Surfaceenhanced Raman scattering (SERS) is a powerful analysis technique that allows both the identification and detection of analytes at trace levels. However, the low rate of charge transfer (CT) between noblemetal nanoparticles and several analytes prevents them from being effectively detected by SERSbased sensors. They are regarded as low Raman crosssection molecules. In this study, we enhanced the performance of the silver nanoparticles (AgNPs)based SERS sensing platform for a low Raman crosssection molecule, urea, focusing on improving the rate of CT. First, a set of Agtitanium dioxide (TiO2) nanocomposites were synthesized. The presence of TiO2 improved the intensity of the SERS signal of urea, in comparison to the use of bare AgNPs. Second, a photoinduced enhanced Raman spectroscopy (PIERS) technique was employed to further elevate the Raman signal of urea. Thanks to the step of preirradiation using UV light at λ = 365 nm, with the use of the substrates containing 25%, 33%, and 50% TiO2 content, enhancements of 1.93, 3.42, and 7.45 times were achieved, respectively, compared to the use of AgTiO2 composites without UV irradiation. Through modification of the substrate, combined with application of the PIERS technique, the SERS system for urea detection using Ag3TiO2 (50% TiO2) achieved a competitive detection limit of 4.6 × 10−6 M. It also allowed the detection of urea in milk at concentrations down to 10−5 M. This substrate modification and PIERS technique are promising for improvement of the sensing performance of other lowcrosssection molecules.
www.acsanm.org Article Photoinduced Enhanced Raman Spectroscopy for the Ultrasensitive Detection of a Low-Cross-Section Chemical, Urea, Using Silver−Titanium Dioxide Nanostructures Quan Doan Mai,† Ha Anh Nguyen,*,† Thi Lan Huong Phung, Ngo Xuan Dinh, Quang Huy Tran, Tri Quang Doan, Anh Tuan Pham, and Anh-Tuan Le* Downloaded via Ha Anh Nguyen on October 5, 2022 at 00:26:37 (UTC) See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles Cite This: https://doi.org/10.1021/acsanm.2c03524 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Surface-enhanced Raman scattering (SERS) is a powerful analysis technique that allows both the identification and detection of analytes at trace levels However, the low rate of charge transfer (CT) between noble-metal nanoparticles and several analytes prevents them from being effectively detected by SERS-based sensors They are regarded as low Raman crosssection molecules In this study, we enhanced the performance of the silver nanoparticles (AgNPs)-based SERS sensing platform for a low Raman cross-section molecule, urea, focusing on improving the rate of CT First, a set of Ag/titanium dioxide (TiO2) nanocomposites were synthesized The presence of TiO2 improved the intensity of the SERS signal of urea, in comparison to the use of bare AgNPs Second, a photoinduced enhanced Raman spectroscopy (PIERS) technique was employed to further elevate the Raman signal of urea Thanks to the step of preirradiation using UV light at λ = 365 nm, with the use of the substrates containing 25%, 33%, and 50% TiO2 content, enhancements of 1.93, 3.42, and 7.45 times were achieved, respectively, compared to the use of Ag/TiO2 composites without UV irradiation Through modification of the substrate, combined with application of the PIERS technique, the SERS system for urea detection using Ag/3TiO2 (50% TiO2) achieved a competitive detection limit of 4.6 × 10−6 M It also allowed the detection of urea in milk at concentrations down to 10−5 M This substrate modification and PIERS technique are promising for improvement of the sensing performance of other low-cross-section molecules KEYWORDS: PIERS, SERS, urea, low Raman cross-section molecules, silver−titanium dioxide nanostructure INTRODUCTION Surface-enhanced Raman spectroscopy (SERS) has been recognized as one of the most sensitive analysis techniques that magnifies Raman signals of analytes, allowing them to be detected even at the single-molecule level.1,2 Moreover, originating form inelastic light scattering of analytes, SERS provides information about their molecular structures, therefore, working as a powerful identification tool in analytical chemistry.1,3 Various SERS-based applications have been developed for environmental,4,5 food,6,7 and health safety8−10 monitoring The adsorption of analytes on the surface of noble metals such as Au and Ag has been proven to enhance their Raman signals, thanks to localized surface plasmon resonance of those metals.11 Under light excitation, the collective oscillation of conductive electrons of the plasmonic materials creates a strong magnetic field on the materials surface, coupling with the vibrational modes of the analytes and, therefore, enhancing their Raman signals.1,11 It is also the principle of the most important contribution of Raman enhancement in the SERS effect, the electromagnetic © XXXX American Chemical Society mechanism In addition, despite a smaller contribution, the formation of a chemical complex between the analytes and metal surface, also known as the chemical mechanism (CM), cannot be ignored because charge-transfer (CT) transitions between the substrate and the molecule are also essential for the SERS phenomenon to occur.12−14 These two mechanisms generate a giant enhancement in the Raman signal, allowing many kinds of analytes, including pesticides, food additives, biomarkers, etc., to be detected at trace levels.15−17 Unfortunately, such a powerful analytical tool is not effective at detecting every organic analyte In a recent study, we reported on the role of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) Received: August 9, 2022 Accepted: September 28, 2022 A https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Scheme Schematic Illustration of the Synthesis Process of Ag/TiO2 Nanocomposites energy levels of the analyte on the SERS signal and CT process.13 Our study proved that a large gap between the LUMO energy of the analyte and the Fermi level of the substrate could prevent the CT process For example, 4nitrophenol (4-NP), which possesses a nitro group to directly adsorb onto silver nanoparticles (AgNPs), did not show a large enhancement in the SERS spectra, while a significant enhancement was observed in the SERS spectra of another nitrophenyl-substituted molecule, chloramphenicol (CAP) The LUMO levels of 4-NP and CAP were calculated to be −3.55 and −3.84 eV, respectively Therefore, the gap between the LUMO level of 4-NP and the Fermi level of Ag (−4.26 eV) was 0.71 eV, which was larger than that of CAP (0.42 eV).13 With a low level of CT, 4-NP can be regarded as a low Raman cross-section molecule.18,19 Several other molecules such as cysteine, CO2, adenosine triphosphate, and epidermal growth factor receptor peptide have been reported to be low-crosssection, resulting in low SERS signals.14,20,21 However, improving the SERS enhancement of the molecules with low CT levels is not impossible In the report mentioned above, we suggested modification of the nanomaterial to increase the Fermi level.13 In a 2018 study, Tao et al were successful in improving the rate of CT and enlarging the cross section of the analyte by modifying the substrate with WTe2, instead of employing the initial substrate of graphene.22 In addition, in a 2016 study, Parkin’s group reported on a technique called photoinduced enhanced Raman spectroscopy (PIERS), in which a step of preirradiation by ultraviolet (UV) light for a period of time on gold nanoparticles (AuNPs) or AgNPs deposited on a titanium dioxide (TiO2) rutile surface was carried out before the SERS experiments.23 This preirradiation step led to a several times enhancement of the SERS signal This phenomenon is based on the interaction between UV light and TiO2, leading to oxygen vacancy states in the metal oxide semiconductor and an electron donor below the conduction band edge Under laser excitation, electrons can be injected into the Fermi level of the metal, increasing the electron density on the surface of the nanoparticles (NPs) and allowing more electrons to be transferred to the analytes via the CT process.23 Urea, CO(NH2)2, is a redundant product of many organisms; therefore, it is naturally created inside their bodies and then released to the environment However, containing a high content of nitrogen, it has been synthesized and utilized in both agriculture and industry In agriculture, urea has been widely employed as a nitrogen-releasing fertilizer In industry, it is used to produce cleaning products As a result, a high amount of urea has been released into the aquatic environment, including both surface water and groundwater, every year.24 More dangerously, urea has been added illegally into dairy products to inflate the protein contents.25 Excessive amounts of urea in water and milk may cause kidney diseases in human.26 In contrast, in the body, urea is released into blood from the liver and transferred to the kidneys, becoming a part of the urine In humans, the normal level of urea in blood is reported to be 2.5−6.7 mM, while a high level of 30−150 mM represents pathophysiological states.26 Therefore, urea also plays the role of a biomarker for kidney diseases in humans Overall, the detection of urea is important for both environmental and food safety and health monitoring Analytical methods, such as gas chromatography27 and calorimetry,28 can detect urea at low levels Although they can provide sensitive, accurate, and stable results, they are time-consuming and laborious and require expensive instruments For more cost-effectiveness, electrochemical sensors for urea detection have been designed based on the use of urease.29 In the presence of this enzyme, the sensors are sensitive and specific; however, strict requirements of the experimental environment such as the temperature and pH have prevented them from being applied for a wide range of samples Nickel-based materials are excellent for nonenzymatic urea sensors; however, they were reported to involve reducing and expanding structure during the oxidation reaction to the target molecules.30 Containing two primer amino groups and a B https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article RAM) with 785 nm laser excitation The optical properties of Ag/ TiO2 were also performed using photoluminescence (PL) spectroscopy with a 380 nm excitation wavelength 2.3 CV Measurement CV measurements were performed using a Palmsens electrochemical workstation under ambient conditions The experiment was set up based on an established study with a Pt working electrode and an Ag/AgCl reference electrode.37 A 0.1 M phosphate buffer solution (PBS) served as the electrolyte All electrochemical potentials were referenced to an Fc/Fc+ internal standard Cyclic voltammograms of the analytes were performed at a scan rate of 50 mV s−1 in the potential range from −2 to +2 V 2.4 Substrate Preparation and SERS and PIERS Measurements Aluminum (Al) substrates were fabricated with dimensions of cm × cm × 0.1 cm with a surface-active area with a diameter of 0.2 cm The substrates were washed with ethanol and then dried naturally at room temperature (RT) A SERS active substrate (AgNPs or Ag/TiO2) solution was then drop-casted onto the surface-active area and dried at RT Solutions with various concentrations (10−6−10−3 M) of urea were prepared in distilled water For each SERS measurement, the sample was prepared as follows: μL of an analyte solution was dropped directly onto the prepared substrate and dried naturally at RT SERS spectra were recorded by a MacroRaman Raman spectrometer (Horiba) with 785 nm laser excitation Raman measurements were acquired by means of a 100× objective with a numerical aperture of 0.90 The laser power was set as 45 mW at a 45° contact angle, with a diffraction-limited laser spot diameter of 1.1 μm (1.22λ/NA) and a focal length of 115 nm For each measurement, the exposure time was 10 s with three accumulations The final spectrum was obtained after baseline calibration For each PIERS measurement, the prepared substrate was preirradiated using an UV source of 365 or 400 nm in 30 Subsequently, the analyte solution was dropped directly onto the substrate and dried naturally Measurement was then carried out with the same procedure as that for the SERS measurements For real samples, bottled milk was purchased from a local supermarket in Hanoi, Vietnam, and utilized directly without further preparation Urea-spiked milk was obtained by adding an appropriate volume of an urea solution into the milk samples Subsequently, the spiked samples were dropped onto the prepared substrates for PIERS measurements as described above low steric hindrance, urea possesses structural features that allow them to effectively adsorb on the surface of noble-metal NPs Therefore, it is expected that it can be detected at trace levels by SERS sensors Surprisingly, in the literature, there are a only few urea sensors based on the SERS technique.25,31−34 Moreover, their detection limits were not impressive Therefore, urea should be one of the low-cross-section molecules, which can be “unsuitable” for SERS detection In this study, we aim to improve the performance of the AgNPs-based SERS sensing platform for urea detection The LUMO level of urea was calculated using electrochemical cyclic voltammetry (CV) and compared to the Fermi level of Ag and the LUMO levels of the published organic molecules to prove that urea is a low Raman cross-section molecule This was confirmed by the SERS signal of urea on AgNPs To improve the enhancement of the SERS signal of urea, we first modified the substrate by using Ag/TiO2 composite nanomaterials instead of single AgNPs Four types of Ag/TiO2 with varied TiO2 contents, including 25%, 33%, 50%, and 75%, were synthesized and employed for SERS measurement of urea The utilization of composite nanomaterials containing 25%, 33%, and 50% TiO2 resulted in better enhancement compared to the use of AgNPs These functional nanomaterials were then employed for the PIERS technique to further improve their SERS performance The results showed enhancements of 1.93, 3.42, and 7.45 times the SERS intensity when substrates containing 25%, 33%, and 50% TiO2 were preirradiated with UV light of 365 nm, respectively Therefore, by using a noble metal−metal oxide nanocomposite, combined with the PIERS technique, we have improved the performance of SERS sensors for urea, a low Raman cross-section molecule Urea could be detected at concentrations as low as 4.6 × 10−6 and 10−5 M in distilled water and milk, respectively The results confirmed the importance of the CT process in the SERS signal and suggested a direction for evaluating the rate of CT based on material modification and preirradiation to generate advanced SERS sensors for low Raman cross-section molecules RESULTS AND DISCUSSION 3.1 Urea, a Low-Cross-Section Molecule The HOMO and LUMO energy levels of urea can be estimated based on its onset oxidation and reduction potentials (ϕox and ϕred), respectively, using the equations37−39 MATERIALS AND METHODS 2.1 Chemicals Silver nitrate (AgNO3, ≥99.0 wt %), sodium borohydride (NaBH4, 99 wt %), titanium tetrachloride (TiCl4, ≥99.8 wt %), ammonium hydroxide (NH4OH, 28.0−30.0% NH3), ethanol (C2H5OH, 98 vol %), and urea (CH4N2O, 99 wt %) were purchased from Shanghai Chemical Reagent and used directly without further purification Double-distilled water was used throughout the experiments 2.2 Synthesis of Ag/TiO2 Nanocomposite Materials and Their Characterizations Ag/TiO2 nanocomposites were prepared via a facile wet chemistry method, as described in Scheme and in detail in our previous study.35 TiO2 NPs were synthesized using a modified sol−gel method from TiCl4 precursors.36 An AgNO3 solution was added to a set of variously prepared mixtures containing different amounts of crystalline TiO2 NPs calcined at 400 °C and 50 mL of C2H5OH Subsequently, NH4OH was slowly dropped into the solution to completely reduce Ag+ to Ag0 Finally, a set of Ag/TiO2 nanocomposites was obtained with various of TiO2 contents including 25, 33, 50, and 75 wt % and named Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2, respectively The morphology of Ag/TiO2 nanocomposites was studied using scanning electron microscopy (SEM; Hitachi S-4800) operating under an acceleration voltage of kV The crystal structure of Ag/ TiO2 was analyzed via X-ray diffraction (XRD; Bruker D5005 X-ray diffractometer, Cu Kα, λ = 1.5406 Å) under a voltage of 40 kV and a current of 30 mA The composition and chemical properties of Ag/ TiO2 were investigated by Raman spectroscopy (Horiba Macro- E HOMO = e( ox + 4.8 Fc/Fc+) (1) E LUMO = e( red + 4.8 Fc/Fc+) (2) in which ϕFc/Fc+ is the redox potential of the ferrocene/ ferrocenium couple (Fc/Fc+) in the electrochemical system, assuming that the energy level of Fc/Fc+ is −4.8 eV below the vacuum level Similar to the previous study, we set up an electrochemical system based on the description of Bin et al with a Pt working electrode and a Ag/AgCl reference electrode.37 Therefore, ϕFc/Fc+ was assumed to be 0.44 V versus Ag/AgCl.37 However, as discussed in the previous study, while the distinction in the LUMO levels of the analytes led to a significant difference in their SERS intensities, the effects of the HOMO level were not clear.13 Therefore, in this study, we only focus on the reduction potential to calculate the LUMO level of urea CV measurements of urea were performed in 0.1 M PBS (Figure S1a) In the absence of urea, no redox peak was observed In the presence of urea, an irreversible cathodic peak C https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org appears at −1.32 V It is the onset reduction peak of urea The onset reduction potential (ϕred) of urea was estimated to be −0.86 V Using eq 2, we determined the LUMO level of urea to be −3.50 eV The values are averaged over cycles of CV scans (Figure S2) The LUMO level of urea forms a relatively large gap (0.76 eV) with the Fermi level of Ag (−4.26 eV) This gap is even larger than that of 4-NP (0.71 eV), which was reported to have a low SERS signal on the Ag substrate.13 This large gap suggested a low rate of CT between AuNPs and urea in SERS experiments, which limits the cross section of the molecule In other words, urea is a low-cross-section molecule Another example of a low-cross-section molecule, cysteine, was mentioned in a study of Fu et al., in which the authors claimed to know about its weak SERS signal.20 Therefore, SERS measurements were performed to confirm our hypothesis Figure S1b shows the SERS spectra of urea on AgNPs As a simple organic molecule containing one carbonyl group (>C�O) and two amino groups (−NH2), the presence of urea absorbed on SERS substrates resulted in simple SERS spectra with only one characteristic band at 1010 cm−1, representing the C−N stretching mode.40 Although the presence of −NH2 groups allowed urea to bind directly on the surface of the Ag substrate, the low intensity of this dominant peak can be observed, even at a relatively high concentration of 10−3 M Hence, urea can be regarded as a low-cross-section molecule However, the cross section can be enlarged by increasing the rate of CT.22 In a 2021 study, we designed an experimental model using methylene blue as the analyte to prove that Ag/ TiO2 contacts in Ag/TiO2 nanocomposites could enhance electron transfer, leading to improvement of the SERS intensity.35 Therefore, in the effort of improving the performance of SERS sensors for urea, we utilized a set of Ag/TiO2 nanocomposites with different TiO2 contents 3.2 Characterization of Ag/TiO2 Nanocomposites Four types of Ag/TiO2 composite nanomaterials, including Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2, containing 25%, 33%, 50%, and 75% TiO2 content, respectively, were synthesized as described in section The content of TiO2 in the nanocomposites was calculated based on the weight of TiO2 nanostructures and AgNO3 precursors added during the synthesis Their SEM images show the difference in the TiO2/ Ag ratio of distinct nanocomposites (Figure 1a−d) Two types Article of materials with different sizes of 43 and 125 nm were detected in the SEM images As revealed by the small size of the AgNPs in Figure S3, the smaller nanoobjects in these SEM images can be AgNPs while the larger ones can be TiO2 NPs From Ag/1TiO2 to Ag/4TiO2, the density of the larger nanostructures increases while that of the smaller ones decreases, implying a rise in the TiO2/Ag ratio in those nanocomposites Moreover, their energy-dispersive spectroscopy (EDS) spectra provided qualitative and semiquantitative results of TiO2 and Ag in the nanostructures Figure S4 shows the EDS spectra of Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/ 4TiO2 with the contents of TiO2 estimated to be 23%, 34%, 45%, and 78%, respectively, which are in agreement with the initially calculated TiO2 contents The XRD results of the asprepared materials also indicated the presence of Ag and TiO2 in the nanocomposites Figure 2a shows the XRD pattern of Ag/TiO2 nanocomposites in comparison to the reference patterns of TiO2 and Ag The main peaks corresponding to Ag (ICDD 01-087-0597) as well as TiO2 (ICDD 01-086-1157), both anatase and rutile, appear in the XRD pattern of Ag/ TiO2 This is evidence for the presence of AgNPs in the TiO2 crystal matrix On the basis of their diffraction peaks, the average crystal grain sizes of AgNPs and TiO2 were calculated to be 14 and 19 nm, using the Scherrer formula.41 The presence of AgNPs in the TiO2 crystal matrix was also confirmed by the PL measurements (λext = 380 nm; Figure 2c,d) Upon a decrease of the TiO2 content, from pure TiO2 (100% TiO2) to Ag/1TiO2 (25% TiO2), the Ag content increases, resulting in a decrease of the luminescence intensity of the composite materials The contact between AgNPs and TiO2 NP should have led to luminescence quenching These results also confirmed the difference in the TiO2 (and Ag) content in the composite materials In addition, Figure 2b demonstrates the Raman spectra of Ag/TiO2 nanocomposites in comparison to that of TiO2 NPs A red shift of 4−6 nm can be observed at the dominant peak of TiO2 (146 cm−1 in the TiO2 Raman spectrum) in the presence of AgNPs at different ratios This band represents the Eg optical Raman mode of anatase TiO2.42 Upon an increase of the Ag content in the nanocomposites, this peak is shifted further toward longer wavelengths This indicates the incorporation between AgNPs and the crystal structure of TiO2 NPs Information about other characteristic peaks of the nanocomposite materials will be further discussed in the following part 3.3 SERS Sensing Performance of Ag/TiO2 Nanocomposites to Detect Urea Figure 3a shows the SERS spectra of urea on five different substrates, including AgNPs and Ag/1TiO , Ag/2TiO , Ag/3TiO , and Ag/4TiO composites Compared to AgNPs, with all TiO2-containing substrates, the spectra show characteristic bands of TiO2 NPs The strong band at 150 cm−1 represents the Eg optical Raman mode of anatase TiO2 In addition, the bands at 398, 516, and 638 cm−1 are assigned to the B1g, A1g, and Eg modes of anatase TiO2, respectively.42,43 A band at 238 cm−1 appears in all samples It could be associated with the Ag−N stretching mode.44,45 It can be observed that from Ag/1TiO2 to Ag/ 4TiO2, the intensity of the band at 150 cm−1 increases with a decrease of that at 238 cm−1 These changes are associated with the ratio of the TiO2 and Ag contents in the nanocomposites because Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2 contain 25%, 33%, 50%, and 75% of TiO2, respectively Concerning the characteristic band of urea at 1010 cm−1, it is obvious that employing Ag/1TiO2, Ag/2TiO2, Figure SEM images of (a) Ag/1TiO2, (b) Ag/2TiO2, (c) Ag/ 3TiO2, and (d) Ag/4TiO2 powders D https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure (a) XRD patterns of Ag/3TiO2 (b) Raman spectra of TiO2, Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2 (c) PL spectra of TiO2, Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2 (d) Zoomed-in view of the PL spectra of Ag/1TiO2 and Ag/2TiO2 and Ag/3TiO2 nanocomposites as SERS substrates resulted in a higher intensity of the SERS signal of urea, in comparison to the use of AgNPs The presence of TiO2 is the key for improvement of the CT rate because employing substrates containing high percentages of TiO2 (i.e., 33% and 50%) resulted in a higher SERS intensity than using a substrate containing a lower percentage of TiO2 (i.e., 25%) However, on the Ag/4TiO2 substrate, we could not detect the characteristic peak of urea By increasing the TiO2 content up to 75%, we decreased the Ag content Because the presence of AgNPs is essential for urea to experience the SERS effect, this low Ag content might have limited the SERS performance of the material The disappearance of the band at 238 cm−1 also represents this low Ag content Because the substrate of Ag/4TiO2 did not improve the SERS performance of the urea sensors compared to AgNPs, this material was not employed for the following experiments Because the difference in the SERS intensity of urea while using Ag/2TiO2 and Ag/3TiO2 is not clear, we selected one of those two, Ag/3TiO2, to evaluate the sensitivity of Ag/TiO2 substrates in the detection of urea Five samples of urea in water were prepared at different concentrations, from 10−3 to 10−5 M Subsequently, their SERS spectra were recorded (Figure 3b) As expected, the intensity of the band at 1010 cm−1 increased with an increase of the urea concentration The plot of the logarithmic SERS intensity at 1010 cm−1 against the urea concentration within that range is shown in Figure 3c, representing a good linear relationship with a linear regression of 0.97 Thanks to the presence of TiO2, the urea sensor based on the Ag/3TiO2 nanocomposite had a limit of detection (LOD) of 4.2 × 10−5 M, which is lower than those of most of the reported noble-metal-based SERS sensors for urea (Table 1) The calculation of the LOD is shown in the Supporting Information In addition, the reproducibility of the method was studied as five Ag/3TiO2 substrates were prepared independently, using the same protocol, to measure the SERS signal of urea (10−3 M; Figure 3d) The relative standard deviation (RSD) was calculated to be 8.53%, indicating good reproducibility of the sensor 3.4 PIERS Sensing Performance of AgNPs and Ag/ TiO2 Nanocomposites to Detect Urea In a continuation of the effort to further improve the SERS performance of the urea sensors, the PIERS technique was applied The TiO2containing substrates, including Ag/1TiO2, Ag/2TiO2, and Ag/3TiO2, experienced UV exposure for 30 Subsequently, the SERS signals of urea (10−3 M) were collected on those UV-preirradiated substrates To confirm the importance of TiO2, a similar experiment was carried out on the substrate of AgNPs In the absorption spectrum (Figure S5), TiO2 NPs exhibit a broad spectrum in the UV region In several previous reports, authors selected UVC light (254 nm) for UV irradiation for TiO2-containing nanocomposites.23,46 In this study, we selected the UV source with λ = 365 nm, which lies within the absorption region of TiO2 NPs In addition, the success of E https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure (a) SERS spectra of urea on five substrates of AgNPs and Ag/1TiO2, Ag/2TiO2, Ag/3TiO2, and Ag/4TiO2 nanocomposites (b) SERS spectra of urea (10−5−10−3 M) on Al/3TiO2 (c) Plot of the logarithmic SERS intensity at 1010 cm−1 against the urea concentration (slope 0.71 ± 0.02; intercept 5.58 ± 0.02) (d) Reproducibility of the SERS sensor for urea detection based on Ag/3TiO2/Al substrates Table Several Reported SERS-Based Urea Sensors material functionalization LOD (M) linear range (M) laser (nm) ref Au@AgNP Ag dendrite Au nanostar Au/Cu hybrid nanostructure arrays Ag nanostructures on filter paper Ag/3TiO2 Ag/3TiO2 (UV irradiation, λ = 365 nm) Ag/3TiO2 (UV irradiation, λ = 400 nm) none none 11- mercaptoundecanoic acid + urease none none none none none 0.83 × 10−5 3.3 × 10−3 3.3 × 10−2 10−3 × 10−4 4.2 × 10−5 4.6 × 10−6 4.28 × 10−5 0.83 × 10−5−1.33 × 10−4 3.3 × 10−3−1.7 × 10−2 3.3 × 10−2−3.3 × 10−1 633 532 525 10−5−10−3 10−6−10−3 10−5−10−3 785 785 785 785 25 31 32 33 34 this work this work this work Ke et al using a xenon ozone-free arc lamp with λ > 400 nm for irradiation of an Au-TiO2 NPs system47 triggered us to try another UV light of longer wavelength Thus, we also selected UV light with λ = 400 nm, which is the longest wavelength within the UV range However, the absorption of TiO2 at this wavelength was not as good as that at λ = 365 nm The PL results for each UV source are presented in Figure S6 Concerning the substrates irradiated by UV light with λ = 365 nm, similar to the SERS signals without UV irradiation (Figure 3a), Ag/TiO2 nanocomposites showed a better SERS effect in comparison to AgNPs (Figure 4a) However, it is noticeable that, after UV exposure, the SERS performance of Ag/3TiO2 significantly surpassed that of Ag/2TiO2 This difference was not observed without UV exposure Therefore, it should have been the effect of this UV-irradiation step To clarify the effect of UV irradiation on each substrate, we compared the SERS signal of urea on each substrate with and without the UV-irradiation step Figure 4b demonstrates that UV exposure did not have any effect on AgNPs because no significant change was observed in the SERS signal In contrast, a significant increase in the intensity was observed in the SERS spectra of urea on three TiO2-containing substrates, including Ag/1TiO2, Ag/2TiO2, and Ag/3TiO2, thanks to the UVirradiation step (Figure 4c−e) Therefore, the PIERS phenomenon has occurred Moreover, this phenomenon is directly related to the presence of TiO2 in the nanocomposite materials The intensities of the peak of 1010 cm−1 of urea on the Ag/1TiO2, Ag/2TiO2, and Ag/3TiO2 substrates after UV irradiation were calculated to be 1.93, 3.42, and 7.45 times higher than those without UV irradiation, respectively A F https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure (a) PIERS spectra of urea (10−3 M) on five substrates: AgNPs, Ag/1TiO2, Ag/2TiO2, and Ag/3TiO2 (UV irradiation, λ = 365 nm) Comparison of the PIERS spectra of urea on AgNPs (b), Ag/1TiO2 (c), Ag/2TiO2 (d), and Ag/3TiO2 (e) with (green) and without (blue) UV irradiation intensity at 1010 cm−1 against the urea concentration within that range is demonstrated in Figure 5b with a linear regression of 0.96 The equation in Figure 5b was used to calculate the LOD of this sensor, resulting in a LOD of 4.6 × 10−6 M, which is lower than that without the PIERS effect Moreover, this is also a competitive LOD in comparison to other SERS-based sensors for urea detection (Table 1), and the linear range was also enlarged compared to that of the initial SERS sensors higher content of TiO2 resulted in better enhancement in the SERS signal after UV irradiation With the most impressive enhancement, Ag/3TiO2 was selected for the following experiments Thanks to the PIERS effect, Ag/3TiO2 preirradiated with UV light (λ = 365 nm) exhibited better sensitivity than that without UV exposure The SERS spectra of seven samples of urea in water at different concentrations, from 10−3 to 10−6 M, are shown in Figure 5a The plot of the logarithmic PIERS G https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure (a) PIERS spectra of urea (10−6−10−3 M) on Ag/3TiO2 after UV irradiation (λ = 365 nm) (b) Plot of the logarithm of PIERS intensity versus concentration at 1010 cm−1 (slope 0.71 ± 0.02; intercept 5.58 ± 0.02) (c) Reproducibility of the PIERS sensor for urea detection based on Ag/3TiO2/Al substrates In addition, the reproducibility of the sensor was also investigated using five independently prepared Ag/3TiO2 substrates (Figure 5c) The sensor exhibited good reproducibility because the RSD was calculated to be 8.56% However, with the other UV source (λ = 400 nm), the PIERS phenomenon did not occur Figure S7a compares the SERS spectra of urea (10−5−10−3 M) on Ag/3TiO2 without (Figure S7a) and with (Figure S7b) UV irradiation (λ = 400 nm) No significant change was observed In fact, the LOD of urea detection in this UV-irradiated sensor was calculated to be 4.28 × 10−5 M, which was only slightly higher than that of the non-UV-irradiated sensor Therefore, UV light with λ = 400 nm could not trigger the PIERS effect in our Ag/TiO2-based sensing system In addition to the preirradiation source, the effects of the preirradiation time were also investigated Figure S8 demonstrates the Raman spectra of urea (10−3 M) on Ag/ 3TiO2 substrates without and with UV irradiation (λ = 365 nm) for 10, 20, 30, 40, and 60 It is obvious that elongating the UV exposure time from 10 to 30 improved the performance of Ag/3TiO2 substrates because the intensity of the band at 1010 cm−1 increased rapidly However, longer exposure times, such as 40 and 60 min, did not cause any significant enhancement in the Raman signal, in comparison to 30 of preirradiation The experiments were repeated three times (Figure S9) Hence, elongating the preirradiation time might enhance the PIERS effects on the Ag/3TiO2 nanocomposite; however, it could reach the limit in 30 The persistence of the PIERS phenomenon on the Ag/ 3TiO2 nanocomposite was studied by collecting a series of PIERS spectra of urea at 5, 15, 30, 45, and 60 of UV preirradiation It took at least for the urea solution to completely dry on the substrate; therefore, the first measurement was performed after UV exposure Subsequently, the intensity of the PIERS signal decreased with relaxing time (Figure S10a) A total of 60 after preirradiation, the PIERS phenomenon nearly disappeared because the PIERS intensity was then only slightly higher than the SERS intensity (Figure S10b) The experiments were repeated three times (Figure S11) 3.5 Proposed Mechanism of the PIERS Phenomenon on Ag/TiO Nanocomposites and Effects of the Irradiation Wavelength Figure S12a demonstrates the structure of TiO2 before it was irradiated with UV light Its conduction band minimum (CBM) and valence band maximum (VBM) energy levels are −4.3 and −7.5 eV, creating a band gap of 3.2 eV.23 It has been reported that irradiating UV light (λ = 365 nm) could create “line defects” along the (001) direction of TiO2 because of cooperative oxygen removal,48 causing oxygen vacancies on the surface of the TiO2 semiconductor (Figure S12b) Subsequently, electron donor states are created at approximately 0.7 eV below its conduction band.23 However, only UV sources with the H https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure SERS (a) and PIERS (b) phenomena with substrates of the Ag/TiO2 nanocomposite materials appropriate wavelength could separate the electron−hole pairs and send electrons to these states Almohammed et al proposed that the energy of the incident photons had to be equal or larger than the band gap of the semiconductor to achieve an enhancement in the Raman signal after preirradiation with UV light and then laser excitation in the SERS experiment.49 The energies of two UV sources with λ = 365 and 400 nm were calculated to be 3.4 and 3.1 eV, respectively, using the well-known formula E = hc/ analyte solution However, this step was unavoidable because UV exposure for 30 might have damaged the organic analyte if the analyte solution was drop-casted onto the substrate prior to UV irradiation Therefore, to achieve the most effective enhancement of the PIERS signal, measurement should be performed as soon as the analyte solution is dried on the preirradiated substrate Concerning the CT process, the PIERS effect relies on CM enhancement and contributes to part to this enhancement Thus, the PIERS enhancement could not be too high (less than 10 times); however, it obviously improved the sensitivity (i.e., LOD and linear range) of the SERS-based sensing platform for the detection of a low Raman cross-section molecule, urea 3.6 Selectivity and Practicability of a Ag/3TiO2-Based PIERS Sensor for Urea Detection To investigate the selectivity of a Ag/3TiO2-based PIERS sensor for urea detection, we performed PIERS measurements of urea in the presence of interfering compounds that are possibly present in food and clinic samples, including glucose, ascorbic acid, and hydrogen peroxide (H2O2) A solution containing those three interfering compounds was prepared in distilled water with a concentration of 10−4 M for each compound Subsequently, urea was added to obtain concentrations of 10−4 and 10−5 M Because the nanocomposite was synthesized using a simple procedure without any specific functionalization, the PIERS sensor was not expected to exhibit high specificity and selectivity However, within the PIERS spectrum of the mixture containing 10−4 M urea, the characteristic band of urea (1010 cm−1) was still detectable in the presence of characteristic peaks of the interfering compounds, such as H2O2 (885 cm−1),53 ascorbic acid (1127 cm−1),54 and glucose (928, 1090, and 1127 cm−1)55 (Figure S13a) The PIERS spectra of the mixtures containing 10−4 and 10−5 M urea were compared with the PIERS spectra of urea in water at the same concentrations (10−4 and 10−5 M), as shown in parts b and c of Figure S13, respectively Obviously, the band at 1010 cm−1 can be detected in the complex spectra of the mixture, representing the presence of urea Although the presence of interfering compounds might have prevented urea from accessing the Ag/3TiO2 surface, leading to a slight decrease in the intensity of the 1010 cm−1 band, the nanocomposite still exhibited the ability to detect the target analyte of urea at (3) where λ is the wavelength, c is the speed of light in a vacuum, and h is the Planck constant The first one (λ = 365 nm) has a photon energy larger than the band gap of TiO2 (3.2 eV), while the energy of the other (λ = 400 nm) is lower than that band gap This explains the reason why, despite a small difference in the wavelength (44 nm), UV light at λ = 365 nm could trigger the PIERS effect, while the other could not Preirradiation with UV light at λ = 400 nm can be suitable for other semiconductors with narrower band gaps such as WO3 (∼2.6 eV),50 WS2 (1.0−2.1 eV),51 etc After UV irradiation at a suitable wavelength, the substrate then experienced excitation of a laser source (785 nm) Hence, the CT process in the PIERS phenomenon followed two steps, as demonstrated in Figure 6b (1) Thanks to UV irradiation, the electrons jump from the oxygen vacancy states to the conduction band of TiO2, and then they are injected into the Fermi level of Ag.23,52 As a result, the density of hot electrons on the surface of AgNPs increases (2) Thus, more electrons can be transferred to the analyte on the surface of the plasmonic NPs Therefore, the obtained Raman signal is magnified compared to the normal SERS phenomenon (Figure 6a) Because cooperative oxygen removal only occurs on the surface of the nanocomposites, it gradually increases, leading to a rise in the PIERS intensity However, it reaches the saturation state by 30 of UV irradiation Further elongating the exposure time leads to a negligible change in the intensity of the signal In addition, the defects on the surface of the nanocomposites caused by UV irradiation can be gradually healed upon exposure of the substrate to air, leading to a slow decay of the PIERS phenomenon.23 This surface healing may also have occurred because of the addition of an I https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Article Figure (a) Raman spectrum of a milk sample (red) on a Ag/3TiO2 substrate compared to those of TiO2 (blue) and a milk sample in the absence of Ag/3TiO2 (black) (b) PIERS spectra of urea at different concentrations (10−6−10−3 M) in milk on Ag/3TiO2 substrates Figure (a) SERS spectra of 4-NP (10−5−10−3 M) on Ag/3TiO2 substrates (b) Comparison of the PIERS and SERS spectra of 4-NP on Ag/ 3TiO2 (c) PIERS spectra of 4-NP (10−6−10−3 M) on Ag/3TiO2 substrates (d) Plot of the logarithmic PIERS intensity of 4-NP against the 4-NP concentration at 640 cm−1 concentrations as low as 10−5 M in the presence of interfering compounds The practicability of a Ag/3TiO2-based PIERS sensor was studied by spiking urea into bottled milk samples to obtain different concentrations (10−6−10−3 M) Before spiking with urea, the Raman spectra of the milk sample were recorded In the absence of the Ag/3TiO2 substrate, the milk sample showed no characteristic peak in the Raman spectrum (black; Figure 7a) In the presence of the nanocomposite substrate, several vibrational bands can be detected in the spectrum (red), including characteristic bands of TiO2 and a few new ones at 238, 308, and 551 cm−1 They might be due to the presence of protein in milk Upon the addition of urea, the characteristic band at 1010 cm−1 appeared (Figure 7b) This J https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org band is obviously detectable at concentrations down to 10−5 M The recovery rates range from 71% to 121% with RSDs of 7.24−12.89% (Table S1) The presence of protein in the milk samples might have interfered with and prevented urea residues from accessing the nanocomposite substrates, elevating the LOD of the sensor as well as affecting its accuracy and precision However, it is undeniable that, even with the interference in the real samples, the PIERS technique has allowed urea to be detected in milk at concentrations lower than those using Ag/3TiO2 without UV irradiation to sense urea in distilled water (LOD = 4.2 × 10−5 M) 3.7 Ag/3TiO2-Based PIERS Sensor for Another Low Raman Cross-Section Molecule: 4-Nitrophenol To confirm the potential of the PIERS technique as a promising tool to sense low Raman cross-section molecules, herein, we provide another example of 4-NP In our previous study, 4-NP was proven to be a low cross-section molecule with a large gap between its LUMO level and the Fermi level of Ag (0.71 eV) and low SERS signal.13 To improve the performance of the 4NP sensor, we employed Ag/3TiO2 as the substrate The SERS spectra of 4-NP at different concentrations (10−5−10−3 M) are shown in Figure 8a with characteristic bands at 640, 859, 1150, 1245, and 1324 cm−1 The band at 640 cm−1 represents the bending mode of C−C−C.56,57 The bands at 859 and 1324 cm−1 are assigned to the bending and symmetric stretching modes of the nitro group, respectively.56,57 The band at 1245 cm−1 represents a ring deformation mixed with the stretching mode of the nitro group.56,57 The band at 1155 cm−1 is attributable to the bending vibration mode of C−H.56,57 However, these characteristic bands are nearly undetectable at the concentration of 10−5 M Thanks to the assistance of the PIERS technique, the intensity of the signal of 4-NP (10−3 M) increased by 6.95 times (Figure 8b) As a result, the Ag/3TiO2based PIERS sensor exhibited better sensitivity because the characteristic peaks of 4-NP can be observed at lower concentrations in Figure 8c The plot of the logarithmic PIERS intensity at 640 cm−1 against the 4-NP concentration in the range of 10−6−10−3 M and its equation are shown in Figure 8d with a linear regression of 0.90 The LOD of the sensor was calculated to be 1.4 × 10−6 M Hence, the PIERS technique is also effective for improving the sensing performance of 4-NP sensors This result confirmed our hypothesis about the ability of PIERS as a tool to detect low Raman crosssection molecules Article based on Ag/3TiO2 was calculated to be 4.6 × 10−6 M With lower energy, UV irradiation at λ = 400 nm could not trigger the PIERS effect Thus, the wavelength of UV preirradiation also played a decisive role in the occurrence of the PIERS phenomenon By modifying the substrates and PIERS technique, we improved the sensing performance of AgNPsbased urea SERS sensors In real samples of bottled milk, the Ag/3TiO2-based PIERS sensor also exhibited the ability of urea detection at concentrations as low as 10−5 M PIERS is a promising tool to detect low Raman cross-section molecules ■ ASSOCIATED CONTENT sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.2c03524 Calculations of LOD and RSD, CV curves of urea in PBS and SERS spectra of urea (10−3 M) on AgNPs, CV of urea with five cycles, SEM images of AgNPs, EDS spectra of Ag/TiO2 nanocomposites, absorption spectra of TiO2 NPs, PL of two UV sources, comparison of the urea detection performances using a Ag/3TiO2 nanocomposite without preirradiation and with 400 nm preirradiation, Raman spectra of urea (10−3 M) on Ag/ 3TiO2/Al substrates without and with preirradiation for different periods of time (10−60 min) repeated three times, PIERS spectra of urea (10−3 M) after different relaxing times repeated three times, comparison of the PIERS signal of urea (10−3 M) on a Ag/3TiO2/Al substrate after 60 of relaxing time and the SERS signal of urea on the same substrate without UV irradiation, illustration of the TiO2 nanosrystal structure before and after preirradiation with UV light, PIERS spectra of the mixture (urea, glucose, ascorbic acid, and H2O2), and practicability of a Ag/3TiO2-based PIERS sensor to detect urea in milk samples (PDF) ■ AUTHOR INFORMATION Corresponding Authors Ha Anh Nguyen − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam; orcid.org/ 0000-0002-1183-5041; Email: anh.nguyenha@phenikaauni.edu.vn Anh-Tuan Le − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam; Faculty of Materials Science and Engineering, Phenikaa University, Hanoi 12116, Vietnam; Email: tuan.leanh@phenikaa-uni.edu.vn CONCLUSIONS In this study, urea is regarded as a low-cross-section molecule because the gap between its LUMO level and the Fermi level of Ag is relatively large The low rate of CT was then confirmed by its weak SERS signal on the substrate of AgNPs To improve the performance of the urea SERS sensors, a set of Ag/TiO2 nanocomposite materials were employed as SERSactive substrates instead of single AgNPs Ag/1TiO2, Ag/ 2TiO2, and Ag/3TiO2 containing 25%, 33%, and 50% TiO2, respectively, improving the sensitivity of the SERS sensing platform for urea detection thanks to CT via the contacts between Ag and TiO2 The use of Ag/3TiO2 resulted in a LOD of 4.2 × 10−5 M Thanks to preirradiation with UV light (λ = 365), the PIERS phenomenon occurred, leading to enhancement in the Raman signal compared to the initial SERS experiments Enhancements of 1.93, 3.42, and 7.45 times the signal intensity were achieved with the use of Ag/1TiO2, Ag/2TiO2, and Ag/3TiO2 The LOD of the urea PIERS sensor Authors Quan Doan Mai − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam; orcid.org/ 0000-0003-2931-0822 Thi Lan Huong Phung − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam; Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi 10000, Vietnam Ngo Xuan Dinh − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam Quang Huy Tran − Phenikaa University Nano Institute, Phenikaa University, Hanoi 12116, Vietnam Tri Quang Doan − International Training Institute for Materials Science, Hanoi University of Science and Technology, Hanoi 10000, Vietnam K https://doi.org/10.1021/acsanm.2c03524 ACS Appl Nano Mater XXXX, XXX, XXX−XXX ACS Applied Nano Materials www.acsanm.org Anh Tuan Pham − Faculty of Materials Science and Engineering, Phenikaa University, Hanoi 12116, Vietnam; Vicostone Joint Stock Company, Phenikaa Group, Thach That, Hanoi 10000, Vietnam (10) Haldavnekar, R.; Venkatakrishnan, K.; Tan, B Nonplasmonic semiconductor quantum SERS probe as a pathway for in vitro cancer detection Nat Commun 2018, (1), 3065 (11) Langer, J.; Jimenez de Aberasturi, D.; Aizpurua, J.; AlvarezPuebla, R A.; Auguié, B.; Baumberg, J J.; Bazan, G C.; Bell, S E J.; Boisen, A.; Brolo, A G.; Choo, J.; Cialla-May, D.; Deckert, V.; Fabris, L.; Faulds, K.; García de Abajo, F J.; Goodacre, R.; Graham, D.; Haes, A J.; Haynes, C L.; Huck, C.; Itoh, T.; Käll, M.; Kneipp, J.; Kotov, N A.; Kuang, H.; Le Ru, E C.; Lee, H K.; Li, J.-F.; Ling, X Y.; Maier, S A.; Mayerhöfer, T.; Moskovits, M.; Murakoshi, K.; Nam, J.-M.; Nie, S.; Ozaki, Y.; Pastoriza-Santos, I.; Perez-Juste, J.; Popp, J.; Pucci, A.; Reich, S.; Ren, B.; Schatz, G C.; Shegai, T.; Schlücker, S.; Tay, L.-L.; Thomas, K G.; Tian, Z.-Q.; Van Duyne, R P.; Vo-Dinh, T.; Wang, Y.; Willets, K A.; Xu, C.; Xu, H.; Xu, 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with coffee ring effect Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2019, 36 (6), 851−862 Complete contact information is available at: https://pubs.acs.org/10.1021/acsanm.2c03524 Author Contributions † Q.D.M and H.A.N contributed equally to this work Author Contributions Q.D.M.: conceptualization, validation, investigation, writing� original draft H.A.N.: conceptualization, methodology, formal analysis, writing�original draft T.L.H.P.: validation, investigation N.X.D.: conceptualization, formal analysis Q.H.T.: methodology, validation T.Q.D.: methodology, formal analysis A.T.P.: methodology, validation, supervision A.T.L.: conceptualization, methodology, supervision, project administration, writing�review and editing Notes The authors declare no competing financial interest ■ ACKNOWLEDGMENTS This research was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) through a fundamental research project (103.02-2019.01) The authors 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plays the role of a biomarker for kidney diseases in humans Overall, the detection of urea is important for both environmental and food safety and health... performance of the AgNPs-based SERS sensing platform for urea detection The LUMO level of urea was calculated using electrochemical cyclic voltammetry (CV) and compared to the Fermi level of Ag... improved the sensitivity (i.e., LOD and linear range) of the SERS-based sensing platform for the detection of a low Raman cross- section molecule, urea 3.6 Selectivity and Practicability of a Ag/3TiO2-Based