Preparation of Quasi-Three-Dimensional Porous Ag and Ag-NiO Nanofibrous Mats for SERS Application

In this study, two new quasi-three-dimensional Surface Enhanced Raman Scattering (SERS) substrates, namely porous Ag and Ag-NiO nanofibrous mats, were prepared using a simple, electrospinning-calcination, two-step synthetic process. AgNO3/polyvinyl pyrrolidone (PVP) and AgNO3/Ni(NO3)2/PVP composites serving as precursors were electrospun to form corresponding precursory nanofibers. Porous Ag and Ag-NiO nanofibers were successfully obtained after a 3-h calcination at 500 °C under air atmosphere, and analyzed using various material characterization techniques. Synthesized, quasi-three-dimensional porous Ag and Ag-NiO nanofibrous mats were applied as SERS substrates, to measure the model compound Rhodamine 6G (R6G), and investigate the corresponding signal enhancement. Furthermore, porous Ag and Ag-NiO nanofibrous mats were employed as SERS substrates for melamine and methyl parathion respectively. Sensitive detection of melamine and methyl parathion was achieved, indicating their feasibility as an active SERS sensing platform, and potential for food safety and environmental monitoring. All the results suggest that the electrospinning-calcination, two-step method offers a new, low cost, high performance solution in the preparation of SERS substrates.


Introduction
Raman spectroscopy is an accurate and attractive molecule identification and monitoring method. It probes the chemical contents through molecular vibration, providing a unique, specific chemical or vibration "finger-print" for molecules [1][2][3][4]. However, its relatively low sensitivity poses challenges in trace chemical detection. Therefore, since its discovery in the 1970s surface enhanced Raman scattering (SERS) has become a hot research topic due to its enhanced signal amplification [1,[5][6][7][8]. SERS is a phenomenon originating from a giant enhancement of the electromagnetic field surrounding noble nanostructured materials (such as Ag or Au). Generally, two main mechanisms, namely electromagnetic enhancement and chemical enhancement, are used as reasonable explanations for the observed enhanced Raman signals. On the one hand, excitation of localized, surface plasmon resonance electrospinning-calcination, two-step method offers a new, low cost, high performance route in the preparation of SERS substrates.

Instruments and Apparatus
Scanning electron microscopic (SEM) images were recorded using FEI Tecnai G2 Spirit BioTWIN and FEI Nova NanoSEM 450. X-ray Diffraction (XRD) pattern was recorded by a Rigaku Ultima IV diffractometer. A portable Raman spectrometer (QE Pro, Ocean Optics) was used to collect the Raman spectra coupled with a 785 nm, 499 mW laser. For each measurement, the Raman spectrum was obtained with an integration time of 5 s.

Preparation of Quasi-3D Porous Ag and Ag-NiO Nanofibers Mats
Quasi-3D porous Ag and Ag-NiO nanofibrous mats were synthesized following the procedure in our previous report, with a minor revision [35]. In a typical process for obtaining Ag-NiO nanofibrous mats, 0.2 g AgNO3, 0.2 g Ni(NO3)2·6H2O and 0.8 g PVP were dissolved in 4 mL dimethyl formamide (DMF). The mixture was then stirred for 4 h to form an homogenous solution. Figure 2 shows the electrospinning of nanofibers. The AgNO3/Ni(NO3)2/PVP nanofibers were prepared using the electrospinning setup, in which a 23-gauge needle and flow rate of 0.3 mL/h was employed, with an applied voltage of 20 kV. The collection distance between the needle tip and aluminum foil (serving as nanofibers collector) was 15 cm. In order to acquire the Ag-NiO nanofiber mats, as-synthesized AgNO3/Ni(NO3)2/PVP nanofibers were then thermal-treated at 500 °C for 3 h under air atmosphere. Preparation of porous Ag followed a similar procedure except for the absence of nickel salt (Ni(NO3)2·6H2O).

Instruments and Apparatus
Scanning electron microscopic (SEM) images were recorded using FEI Tecnai G2 Spirit BioTWIN and FEI Nova NanoSEM 450. X-ray Diffraction (XRD) pattern was recorded by a Rigaku Ultima IV diffractometer. A portable Raman spectrometer (QE Pro, Ocean Optics) was used to collect the Raman spectra coupled with a 785 nm, 499 mW laser. For each measurement, the Raman spectrum was obtained with an integration time of 5 s.

Preparation of Quasi-3D Porous Ag and Ag-NiO Nanofibers Mats
Quasi-3D porous Ag and Ag-NiO nanofibrous mats were synthesized following the procedure in our previous report, with a minor revision [35]. In a typical process for obtaining Ag-NiO nanofibrous mats, 0.2 g AgNO 3 , 0.2 g Ni(NO 3 ) 2 ·6H 2 O and 0.8 g PVP were dissolved in 4 mL dimethyl formamide (DMF). The mixture was then stirred for 4 h to form an homogenous solution. Figure 2 shows the electrospinning of nanofibers. The AgNO 3 /Ni(NO 3 ) 2 /PVP nanofibers were prepared using the electrospinning setup, in which a 23-gauge needle and flow rate of 0.3 mL/h was employed, with an applied voltage of 20 kV. The collection distance between the needle tip and aluminum foil (serving as nanofibers collector) was 15 cm. In order to acquire the Ag-NiO nanofiber mats, as-synthesized AgNO 3 /Ni(NO 3 ) 2 /PVP nanofibers were then thermal-treated at 500 • C for 3 h under air atmosphere. Preparation of porous Ag followed a similar procedure except for the absence of nickel salt (Ni(NO 3 ) 2 ·6H 2 O).

Sample Preparation and Measurement Procedure
A methyl parathion ethanolic solution (0.01 M), R6G (0.01 M) and melamine (0.01 M) aqueous solution were prepared and used as stock solutions. Methyl parathion, R6G and melamine solutions with various concentrations were obtained by diluting corresponding stock solutions. Target molecule solutions (2 μL) with certain concentrations were directly dropped onto the surface of porous Ag. After drying of the target solutions the SERS spectra were recorded. To fabricate Ag-NiO nanofiber mats-based substrate, 0.2 mg of Ag-NiO nanofibers was dispersed into 2 mL water and treated with an ultrasonic bath for 30 s. Then, 5 μL of suspension was dropped onto a silicon wafer and left to dry. Two microliters of target solution with various concentrations were dropped onto the Ag-NiO nanofibers SERS substrate, and after being dried on the surface the SERS spectra was recorded.

Nanofber Characterization
To study the morphology of the electrospun nanofibers before and after calcination, SEM characterization was first conducted. Figure 3A shows the typical morphology of the electrospun AgNO3/PVP nanofibers. The AgNO3/PVP nanofibers possess smooth surface and good uniformity. The inset of Figure 3A indicates that the average diameter of AgNO3/PVP nanofibers was about 200 nm. Figure 3B indicates that similar morphology was obtained for the as-prepared AgNO3/Ni(NO3)2/PVP nanofibers with a smaller average diameter (ca. 150 nm). However, there are some very tiny nanowires intertwined with large nanofibers. These observations can mainly be attributed to the difference in electrical conductivity and viscosity between AgNO3/PVP and AgNO3/Ni(NO3)2/PVP precursors. These results indicate the successful synthesis of AgNO3/PVP nanofibers and AgNO3/Ni(NO3)2/PVP nanofibers using electrospinning method.
After 3 h of thermal treatment of the precursory nanofibers at 500 °C in air, the polymer completely decomposed and disappeared. The AgNO3 and Ni(NO3)2 were degraded to yield metal Ag and NiO, respectively, following the reactions below [35,36]:

Sample Preparation and Measurement Procedure
A methyl parathion ethanolic solution (0.01 M), R6G (0.01 M) and melamine (0.01 M) aqueous solution were prepared and used as stock solutions. Methyl parathion, R6G and melamine solutions with various concentrations were obtained by diluting corresponding stock solutions. Target molecule solutions (2 µL) with certain concentrations were directly dropped onto the surface of porous Ag. After drying of the target solutions the SERS spectra were recorded. To fabricate Ag-NiO nanofiber mats-based substrate, 0.2 mg of Ag-NiO nanofibers was dispersed into 2 mL water and treated with an ultrasonic bath for 30 s. Then, 5 µL of suspension was dropped onto a silicon wafer and left to dry. Two microliters of target solution with various concentrations were dropped onto the Ag-NiO nanofibers SERS substrate, and after being dried on the surface the SERS spectra was recorded.

Nanofber Characterization
To study the morphology of the electrospun nanofibers before and after calcination, SEM characterization was first conducted. Figure 3A shows the typical morphology of the electrospun AgNO 3 /PVP nanofibers. The AgNO 3 /PVP nanofibers possess smooth surface and good uniformity. The inset of Figure 3A indicates that the average diameter of AgNO 3 /PVP nanofibers was about 200 nm. Figure 3B indicates that similar morphology was obtained for the as-prepared AgNO 3 /Ni(NO 3 ) 2 /PVP nanofibers with a smaller average diameter (ca. 150 nm). However, there are some very tiny nanowires intertwined with large nanofibers. These observations can mainly be attributed to the difference in electrical conductivity and viscosity between AgNO 3 /PVP and AgNO 3 /Ni(NO 3 ) 2 /PVP precursors. These results indicate the successful synthesis of AgNO 3 /PVP nanofibers and AgNO 3 /Ni(NO 3 ) 2 /PVP nanofibers using electrospinning method.
After 3 h of thermal treatment of the precursory nanofibers at 500 • C in air, the polymer completely decomposed and disappeared. The AgNO 3 and Ni(NO 3 ) 2 were degraded to yield metal Ag and NiO, respectively, following the reactions below [35,36]:  Quasi-3D porous Ag mat was obtained after calcination of AgNO3/PVP nanofibers. However, due to the high temperature applied during calcination, some Ag merged together to form 3D porous structure with a rough rather than a fibrous surface, shown in Figure 4A. By contrast, NiO still maintained the nanofiber structure at 500 °C, leading to well-defined Ag-NiO nanofibers ( Figure 4B). It can be observed that as-synthesized Ag-NiO nanofibers displayed a rough surface, which was attributed to the decomposition of PVP, metal (Ag) crystallization and metal oxide (NiO) formation. High magnification SEM image further confirmed the formation of Ag-NiO nanofibers with rough surfaces (inset of Figure 4B). Quasi-3D porous Ag porous network and Ag-NiO nanofiber mats with rough surface structures potentially offer a large surface area and a number of hot spots, which render them active and efficient SERS substrates for sensing applications. To study the chemical composition and crystallinity of porous Ag network and Ag-NiO nanofibers, XRD study was conducted. Figure 5A shows the XRD spectrum collected from 30° to 90° of porous Ag mat. Five sharp and strong diffraction peaks at 2θ of 38.06°, 44.26°, 64.44°, 77.28° and 81.48° were observed, corresponding to (111), (200), (220), (311) and (222) crystal planes of Ag, respectively. This result indicates the formation of cubic crystalline Ag [35]. The XRD pattern of Ag-NiO composite is shown in Figure 5B  Quasi-3D porous Ag mat was obtained after calcination of AgNO 3 /PVP nanofibers. However, due to the high temperature applied during calcination, some Ag merged together to form 3D porous structure with a rough rather than a fibrous surface, shown in Figure 4A. By contrast, NiO still maintained the nanofiber structure at 500 • C, leading to well-defined Ag-NiO nanofibers ( Figure 4B). It can be observed that as-synthesized Ag-NiO nanofibers displayed a rough surface, which was attributed to the decomposition of PVP, metal (Ag) crystallization and metal oxide (NiO) formation. High magnification SEM image further confirmed the formation of Ag-NiO nanofibers with rough surfaces (inset of Figure 4B). Quasi-3D porous Ag porous network and Ag-NiO nanofiber mats with rough surface structures potentially offer a large surface area and a number of hot spots, which render them active and efficient SERS substrates for sensing applications.  Quasi-3D porous Ag mat was obtained after calcination of AgNO3/PVP nanofibers. However, due to the high temperature applied during calcination, some Ag merged together to form 3D porous structure with a rough rather than a fibrous surface, shown in Figure 4A. By contrast, NiO still maintained the nanofiber structure at 500 °C, leading to well-defined Ag-NiO nanofibers ( Figure 4B). It can be observed that as-synthesized Ag-NiO nanofibers displayed a rough surface, which was attributed to the decomposition of PVP, metal (Ag) crystallization and metal oxide (NiO) formation. High magnification SEM image further confirmed the formation of Ag-NiO nanofibers with rough surfaces (inset of Figure 4B). Quasi-3D porous Ag porous network and Ag-NiO nanofiber mats with rough surface structures potentially offer a large surface area and a number of hot spots, which render them active and efficient SERS substrates for sensing applications. To study the chemical composition and crystallinity of porous Ag network and Ag-NiO nanofibers, XRD study was conducted. Figure 5A shows the XRD spectrum collected from 30° to 90° of porous Ag mat. Five sharp and strong diffraction peaks at 2θ of 38.06°, 44.26°, 64.44°, 77.28° and 81.48° were observed, corresponding to (111), (200), (220), (311) and (222) crystal planes of Ag, respectively. This result indicates the formation of cubic crystalline Ag [35]. The XRD pattern of Ag-NiO composite is shown in Figure 5B  To study the chemical composition and crystallinity of porous Ag network and Ag-NiO nanofibers, XRD study was conducted. Figure 5A

SERS Performance of Porous Ag and Ag-NiO Nanofibers
To study the SERS activity of as-synthesized porous Ag and Ag-NiO nanofibrous mats, R6G was used as a model Raman dye. Figure 6 shows the Raman spectrum of R6G (5 times) with concentration of 1 × 10 −3 M, SERS spectra of R6G with concentration of 5 × 10 −8 M and 3 × 10 −7 M recorded on the porous Ag and Ag-NiO nanofibers mats, respectively (Raman shift in the range from 670 cm −1 to 1670 cm −1 was collected). There was no obvious Raman scattering for both porous Ag and Ag-NiO nanofibers without casting R6G, indicating negligible background interferences from as-fabricated SERS substrates. Raman spectrum of high concentration R6G shows three relatively weak peaks at 1311 cm −1 , 1361 cm −1 and 1509 cm −1 . By contrast, SERS spectrum of R6G with much lower concentration was collected on the porous Ag. Besides three aforementioned peaks, four other distinct peaks appeared at 769 cm −1 , 1123 cm −1 , 1194 cm −1 and 1647 cm −1 , accompanied with a significantly enhanced Raman signal. All these molecule vibration assignments were listed in Table 1 [37][38][39][40]. Similar Raman signal enhancement on Ag-NiO nanofibrous mats was observed, except for the degree of enhancement. The enhancement factor (EF) of porous Ag and Ag-NiO nanofibrous mat was determined using the following expression [41,42]: where and are the integrated SERS and normal Raman scattering (NRS) intensities of R6G at the same Raman band, respectively. and are the concentrations of probed molecules in the SERS and NRS measurements, respectively. In this study, Raman intensities with baseline correction of R6G at 1509 cm −1 were extracted to serve as and . Values of , , and for both of porous Ag and Ag-NiO nanofibers were summarized in Table 2. The as-prepared porous Ag and Ag-NiO nanofibers show EF of 1.59 × 10 5 and 2.89 × 10 4 for R6G. A relative lower EF obtained for Ag-NiO nanofibers can be attributed to the distribution of NiO phase on the surface of Ag-NiO nanofibers [35].

SERS Performance of Porous Ag and Ag-NiO Nanofibers
To study the SERS activity of as-synthesized porous Ag and Ag-NiO nanofibrous mats, R6G was used as a model Raman dye. Figure 6 shows the Raman spectrum of R6G (5 times) with concentration of 1 × 10 −3 M, SERS spectra of R6G with concentration of 5 × 10 −8 M and 3 × 10 −7 M recorded on the porous Ag and Ag-NiO nanofibers mats, respectively (Raman shift in the range from 670 cm −1 to 1670 cm −1 was collected). There was no obvious Raman scattering for both porous Ag and Ag-NiO nanofibers without casting R6G, indicating negligible background interferences from as-fabricated SERS substrates. Raman spectrum of high concentration R6G shows three relatively weak peaks at 1311 cm −1 , 1361 cm −1 and 1509 cm −1 . By contrast, SERS spectrum of R6G with much lower concentration was collected on the porous Ag. Besides three aforementioned peaks, four other distinct peaks appeared at 769 cm −1 , 1123 cm −1 , 1194 cm −1 and 1647 cm −1 , accompanied with a significantly enhanced Raman signal. All these molecule vibration assignments were listed in Table 1 [37][38][39][40]. Similar Raman signal enhancement on Ag-NiO nanofibrous mats was observed, except for the degree of enhancement. The enhancement factor (EF) of porous Ag and Ag-NiO nanofibrous mat was determined using the following expression [41,42]: where I SERS and I NRS are the integrated SERS and normal Raman scattering (NRS) intensities of R6G at the same Raman band, respectively. C SERS and C NRS are the concentrations of probed molecules in the SERS and NRS measurements, respectively. In this study, Raman intensities with baseline correction of R6G at 1509 cm −1 were extracted to serve as I SERS and I NRS . Values of I SERS , I NRS , C SERS and C NRS for both of porous Ag and Ag-NiO nanofibers were summarized in Table 2. The as-prepared porous Ag and Ag-NiO nanofibers show EF of 1.59 × 10 5 and 2.89 × 10 4 for R6G. A relative lower EF obtained for Ag-NiO nanofibers can be attributed to the distribution of NiO phase on the surface of Ag-NiO nanofibers [35].

SERS Detection for Melamine and Methyl Parathion
To further demonstrate the applicability of porous Ag and Ag-NiO nanofibers mat in SERS sensing, porous Ag was employed for melamine detection, while Ag-NiO nanofibrous mat was used for methyl parathion monitoring. Figure 7 shows the corresponding Raman spectra results. It is well-noted that no obvious peak could be observed for both porous Ag and Ag-NiO nanofibrous mat. Figure 7A shows SERS spectra of melamine at various concentrations (0 to 5 × 10 −4 M) recorded on porous Ag. One can see that after loading 2.5 × 10 −6 M of melamine on the SERS substrate, a prominent peak at 684 cm −1 was observed, corresponding to the characteristic peak of melamine (ring breathing) [43]. The Raman intensities at 684 cm −1 gradually increased with the increasing of melamine concentration. The results demonstrated that the porous Ag network displayed good sensitivity (down to micromolar level) towards melamine detection. SERS spectra of methyl

SERS Detection for Melamine and Methyl Parathion
To further demonstrate the applicability of porous Ag and Ag-NiO nanofibers mat in SERS sensing, porous Ag was employed for melamine detection, while Ag-NiO nanofibrous mat was used for methyl parathion monitoring. Figure 7 shows the corresponding Raman spectra results. It is well-noted that no obvious peak could be observed for both porous Ag and Ag-NiO nanofibrous mat. Figure 7A shows SERS spectra of melamine at various concentrations (0 to 5 × 10 −4 M) recorded on porous Ag. One can see that after loading 2.5 × 10 −6 M of melamine on the SERS substrate, a prominent peak at 684 cm −1 was observed, corresponding to the characteristic peak of melamine (ring breathing) [43]. The Raman intensities at 684 cm −1 gradually increased with the increasing of melamine concentration. The results demonstrated that the porous Ag network displayed good sensitivity (down to micromolar level) towards melamine detection. SERS spectra of methyl parathion at various concentrations on Ag-NiO nanofibrous mat were collected and are shown in Figure 7B. One peak at 1344 cm −1 (bending vibration of C-H) [44] appeared upon addition of 1 × 10 −5 M of methyl parathion. Raman intensities increased significantly with the increase of methyl parathion concentrations. At higher concentrations, a new peak at 1111 cm −1 was also observed, corresponding to the stretching vibration of C-N [44]. An acceptable sensitivity of Ag-NiO nanofiber-based SERS substrate was also acquired. These results suggest that as-prepared porous Ag and Ag-NiO nanofibrous mat as SERS substrates display good sensitivities towards target molecules, indicating that the electrospinning-calcination, two-step method offers a new, high performance route in the fabrication of SERS substrates. parathion at various concentrations on Ag-NiO nanofibrous mat were collected and are shown in Figure 7B. One peak at 1344 cm −1 (bending vibration of C-H) [44] appeared upon addition of 1 × 10 −5 M of methyl parathion. Raman intensities increased significantly with the increase of methyl parathion concentrations. At higher concentrations, a new peak at 1111 cm −1 was also observed, corresponding to the stretching vibration of C-N [44]. An acceptable sensitivity of Ag-NiO nanofiber-based SERS substrate was also acquired. These results suggest that as-prepared porous Ag and Ag-NiO nanofibrous mat as SERS substrates display good sensitivities towards target molecules, indicating that the electrospinning-calcination, two-step method offers a new, high performance route in the fabrication of SERS substrates.

Conclusions
In conclusion, we fabricated two new SERS substrates, porous Ag and Ag-NiO nanofiber, by using a simple, electrospinning-calcination two-step method with AgNO3/PVP and AgNO3/Ni(NO3)2/PVP as precursors, respectively. Formation of porous Ag was attributed to partial melting of silver at 500 °C. By contrast, the introduction of Ni(NO3)2 maintained the nanofibrous structure due to the formation and presence of NiO. The good SERS performances of as-synthesized quasi-three-dimensional porous Ag and Ag-NiO nanofibrous mat were first demonstrated using R6G as a model compound. The feasibility of a porous Ag and Ag-NiO nanofiber-based SERS sensing platform was further demonstrated for monitoring melamine and methyl parathion, respectively, indicating their potential application in food safety and environmental monitoring. These results demonstrate that the electrospinning-calcination two-step method offers a new strategy in the preparation of highperformance SERS substrates.

Conclusions
In conclusion, we fabricated two new SERS substrates, porous Ag and Ag-NiO nanofiber, by using a simple, electrospinning-calcination two-step method with AgNO 3 /PVP and AgNO 3 /Ni(NO 3 ) 2 /PVP as precursors, respectively. Formation of porous Ag was attributed to partial melting of silver at 500 • C. By contrast, the introduction of Ni(NO 3 ) 2 maintained the nanofibrous structure due to the formation and presence of NiO. The good SERS performances of as-synthesized quasi-three-dimensional porous Ag and Ag-NiO nanofibrous mat were first demonstrated using R6G as a model compound. The feasibility of a porous Ag and Ag-NiO nanofiber-based SERS sensing platform was further demonstrated for monitoring melamine and methyl parathion, respectively, indicating their potential application in food safety and environmental monitoring. These results demonstrate that the electrospinning-calcination two-step method offers a new strategy in the preparation of highperformance SERS substrates.