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Abstract
The abuse of antibiotics in animal husbandry has been regarded as a daunting public health risk, facilitating the emergence and spread of resistant pathogens to humans. Herein, bimetallic Au@Ag core-shell nanorods (NRs) with precise, controllable Ag shell-thickness (2.1~14.1 nm) were fabricated and developed for ultralow detection of levofloxacin molecules using surface enhanced Raman scattering spectroscopy (SERS). We found that the Au@Ag NRs with 7.3 nm Ag shell-thickness provided maximized SERS activity in comparison with other as-prepared nanosubstrates in this paper. The detection limit of levofloxacin molecules was achieved at a nanomole (nM) level of 0.37 ng/L (10−9 M), providing ultrasensitive assessment of antibiotics in natural ecosystems.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Over the past decades, antibiotics have been used for reducing morbidity and mortality of infectious diseases, which are regarded as the most successful drugs to save millions of lives and improve human health and wellbeing. In addition to the human therapy, the antibiotics (especially antimicrobials) have also been extensively used in animal husbandry [1–5]. For instance, the wide use of antibacterial agents in chickens, swine or other food-producing animals shows great practical potential for treating animal infections as well as promoting growth in animal farming. Especially in less-developed counties, the abuse of antibiotics was served as a main and effective tool for production of abundant quantities of high-quality and low-cost food for human consumption [1]. However, it has to be stressed that animals do not effectively metabolize antibiotics and largely pass them back into natural ecosystems, resulting in serious environmental contaminations with original substances or derivatives [2]. Moreover, the antibiotic residues in food animal tissues will directly cause a daunting public health risk, facilitating the emergence of resistant bacteria in human being, and even the spread/transmission of such resistant pathogens within the human population [3]. Therefore, ultrasensitive detection of antibiotic molecules is highly designable for precise assessment of food security, ensuring nutritious, safe and healthy products for human consumption.
Recently, surface-enhanced Raman scattering spectroscopy (SERS) has been established as a simple, rapid, nondestructive and powerful spectroscopic analysis for ultrasensitive detection and precise identification of various molecules at single molecule level (<10−9 M) [6–12]. Under laser irradiation, the plasmonic silver (Ag), gold (Au) or copper (Cu) nanostructures with unique localized surface plasmon resonance (LSPR) can provide enhanced electromagnetic (EM) field and then significantly magnify the Raman signals of adsorbed molecules on the surfaces. In this way, the generating SERS signals will provide enriched “molecular fingerprint” information originated from specific vibration energies of chemical bands. Moreover, the SERS signals can be obviously further improved by optimizing the shape, composition and size of plasmonic nano-substrates, giving rise to the ultralow detection of probe molecules in range of nanomole (nM) to even femtomole (fM) level. Most recently, for example, we have demonstrated that the SERS detection limits of 4-aminothiophenol (4-ATP) molecules and triphenylmethane dyes (crystal violet) can be achieved at 10−13~10−14 M and 10−13 M by using Ag@Au hollow-shaped nanourchins and branched nanodendrites, as well as Au/Ag self-assembled monolayers (SAMs) [13–15]. As for antibiotic molecules, some pioneering works based on lab-on-a-chip SERS and optical fiber nanoprobe-based SERS have illustrated that the levofloxacin molecules served as a typical third-generation fluoroquinolone antibiotic drug can be sensitively monitored in human urine or mouse blood [16,17]. On the other hand, the detection limits of levofloxacin molecules were ~0.8 μm by using Ag nanoparticles and 1.29 × 10−7 M via Zn-TiO2 nanoparticles [18,19]. The above works confirm that the SERS technique shows great practical potential for monitoring and detection of antibiotic molecules in vitro or even in vivo.
It should be noted that although SERS analyses of levofloxacin have been carried out in recent years, while the SERS sensitivities of previous works are less than 10−7 M. The SERS detection limit of antibiotic levofloxacin molecules-appears far away from the ultralow detection realized by other time-consuming and expensive developed methods. For example, the detection limits of enrofloxacin, ciprofloxacin, norfloxacin, and difloxacin were located in the range of 1~2 ng/g by using specific reversed-phase high-performance liquid chromatography (HPLC) [20]. Moreover, the multiresidue determinations of sulfonamides in bovine liver and kidney tissues were about 3.3~10 ppb via HPLC coupled to a mass spectrometer (MS) [21]. Recently, based on hollow fiber liquid-phase microextraction (HF-LPME) and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME), the ultra-trace determination of eight drugs of abuse in urine and blood samples were calculated about 0.5~5 ng/ml [22]. Therefore, it is highly desirable for further improving SERS detection limit of antibiotics by optimizing SERS-active nanostructures, which is very important for precise assessing the threat to human health.
Herein, we demonstrate a novel and prominent SERS nanosubstrates based on the fabrication of well-defined bimetallic Au@Ag core-shell nanorods (NRs) with precise controllable Ag shell-thickness (2.1~14.1 nm). The uniform Ag shell-decorated Au nanorods were obtained by seeded-growth of Au rod-shaped nano-cores and then homogeneous growth of Ag shells via wet chemical synthesis in a hexadecyltrimethylammonium bromide (CATB) solution. The Ag shell thickness-dependent SERS analysis of crystal violet (CV) molecules revealed that the Au@Ag NRs with 7.3 nm Ag shell provide maximized SERS activity in comparison with Au@Ag NRs with other Ag shell thickness. Based on finite-difference time-domain (FDTD) method, the simulation results confirmed that the 7.3 nm Ag shell-decorated Au nanorods exhibit enhanced electric filed compared with other Au@Ag NRs, verifying the presence of optimal SERS-active nanosubstrates. The maximized SERS activity is highly related to the pronounced intermetallic synergies between Ag and Au metals at appropriate core-shell NRs structure. More importantly, the optimal Au@Ag NRs-based SERS analyses demonstrate that the dominating characteristic bands of levofloxacin molecules can be clearly distinguished even with the concentration decreased to as low as 0.37 ng/L (10−9 M), which is superior to many exciting reports (<10−7 M) [16–19]. Moreover, the SERS detection limit of antibiotic molecules at nanomole (nM) level can be comparable with the ultralow results by using high-sensitive developed techniques such as HPLC, HPLC-MS, HF-LPME, and UA-LDS-DLLME, etc [20–22]. Finally, the corresponding SERS analyses also illustrate that well-defined linear relationships can be established between SERS peak intensities and logarithmical scale of levofloxacin concentrations in a wide range of 10−4~10−9 M. The ultralow SERS detection limit and established linear relationships will provide more important internal standard for exact quantitatively analyzing and ultrasensitive monitoring the slight changes of antibiotic residual in food-animal products or environmental contaminations.
2. Experimental setup
Preparation of Au NRs. Au NRs were prepared by a seed-mediated growth method, which is very similar to previous works [23–26]. A 10ml mixed aqueous solution containing 0.1 M cetyltrimethylammonium bromide (CTAB) and 0.25 mM HAuCl4 was added in a glass dish. Then 0.6 ml of freshly prepared NaBH4 (0.01 M) was added to the above solution with vigorous stirring for 2 min, which immediately resulted in the formation of a brownish yellow solution. The prepared seed solution was kept in a water bath maintained at 30 °C for 2 h. As for preparation of the growth solution, in a clean test tube, 5 ml of 0.2 M CTAB, 33 μl of 0.04 M AgNO3, 5 ml of 1mM HAuCl4 and 37.5 μl of 0.1 M ascorbic acid (AA) were taken one by one in the order given under gentle stir. AA as a mild reducing agent makes color of solution changed from deep yellow to colorless. The next step was to add an amount of 12 μl prepared seed solution into the growth solution under slight stirring and the color of mixture changed to pink within 10 min. Then the mixture was placed in a water bath maintained at 30 °C for 6 hours and the final solution color was close to pale red, indicating the successful synthesis of Au NRs.
Preparation of Au@Ag core-shell NRs. 10ml as-prepared Au NRs were washed by centrifugation at 10000 rpm during 15 minutes, the supernatant was discarded and precipitate redispersed in 3 ml of 0.02 M cetyltrimethylammonium chloride (CTAC). Next, using the original Au NRs as seeds, different amount of 0.1 M AgNO3 (2, 8, 12, 16, 20, 24 μl) and corresponding variation of AA (0.1 M) were added into the solution and stirred 1 min. The mixture was incubated at 65 °C for 2.5 h without stirring, which grew into Au@Ag core-shell NRs with different thickness of Ag shell (2.1~14.1 nm). The obtained solution was centrifuged at 10000 rpm for 10min and the sediment was re-suspended in distilled water and centrifuged again.
Characterization and SERS testing of Au@Ag NRs. The obtained precipitates were dropped on a copper mesh and dried in an oven for observation via transmission electron microscopy (JEOL-JEM-2100F). The morphologies of the samples were recorded though field emission scanning electron microscopy (SEM, Hitachi S-4800). Crystallographic information about as-prepared products were obtained on a Bruker D8 advanced X-ray diffractometer (XRD, Cu Kα radiation, λ = 1.5418 Å). In addition, absorption spectra of the solutions were recorded by a UV-vis NIR spectrophotometer (UV-1800, Shimadzu). As for the SERS analysis of probe molecules, the preparation ethanol solutions of CV probe molecule illustrated as follows. 0.004 g CV powder was dispersed in 10ml ethanol by ultrasonic vibration equipment to form 10−3 M CV solutions. And then, the solution will be separately diluted with ethanol to prepare CV samples with different concentrations 10−3~10−6 M. Similarly, levofloxacin solutions with different concentrations (10−4~10−9 M) were obtained in the same dilution process. In a typical Raman spectroscopic analysis, 0.1 mg sediment of Au NRs or Au@Ag core-shell NRs were suspended in 1 ml solutions with different probe molecules concentration under stirring with a constant speed of 200 rpm for 3 h. It ensures that more effective probe molecules can be attached to the surface of as-prepared products. Then the solutions were centrifuged at 10000 rpm for 10 min in an ultracentrifuge. The precipitates were re-suspended in distilled water (0.5 ml) for SERS measurements. All the SERS signals were collected by a confocal microprobe Raman spectrometer (LabRAM HR 800 spectrograph) with an excitation of 633 nm (power at the sample ~50 μW) and 10 s in integral time.
3. Results and discussion
The Au NRs were generated by seed-mediated growth method, which will be served as precursors for further fabrication of Au@Ag core-shell NRs. The morphology of as-prepared Au NRs was separately illustrated by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in Fig. 1. The typical TEM image in Fig. 1(a) shows that numerous well-defined cylindrical Au NRs with fairly good uniformity possess smooth surfaces and spherical heads. The uniformly mono-dispersed Au NRs with high quantity (yield>98%) can be also verified by the SEM image in Fig. 1(b). Based on the measurements of more than 500 nanorods in sight on the TEM and SEM images, the average length and width of as-prepared NRs were calculated ~53.6 ± 4.1 nm and 15.8 ± 2.5 nm, respectively. The high resolution TEM (HRTEM) image in Fig. 1(c) further provides the detailed structure of an individual Au NR. As shown in Fig. 1(c), the Au NR is found to be well crystalline according to the clear lattice fringes. Correspondingly, the parallel lattice fringes with a d- spacing of 0.232 nm could be assigned to the (111) plane in the Au face-centered cubic crystal structure.
Fig. 1 The typical low-magnification TEM (a) and SEM (b) images of Au NRs prepared by seed-mediated growth method. (c) The HRTEM image of an individual Au NRs.
During the process of adding 0.1 M AgNO3 (2, 8, 12, 16, 20, 24 μl) and 0.1 M AA (8, 32, 48, 64, 80, 96 μl), homogeneous over growth of Ag shell on as-prepared Au NRs will result in the formation of Au@Ag core-shell NRs. Based on 12 μl AgNO3 and 48 μl AA, the structures of obtained Au@Ag NRs are shown in Fig. 2(a)-2(d). The TEM image [Fig. 2(a)] shows that numerous mono-dispersed Au@Ag NRs with uniform cylindrical structures were successfully formed by wet chemical approach. The corresponding elemental mapping images [Fig. 2(b)] of an individual NR further clearly reveal that the nano-products are indeed composed of Au core and Ag shell structures. Evidently, the obvious bounds between Au core and Ag shell are shown as contrasting light image with the central core as bright white and shell region as dark white. Moreover, the distributions of Au and Ag elements are separately located in core and shell regions, confirming the formation of Au@Ag core-shell NRs in this paper. Based on numerous Au@Ag NRs with homogeneous structures [Fig. 2(a), and SEM image in Fig. 2(c)], the average thickness of Ag shell is calculated about 7.3 nm. The HRTEM image in Fig. 2(d) shows a typical structural detail of the cross region between Au core and Ag shell. It can be found that the measured lattice-spacing of Au core is about 0.232 nm, which is agreed with the Au (111) plane in original Au NRs structure. While, the lattice fringes with a d-spacing of 0.205 nm at shell region should be belonged to the Ag (200) structure. The preferential growth of Ag (200) orientation will be also verified in Au@Ag NRs structures with much thicker Ag shell as follows.
Fig. 2 The representative structures of Au@Ag core-shell NRs with different Ag shell thickness produced by wet chemical method. [(a)-(d), (e)-(h)] TEM, elemental mapping, SEM, and HRTEM image of 7.3 nm and 14.1 nm Ag shell-decorated Au NRs, respectively.
We further increased the amounts of AgNO3 and AA to 24 μl and 96 μl, respectively, and found much thicker Ag shell decorated on Au@Ag NRs in Fig. 2(e)-2(h). As shown Fig. 2(e)-2(g), the Au@Ag NRs were already transformed into cuboid-shaped structure, which is different from the cylindrical NRs with spherical heads. The corresponding elemental mapping images of an individual NR in Fig. 2(f) also clearly reveal the obvious Au-core and Ag shell structure. As illustrated in Figs. 2(b) and 2(f), it can be deduced that the lateral growth of Ag species should dominate the over growth of Au@Ag NRs structure, since the Ag thickness along longitudinal direction nearly maintain at 3.2 nm during the reaction process. While, the Ag shell thickness along lateral direction significantly increased from ~7.3 nm to ~14.1 nm. Moreover, HRTEM image in Fig. 2(h) also illustrates the preferential growth of Ag (200) orientation at thicker shell region since the marked d-spacings are calculated about 0.205 nm along longitudinal cross region. The XRD patterns of Au NRs and Au@Ag NRs with 7.3 nm Ag shell are shown in Fig. 3. As for Au NRs, the primary peak of (111) plane dominant the XRD pattern in Fig. 3. Meanwhile, in addition of original primary Au (111) plane, the XRD pattern of Au@Ag NRs also reveals that the relative higher diffraction intensity of (200) were observed at 44.3°(marked with yellow color), confirming the preferential growth of Ag (200) at shell region. Additionally, the detailed evolutions of Ag shell thickness are summarized in Table 1. In this way, it can be deduced that the Au@Ag NRs with controllable Ag shell thickness can be obtained by simply turning the amounts of AgNO3 and AA solution.
Table 1. Amounts of AgNO3 and AA solution in Au@Ag core-shell NRs synthesis with varying Ag shell thickness.
The well-defined Au@Ag NRs with precise controllable Ag shell structures will provide tunable localized surface plasmon resonances (LSPR) on the surfaces, which can be monitored by using UV-visible absorption spectra. Therefore, seven groups of solutions including Au NRs and Au@Ag NRs with different Ag shell thicknesses were prepared in this work. The corresponding direct photographs and geometrical models of the seven products are show in Fig. 4(a). The increase of Ag shell thickness leads to a significant change in the solution color from pink for Au NRs to green for 7.3 nm Ag shell and then to orange for 14.1 nm Ag shell condition. The UV-visible absorption spectra clearly describe the variations of plasmon bands (A-D, from low to high energies) as the thickness of Ag shell increases from 0nm to 14.1 nm, as illustrated in Fig. 4(b). Increasing Ag shell thickness gradually enhances the intensities of four prominent LSPR bands. Meanwhile, the evolutions of these plasmon band positions are also illustrated in Fig. 4(c). With increasing Ag shell thickness, the peak A originated from the longitudinal dipolar plasmon mode [27,28] significantly blue-shift from 841 to 546 nm [Fig. 4(c)], owing to both the reduction aspect ratio of NRs and a contribution of optical behavior of Ag different from Au. The peak B should be attributed to the positions of two over-lapped resonance modes including both the lowest transverse dipolar mode and the second-lowest longitudinal quadrupole mode [27–30]. It starts blue shifting (from 520 to 442 nm) until the Ag shell thickness reached 7.3 nm, and then a slightly red-shift (from 442 to 456 nm) as Ag thickness further increased to 14.1 nm [Fig. 4(c)]. The first blue shifting is due to the dominated metal of surface changed from gold to silver [27], and the subsequent red-shifting can be explained by the nanorods developed into nanocuboids via increasing edge sharpness, leading to an increase in volume. In addition, two highest-energy peaks, located at peak C and D in Fig. 4(b), should be ascribed to octupole plasmon modes [27], which became evident when the Ag shell gets thicker up to ~4.1 nm [Fig. 4(c)]. Moreover, peak C slight red-shift from 392 to 403 nm and analogously for peak D (from 339 to 343 nm), because of the formation of a cuboid mentioned above. Herein, the characteristic LSPR peaks of obtained Au@Ag NRs were analyzed in detail way.
Fig. 4 (a) Direct photographs of Au NRs and Au@Ag NRs solutions with different Ag shell thicknesses (on the left) and geometrical models of the corresponding shapes (on the right). (b) The UV-visible absorption spectra of the corresponding Au NRs and Au@Ag NRs solutions, (c) Evolutions of the peak positions (A, B, C and D plasmon bands) versus the Ag shell thickness.
It is well known that the controllable bimetallic Au@Ag nanocomposites with appropriate structures have a promising potential for significantly enhancing SERS activity. Therefore, in order to obtain maximized SERS activity, the SERS performances of Au@Ag NRs with different Ag shell thicknesses were illustrated by using crystal violet (CV) as probe molecules in this paper. The inset in Fig. 5(a) shows CV structural diagram, which is composed of complex aromatic molecular structure. Figure 5(a) shows the SERS spectra of 10−6 M CV molecules adsorbed on five groups of Au@Ag NRs with Ag shell thickness of 0, 2.1, 7.3, 11.9 and 14.1 nm, respectively. As shown in Fig. 5(a), the dominating characteristic bands of CV molecules at 738, 769, 807, 916, 948, 985, 1178, 1304, 1379, 1445, 1592 and 1621 cm−1 are all clearly detected in SERS spectra, providing much enriched “molecular fingerprint” information. In detail, the prominent characteristic bands of CV molecules exhibit well match with previous reports [14,15,31,32], which are identified as follows: peaks located at 1621, 1592, 1542, 1445 and 1304 cm−1can be attributed to ring C-C stretching vibration; another peak at 1379 cm−1 is related to N-phenyl stretching; the peaks at 985, 948 and 916 cm−1 should be originated from the ring skeletal vibration of radical orientations; the final peaks at 1178, 807, 769 and 738 cm−1 are corresponded to ring C-H bends. In addition to the specific vibrational information, moreover, the evolutions of Raman signal intensities reveal that the Au@Ag NRs with 7.3 nm Ag shell thickness provide maximized SERS activity in comparison with other four samples. As shown in Fig. 5(a), the corresponding Raman signal intensities are much higher than that originated from other four SERS substrates. The detailed information obtained from the variations of SERS intensities at 807, 916, 1178, 1379 and 1621 cm−1 versus different Ag shell thicknesses decorated Au@Ag NRs were separately shown in Fig. 5(b). The curves of the five signal intensities show very similar behavior, such as the obvious rising trend for Ag shell thickness in the region of 0~7.3 nm and the declining trend for further increasing thickness to 14.1 nm. In a typical curve, the Raman signal intensity at 1621 cm−1 significantly increased from ~1132.6 a.u for monometallic Au NRs to ~28251.2 a.u for Au@Ag NRs with 7.3 nm Ag shell, and then dropped to ~8381.4 a.u for Au@Ag NRs with thicker Ag shell of 14.1 nm. It is clearly seen that the SERS intensity originated from the optimal Au@Ag NRs is about 25 times and 3.3 times higher than that of monometallic Au NRs and 14.1 nm Ag shell decorated Au NRs, respectively.
Fig. 5 (a) SERS spectra of Au@Ag NRs with different thickness of Ag shell (0, 2.1 nm, 7.3 nm, 11.9 nm, 14.1 nm) in 10−6 M CV. The inset is the CV molecular structure. (b) Variations of diverse Raman signal peaks with Ag shell thickness.
The maximized SERS activity of Au@Ag NRs with appropriate Ag shell thickness should be highly related to the unique pronounced electromagnetic (EM) enhancement, which will be illustrated as follows. As for Au or Ag nanostructures, it has been well known that the EM enhancement originated from the interaction of a laser beam with plasmonic Au or Ag nanomaterials plays a dominating role for improving SERS activity in comparison with the charge-transfer (CT) mechanism between nanosubstrate and probe molecules [6–9,13–15]. As illustrated in Fig. 4(b), the increasing Ag shell thickness from 0 to 7.3 nm enables the LSPR of Au@Ag NRs to be blue shifted from 841 nm to ~627 nm, and then further shifted to 546 nm for 14.1 nm Ag shell condition. Compared with other NRs in this paper, the 633 nm laser beam used in SERS measurement is more commensurate with the LSPR position of Au@Ag NRs with 7.3 nm Ag shell. In our previous work, we have demonstrated that the matching degree between the plasmon resonance and the incident laser wavelength played an important role in the variation of SERS activity [15], which is also coincident with this case. Therefore, the 633 nm laser excitation of Au@Ag NRs with 7.3 nm Ag shell will facilitate the creation of much more localized free electrons, forming an enhanced EM field around the nanoparticle. Additionally, another contribution should be also considered for further increasing EM enhancement in this work. Superior to monometallic Au nanomaterials, the bimetallic Au@Ag with appropriate Ag composition is believed to synergistically enhance the intrinsic EM field of each component, giving rise to the formation of much more SERS hotspots for SERS detection [13,14,33–37]. In this paper, the intermetallic synergy will be enhanced as the coating of Ag shell increases in the initial overgrowth of Ag species on Au NRs surfaces. As illustrated in previous works [30,38], the thickness of the out layer ranging from 5~10 nm is most interesting intermediate region, since the internal and external energy modes are closer to each other, providing appreciable greater coupling effect between Au and Ag core-shell structure. In this way, the Au@Ag NR with 7.3 nm Ag shell thickness has a promising potential for appreciably increasing EM field of bimetallic structures. Subsequently, further increasing the Ag shell thickness (>10 nm), the intermetallic synergy can be significantly shielded by the thick outer layer when the laser light cannot reach at the inner Au region. The LSPR of Ag shell grows as strong as that of single Ag structure with laser energy dissipation in Au core [30]. In this way, the excited EM field will obviously decrease owing to the weaker interaction between thicker Ag shell and Au core. In order to verify these mechanism of EM enhancement in this paper, we further carried out the finite-difference time-domain (FDTD) simulations using comsol software to investigate the relative electrical field intensities (|E|/|E0|, the ratio of electrical field |E| to incident field |E0|) at the surfaces of individual Au NRs, Au@Ag NRs with 7.3 nm Ag shell, and Au@Ag NRs with 14.1 nm Ag shell, respectively. The incidence wavelength in FDTD simulations was selected at 633 nm, according to the laser wavelength used in SERS experiments. For comparison, the relative intensity is recorded on the same color scale. The |E|/|E0| distributions of longitudinal (parallel to the nanorod length axis) and transverse (perpendicular to the length axis) plamon excitations of three samples are separately displayed in Fig. 6(a)-6(b), 6(c)-6(d) and 6(e)-6(f), respectively. As the FDTD simulations presented in Fig. 6, the |E|/|E0| distributions clearly reveal that the 7.3 nm Ag shell decorated Au NRs provide significantly enhanced intense electric fields at both longitudinal and transverse axes, which are about 5 and 1.8 times higher than that of monometallic Au NRs and Au@Ag NRs with thicker Ag shell, respectively. The obvious variation of electric field intensities of three samples has a direct influence on the SERS activity. Therefore, the FDTD simulations clearly suggest that the optimal Au@Ag NRs with appropriate 7.3 nm Ag shell thickness can provide maximized EM enhancement and then greatly enhanced SERS activity in this paper.
Fig. 6 FDTD calculations of relative electric field intensities for longitudinal and transverse plasmon excitation of individual Au NRs (a-b), Au@Ag NRs with 7.3 nm Ag shell (c-d), and Au@Ag NRs with 14.1 nm Ag shell (e-f), respectively.
Finally, the optimal Au@Ag NRs with 7.3 nm Ag shells were served as an advanced SERS substrate for ultralow spectral analysis of antibiotic levofloxacin molecules. The inset in Fig. 7(a) shows the detailed molecular structure of levofloxacin. The corresponding SERS analyses of levofloxacin molecules with different concentrations (10−4~10−9 M) are shown in Fig. 7. The dominating characteristic bands of levofloxacin in the range of 600~1800 cm−1 are clearly identified in SERS spectra, which are consistent with previous works [16–19]. In detail, the main characteristic peak located at 1337 cm−1 should be originated bending vibrations of (CH + CH2 + CH3). Another prominent band at 1396 cm−1 could be related to the quinolone stretching vibration of the ring. In the region of 1600~1700 cm−1, the SERS peak at 1614 cm−1 can be attributed to the C = C asymmetrical stretching vibration of the aromatic rings. Moreover, based on the optimal Au@Ag NRs-based SERS nanosubstrate, it should be noted that the three characteristic bands of levofloxacin molecules are also clearly distinguishable even the concentration decreased to as low as 10−9 M, which is much better than that (<10−7 M) in previous works [16–19]. More importantly, the SERS detection limit of antibiotic levofloxacin molecules can reach at nM level of 0.37 ng/L (10−9 M), which is comparable with the ultralow results by using high-sensitive developed techniques such as HPLC, HPLC-MS, HF-LPME, and UA-LDS-DLLME, etc [20–22]. Superior to these developed techniques, the distinctive advantage of ultralow SERS analysis in this work is to overcome the shortcomings of these time-consuming and expensive methods including complicated pretreatment, long assay duration and high-cost samples, etc. On the other hand, if the SERS spectral lines can correctly and precise proportional to the levofloxacin concentrations, the corresponding linear relationships will provide more important internal standard for exact quantitatively and sensitively monitoring the slight changes of antibiotic residuals in animal husbandry. So, the quantitative relationships between the intensities of SERS peaks at 1337, 1396, 1614 cm−1 and logarithm concentrations of levofloxacin molecules (10−4-10−9 M) were separately depicted in Fig. 7(b). It can be found that three well-defined linear relationships (R2 = 0.987 at 1337 cm−1, R2 = 0.9891 at 1396 cm−1 and R2 = 0.9899 at 1614 cm−1) were obtained by plotting the different SERS peak intensities versus the logarithm concentrations of levofloxacin solutions. The established linear relationships will provide unprecedented opportunity for precise assessment of antibiotic residuals in food-animal products and environmental antibiotics-based contaminations.
Fig. 7 (a) SERS spectra of levofloxacin solutions with different concentrations (10−4-10−9 M) absorbed on the optimal Au@Ag NRs with7.3 nm Ag shell. The inset is the levofloxacin molecular structure. (b) The plot of intensities of the SERS peaks at 1337, 1396, 1614 cm−1 versus the concentration.
4. Conclusions
In summary, well-defined Au@Ag core-shell nanorods with precise controlled Ag shell thickness (2.1~14.1 nm) were fabricated and optimized for ultrasensitive SERS analyses of antibiotic levofloxacin molecules. Ag shell thickness-dependent SERS measurements of CV molecules revealed that the Au@Ag NRs with 7.3 nm Ag shell provide maximized SERS activity in comparison with original Au NRs and other Au@Ag NRs. The unique pronounced EM enhancement mechanisms behind these phenomena have been discussed in detail. The incident laser light (633 nm) overlapped better with the LSPR (627 nm) of Au@Ag NRs with 7.3 nm Ag shell, enabling the creation of much more localized free electrons to form an enhanced EM field. Moreover, the excited EM field will be also further increased in Au@Ag core-shell NRs with appropriate Ag shell thickness, owing to the stronger intermetallic synergy between optimal Ag shell and Au core. More importantly, based on the optimized SERS substrate, the detection limit of antibiotic levofloxacin molecules was realized at nM level of 0.37 ng/L (10−9 M) in this work, approaching the requirement for ultralow antibiotics analysis. Finally, the well-defined linear response were established between SERS signal intensities and logarithmical scale of levofloxacin concentration in a wide range of 10−4~10−9 M, which will provide a promising potential for ultrasensitive analysis of antibiotic residuals ecological environment.
Funding
National Natural Science Foundation of China (NSFC) (11575102, 11105085, 11775134). The Fundamental Research Funds of Shandong University (2018JC022).
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