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Abstract
The blue laser beam-excited surface-enhanced Raman scattering (SERS)-based pH sensing holds great promise for avoiding undesired thermal heating effect on some special temperature-vulnerable molecules, as compared to the vast majority studies by exciting in the red or near-infrared (NIR). Herein, we report an ingenious approach to support core-shell Au@Ag nanodendrites (NDs) on TiO2 nanowires, which can possess enhanced SERS activity under 473 nm laser excitation, owing to the improved charge-transfer effect on modified TiO2 support by inserting plasmonic Au@Ag. By using pH-indicating 4-mercaptobenzoic acid (4-MBA), the obtained TiO2/Au@Ag NDs can not only exhibit high sensitive linear-responses of pH changes ranging from pH 4.0 to 9.0 in different solutions (deionized water, NaCl, CaCl2, and MgCl2) but also provide excellent temperature stability under 4°C, 25°C and 37°C temperatures as well as good time stability after storage for 10 days. The established SERS-pH sensing by using shorter wavelength laser excitation is highly desirable for understanding physiological process in temperature-vulnerable microenvironment.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
In biological systems, it is well known that the bioenvironmental monitoring and biomedical diagnostic of molecules processes such as cell cycle progression, proliferation, endocytosis, metabolism, phagocytosis, and differentiation, etc. are highly related to the extracellular and/or intercellular pH homoeostasis [1–8]. Especially, the pH changes originated from the protonation-deprotonation reaction can also give rise to understand the cell status, metabolic activity, and the development of many diseases including cancer, Parkinson’s and Alzheimer’s diseases, etc. [1,3]. Therefore, the precise measurement pH value in biological system by an efficient technique is one of critical urgent issue for assessing physiological activities. Compared with other traditional analytical techniques such as absorption/fluorescence spectroscopy [9], electrochemistry [10], nuclear magnetic resonance (NMR) [11], surface-enhanced Raman scattering (SERS) spectroscopy with ultrasensitive vibrational spectroscopic property has been served as an attractive and promising strategy for quantitative detection of local microenvironment pH values in various complex mediums [1–6]. The unique efficient SERS provides nondestructive/noninvasive and reliable molecular fingerprinting information even at single-molecule level, enabling the Raman marker peaks of pH-indicating molecules to sensitively response to the pH fluctuation in complex medium. Moreover, on the basis of intense optical scattering between adsorbed probe molecules and plasmonic metallic silver (Ag), gold (Au), copper (Cu) nano-substrates or some semiconductors-based nanosubstrates, the SERS signals can be also resist the photobleaching or quenching that derived from many other optical techniques in liquid medium, which is very suitable for practical biological pH detections in clinical diagnosis.
In the past few years, great SERS-based pH sensing achievements have been obtained by using Au and/or Ag-based nanocomposites (NCs) with various morphologies, including Au@Ag/multi-walled carbon nanotubes (MWCNTs) [11], functionalized Ag nanoparticles (NPs) [12], hollow Au nanoparticles (NPs) [13], modified Au nanorods [4,14], Au@Ag nanostars [15], and Au quasi three-dimensional plasmonic nanostructure array (Q3D-PNA) [7], etc. While it should be noted that most of previous works principally focused on the longer wavelength laser-excited SERS spectra of pH sensing, and the excitation wavelength was almost restricted from red region of 633 nm, 785 nm to even near-infrared (NIR) 1064 nm. The main reason is that the localized surface plasmon resonance (LSPR) of Au and/or Ag-based NCs is generally located at the red and NIR region (>500 nm). The improved SERS enhancement originated from pronounced electromagnetic (EM) field could be simply achieved under suitable optical resonant condition between LSPR of SERS substrate and incident laser wavelength, providing ultrahigh SERS activity for pH sensing. However, the undesirable effect of thermal heating generated by exciting in the red and NIR laser should be not ignored in SERS-based pH detection [16,17], which can be further significantly enhanced in the presence of Au and/or Ag NCs under optical resonant condition [18]. In this way, there are two disadvantages should be considered as follows. Firstly, the probe molecules heated by this thermal effect upon red or NIR excitation can result in unpredictable influence on SERS spectra, giving rise to unpredictable errors in pH detection. For instance, the red (633 nm, 130 µW) laser excitation enable the benzenethiol (BT) molecules to loss the original SERS signal intensity of about 90% on a Ag island film [19], owing to longer wavelength laser-induced photosublimation of probe molecules. Moreover, the undesired thermal heating can also enable the molecular diffusion of nonvolatile molecules across the metallic nano-surfaces to occur at low analyte concentration, resulting in obvious fluctuation of SERS spectra [20,21]. Secondly, the inevitable thermal heating under longer laser excited SERS pH sensing can also bring destructive/invasive effect on some special surrounding temperature-vulnerable biomolecules/cells/proteins in bioenvironmental medium [16,17]. For example, amelogenin molecules with sizes of 15∼18 nm can be formed at liquid temperature of 27∼35°C, while that of 60∼70 nm after the temperature was elevated to 37∼40°C [17]. As for the SERS-based pH detection of such special biological systems, this thermal effect originated from red or NIR laser excitation should be reduced and avoided. Therefore, several strategies have been applied to overcome that challenge. Compared to some developed ways, such as defocusing the sample, decreasing laser power or adopting lower-magnification objective, etc. another promising alternative is proposed to switch the excitation source to shorter wavelengths (blue or UV laser beam) that possess obviously lower thermal effect in comparison with that of red or NIR. Because of very modest SERS enhancement and the risk of fluorescence by using longer wavelength source on Au and/or Ag-based traditional composites, the shorter wavelength laser- excited SERS pH sensing based on a novel SERS-active nanosubstrate has not been reported up to now.
Herein, we report an efficient blue laser (473 nm) excited SERS-based pH sensing by using core-shell Au@Ag nanodendrites (NDs) supported on semiconductor TiO2 nanowires as SERS-active substrate. The Au@Ag/TiO2 nanocomposites (NCs) were fabricated by UV-laser assisted deposition of Ag NPs on TiO2 supports and then in-site anisotropic growth of Au branched shell on Ag cores. The SERS spectra of crystal violet (CV) molecules confirm that besides electromagnetic (EM) enhancement originated from Au@Ag NDs, the pronounced chemical (CM) enhancement derived from charge-transfer (CT) between probe molecules and modified TiO2 supports obtained by inserting bimetallic Au@Ag NDs provide an important contribution for improving SERS activity under 473 nm laser excitation. As expected, the Au@Ag/TiO2 combined with pH-indicating 4-MBA molecules exhibits a characteristic response to the pH in different surrounding solution (deionized water, NaCl, CaCl2, and MgCl2) and is sensitive to pH changes in the range of 4.0-9.0. Moreover, it provides an excellent reliability under different temperatures (4°C, 25°C and 37°C), and can be also well maintained after storage for 10 days. The proposed study is highly desirable for stable SERS-based pH sensing by using shorter wavelength excitation and is important for understanding physiological and biological processes of some special temperature-vulnerable molecules/cells/ proteins/DNAs.
2. Experimental
2.1 Chemicals
Lithium acetate dihydrate (LiAc•2H2O), acetic acid (HAc), ascorbic acid (AA) and 4-mercaptobenzoic acid (4-MBA) were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). Titanium butoxide, N, N-dimethylformamide (DMF), silver nitrate (AgNO3), dibasic sodium phosphate (Na2HPO4•12H2O), sodium dihydrogen phosphate (NaH2PO4•2H2O) and anhydrous phosphoric acid (H3PO4) were obtained from Sinopharm Chemical Reagent Co., Ltd. Chloroauric acid (HAuCl4) and crystal violet (CV) were purchased from Sigma. Ethanol, sodium chloride (NaCl), calcium chloride (CaCl2) and magnesium chloride (MgCl2) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. All the above chemicals were of reagent grade and used as received without further purification. The pH of the phosphate buffer (PB) solution was adjusted with Na2HPO4•12H2O, NaH2PO4•2H2O and H3PO4 and measured with a digital pH/ion meter.
2.2 Fabrication of TiO2 nanowires and Ag/TiO2 nanocomposites (NCs)
TiO2 nanowires were prepared via hydrothermal method described in detail elsewhere [22]. In a typical process, 0.2 g LiAc•2H2O was dispersed in 5 mL HAc and 5 mL DMF mixed solution by ultrasonication. Then, 2 mL titanium butoxide was added to this mixture, which was subsequently transferred into a 20 mL stainless steel Teflon-lined autoclave and heated at 200°C for 20 h. Finally, the as-prepared TiO2 sediments were washed by deionized (DI) water for three times and then dried in vacuum oven at 80°C for 12 h. The UV (375 nm) laser-induced overgrowth of Ag species on TiO2 nanostructures is similar to our previous works [23,24]. In brief, 2 mg TiO2 powder was diluted with 5 mL distilled water by 5 min ultrasonic vibration. Then, 1 mL 0.01 M AgNO3 and 1 mL ethanol solutions were added into 5 mL TiO2 solution. Under magnetic stirring condition, the deposition of Ag NPs on TiO2 nanowires was obtained via laser irradiation (400 mW) at a wavelength of 375 nm. After the irradiation of 0-60 min, the as-synthesized samples were centrifuged by 8000 rpm for 10 min in an ultracentrifuge.
2.3 Fabrication of core-shell Au@Ag/TiO2 nanocomposites (NCs)
In a typical experiment of synthesizing core-shell Au@Ag/TiO2 NCs, the precipitate was dispersed into deionized water for preparation of 0.1 M/2 mL Ag/TiO2 nanomaterials. Then, the Au@Ag/TiO2 NCs can be obtained by adding 0.08 M/2 mL ascorbic acid (AA) solution into the 200 µL HAuCl4 solution (0.05 M) diluted with 10 mL deionized water in the present of 0.1 M/2 mL Ag/TiO2 nanomaterials. Then, the obtained nanoproducts were centrifuged by 5000 rpm for 10 min in an ultracentrifuge.
2.4 Materials characterization
The morphological structures and corresponding elemental mapping images of the nanoproducts were characterized by transmission electron microscopy (JEOL-JEM-2100F) equipped with energy dispersive X-ray (EDX) spectroscopy via a scanning transmission electron microscopy (STEM) unit. Moreover, the nanostructure of the samples were further investigated by field emission scanning electron microscope (SEM, JSM-7610F) equipped with energy-dispersive X-ray spectroscopy (EDS). The detailed sample surface structures of the products were studied by X-ray photoelectron spectra (XPS) using a PHI Quantera SXM with an Al Kα = 280.00 eV excitation source. The absorption spectra were recorded by a UV-Vis-IR spectrometer (UV-1800, Shimadzu).
2.5 SERS measurement
In a typical SERS procedure, the nanomaterials-based substrates were prepared by dropping 0.3 M/15 µL sediments on clean silicon wafers and dried at room temperature for 4 h. Then, the SERS nanosubstrates were totally immersed into certain concentration of crystal violet (CV) molecules ethanol solution (10−6 M-10−9 M) and 4-mercaptobenzoic acid (4-MBA) ethanol solution (10−3 M) under lucifugal circumstance for 12 h and then dried spontaneously. In order to investigate the pH-dependent SERS characteristics of Au@Ag/TiO2 NCs, PB solution with different pH values (4.0-9.0) were prepared. 10 µL different pH values of PB solution were added to the silicon wafers before SERS measurements. The SERS measurements were performed on the LabRAM HR 800 spectrograph with 473 nm laser beam at room temperature. Figure 1 provide the direct photograph and schematic description of using blue laser (473 nm) beam as an excitation source for SERS analysis, which is significantly different from the red laser-excited SERS detection. The exciting laser power on the sample was located at about 50 µW, and the acquisition time used for each spectrum was 20 s.
Fig. 1. The direct photograph and schematic description of blue laser (473 nm) beam excited SERS analysis by using Au@Ag/TiO2 as efficient substrate.
3. Result and discussion
The typical TEM image of TiO2 nanowires synthesized by hydrothermal method is presented in Fig. 2(a). Numerous elongated wires with length of ∼200 nm and diameter of ∼17 nm are prone to bend and connect with each other. After 60 min UV laser irradiation of TiO2 support in Ag+ solution, the morphologies of obtained nanoproducts are illustrated by TEM images with different magnifications, as shown in Figs. 2(b)–2(d). It can be seen that there are abundant well-dispersed Ag NPs with diameter ranging from 5∼11 nm formed on sides of bended wire-like TiO2 walls. Moreover, the high-resolution (HR) TEM image (Fig. 2(d)) exhibits a representative structural detail of the enlarged region between Ag NPs and TiO2 nanowire. Evidently, it can be observed that the Ag NPs are indeed accreted/inserted on TiO2 supports instead of physical mixture of two different nanomaterials, owing to the obvious interconnected structure formed at boundary region. Furthermore, the crystal structure of an individual supported nanoparticle with lattice-spacing value of 0.237 nm can be indexed as (111) in the Ag face-centered cubic (fcc) crystal structure. To further investigate the morphology in detail, the SEM images and corresponding EDS pattern of Ag/TiO2 were further carried out, as shown in Figs. 3(a)–3(b). Clearly, these TiO2 nanowires are completely covered by plentiful well-dispersed and dense NPs on wall-surfaces. The relative Ag ratio in the NCs is about 4.4% based on the EDS result (inset in Fig. 3(a). On the other hand, the laser-induced deposition of Ag NPs on TiO2 supports was monitored by UV-visible absorption spectra of Ag/TiO2 solution irradiated by 375 nm laser beam with different times. As shown in Fig. 3(c), the absorption band shoulder of original TiO2 is located at UV region. The photo energy of adopted 375 nm laser beam will be significantly absorbed by semiconductor TiO2, resulting in the formation of electron-hole pairs on surface. The photon-excited electrons will provide active sites for reduction of surrounding Ag+ ions, resulting in the nucleation of Ag NPs on TiO2 supports. As illustrated in Fig. 3(c), the absorption peak at ∼420 nm is attributed the LSPR of Ag NPs, which gradually increase with an increase of irradiation time (0∼60 min). The increasing intensity of Ag LSPR is also an evidence for the higher yield deposition of Ag NPs on TiO2 supports, which enable the solution color to change from milk white for original TiO2 solution to brownish yellow for Ag/TiO2 fabricated by 60 min laser irradiation (inset in Fig. 3(c)). Based on the EDS patterns of Ag/TiO2 obtained by laser irradiation with different times, the variation of Ag content in nanoproducts versus irradiation times is shown in Fig. 3(d). It is clearly shown that the relative ratio of Ag obviously increases to 4.35% as the irradiation time increases to 45 min, and then slightly reaches at maximum value of 4.4% after 60 min reaction. While, further increasing irradiation time also result in the large-scaled aggregation of Ag NPs. Then, these dispersed Ag NPs on TiO2 walls will be served as nanoseeds for further anisotropic overgrowth of Au nanodendrites on Ag cores.
Fig. 2. (a) TEM image of original TiO2 nanowires. (b-d) Different magnified TEM images of as-prepared Ag/TiO2 NCs fabricated by 375 nm laser irradiation of TiO2 supports in AgNO3 solution. The irradiation time is 60 min.
Fig. 3. (a-b) The typical low and enlarged SEM images of as-prepared Ag/TiO2 NCs. The inset is the corresponding EDS pattern. (c) The absorption spectra of Ag/TiO2 solution generated by 375 nm laser irradiation with different times. The inset shows the color change of original TiO2 and Ag/TiO2 NCs fabricated by 60 min laser irradiation. (d) The curve of Ag content in nanocomposites versus laser irradiation time.
After the subsequent process of adding HAuCl4 and AA into as-prepared Ag/TiO2 precursors, the morphologies of these obtained nanoproducts are carefully investigated by TEM with different magnifications in Figs. 4(a)–4(c). As shown in Fig. 4(a), a great deal of branched nanomaterials with obvious interconnected multi-tips are formed on wire-shaped TiO2 supports, which is drastically different from that of the as-prepared well-dispersed Ag NPs on TiO2 precursors. The enlarged TEM images in Figs. 4(b)–4(c) further confirm that the formed nanodendrites are composed of a solid body (relative dark one) with multiple branched shell structures (relative light ones) on the nano-core. The estimated lattice spacing distance of branched shells (Figs. 4(b)–4(c)) is about 0.235 nm, which is attributed to the (111) plane of Au (fcc) crystal structure, confirming the formation of branched Au shells on Ag core. Moreover, the dendritic nature of Au shells was also visualized by the change of LSPR peak, as the absorption spectra shown in Fig. 4(d). The LSPR peak of Au@Ag/TiO2 is located at ∼715 nm and obviously longer than that of Ag/TiO2 at 421 nm. Meanwhile, the solution color also changed from brownish yellow to dusty blue, confirming the anisotropic overgrowth of Au species. On the other hand, high-angle annular dark-field scanning TEM (HAADF-STEM) of Au@Ag/TiO2 and corresponding elemental mapping images are shown in Fig. 4(e). It can be illustrated that the obtained nanoproducts are indeed composed of uniform Ti and O species as well as isolated Au and Ag elements. The calculated relative ratio of Au to Ag is about 10:1, confirming the forming of thick Au shell in this work.
Fig. 4. (a-c) The low and enlarged TEM images of obtained Au@Ag/TiO2 by adding 0.05 M, 200 µL HAuCl4 into as-prepared Ag/TiO2 precursors. The inset is the corresponding EDS pattern. (d) The absorption spectra of as-prepared Ag/TiO2 and Au@Ag/TiO2. The inset shows the direct photograph images of two different solutions. (e) the HAADF-STEM image of a typical Au@Ag/TiO2 nanostructures and the corresponding elemental mapping images.
To further evaluate the electronic structures and chemical states of obtained Au@Ag/TiO2, as-prepared Ag/TiO2, and original TiO2 nanowires, X-ray photoelectron spectroscopy (XPS) analyses were carried out in this paper. As shown in Fig. 5(a), the full survey XPS spectra illustrate that these three samples all have Ti2p and O1s peaks originated from TiO2 supports as well as the weak C1s peak at 285 eV. The high resolution XPS patterns of Ti element in Fig. 5(b) show that the double peaks of Ti2p detected at 458.5 eV and 464.5 eV are assigned to the 2p3/2 and 2p1/2 binding energies of Ti4+ oxidation states respectively. The two peak intensities gradually decrease as the deposition of Ag NPs and then overgrowth of Au@Ag on TiO2. Moreover, the high-resolution XPS spectra of Ag3d5/2 and Ag3d3/2 originated from Ag/TiO2 and Au@Ag/TiO2 in the region of 360∼380 eV are illustrated in Fig. 5(c). As for Au@Ag/TiO2, the double peaks of atomic Ag3d5/2 and Ag3d3/2 are located at 367.7 and 373.7 eV, which are obviously blue-shifted than that of Ag/TiO2 at 368.3 and 374.4 eV, respectively. As illustrated in our previous work [25], the blue-shifted of Ag atoms in Fig. 5(c) is attributed to the interaction between Au and Ag in Au@Ag/TiO2 NCs, providing bimetallic synergistic effect for enhancing SERS performance. Meanwhile, as for Au@Ag/TiO2 NCs, the presence of strong Au double peaks (Au4f7/2: 84.2 eV and Au4f5/2: 87.9 eV in Fig. 5(a)) is accompanied by the significant decreased intensities of double peaks of Ag3d5/2 and Ag3d3/2 in Fig. 5(c), implying the formation of Au shell and Ag core structure. On the other hand, the loading metallic NDs can also give rise to the structure change of TiO2 supports. As illustrated in Fig. 5(d), by using a Gaussian-Lorentzian fitting method, the XPS spectra of O1s can be de-convoluted into three couples of peaks, attributing to absorbed O2 (peak 3: 532.4 eV), surface hydroxyl oxygen in TiO2 (peak 2: 531.2 eV) and surface bridging oxygen in TiO2 (peak 1: 529.9 eV) [26,27]. The enhanced XPS peaks of O1s at 532.4 and 531.2 eV originated from Au@Ag/TiO2 NCs in Fig. 5(d) suggests that the surface structure of TiO2 support can be effectively modified by accreting bimetallic Au@Ag NDs. In this way, the conduction band electrons of TiO2 can easily transfer to the inserted bimetallic NDs by external laser excitation during SERS experiment, providing enhanced CM enhancement for improving SERS activity.
Fig. 5. XPS spectra of obtained Au@Ag/TiO2, as-prepared Ag/TiO2, and original TiO2 nanowires, respectively. (a) survey spectra. (b-d) Ti2p and metallic Ag3d as well as O1s spectra, respectively.
Based on the above discussions, the obtained Au@Ag/TiO2 NCs are expected to provide improved SERS activity in comparison with Au@Ag or TiO2 nanomaterials. To verify it, the SERS analyses of 10−8 M CV molecules adsorbed on Au@Ag/TiO2 NCs and core-shell Au@Ag NDs were performed via 473 nm blue laser excitation. As shown in Fig. 6(a), it can be observed that the Au@Ag/TiO2 NCs exhibit improved SERS performance with much enriched “molecular fingerprint” and enhanced SERS activity in comparison with Au@Ag NDs. For instance, the characteristic bands of ring C-C stretching vibrations at 1622, 1591 and 1302 cm−1, N-phenyl stretching model at 1378 cm−1, the ring skeletal vibration of radical orientation at 916 cm-1, and ring C-H bend at 810 cm−1 are all clearly detected on Au@Ag/TiO2 substrate, while that of Au@Ag NDs is only 1591 cm−1. Furthermore, based on the SERS spectra at 1591 cm−1, the enhancement factor (EF) of CV molecules adsorbed on obtained Au@Ag/TiO2 NCs and core-shell Au@Ag NDs can be further estimated according the equation:
Where ISERS and INOR are the signal intensities of SERS and normal Raman spectrum of the same vibration peak for the CV molecule, respectively. And NSERS and NNOR represent the corresponding number of CV molecules exposed to the laser spot focused area. Because the laser parameters adopted in the SERS measurements were the same, NSERS and NNOR can be approximately determined by the concentration of CV, this empirical calculation has been verified in many previous works [28–31]. According to this equation, the EFs of SERS for CV molecules adsorbed on Au@Ag/TiO2 NCs and core-shell Au@Ag NDs were estimated to be about 6.34×107 and 1.12×107, respectively. The corresponding enhanced SERS activity of Au@Ag/TiO2 NCs is higher than that of core-shell Au@Ag NDs as well as many previous reports [29,32–34]. The modest enhancement of Au@Ag NDs in Fig. 6(a) is attributed to that the LSPR maximum of Au@Ag at red region (>650 nm) is obviously red-shifted with respect to the blue laser excitation at 473 nm. Because of very similar geometric structures of sole Au@Ag NDs (inset in Fig. 6(a)) and branched Au@Ag loaded on TiO2, the obvious enhanced SERS activity in Fig. 6(a) should be attributed to the modified semiconductor TiO2 support. The CM (chemical) enhancement originated from the charge-transfer (CT) mechanism between semiconductor TiO2 and probe CV molecules is confirmed in Fig. 6(b). The SERS spectra of CV molecules adsorbed on original TiO2 nanowires can be clearly detected at 10−5 M concentration. Compared to red or NIR laser source, the semiconductor TiO2 nanowires can be efficiently excited by blue 473 nm laser excitation, resulting in enhanced CT effect for improving SERS intensity. Most importantly, the electronic interaction between Au@Ag NDs and TiO2 in this work gives rise to the formation internal electric field and will provide another additional CM contribution for increasing SERS signals, which has been verified in previous works [35,36]. In this way, the obtained Au@Ag/TiO2 have both modest electromagnetic (EM) enhancement from bimetallic Au@Ag and pronounced CM effect from modified TiO2 by accreting metallic NDs. The unique feature enables the SERS limit of detection (LOD) to achieve at nanomole level ∼10−9 M, since the dominating characteristic bands of CV molecules are also clearly distinguishable even the concentration decreased to as low as 10−9 M (Fig. 6(c)). On the other hand, Figure 6(d) exhibits the SERS spectra of CV molecules adsorbed Au@Ag/TiO2 substrate before and after storage for 10 days at room temperature, supporting that there is no obvious change in peak position and intensity after storage for 10 days. For instance, the Raman signal intensity of 1624 cm−1 slightly decreased from about 6130 a.u for a flesh substrate to 5010 a.u after storage for 10 days, 81.7% SERS intensity can be maintained during this process. We also performed the SERS signal changes on Ag/TiO2 substrate, and found only 22% Raman signal intensity can be maintained after the same storage for 10 days. It can be confirmed that the Au shell in Au@Ag/TiO2 can significantly improve the stability of SERS substrates.Fig. 6. (a) SERS spectra of 10−8 M CV molecules adsorbed on obtained Au@Ag/TiO2 NCs and core-shell Au@Ag NDs and 0.1 M CV without any nanosubstrate. (b-c) SERS spectra of CV molecules with different concentrations adsorbed on TiO2 nanowires and Au@Ag/TiO2 NCs, respectively. (d) Based on Au@Ag/TiO2 NCs substrate, SERS spectra of 10−6 M CV molecules before and after storage for 10 days at room temperature.
Finally, we demonstrate the potential of Au@Ag/TiO2 NCs as SERS substrate for pH-sensing application based on blue wavelength (473 nm) laser excitation. As shown in Fig. 7(a), it can be observed that SERS spectrum of 4-MBA molecules adsorbed on Au@Ag/TiO2 exhibits well-defined and high resolution Raman spectral lines in the absence of any background fluorescence or interference from photobleaching. The 473 nm laser-excited SERS spectral lines in this paper are consistent with many previous reports by using red or NIR laser excitations [6–8], which are identified as follows: the two stronger peaks located at 1585 and 1075 cm−1 can be attributed to v8a and v12 aromatic ring vibrations [37]; Other two important peaks detected at 1690 and 1406 cm−1 should be originated from the C = O and COO- stretching modes [7,37,38]; The residual weak peaks such as 1481, 1180, 848, 718 and 521 cm−1 match with five functional groups including β19a, v9a, βCOO-, v4b, Si + Vcs, respectively [39]. Moreover, the corresponding SERS spectrum of 4-MBA molecules with pH from 4.0 to 9.0 in steps of 1.0 pH unit are normalized at the intense peak at 1585 cm−1 and displayed in Fig. 7(b). It can be found that the COO− and C = O stretching modes at 1406 and 1690 cm−1 change obviously at different pH conditions, attributing to the sensitive changed structures of 4-MBA in the different pH environments. In detail, the COO- stretching mode at 1406 cm−1 increases with the pH increases, while the C = O stretching mode at 1690 cm−1 decreases with the pH increases. In this way, the relative intensity ratio of 1406 cm−1 to 1585 cm−1 can be used to determine the surrounding pH value, which is also verified in previous works [6–8]. The detailed results of I(vCOO-)/I(v8a) under different pH conditions are summarized in Fig. 7(c), and the corresponding variations as a function of pH values are plotted in Fig. 7(d). It should be noticed that the relative result of I(vCOO-)/I(v8a) based on blue 473 nm laser-excited SERS spectra of 4-MBA (10−3 M) provide well-defined linear relationships at different pH conditions, supporting that the obtained Au@Ag/TiO2 have a good and sensitive SERS-based pH response. Furthermore, in order to evaluate the stability of this Au@Ag/TiO2 SERS-based pH sensing, the SERS spectra obtained at temperatures of 4°C and 37°C were carried out in this paper. The corresponding pH curves are shown in Fig. 7(e), which are very close to that obtained at room temperature (25°C), supporting that the established pH sensing has a good temperature stability. On the other hand, an excellent SERS-based pH sensing should provide sensitive pH response in complex mixed environments including the presence of different cations, which is highly desirable for practical extracellular and/or intercellular pH sensing. For instance, Na+ ions are the well-known cations in cell culture media, moreover, a small amount of Ca2+ and Mg2+ are also required in many tissues. In this way, the SERS spectra of 4-MBA molecules in deionized (DI) water, Na+, Ca2+ and Mg2+ cations (0.01M, respectively) were carried out under different pH values. The corresponding Raman peaks of the COO- stretching mode (1406 cm−1) at pH 7.0 (Fig. 7(f)) show that the Ca2+ and Mg2+ divalent cations provide more influences on the peak of 1406 cm−1 in comparison with the Na+ monovalent cations due to their higher binding affinities to the carboxylate group, which is also consistent with previous report [7]. Moreover, the corresponding pH curves in aforementioned four solutions are illustrated in Fig. 7(g). Although there are different influences on SERS spectral intensities of 4-MBA in four solutions, the four curves in Fig. 7(g) exhibit very similar behavior with well-linear relationships ranged from pH 4.0 to pH 9.0. Therefore, the SERS-based pH sensing with good accuracy in this work can provide reliable pH detection under different solutions, which is suitable for real-world bioenvironmental monitoring and biological applications. Meanwhile, the stability of this SERS-based pH sensing is also illustrated in Fig. 7(h), in which one of pH curves was derived from a freshly prepared Au@Ag/TiO2 and the other was originated from that preserved for several days (3, 7 and 10 days). It can be observed that the obtained SERS pH sensing possesses excellent stability with only slightly deviations ranging from pH 4.0 to pH 9.0 before and after storage for 10 days. Moreover, the SERS spectra of 4-MBA (10−3 M, pH = 7.0) adsorbed on Au@Ag/TiO2 before and after storage for several days (3, 7 and 10 days) are illustrated in Fig. 8 (Appendix). It reveals that no significant change can be observed in both the peak position and the intensity, where 80.8% SERS intensity can be maintained after storage for 10 days. In summary, all these experimental results in this paper confirm that the core-shell Au@Ag/TiO2 NCs are promising to serve as efficient and stable SERS-based pH sensing by using shorter wavelength laser as excitation source.
Fig. 7. (a) Blue laser (473 nm) excited the SERS spectra of 4-MBA molecules (10−3 M) adsorbed on Au@Ag/TiO2 NCs at pH = 7.0 condition. (b) The corresponding SERS spectra of Au@Ag/TiO2 SERS-based pH sensor in 4-MBA (10−3 M) of various pH values ranging from pH 4.0 to pH 9.0 in steps of 1.0 pH unit, respectively. (c-d) the variation of I(vCOO-)/I(v8a) versus different pH values. (e) pH calibration curves of SERS-based pH sensor at temperatures of 4°C, 25°C and 37°C. (f) the Raman peak of the 1406 cm−1 versus different cations in DI water at pH 7.0. (g) the pH curves for different SERS detection solutions. (h) SERS-based pH sensor before and after storage for several days (3, 7 and 10 days).
4. Conclusion
In summary, we have demonstrated an efficient blue laser-excited SERS-active nanosubstrate by loading core-shell Au@Ag NDs on TiO2 nanowires. The SERS analyses of CV molecules adsorbed on Au@Ag/TiO2 NCs confirm that the novel hybrid nanostructure possesses both EM enhancement originated from plasmonic Au@Ag and pronounced CM enhancement derived from modified semiconductor TiO2 by accreting bimetallic NDs. Based on the SERS spectra of 4-MBA molecules by using 473nm laser beam as excitation source, the obtained Au@Ag/TiO2 NCs exhibit high sensitive responses to pH change ranging from pH 4.0 to pH 9.0 under DI, Na+, Ca2+ and Mg2+ solutions. Moreover, the temperature (4°C, 25°C and 37°C) and time (10 days) stabilities of this blue laser excited SERS-pH sensing have been illustrated in this paper. Thus, this work provides an effective SERS-based pH sensing by using shorter wavelength laser beam as excitation source, which can overcome the thermal heating generated from red or NIR laser-excited SERS analysis. It will be an alternative solution for SERS-based pH sensing in some special bio-environments including temperature-vulnerable molecules/cells/proteins.
Appendix
The SERS spectra of 4-MBA adsorbed on Au@Ag/TiO2 before and after storage for several days (3, 7 and 10 days)
Funding
Fundamental Research Fund of Shandong University (2018JC022); Natural Science Foundation of Shandong Province (ZR2016CM02); National Natural Science Foundation of China (11105085, 11375108, 11575102, 11775134).
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