Niobium pentoxide: a promising surface-enhanced Raman scattering active semiconductor substrate

Surface-enhanced Raman scattering technique, as a powerful tool to identify the molecular species, has been severely restricted to the noble metals. The surface-enhanced Raman scattering substrates based on semiconductors would overcome the shortcomings of metal substrates and promote development of surface-enhanced Raman scattering technique in surface science, spectroscopy, and biomedicine studies. However, the detection sensitivity and enhancement effects of semiconductor substrates are suffering from their weak activities. In this work, a semiconductor based on Nb2O5 is reported as a new candidate for highly sensitive surface-enhanced Raman scattering detection of dye molecules. The largest enhancement factor value greater than 107 was observed with the laser excitation at 633 and 780 nm for methylene blue detection. As far as literature review shows, this is in the rank of the highest sensitivity among semiconductor materials; even comparable to the metal nanostructure substrates with “hot spots”. The impressive surface-enhanced Raman scattering activities can be attributed to the chemical enhancement dominated by the photo-induced charge transfer, as well as the electromagnetic enhancement, which have been supported by the density-functional-theory and finite element method calculation results. The chemisorption of dye on Nb2O5 creates a new highest occupied molecular orbital and lowest unoccupied molecular orbital contributed by both fragments in the molecule-Nb2O5 system, which makes the charge transfer more feasible with longer excitation wavelength. In addition, the electromagnetic enhancement mechanism also accounts for two orders of magnitude enhancement in the overall enhancement factor value. This work has revealed Nb2O5 nanoparticles as a new semiconductor surface-enhanced Raman scattering substrate that is able to replace noble metals and shows great potentials applied in the fields of biology related. A semiconductor that makes recognizing molecules easier is identified by researchers in China. Yong Yang and co-workers from the Shanghai Institute of Ceramics show that niobium pentoxide can strongly enhance the optical signature of the colored dyes used in biomedical applications. Nanometer-sized features on a rough surface can increase optical fields. This phenomenon can enhance the optical signature used to identify a specific molecule in a technique called surface-enhanced Raman spectroscopy (SERS). But only a few materials, notably noble metals such as gold and silver, have demonstrated useful levels of enhancement. Yang et al. find that niobium pentoxide nanoparticles can be used as a most-active SERS semiconductor substrate to detect the dyes methylene blue, methyl violet and methyl blue. They measured a SERS enhancement factor of over ten million using 633 and 780 nano meter light to detect methylene blue.


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 TiO 2 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 × 10 6 . 17 Other semiconductors, such as V 2 O 5 , 18 CuO, 19 AgFeO 2 , 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 10 3~1 0 6 . 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 MoO 3 ribbon 14 and near-field EM in Cu 2 O nanostructure, 19 has been seldom reported in semiconductor SERS materials. Especially for TiO 2 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 (Nb 2 O 5 ), 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 Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 would exhibit excellent SERS activity.
In this work, the Nb 2 O 5 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 Nb 2 O 5 semiconductor substrate were observed with the SERS EF values over 10 7 under the excitation waves of 633 and 780 nm for MeB detection. It is of great interest that the SERS activity of Nb 2 O 5 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.

Characterization of Nb 2 O 5 NPs
The X-ray diffraction (XRD) pattern of Nb 2 O 5 NPs (Fig. 1a) indicates that all the peaks can be indexed to orthorhombic Nb 2 O 5 (PDF #71-0336). 29 The Nb 2 O 5 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 Nb 2 O 5 phase.

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 Nb 2 O 5 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 Nb 2 O 5 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 To reasonably illustrate the superior SERS activity of semiconductive Nb 2 O 5 , the enhancement ability of anatase TiO 2 and Ag colloids for MeB molecules were further investigated. Fig. S1 presents the SEM images of Nb 2 O 5 , TiO 2 and Ag NPs respectively. Nb 2 O 5 NPs show irregular shapes with an average particle size of 100 nm, TiO 2 NPs show a unique cage-like nanostructure with 50 nm in diameter, and Ag NPs have an average particle size of 27 nm. TiO 2 has similar energy levels as Nb 2 O 5 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 TiO 2 surface. Fig. 2b-d presents the Raman spectra of MeB collected on TiO 2 and Ag colloid substrates with 532, 633, and 780 nm laser, respectively. TiO 2 substrate delivers three Raman bands at 396, 516, and 638 cm -1 assigned to anatase TiO 2 . 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 Nb 2 O 5 is even stronger than that collected on Ag colloid substrate (with a calculated EF value of 6.9 × 10 5 with 532 nm laser) under the excitation of 532 and 780 nm lasers.
It has been reported that Nb 2 O 5 provides various adsorption sites, including the coordination adsorption of Nb 5+ 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 Nb 5+ . To disclose the adsorption behavior of MeB, MV, and MB molecules on Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 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 × 10 6 , 1.9 × 10 7 , and 7.1 × 10 7 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). The reproducibility of SERS signals for MeB molecule on the semiconductor substrates was further investigated. Eighteen Raman curves were obtained from six Nb 2 O 5 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 Nb 2 O 5 semiconductor substrates.  Table 1 (calculation details in Supporting Information) reveal the largest EF value as large as 4.7 × 10 6 .
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 Nb 2 O 5 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 Nb 2 O 5 substrate. The EF value was found as large as 2.3 × 10 6 according to the results presented in Fig. S5b (details in Supporting Information). The adsorption capability of MB molecules to Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 . 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 semiconductormolecule system has to be taken into account. The extra bonding between Nb 2 O 5 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 Nb 2 O 5 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-Nb 2 O 5 composite. To reduce computational cost, Nb 4 O 10 cluster with H-passivation was used to simulate   (Fig. 2b)  The chemical bonding induced CM enhancement is closely related to the excellent adsorption ability (uptake of 5.96 × 10 18 MeB molecules or 4.18 × 10 18 MV molecules per gram of Nb 2 O 5 powder) which was confirmed by the UV-vis adsorption curves shown in Fig. S2. Prado et al. 36 calculated the pK 1 and pK 2 of Nb 2 O 5 from the density surface charge as a function of pH and observed that Nb 2 O 5 surface was protonated corresponding to Nb-OH 2 + 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 Nb 2 O 5 is fully deprotonated in the alkalescent MeB solution (Scheme S2b). The resulting excess negative charge on the Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 surface. These results demonstrate that the deprotonated sites with one negative charge play a crucial role for the excellent SERS detection of MeB molecules.
Similarly, the bonding effect between MV molecules and Nb 2 O 5 also promotes significantly electron transfer, which was revealed by a broader MV adsorption for the MV molecules modified Nb 2 O 5 powder than MV solution (Fig. S7a). However, MB molecule has a poorer adsorption behavior on the surface of Nb 2 O 5 than MeB and MV owing to the same-charged character between the coloring   (Fig. S7b). Therefore, there is no effective bonding effect to produce the extra hybrid surface states between VB and CB of Nb 2 O 5 , and longer irradiation wavelength could not achieve feasible electron transfer between dye molecules and Nb 2 O 5 , 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 10 5 , thus, the electromagnetic enhancement here should be investigated using finite element method (FEM). 37 Fig. 6 presents the electric field distribution around a single Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 with EFs as high as 10 7 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.
In summary, the Nb 2 O 5 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 10 7 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 Nb 2 O 5 and molecule. Two hybrid energy orbitals as new HOMO and LUMO are contributed by both dye and Nb 2 O 5 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 Nb 2 O 5 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 10 2 . Our results demonstrate that Nb 2 O 5 nanocrystals could be highly-active SERS substrate as substitution of noble metals for rapid detection of organic molecules.

Materials and instruments
The Nb 2 O 5 powders with an analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd, while anatase TiO 2 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 Nb 2 O 5 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 Nb 2 O 5 powders and 20 ml analyte solutions were mixed thoroughly with ultrasonic dispersion for 30 min. Finally, Nb 2 O 5 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 Nb 2 O 5 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 Nb 2 O 5 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.