Introduction

Surface-enhanced Raman scattering (SERS) has inspired researchers’ great interests because of its highly sensitive, efficient, and nondestructive characters for detection of traces amount of target molecules. This enables promising applications in the fields of surface science, spectroscopy, chemical analysis, and biochemical detection.1,2,3,4,5 Generally, the noble metals of Au and Ag, because of their unique and remarkable surface plasmon resonance effect, are widely used as SERS sensors with high sensitivity.6,7,8,9 However, the SERS technique as a new analytical methodology has not vanished as far as initially predicted.10 It is one of the most profound difficulties that only several limited noble metals possess a significant Raman enhancement capability, but can hardly satisfy the applications involving other non-metal materials. In addition, the noble metal nanostructures are difficult and costly to fabricate with high uniformity.11 They are also poor in stability and reproducibility because of their readily oxidizability and agglomeration.12 These limitations for noble metal SERS substrates seriously hinder the exploitation of SERS technique in practical applications. Accordingly, it is of great significance to explore alternative non-metal SERS substrates for a better applicability.

Recent achievements in the semiconductor SERS-active substrates have endowed semiconductors as potential candidates for new generation of SERS sensors.10, 12,13,14,15 Compared with the noble metal SERS substrates, many semiconductor substrates are more stable, reproducible, and biocompatible. Meanwhile, the mature synthesis method also enables to manufacture semiconductors with better uniformity at lower cost, which makes them an immense prospect in the fundamental studies as well as the practical applications. Liu et al. detected the Raman signals of 4-mercaptobenzoic acid at a small concentration of 10–6 M on porous ZnO.15 Qi et al. applied TiO2 inverse opals SERS substrates for sensitive detection of methylene blue (MeB) molecules.16 Islam et al. observed the SERS spectra of 4-mercaptopyridine (4-MPy) from a chemically etched ZnSe surface with the greatest enhancement factor (EF) value for semiconductor substrate up to ~2 × 106.17 Other semiconductors, such as V2O5,18 CuO,19 AgFeO2,20 ZnS,21 CdS,22 CuTe,23 CdTe,22 GaP,24 Si and Ge,10 were also applied as SERS substrates with EF values in the range of 103~106. However, although semiconductors deliver significant advantages in SERS examination, the relatively worse enhancement effect originated from the chemical enhancement (CM) is the most prominent problem that the overwhelming majority of semiconductor SERS sensors are suffering. The electromagnetic enhancement (EM), except when caused by the remote excitation of surface plasmon in MoO3 ribbon 14 and near-field EM in Cu2O nanostructure,19 has been seldom reported in semiconductor SERS materials. Especially for TiO2 and ZnO, the most popular semiconductors that have exhibited excellent performance in many fields, however, the SERS behavior for them is rather unsatisfactory.15, 16 Therefore, it is of great challenge to seek novel semiconductors instead of noble metals for high-sensitive SERS sensors.

Niobium pentoxide (Nb2O5), one of the most important transition metal oxides, has always been an interesting research subject due to its potential applications in gas sensors, catalysts, electrochemistry, lithium batteries, microelectronics, and optoelectronics.25, 26 It is known that there are rich adsorption sites on Nb2O5 surface, including complexible Lewis sites and charged Brønsted sites, which could promote a strong interaction with dye molecules carrying nucleophilic groups or opposite charges.27, 28 All the effective adsorption of analyte molecules on the surface of Nb2O5 would further change the electron density distribution of the molecules and result in an increased Raman scattering cross section. Therefore, we expected that the semiconductor of Nb2O5 would exhibit excellent SERS activity.

In this work, the Nb2O5 nanoparticles (NPs) were used as active SERS substrate to detect dyes of MeB, methyl violet (MV), and methyl blue (MB) (Scheme S1). The greatly enhanced SERS effects on the Nb2O5 semiconductor substrate were observed with the SERS EF values over 107 under the excitation waves of 633 and 780 nm for MeB detection. It is of great interest that the SERS activity of Nb2O5 is even comparable to the noble metal substrate with “hot spots”, foreseeing a substitution of metal substrate by semiconductors for various applications, especially in the fields of biological diagnosis and treatment.

Results

Characterization of Nb2O5 NPs

The X-ray diffraction (XRD) pattern of Nb2O5 NPs (Fig. 1a) indicates that all the peaks can be indexed to orthorhombic Nb2O5 (PDF #71-0336).29 The Nb2O5 powder was found consistent with irregular NPs with an average size of 104 nm (Fig. 1b). The high resolution transmission electron microscope (HRTEM) micrograph in Fig. 1c further indicates the spacing of adjacent lattice fringes as 0.391 nm, which is corresponding to the (001) lattice planes. The selected area electron diffraction (SAED) pattern in Fig. 1d from one nanoparticle shows a single diffraction pattern corresponding to a single nanocrystal. The calculated lattice parameters were also well matched with the orthorhombic Nb2O5 phase.

Fig. 1
figure 1

a XRD pattern for the commercial Nb2O5, b transmission electron microscope (TEM) micrograph, c HRTEM micrograph, and d SAED pattern of Nb2O5

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SERS enhancement for dyes detection

The MeB is a well-known dye with applications in biotherapy and medical treatment. It is not only a good phenothiazine photosensitizer with high quantum yields of singlet oxygen production, but also a common cationic dye for histological and bacteriologic staining because of its prominent blue color.28 It can also be used as a therapeutic drug to treat methemoglobinemia, urinary tract infections, and malaria infection, and as an effective antagonist against heat-shock response gene expression in cancer cells.30,31,32 Therefore, it is of crucial importance to seek more biocompatible substrate materials instead of noble metals to detect and record the track of MeB molecules and make an insight into its functionality in organisms.

A series of Raman spectra of MeB molecules with concentrations in the range of 1 × 10–4 to 1 × 10–6 M were collected on the Nb2O5 substrates with the irradiation of 532 nm laser (Fig. 2a) and the detection limitation of as small as 10–6 M. The influences of the excitation wavelength on the SERS activity were also studied. Graphs in Fig. 2b–d display the Raman spectra of solid MeB powders and SERS spectra collected on Nb2O5 substrates under the excitation of 532, 633, and 780 nm lasers, respectively. The Raman shifts and assignments of the main vibrational modes observed in the solid MeB powder and SERS spectra collected with different lasers are presented in Table S1.28, 33 The main bands at around 1630 and 453 cm–1 are attributed to ν(C–C) ring stretching vibration and C–N–C skeletal deformation mode. The asymmetric and symmetric C–N stretches give birth to the bands around ~1445 and 1402 cm–1. An extra band at ~250 cm–1, which occurs only in SERS spectra, is ascribed to the Nb–N stretching of the Nb–MeB complex. Several of the bands in the SERS spectra revealed significant shifts relative to the corresponding bands in solid MeB, which implies some kind of chemical interactions between Nb2O5 substrates and analytes. The variety of the irradiation laser also has an important effect on the intensity and position of vibrational band. Because of the fluorescence effect at 660 nm for MeB as well as the weakened and non-resonant excitation by 632 and 780 nm lasers, many bands for solid MeB did not appear in the spectra. Especially, for 780 nm irradiation laser, only two bands located at 1614 and 1394 cm–1 can be recognized for solid MeB. However, the situation has improved greatly as most of the characteristic bands can be clearly identified in the SERS spectra. Thus, the Nb2O5 semiconductor substrate could effectively attenuate the fluorescence effects and enormously enhance the Raman signals of MeB molecules.

Fig. 2
figure 2

a SERS signals of MeB molecules with various concentrations excited by laser of 532 nm on Nb2O5 substrates. b–d Raman spectra of solid MeB powders and MeB molecules at a concentration of 5 × 10–5 M collected on Nb2O5, TiO2 semiconductor, and Ag substrates with the incident laser of 532, 633 and 780 nm lasers, respectively

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To reasonably illustrate the superior SERS activity of semiconductive Nb2O5, the enhancement ability of anatase TiO2 and Ag colloids for MeB molecules were further investigated. Fig. S1 presents the SEM images of Nb2O5, TiO2 and Ag NPs respectively. Nb2O5 NPs show irregular shapes with an average particle size of ~100 nm, TiO2 NPs show a unique cage-like nanostructure with ~50 nm in diameter, and Ag NPs have an average particle size of ~27 nm. TiO2 has similar energy levels as Nb2O5 and their isoelectric points have been reported in the range of 2.7–6.0.34 Therefore, the cationic coloring regents of MeB dye are apt to adsorb onto TiO2 surface. Fig. 2b-d presents the Raman spectra of MeB collected on TiO2 and Ag colloid substrates with 532, 633, and 780 nm laser, respectively. TiO2 substrate delivers three Raman bands at 396, 516, and 638 cm–1 assigned to anatase TiO2. Only an extremely weak Raman band at 1632 cm–1 assigned to MeB is nearly negligible under the excitation of 532 nm laser. It is noteworthy that the intensity of SERS spectra collected on Nb2O5 is even stronger than that collected on Ag colloid substrate (with a calculated EF value of 6.9 × 105 with 532 nm laser) under the excitation of 532 and 780 nm lasers.

It has been reported that Nb2O5 provides various adsorption sites, including the coordination adsorption of Nb5+ ions (Lewis acidic sites) and electro-adsorption of surface hydroxyl species (Brønsted sites). As shown in Scheme S1, both MeB and MV are base dyes that can be dissociated into cationic pigments in aqueous solutions, whereas MB is an acid dye with anionic coloring agents. The characteristic nucleophilic groups (N or S) make the dyes more inclined to bind with Nb5+. To disclose the adsorption behavior of MeB, MV, and MB molecules on Nb2O5 powder, we measured the UV-vis absorption spectra in Fig. S2 of the original analyte solutions with concentration of 5 × 10–5 M and the centrifugal solutions out of the Nb2O5 suspensions. The adsorption ratios reach up to 19.8 and 13.9% for MeB and MV solutions, respectively, whereas it was observed smaller than 2% for MB solution. The data of adsorption ratios for dyes were further used to evaluate the enhancement capability of Nb2O5 semiconductor substrates under the excitation of different laser beams. According to the results presented in Fig. 2b-d, the bands centered at 1402 cm–1 were selected to calculate the Raman EFs (details in Supporting Information). The resulted EFs are listed in Table 1, which are incredibly as large as 2.6 × 106, 1.9 × 107, and 7.1 × 107 for 532, 633, and 780 nm laser beams, respectively. The EF values for MeB molecules excited at 633 and 780 nm were observed as one order of magnitude greater than the largest EF value for all the reported semiconductor SERS materials (Table 2).

Table 1 Calculated EF values for MeB, MV, and MB detection irradiated by different lasers
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Table 2 Performance of semiconductor SERS active materials from literatures
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The reproducibility of SERS signals for MeB molecule on the semiconductor substrates was further investigated. Eighteen Raman curves were obtained from six Nb2O5 substrates and three spots for each sample were randomly selected to collect the Raman signals (Fig. S3). The calculated relative standard deviation (RSD) values for the main Raman peaks for MeB were observed smaller than 0.1 (Table S2), suggesting an excellent reproducibility. Semiconductor SERS substrates always delivered a much better repeatability than noble metal SERS substrates, as a uniform adsorption of analyte molecules onto semiconductors was more feasible to realize in comparison to an even distribution of “hot spots” for metal SERS substrates.

MV molecules with positively charged coloring agents were further applied as the analyte to evaluate the SERS performance of Nb2O5 semiconductor substrates. Fig. S4 displays the enhanced Raman spectra of MV molecules with various dye concentrations and lasers. The number as well as the intensity of the bands for solid MV powders decreased with the increase of irradiation laser wavelength due to reduced electronic cross sections. Much more bands were recognized for the SERS spectra of MV molecules collected on Nb2O5 even under the 780 nm laser excitation. The EF values are calculated from Fig. S4b–c and listed in Table 1 (calculation details in Supporting Information) reveal the largest EF value as large as 4.7 × 106.

Different from the basic dyes of MeB and MV, the coloring agent for MB was electronegative (Scheme S1c). The detection limitation for MB molecules on Nb2O5 was observed as 10–6 M (Fig. S5a). However, only the SERS spectra of MB with an excitation laser of 532 nm was achieved on the Nb2O5 substrate. The EF value was found as large as 2.3 × 106 according to the results presented in Fig. S5b (details in Supporting Information). The adsorption capability of MB molecules to Nb 2 O5 was much weaker than those for MeB and MB (Fig. S2c). In addition, there was not any identifiable Raman peak with 633 and 780 nm lasers irradiation, indicating a distinction with the basic dyes. The obvious contrast of the SERS performance for Nb2O5 substrate in detection of basic dyes (MeB and MV) and acid dye (MB) would be further discussed in the enhancement mechanism.

Discussion

Though the exact enhancement mechanism is still a controversial issue, two widely accepted theories have been proposed to interpret the SERS phenomenon in most cases.35 One is EM, which is based on the enhancement of optical fields at the surface originated from the localized surface plasmon resonance of substrate materials. The other is CM, predicting that the enhancement is introduced by the charge transfer between chemisorbed molecules and substrate. Both the CM and EM mechanisms will be discussed as follows.

Photo-induced charge transfer (PICT) mechanism is one of the most popular CM for semiconductor substrates. Based on the Born-Oppenheimer and Herzberg-Teller theories, the contribution of PICT process to the molecular polarizability tensor in semiconductor-molecule system has been calculated (see Supporting Information), where the vibrational coupling between the semiconductive energy states (conduction band state S and valence state S′) and the molecular energy states (excited state K and ground state I) are included. The energy level of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) should match with the valence band (VB) and conduction band (CB) of semiconductor. Thus thermodynamically allowed charge transfer would be either from semiconductor to molecule or from molecule to semiconductor (see Scheme S3). The HOMO and LUMO of MeB molecule are located at −6.26 and −4.55 eV, and the CB and VB of semiconductor Nb2O5 are located at −4.00 and −7.10 eV, respectively (see Fig. S6a). As a result, the related thermodynamically feasible charge transfer process with the excitation of 532 nm laser includes the photon-induced charge transfer resonance from the HOMO level of MeB to the CB of Nb2O5. The charge transfer process would create a modified polarizability tensor and electron density distribution of the MeB molecules, leading to the observation of non-totally symmetric SERS modes.

It is noted that the theoretical minimum matching photon energy is greater than 2.0 eV for all dyes (see Fig. S6), and the incident lasers with wavelengths of 633 and 780 nm seem unable to induce the charge transfer between molecules and semiconductor. However, different from the PICT mechanism derived from metal-molecule system disregarding of the chemisorption effect, the adsorption effect of analyte molecules for semiconductor-molecule system has to be taken into account. The extra bonding between Nb2O5 and dye molecules through chemisorption would readjust the positions of molecular HOMO and LUMO as well as semiconducting VB and CB, which would allow the PICT process excited by 633 and 780 nm lasers.

Taking the chemical bonding effect between Nb2O5 and MeB into consideration, the density-functional-theory (DFT) calculations were performed to study the chemical enhancement mechanism including charge transfer and light adsorption capacities of MeB-Nb2O5 composite. To reduce computational cost, Nb4O10 cluster with H-passivation was used to simulate Nb2O5 nanostructure that further interacts with MeB molecule by Nb-S chemical bonding and the Nb-S bond length (2.45 Å) in MeB-Nb2O5 composite. Therefore, Raman vibrational modes associated with S atom in MeB were considered and compared with experimental values. More interaction modes between Nb2O5 surfaces and adsorbed molecule MeB such as Nb–N bonding were not considered in this work. Fig. 3 displays the optimized structures and computed normal-mode Raman spectra of MeB and MeB-Nb4O10. It is found that several dominant bands located in 1410, 660, and 1445 cm−1 in MeB-Nb4O10 are obviously enhanced by compared with corresponding spectra of MeB. Our calculated results are in rough agreement with the observed Raman enhancement around 1402, 667, and 1441 cm−1 (Fig. 2b) in the experimental measurement. The strongest enhancement located in 1630 cm−1 was not found in our DFT-calculated spectra, which may be attributed to different chemical bonding (Nb–N, Nb–S, N–O, and S–O) between MeB and Nb2O5. Since the Raman spectra of 1630 cm−1 was experimentally assigned to the C–N bond stretching, it is anticipated to be enhanced resonantly by chemical bonding of Nb–N.

Fig. 3
figure 3

Calculated Raman spectrum of a MeB, and b MeB-(Nb2O5)2 complex. The inset shows Optimized geometry of MeB, and MeB-(Nb2O5)2 complex, respectively

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Under a ground state, the Raman scattering from MeB was enhanced by charge transfer and orbital overlap between MeB and Nb2O5 as a result of the chemical bonding effect. Fig. 4a presents the UV–vis optical absorption spectra of neat Nb2O5, MeB molecule modified Nb2O5, and MeB aqueous solution. An apparent shift of adsorption peak occurs for the MeB molecule adsorbed on Nb2O5 in comparison with the pure MeB, indicating a strong CT process between MeB molecules and Nb2O5 NPs. Based on the optimized geometries, we further carried out molecular orbital analysis (energy level and orbital structure) for MeB, Nb4O10, and MeB-Nb4O10 in order to elucidate charge transfer mechanism. As shown in Fig. 4b–d, both HOMO and LUMO of MeB-Nb4O10 are formed by combining HOMO of MeB with LUMO of Nb4O10. Therefore, two active molecular orbitals participating electronic transition in the complex are averagely distributed in two fragments, which is significantly favorable to increase efficiency of charge transfer. In addition, the energy gap (1.07 eV) between occupied and unoccupied MOs in MeB-Nb4O10 is significantly reduced by compared with MeB (3.58 eV) and Nb4O10 (2.83 eV). The reduction can increase light adsorption capacity, which favors Raman enhancement of MeB-Nb4O10. Therefore, the calculation results have verified the allowance of the PICT process between MeB and Nb2O5 with 633 and 780 nm excitation lasers.

Fig. 4
figure 4

a The UV-vis optical absorption spectrum of pure Nb2O5 powders, MeB modified Nb2O5 powders and MeB aqueous solution. b–d The molecular orbital illustrations of the molecule, Nb2O5 cluster and MeB-(Nb2O5)2 complex

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The chemical bonding induced CM enhancement is closely related to the excellent adsorption ability (uptake of 5.96 × 1018 MeB molecules or 4.18 × 1018 MV molecules per gram of Nb2O5 powder) which was confirmed by the UV–vis adsorption curves shown in Fig. S2. Prado et al.36 calculated the pK1 and pK2 of Nb2O5 from the density surface charge as a function of pH and observed that Nb2O5 surface was protonated corresponding to Nb–OH2 + acid sites at pH below 4. At pH greater than 5.4, the surface is covered with fully deprotonated base sites of Nb–O– (Scheme S2a). For the detection of base dyes, the surface of Nb2O5 is fully deprotonated in the alkalescent MeB solution (Scheme S2b). The resulting excess negative charge on the Nb2O5 surface further promotes strong interactions with the coloring agents through the positively charged S or N atom. We collected the Raman spectra of MeB at different pH values (Fig. 5) in which the HCl and NaOH solutions were used to adjust pH of MeB solutions to the target value. As more deprotonated base sites on the surface of Nb2O5 are produced in the basic dye solution with a higher pH value, MeB color regent with positive charges will be more easily bonded to these sites. Therefore, the largely enhanced Raman spectra were obtained with a high pH of 10 and 12. Little attenuated Raman signal at pH 4 is ascribed to the deceased base sites. There is not any characteristic Raman band at pH 1 and 2 as a result of fully protonated acid sites coverage on Nb2O5 surface. These results demonstrate that the deprotonated sites with one negative charge play a crucial role for the excellent SERS detection of MeB molecules.

Fig. 5
figure 5

Raman spectra of MeB at different pH values collected on Nb2O5 substrates

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Similarly, the bonding effect between MV molecules and Nb2O5 also promotes significantly electron transfer, which was revealed by a broader MV adsorption for the MV molecules modified Nb2O5 powder than MV solution (Fig. S7a). However, MB molecule has a poorer adsorption behavior on the surface of Nb2O5 than MeB and MV owing to the same-charged character between the coloring agent of MB and Nb2O5 surface. Only one broad peak appears in the curve of MB modified Nb2O5 with respect to the double peaks adsorption of MB aqueous solution (Fig. S7b). Therefore, there is no effective bonding effect to produce the extra hybrid surface states between VB and CB of Nb2O5, and longer irradiation wavelength could not achieve feasible electron transfer between dye molecules and Nb2O5, which also explains the disappearance of the SERS activity with the irradiation lasers of 633 and 780 nm in our experiments.

PICT mechanism could only account for an EF value of smaller than 105, thus, the electromagnetic enhancement here should be investigated using finite element method (FEM).37 Fig. 6 presents the electric field distribution around a single Nb2O5 crystal with the excitation of 526 nm laser. A quasi-hot spot with the highest electric field amplitude (E) of 5.25 is located around Nb2O5 nanocrystal. As the EF value is proportional to |E|4, EM contributes as high as two orders of magnitude to EF. The calculated results by combining CM and EM are in well consistent with our experimental results. This work has delivered a new semiconductor substrate of Nb2O5 with EFs as high as 107 that is comparable with noble metal (Ag) substrates with “hot spots”, indicating a substituted application of metal substrate by semiconductors in some fields where metals are forbidden.

Fig. 6
figure 6

FEM simulation of electric field distribution on XOZ plane for a Nb2O5 nanocrystal with the diameter of 400 nm (the aggregated nanoparticles) as the incident light propagates along X axis. (λ = 526 nm)

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In summary, the Nb2O5 NPs were applied as a highly active semiconductor SERS substrate for detection of MeB, MV, and MB molecules. The EFs for this application were observed incredible greater than 107 for MeB detection excited at 633 and 780 nm, one order of magnitude lager than the largest EF value for all reported semiconductor SERS materials; even comparable to noble metals nanostructures with “hot spots”. An excellent reproducibility of the Raman spectra was further exposed with RSD values smaller than 0.1. The contributions of CM and EM to the SERS enhancement are further analyzed respectively by DFT and FEM calculations. The former discloses that several of Raman bands for dyes are observably enhanced through a certain chemical bonding between Nb2O5 and molecule. Two hybrid energy orbitals as new HOMO and LUMO are contributed by both dye and Nb2O5 in the chemisorption system, which means the charge transfer more efficient. Besides, the reduced energy gap between two new MOs would further make it feasible to induce a PICT process with a longer irradiation laser of 633 and 780 nm. The chemisorption behavior between dye and Nb2O5 plays a significant role for chemical enhancement, which has been proved by the dependence of pH and SERS intensity. FEM results indicate that the electromagnetic enhancement also delivered an EF value of 102. Our results demonstrate that Nb2O5 nanocrystals could be highly-active SERS substrate as substitution of noble metals for rapid detection of organic molecules.

Methods

Materials and instruments

The Nb2O5 powders with an analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd, while anatase TiO2 NPs, MeB, MV, and MB were supplied by Aladdin Co., Ltd. All the reagents are received without further purification. The XRD analysis was carried out using Cu Kα target at a working voltage 40 kV and current 200 mA on a powder X-ray diffractometer (D8, ADVANCE-TXS, Bruker AXS). The size and shape of Nb2O5 powders were studied using the JEOL JEM-2100F field emission source transmission electron microscope with an accelerating voltage of 200 KV.UV–Vis diffuse reflectance spectra were recorded on a PE lambda 950 instrument. SERS spectra were collected on a DXR Raman microscope (Thermo Scientific) with the excitation wavelengths of 532, 683, 780 nm, respectively.

Sample preparation

MeB, MV, and MB solutions were diluted with deionized water to various concentrations of 1 × 10−4, 5 × 10−5, 1 × 10−5, 5 × 10−6, and 1 × 10−6 M. Then 20 mg Nb2O5 powders and 20 ml analyte solutions were mixed thoroughly with ultrasonic dispersion for 30 min. Finally, Nb2O5 was separated from the suspensions by centrifugation and further transferred to the object slice. After dried in a 60 °C oven for 10 min, the Nb2O5 substrate was ready for the Raman measurement.

Silver colloids were prepared via a solvothermal method as reported in our previous work. In a typical procedure, 0.3 mM silver oleate was dissolved in 30 ml octadecylene with stir overnight, and during this process, 0.5 ml oleic acid was injected as surfactant. Finally, the solution was transferred to a 50 ml Teflon-lined stainless steel autoclave and heated at 150 °C in an oven for 5 h. After the autoclave was cooled down to room temperature naturally, the black precipitate was washed with mixed solvent of hexane and isopropanol (volume ratio 1:4) for 4 times. Then the precipitate was washed with acetone for two times to remove remaining hexane and re-dispersed in ethanol again.

Raman measurements

The SERS spectra were measured with a DXR confocal microscopy Raman spectrometer. Three different wavelengths of lasers (532, 633, and 780 nm) were selected to evaluate the SERS activity of Nb2O5 substrates. The diameter of laser spot was estimated by the formula: d = 1.22/D, where D is referred to the numerical aperture of objective lens. For a 10× lens, D is 0.25 μm, so the calculated diameters for focused laser spot are 2.6, 3.1, and 3.8 μm for 532, 633, and 780 nm lasers, respectively.