confirm the formation (as shown in Fig. S4) and distribution of NCs on fibAu_NR. Ag NCs on fibAu_NR were highly aggregated compared to Au NCs on fibAu_NR due to their varied deposition rates, e.g., by measuring the decrease of Au element from fibAu_NR, as listed in Table S2. In addition, Ra values, measured from AFM, for Ag NCsfibAu_NR ranged from 18 to 33 nm and followed the sequence S_1fibAu_NR 4S_2fibAu_NR 4S_3fibAu_NR. These values were higher than the corresponding values for Au NCsfibAu_NR samples G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR (13– 21 nm). Notably, the EM effect, and thus the SERS effect, is significantly increased by surfaces with a relatively high Ra (Costa et al., 2009; Kleinman et al., 2013; Rycenga et al., 2009; Shao et al., 2014; Sivashanmugan et al., 2014, 2015, 2013a, 2013b; Yamamoto
Fig. 2. FE-SEM top- and lateral-view (insets) images of Ag NCs on fibAu_NR: (a) S_1fibAu_NR, (b) S_2fibAu_NR, and (c) S_3fibAu_NR (inset image scale bar: 50 nm). HRFETEM images of Ag NCs on fibAu_NR: (a-i) S_1fibAu_NR, (b-i) S_2fibAu_NR, and (c-i) S_3fibAu_NR. (a c-ii) HR-FETEM images of Ag NC distribution on fibAu_NR. Marked regions in (a c-ii) indicate thickness of Ag NCs on fibAu_NR surface in (a c-iii).
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et al. 2013; Yang et al. 2014). Fig. 2(a c-i) and S3(a c-i) show HR-FETEM images of Ag or Au NCsfibAu_NR. A relatively high density of NCs on NRs was obtained by varying the deposition angle (e.g., the Au or Ag target was placed perpendicular to fibAu_NR). The spacing between adjacent NRs was not fully enclosed by Ag or Au NCs, as shown in Fig. 2(a–c-ii) and S3(a–c-ii). As shown in Fig. 2(a–c-iii) and S3(a–ciii), a small gap (e.g., o2 nm) formed between NCs on the side surface of NRs, which may greatly enhance Raman scattering around NCs. The geometry of NCs on NRs is also an important factor: increasing the size and dimension of Ag or Au NCs on Au NRs tended to decrease the D-spacing of NRs, as summarized in Table S2. The optimized sample S_3fibAu_NR had lattice spacings of 0.26 and 0.24 nm for Au and Ag, respectively, which match the lattice spacing of (111) face-centered cubic planes (Fig. S5) (Rycenga et al., 2009; Yang et al., 2014). Moreover, a tiny gap (e.g., o1 nm) formed at the interface between NCs (intra-NCs) and NRs (NCs on NRs), greatly increasing the effect of LSPR around NCs and NCs on NRs and therefore increasing the sensitivity of SERS (Rycenga et al., 2009; Yamamoto et al., 2013; Yang et al., 2014; Yilmaz et al., 2014). 3.2. SERS effect on fibAu_NR and Au or Ag NCsfibAu_NR The sensitivity and enhancement effect of CV were determined at a Raman laser wavelength of 785 nm. The insets in Fig. 3(a) and (b) show 10 5 M CV solution dispersed on fibAu_NR, Au NCs®bAu_NR (G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR), and Ag NCsfibAu_NR (S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR). For comparison, Raman spectra of 10 5 M CV solution dispersed over Au or Ag NCs on an Si substrate are shown in Fig. S6(a) and (b). The Raman enhancement effect was intensified by increasing the size of NCs. In addition, Ag NCs were adsorbed and resulted in a higher Raman scattering effect as compared to Au NCs, presumably attributable to the adjustments of surface roughness and distribution of NCs. The strongest CV Raman scattering was observed for Ag NCsfibAu_NR. The most intense Raman shift from the characteristic peaks of CV usually appears at 1618 cm 1 (CVpeak), which is assigned to the ring C–C stretching modes. The other Raman band shifts are listed in Table S3 (Sivashanmugan et al. 2015, 2013a, 2013b). Based on Eq. (1), the estimated EF values for the samples are shown in Fig. 3(a) and (b). Ag NCsfibAu_NR sample S_3fibAu_NR exhibited the highest Isers and EF (6.92 108). Since the intra-Ag NC and Au NR plasmon energies are most probably matched that increase the electron oscillation at Raman-active sites, the intensity of Raman signals and the value of EF thus increased (Lee et al., 2013; Rycenga et al., 2009; Sivashanmugan et al., 2015, 2013a, 2013b; Yamamoto et al. 2013; Yang et al. 2014; Yilmaz et al. 2014). In Fig. S7, strong Raman intensities at 1618 cm 1 for low concentrations of 10 7, 10 9, 10 13, and 10 15 M of CV on S_3fibAu_NR can be seen. Presumably, the high Ra value of NCs on the surfaces of NRs, along with the SPR effect around NCs and the NC/NR interface and the LSPR effect around NRs, contributes to the improved low-concentration single-molecule detection. 3.3. Quality of 4-ABT-coated Au or Ag NPs on S_3fibAu_NR The optimized Ag NCsfibAu_NR sample S_3fibAu_NR was utilized as the substrate for attachment of 4-ABT-coated Au or Ag NPs. The shape-controlled Au or Ag NPs were analyzed using UV– vis absorption spectroscopy and HR-FETEM. Fig. S8(a) and (b) shows the UV–vis spectra peak position for Au or Ag NP solution, which appeared at 525 nm for Au and 425 nm for Ag. The results correspond well to characteristic nanometer-scale sizes of Au or Ag NPs. HR-FETEM images of Au or Ag NPs are shown in the
Fig. 3. Raman-active peaks of 10 5 M CV molecules on (a) Au NCs on fibAu_NR (G_1fibAu_NR, G_2fibAu_NR, and G_3fibAu_NR) and (b) Ag NCs on fibAu_NR (S_1fibAu_NR, S_2fibAu_NR, and S_3fibAu_NR) examined at Raman laser wavelength of 785 nm (insets show corresponding Raman spectra). Enhancement factors and relative Raman intensities for Au or Ag NCs on fibAu_NR were compared. CV peak at 1618 cm 1 was used as index for relation of relative Raman intensities with respect to Au or Ag NCs on fibAu_NR.
insets of Figs. 4(a) and (b) and S8. Based on the HR-FETEM images, Au or Ag NPs were spherical with an average diameter of 20 nm. In Fig. 4(a) and (b), the likely interaction (or slight aggregation, as shown in the insets) of 4-ABT-coated Au or Ag NPs is shown in the HR-FETEM images. The 10 7 M 4-ABT mixed with Au or Ag NP solution after 10 h is shown in the insets of Fig. 4(a-i) and (b-i). The interactions of thiol and NH2 groups of 4-ABT with Au or Ag NPs are illustrated in Fig. 4(a-i) and (b-i) (Abdelsalam, 2009; Kim et al., 2012). In general, the newly formed bonds between sulfur/ Au and NH2/Ag are very strong; however, the bond strength differs from that of metals. Notably, 4-ABT-coated Ag NPs exhibit more aggregation than that of 4-ABT-coated Au NPs, since a relatively strong covalent bond is formed between Ag and NH2. On the other hand, few layer of surfactant citrate may significantly influence the aggregations of sample with 4-ABT. Fig. 4(c) and (d) show HR-FETEM images of the top surface of 4-ABT-coated Au and Ag NPs, respectively, on S_3fibAu_NR. The area within the dotted lines has small gaps between 4-ABT-coated Au or Ag NPs and Ag NCs/Au NR (o2 nm). The 4-ABT-coated Ag NPs exhibited a relatively large aggregation on S_3fibAu_NR as compared in Figs. 4(c-i) and (d-i) owing to the increased surface
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Fig. 4. HR-FETEM images of as-fabricated 4-ABT-coated (a) Au and (b) Ag NPs. Insets show HR-FETEM images of Au and Ag NPs. Schematic diagrams of 4-ABT-coated (a-i) Au and (b-i) Ag NPs. Insets show 4-ABT-coated Au or Ag NPs in solution. HR-FETEM images of 4-ABT-coated (c) Au and (d) Ag NPs on S_3fibAu_NR. High-density NPs distributed on top surfaces of NR (c d-i) are shown. The two inter-junction gaps between (d-ii) Ag NCs on Au NR and (d-iii) Ag NPs on Ag NCs attached on Au NR surface are shown. (e) Two- and (e-i) three-dimensional AFM images of optimized 4-ABT-coated Ag NPs on S_3fibAu_NR (scale bar: 170 nm).
chemistry of Ag (relatively dark) caused by 4-ABT (Kim et al., 2011a, 2011b). In Fig. 4(d-ii) and (d-iii), the inter-junction gap between Ag NCs and Au NR ( o3 nm) and that between 4-ABTcoated Ag NPs and Ag NCs ( o3 nm) can be seen. In Figs. 4(e) and (e-i) and S9, two- and three-dimensional AFM and HRTEM images of 4-ABT-coated Ag NPs on S_3fibAu_NR are shown, respectively; the distribution of 4-ABT-coated Ag NPs was suggested to the resulted HR-FETEM image in Fig. 4(d). In the 4-ABT-coated Au or Ag NPs on Ag NCs (or S_3) fibAu_NR system, the effect of SERS with low-concentration (10 7 M) 4-ABT was evaluated at a Raman laser wavelength of 785 nm. In Fig. 5 (a) and (b), strong Raman bands appear, which are attributed to the b2-type bands of 4-ABT (as listed in Table S4), in particular for sample N_6 (i.e, 4-ABT-coated Ag NPs on S_3fibAu_NR). The most intense Raman shift appeared at 1586 cm 1, which is assigned to the ring C–C stretching modes (Abdelsalam, 2009; Cao and Che, 2014; Kim et al., 2012, 2011a, 2011b; Shao et al., 2014; Uetsuki et al., 2010), most probably due to a strong electronic charge transfer between 4-ABT-coated Ag NPs and Ag NCsfibAu_NR at the small gaps, as shown in Fig. 4(d-ii) and (d-iii) (Abdelsalam, 2009; Cao and Che, 2014; Kim et al., 2012, 2011a, 2011b; Shao et al., 2014; Uetsuki et al., 2010). In Fig. 5(c) and (d), Isers at 1586 cm 1 and they integrated surface areas are compared. The results
indicate that the increase in the SERS effect is most likely due to the excitation of electrons oscillation at the nanosized gaps of NPs/ NCs and NCs/NR (Abdelsalam, 2009; Dendisová et al., 2013; Kim et al., 2012; Kim and Lee, 2005; Shao et al., 2014; Sivashanmugan et al., 2015, 2013b). 3.4. Quality assessment of Col-I fibers As illustrated in Fig. 6(a), Col-I fibers form a triple helical structure that consists of three polypeptide chains (R) that repeat a triplet amino acid sequence (Jalan et al., 2014; Kadler et al., 1996; Shoulders and Raines, 2009). The three R chains twist together into a unique triple helical structure, whose length and diameter range from 100 to 500 nm and 30 to 100 nm, respectively. The length and diameter of Col-I fibers strongly depend on the incubation time (Capaldi and Chapman, 1984; Jalan et al., 2014). To verify the sensitivity of SERS on Ag NCsfibAu_NR (S_3), Ag NP-coated Col-I fibers were placed on the substrate and measured. Since the peptides are easily incorporated through their biochemical immobilization at the Ag surface without additional modification of the Col-I fiber structure (Orza et al., 2011; Sun et al., 2008a, 2008b), Ag NPs and the Col-I fiber surface exhibit cross-linking, as shown in Fig. S10(a) and (b). The incorporation of
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Fig. 5. Raman-active peaks of 10 7 M 4-ABT and that coated on (a) Au and (b) Ag NPs on S_3fibAu_NR examined at Raman laser wavelength of 785 nm. (c, d) Peak position at 1586 cm 1 (4-ABT) was used as index for comparison between integrated surface area of Raman peak position and relative Raman intensities of samples. T_1 and N_1: 4-ABT on Au; T_2 and N_2: 4-ABT-coated Au and Ag NPs on Au; T_3 and N_3: 4-ABT on fibAu_NR; T_4 and N_4: 4-ABT-coated Au and Ag NPs on fibAu_NR; T_5 and N_5: 4-ABT on S_3fibAu_NR; T_6 and N_6: 4-ABT-coated Au and Ag NPs on S_3fibAu_NR.
Ag NPs leads to an increase of structural disorder on Col-I fibers. For comparison, Fig. S11(a) shows Rama spectra for Col-I fibers, Ag NP-coated Col-I fibers on S_3fibAu_NR, Col-I fibers on a glass substrate, Ag NP-coated Col-I fibers on a glass substrate, Col-I fibers on fibAu_NR, and Ag NP-coated Col-I fibers on fibAu_NR. Among them, Ag NP-coated Col-I fibers on S_3fibAu_NR exhibited the highest Raman signals, i.e., those for amides I and II, Pro or Hyp, and (Gly-X-Y)n (Gullekson et al., 2011; Lee et al., 2010), and the highest integrated area of the Raman peak at 810 cm 1 (Fig. S11(b)). The characteristic Raman peak at 810 cm–1 is assigned to the ʋ(C–C) in Pro and Hyp rings (other Raman bands are listed in Table S5) (Gullekson et al., 2011; Lee et al., 2010). The results suggest that the coupling of Ag NP-coated Col-I fibers attached to NCsfibAu_NR enhances the LSPR near the NPs/NCs-NR system. The sensitivity of SERS for the detection of digested Col-I fibers was verified. In general, the proteolysis of the MMP family can cleave Col and native Col- I, II, and III fibers at a specific site in all three chains of the triple helix structure, with the cleavage starting at approximately the end of C- and N-terminus (Knauper et al., 1997; Kridel et al., 2001; Tam et al., 2002; Verma and Hansch, 2007). Among the MMP family, MMP-9 is most important because it is generated on diseased cell surfaces (Kridel et al., 2001). MMP9 has three common domains, namely propeptide, catalytic, and hemopexin-like domains, the latter of which is linked to the catalytic domain by a flexible hinge region, as illustrated in Fig. 6(b). In this study, Col-I fibers were digested by MMP-9 at concentrations of 0.005 and 0.01 mg/mL. Fig. 6(c) and Fig. S12 shows the two-dimensional AFM topography of Col-I fibers (i and ii), 0.005 mg/mL MMP-9-digested Col-I
fibers (iii and iv), and 0.01 mg/mL MMP-9-digested Col-I fibers (v and vi). The inset of Fig. 6(c-i and ii) shows an AFM image of the as-formed twisted triple helical structure of a Col-I fiber. Since MMP-9 contains catalytic and hemopexin C domains, the ability to bind with Col-I fibers and slowly start to cleave Col-I fibers are thought to be mediated (Aimes and Quigley, 1995; Knauper et al., 1997; Sun et al., 2008a; Verma and Hansch, 2007), as shown in Fig. 6(c-v and vi). Fig. 6(d) shows Raman spectra of Col-I fibers, 0.005 mg/mL MMP-9-digested Col-I fibers, and 0.01 mg/mL MMP-9-digested Col-I fibers on S_3fibAu_NR. Strong Raman signals from amide I (1664 cm 1), Pro and Hyp (1562 cm 1), amide III (1230 cm 1), and ʋ(C–C) in Pro and Hyp rings (810 cm 1) for Col-I fibers on S_3fibAu_NR can be seen in Fig. 6(d-i) and are listed in Table S5 (Cárcamo et al., 2012; Gullekson et al., 2011; Lee et al., 2010). This confirms that S_3fibAu_NR is the most SERS-active substrate, which likely creates a strong local EM effect between intra-NCs and at the NCs/NR interface. In Fig. 6 (d-ii and iii), Raman signals of amides I and III and Pro and Hyp from Col-I fibers slightly decreased with increasing concentration of MMP-9. When MMP-9 was added to Col-I fibers, they gradually lost their integrated structure due to the breaking of hydrogen bonds between Gly peptide chains, as indicated in Fig. 6 (c) and (d) (Aimes and Quigley, 1995; Fini et al., 1992; Knauper et al., 1997; Verma and Hansch, 2007). Raman spectra for 0.005 mg/mL MMP-9 interacting with Col-I fibers (Fig. 6 (d-ii)) show strong aromatic ring breathing at 1004 cm–1 and reduced amides I and II and Gly-X-Y Raman bands. Furthermore, the ends of C- and N-terminal peptides in Col-I fibers were cleaved when
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Fig. 6. Schematic diagrams of structures of (a) Col-I fiber and (b) MMP-9. (c) Two-dimensional and color quantitative AFM topography images of (i and ii) Col-I fibers, (iii and iv) Col-I digested by 0.005 mg/mL MMP-9, and (v and vi) Col-I digested by 0.01 mg/mL MMP-9 on S_3fibAu_NR. Triple helix structure of Col-I fiber and its possibly denatured structure are respectively shown in insets of AFM images (i) and (v) (scale bar: 60 nm). (d) Raman-active peaks of (i) Col-I fibers, (ii) Col-I fibers digested by 0.005 mg/mL MMP-9, and (iii) Col-I digested by 0.01 mg/mL MMP-9 on S_3fibAu_NR.
the MMP-9 concentration was increased to 0.01 mg/mL, as shown in Fig. 6 (d-iii). It is known that an increased interaction of the MMP-9 hemopexin domain with Col-I fibers leads to highly expressed Col-I fiber end terminals as hydrogen bonds between the three chains are thoroughly broken (Aimes and Quigley, 1995; Fini et al., 1992; Knauper et al., 1997; Verma and Hansch, 2007), which is confirmed by the disappearing Raman band, shown in Fig. 6 (diii), i.e., amide bands. Temperature-denatured Col-I fibers on S_3fibAu_NR were studied. The Col-I fibers were rapidly denatured by temperature due to lowered strength and fusion. Their morphology and Raman spectra are shown in Fig. S13. It is difficult to distinguish temperature-denatured Col-I fibers from MMP-9-digested Col-I fibers from Raman spectra. Further study is required. The proposed EM mechanism of Ag NP-coated Col-I fibers on NCsfibAu_NR is illustrated in Fig. 7. In Fig. 7(a), a relativity strong LSPR effect is obtained on the fibAu_NR surface, and the spacing between adjacent NRs and the length of NRs contribute to LSPR. The high SERS may be induced by the combined NC SPR and NR LSPR effects from NCsfibAu_NR, which occurred at the interface of NCs and NRs, as shown in Fig. 7(b). In addition, the intra-NC effect increased the local field effect around NCs and NRs. Interestingly, multi-polar
excitations are mostly generated at the surfaces of Ag NP-coated 4-ABT or Col-I fibers on NCsfibAu_NR, as shown in Fig. 7(c) and (d). The generations of large plasmonic fields around nanogaps in NSs systems, contributing to the detection of single molecules, were also theoretically and experimentally evidenced and thereafter suggested that around nanogaps, the EM effect is significantly enhanced (Qian et al., 2010; Chen et al., 2013). A similar effect was obtained for 4-ABT-coated Ag NPs on NCsfibAu_NR. Notably, Ag NP-coated 4-ABT on NCsfibAu_NR SERS effect systematically enhances the LSPR effect as compared to the Ag NP-coated Col-I fibers on NCsfibAu_NR due to the size effect of target species.
4. Conclusion Highly SERS-active substrates, Au or Ag NCs on fibAu_NR, are fabricated by combining FIB and electron beam deposition methods. The diameter of clusters and the formation of NCs on NR affect the SERS effect, which was evaluated using CV as a molecular probe at low concentration. In this work, the EF of S_3 (Ag NCs) fibAu_NR significantly increased with an Ag cluster size of 8 nm (to 6.92 108). It is very likely that the intra-NC LSPR effect on the
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Fig. 7. Proposed schematic diagrams of SERS effect on (a) Au NR, (b) Ag NCs on Au NR, (c) 4-ABT-coated Au NPs on Ag-NC-coated Au NR, and (d) Ag-NP-coated Col-I fibers on Au NR.
surface of NRs contributes to the detection of single molecules. In addition, a double inter-juction hotspot effect resulted from 4-ABT-coated Au or Ag NPs on S_3fibAu_NR, which provides more hotspot areas at the interface of NPs/NCs and NCs/NRs and significantly increases the number of SERS-active sites. The optimized SERS-active substrate S_3fibAu_NR was applied to detect MMP-9digested Col-I fibers under various MMP-9 concentrations. The results indicate that the Raman band signals of Col-I fibers decrease with increasing MMP-9 concentration. Presumably, MMP9-digested Col-I fibers may gradually degrade the Col-I fiber structure through the scission of hydrogen bonds between Gly peptide chains. Au or Ag NCsfibAu_NR thus has potential for investigating tissue growth in various bio-environments.
Acknowledgment This work was financially supported by the Ministry of Science and Technology, Taiwan under grant 103-2221-E-006-067-MY3.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.091.
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