Detection of 3,4-Methylene Dioxy Amphetamine in Urine by Magnetically Improved Surface-Enhanced Raman Scattering Sensing Strategy

Abuse of illicit drugs has become a major issue of global concern. As a synthetic amphetamine analog, 3,4-Methylene Dioxy Amphetamine (MDA) causes serotonergic neurotoxicity, posing a serious risk to human health. In this work, a two-dimensional substrate of ITO/Au is fabricated by transferring Au nanoparticle film onto indium–tin oxide glass (ITO). By magnetic inducing assembly of Fe3O4@Au onto ITO/Au, a sandwich-based, surface-enhanced Raman scattering (SERS) detection strategy is designed. Through the use of an external magnet, the MDA is retained in the region of hot spots formed between Fe3O4@Au and ITO/Au; as a result, the SERS sensitivity for MDA is superior compared to other methods, lowering the limit of detection (LOD) to 0.0685 ng/mL and attaining a corresponding linear dynamic detection range of 5–105 ng/mL. As an actual application, this magnetically improved SERS sensing strategy is successfully applied to distinguish MDA in urine at trace level, which is beneficial to clinical and forensic monitors.


Introduction
Abuse of illicit drugs poses significant adverse impact on human health and has also resulted in a serious social security problem [1,2] since the late 1970s. Recently, MDA as a synthetic amphetamine analog, which is structurally related to a wide variety of other naturally and synthetic compounds such as amphetamine (a psychomotor stimulant), catecholamines, and mescaline (a potent hallucinogen), has been an important issue of global concern [3]. MDA is included in category I of psychotropic drugs in China [4] and in schedule I of controlled substances in America [5], but is permitted to be used for medical purposes. As an actual situation, MDA is currently one of the most popular substances used recreationally in North America [6]. However, there are many clinical cases indicating that MDA would cause serotonergic neurotoxicity [7] due to stimulant effect on the central nervous system [8]. MDA is more toxic than its close congener, 3,4-methylenedioxy-methamphetamine (MDMA, "Ecstasy") [9], and has significant addictive potential [10][11][12][13][14].

Instrumentation
UV−visible absorption spectra were recorded with a UV−visible spectrophotometer (Shimadzu, Kyoto, Japan UV-1800). SERS spectra were collected by a Raman imaging microscope (Thermo DXR2xi, Madison, WI, USA). A field-emission scanning electron microscope (SEM, JEOL6380LV) and transmission electron microscopy (TEM, JEOL JEM-2000 FX) were used to observe the morphology of nanomaterials. The morphology and structures of ITO/Au substrate were characterized by an atom-force microscope (AFM, Bruker Dimension Icon). The magnetic properties of the observed nanocomposites were evaluated by using a vibrating sample magnetometer (VSM, Lake Shore VSM-736). Ultivo Triple Quadrupole LC/MS (Agilent, No.1 Yishun Ave 7, Singapore) and Mas-sHunterC1.1 system were used to validate the SERS results.

Preparation of ITO/Au Two-Dimensional Substrate
Au NPs with an average size of 30 nm were prepared by citrate reduction according to Frens' method [45]. In brief, 250 μL HAuCl4·4H2O (0.1 mol/L) was boiled for 10 min in 100 mL ultrapure water. Then, 1 mL of freshly prepared 1% sodium citrate solution was dripped rapidly and stirred for 30 min. The volume of Au NPs was concentrated to one-tenth of the original volume for later use. Briefly, as-prepared Au NPs solution was centrifuged at 6000 r/min for 10 min to obtain different Au NPs sols with 0-, 2-, 5-, 10-, and 20-fold concentrations by adding required volumes of ultrapure water. Then, 1 mL raw or concentrated Au NPs solution was injected into a 10 mL beaker, followed by adding 1 mL of CYH as the driving agent. After adding 1 mL of ethanol into the solution, a dense gold nanofilm with a metallic luster was formed by assembly at the interface of

Instrumentation
UV−visible absorption spectra were recorded with a UV−visible spectrophotometer (Shimadzu, Kyoto, Japan UV-1800). SERS spectra were collected by a Raman imaging microscope (Thermo DXR2xi, Madison, WI, USA). A field-emission scanning electron microscope (SEM, JEOL6380LV) and transmission electron microscopy (TEM, JEOL JEM-2000 FX) were used to observe the morphology of nanomaterials. The morphology and structures of ITO/Au substrate were characterized by an atom-force microscope (AFM, Bruker Dimension Icon). The magnetic properties of the observed nanocomposites were evaluated by using a vibrating sample magnetometer (VSM, Lake Shore VSM-736). Ultivo Triple Quadrupole LC/MS (Agilent, No.1 Yishun Ave 7, Singapore) and MassHunterC1.1 system were used to validate the SERS results.

Preparation of ITO/Au Two-Dimensional Substrate
Au NPs with an average size of 30 nm were prepared by citrate reduction according to Frens' method [45]. In brief, 250 µL HAuCl 4 ·4H 2 O (0.1 mol/L) was boiled for 10 min in 100 mL ultrapure water. Then, 1 mL of freshly prepared 1% sodium citrate solution was dripped rapidly and stirred for 30 min. The volume of Au NPs was concentrated to one-tenth of the original volume for later use. Briefly, as-prepared Au NPs solution was centrifuged at 6000 r/min for 10 min to obtain different Au NPs sols with 0-, 2-, 5-, 10-, and 20-fold concentrations by adding required volumes of ultrapure water. Then, 1 mL raw or concentrated Au NPs solution was injected into a 10 mL beaker, followed by adding 1 mL of CYH as the driving agent. After adding 1 mL of ethanol into the solution, a dense gold nanofilm with a metallic luster was formed by assembly at the interface of water/CYH. The gold film could be easily transferred from the water/CYH interface onto abluent ITO glass (5 mm × 10 mm), which had been pretreated in boiling activation

Synthesis of Fe 3 O 4 @Au Nanoparticles
Fe 3 O 4 @Au nanoparticles were synthesized in accordance with our previously reported method [44]. In short, the mixture of 0.318 g FeCl 3 ·6H 2 O and 0.130 g FeCl 2 ·4H 2 O was dissolved in boiled IP 6 ultrapure solution. After stirring for 1 h, 1.2 mL NaOH (0.4 mol/L) and another 5 mL IP 6 was added successively into this mixture. The Fe 3 O 4 @Au nanoparticles were collected with a magnet, and again carefully rinsed and dispersed in the required volume of water. Then, 2.5 mL HAuCl 4 ·4H 2 O (1% wt) was injected into the solution and 5 mL sodium citrate (1% wt) was rapidly added to the solution after refluxing for 15 min. After heating for 45 min, Fe 3 O 4 @Au nanoparticles were collected by using a magnet.

Magnetically Improved SERS Detection
Fe 3 O 4 @Au nanoparticles and MDA solution were fully mixed in a volume ratio of 1:2; then, 30 µL of the mixture was dropped to the surface of the ITO/Au with the assistance of a magnet under the ITO/Au. Raman spectra were recorded by using a DXR2xi Raman microscope with a 50× objective and excitation laser at 785 nm with 6.0 mW power. An acquisition time of 0.1 s was applied to avoid the heat effect of the laser on the sample; 1000 accumulations were used so that more target molecules would approach the vicinity of hotspots, thus obtaining a good SERS signal-noise ratio. The whole SERS detection time for each sample required 100 s.

Pretreatment of Actual Samples
One milliliter of urine sample was spiked with 10 µL MDA standard solution to mimic an actual sample. Before the Raman test, 1 mL methanol was added to the urine sample and given a full shake. The mixture was centrifuged at 6000 r/min for 10 min after showing white precipitate of proteins. Nitrogen was then purged to remove excess methanol. The final volume of residue was maintained at 1 mL for later experiments. Figure 2A,C show the SEM and AFM images of ITO/Au, respectively. Clearly, continuous film is successfully self-assembled by~30 nm Au NPs over the whole surface of ITO in uniform distribution. The UV-visible spectra of pure Au NPs and ITO/Au are shown in Figure 3A. The novel peak at 650 nm of ITO/Au with respect to Au NPs means the certain aggregation of Au NPs in the assembly film to generate numerous SPR hot spots on the surface, which is very beneficial to amplification of SERS signals in the subsequent experiments.

Characterization of Materials
The morphology of the Fe 3 O 4 @Au was characterized by TEM. In Figure 2B, Au NPs with an average size of about 80 nm could be bound to the magnetic network nanostructure of Fe 3 O 4 (gray composite) via phosphate groups in dispersive way due to the presence of IP 6 . The Fe 3 O 4 @Au NPs were collected by a magnet and washed several times with ultrapure water to remove excess organic compounds. Additionally, the Raman scattering section of IP6 is quite small and has little effect on the SERS detection of MDA. The magnetic property of Fe 3 O 4 @Au was investigated with a vibrating sample magnetometer. As shown in Figure 2D, the curve with minor hysteresis loops indicates Fe 3 O 4 @Au has superior magnetic behavior. In Figure 3B

Optimization of Self-Assembly for ITO/Au
As noted above, the Au NPs monolayer in a large area was constructed by using a water/CYH interface. The reversible aggregation of Au NPs severely influences density of Au NPs in film, which is closely related to the SERS properties. In this work, different concentrations of Au NP sols were obtained by centrifugation and used for optimizing the assembly of Au nanofilms and finally preparing ITO/Au substrates. Figure 4 shows the SERS spectra of 10 −6 mol/L R6G solution recorded for different ITO/Au substrates. It indicates that the strongest SERS signal can be achieved when Au sol is concentrated 10 times, for instance, 10 mL to 1 mL. It is due to formation of an imperfect monolayer of An NPs at the ITO surface, which sufficiently generates numerous hot spots.

Optimization of Self-Assembly for ITO/Au
As noted above, the Au NPs monolayer in a large area was constructed by using a water/CYH interface. The reversible aggregation of Au NPs severely influences density of Au NPs in film, which is closely related to the SERS properties. In this work, different concentrations of Au NP sols were obtained by centrifugation and used for optimizing the assembly of Au nanofilms and finally preparing ITO/Au substrates. Figure 4 shows the SERS spectra of 10 −6 mol/L R6G solution recorded for different ITO/Au substrates. It indicates that the strongest SERS signal can be achieved when Au sol is concentrated 10 times, for instance, 10 mL to 1 mL. It is due to formation of an imperfect monolayer of An NPs at the ITO surface, which sufficiently generates numerous hot spots.

Optimization of Self-Assembly for ITO/Au
As noted above, the Au NPs monolayer in a large area was constructed by using a water/CYH interface. The reversible aggregation of Au NPs severely influences density of Au NPs in film, which is closely related to the SERS properties. In this work, different concentrations of Au NP sols were obtained by centrifugation and used for optimizing the assembly of Au nanofilms and finally preparing ITO/Au substrates. Figure 4 shows the SERS spectra of 10 −6 mol/L R6G solution recorded for different ITO/Au substrates. It indicates that the strongest SERS signal can be achieved when Au sol is concentrated 10 times, for instance, 10 mL to 1 mL. It is due to formation of an imperfect monolayer of An NPs at the ITO surface, which sufficiently generates numerous hot spots.

SERS Performance of Sandwich Structure of ITO/Au and Fe3O4@Au
SERS spectra of 10 −6 mol/L R6G on ITO/Au, Fe3O4@Au and sandwich ITO/Au and Fe3O4@Au were acquired. As clearly shown in Figure 5A wich-structure-based detection strategy contributes the greatest enhancem cated in Figure 5B, by magnetically inducing the sandwich structure, the l detection of R6G can be as low as 10 −10 mol/L. The homogeneity and reproducibility of the Raman signals from the SE are crucial aspects in the subsequent detection. The SERS intensities of R6G at 30 random sites across the entire sandwich structure of ITO/Au and Fe3O sically same as presented in Figure 6A. In Figure 6B, based on calculatio typical Raman peaks at 622 and 786 cm −1 , the relative standard deviations (R 5.25% and 6.83%, respectively, displaying good signal uniformity on 2D su benefits the subsequent qualitative detection of drugs.

SERS Performance of Sandwich Structure of ITO/Au and Fe 3 O 4 @Au
SERS spectra of 10 −6 mol/L R6G on ITO/Au, Fe 3 O 4 @Au and sandwich structure of ITO/Au and Fe 3 O 4 @Au were acquired. As clearly shown in Figure 5A, the sandwichstructure-based detection strategy contributes the greatest enhancement. As indicated in Figure 5B, by magnetically inducing the sandwich structure, the limit of SERS detection of R6G can be as low as 10 −10 mol/L.

SERS Performance of Sandwich Structure of ITO/Au and Fe3O4@Au
SERS spectra of 10 −6 mol/L R6G on ITO/Au, Fe3O4@Au and sandwich structure of ITO/Au and Fe3O4@Au were acquired. As clearly shown in Figure 5A, the sandwich-structure-based detection strategy contributes the greatest enhancement. As indicated in Figure 5B, by magnetically inducing the sandwich structure, the limit of SERS detection of R6G can be as low as 10 −10 mol/L. The homogeneity and reproducibility of the Raman signals from the SERS substrate are crucial aspects in the subsequent detection. The SERS intensities of R6G (10 −6 mol/L) at 30 random sites across the entire sandwich structure of ITO/Au and Fe3O4@Au are basically same as presented in Figure 6A. In Figure 6B, based on calculations using the typical Raman peaks at 622 and 786 cm −1 , the relative standard deviations (RSDs) were of 5.25% and 6.83%, respectively, displaying good signal uniformity on 2D substrate. This benefits the subsequent qualitative detection of drugs. The homogeneity and reproducibility of the Raman signals from the SERS substrate are crucial aspects in the subsequent detection. The SERS intensities of R6G (10 −6 mol/L) at 30 random sites across the entire sandwich structure of ITO/Au and Fe 3 O 4 @Au are basically same as presented in Figure 6A. In Figure 6B, based on calculations using the typical Raman peaks at 622 and 786 cm −1 , the relative standard deviations (RSDs) were of 5.25% and 6.83%, respectively, displaying good signal uniformity on 2D substrate. This benefits the subsequent qualitative detection of drugs.
For checking preparation reproducibility, the SERS intensities at 622 cm −1 of 10 −6 mol/L R6G solution were recorded from three random points on one ITO/Au of three different batches. As shown in Figure 7A, it shows promising fabrication reproducibility with an RSD of 6.32%. For checking preparation reproducibility, the SERS intensities at 622 cm −1 of 10 −6 mol/L R6G solution were recorded from three random points on one ITO/Au of three different batches. As shown in Figure 7A, it shows promising fabrication reproducibility with an RSD of 6.32%.
In addition, the storage stability of the SERS substrates is a crucial factor for practical applications. Herein, the long-term stability by SERS investigation is demonstrated in Figure 7B. Variation of SERS intensity recorded for 10 days is just 2.03%, showing excellent storage stability of this SERS substrate. Furthermore, the Fe3O4@Au also kept a commendable stability for ten days, as shown in Figure 7B. In all, magnetically inducing sandwich structure exhibits improved SERS sensing performance, including high sensitivity, signal homogeneity, acceptable preparation reproducibility, and long shelf-time stability.

Sensing Optimization
The different mixtures (volume ratios of 1:1, 1:2, and 2:1) of Fe3O4@Au and R6G (10 −6 mol/L) were explored for reaching an optimal SERS sensing condition. As shown in Figure 8, the strongest Raman signal was obtained for the 1:2 volume ratio.  For checking preparation reproducibility, the SERS intensities at 622 cm −1 of 10 −6 mol/L R6G solution were recorded from three random points on one ITO/Au of three different batches. As shown in Figure 7A, it shows promising fabrication reproducibility with an RSD of 6.32%.
In addition, the storage stability of the SERS substrates is a crucial factor for practical applications. Herein, the long-term stability by SERS investigation is demonstrated in Figure 7B. Variation of SERS intensity recorded for 10 days is just 2.03%, showing excellent storage stability of this SERS substrate. Furthermore, the Fe3O4@Au also kept a commendable stability for ten days, as shown in Figure 7B. In all, magnetically inducing sandwich structure exhibits improved SERS sensing performance, including high sensitivity, signal homogeneity, acceptable preparation reproducibility, and long shelf-time stability.

Sensing Optimization
The different mixtures (volume ratios of 1:1, 1:2, and 2:1) of Fe3O4@Au and R6G (10 −6 mol/L) were explored for reaching an optimal SERS sensing condition. As shown in Figure 8, the strongest Raman signal was obtained for the 1:2 volume ratio. In addition, the storage stability of the SERS substrates is a crucial factor for practical applications. Herein, the long-term stability by SERS investigation is demonstrated in Figure 7B. Variation of SERS intensity recorded for 10 days is just 2.03%, showing excellent storage stability of this SERS substrate. Furthermore, the Fe 3 O 4 @Au also kept a commendable stability for ten days, as shown in Figure 7B. In all, magnetically inducing sandwich structure exhibits improved SERS sensing performance, including high sensitivity, signal homogeneity, acceptable preparation reproducibility, and long shelf-time stability.

Sensing Optimization
The different mixtures (volume ratios of 1:1, 1:2, and 2:1) of Fe 3 O 4 @Au and R6G (10 −6 mol/L) were explored for reaching an optimal SERS sensing condition. As shown in Figure 8, the strongest Raman signal was obtained for the 1:2 volume ratio. It is well known that SERS intensities also depend on the excitation laser wavelength. Herein, three lasers with different wavelengths, 532, 633, and 785 nm, were used to record the SERS signals of MDA (100 μg/mL). As shown in Figure 9, when using 532 It is well known that SERS intensities also depend on the excitation laser wavelength. Herein, three lasers with different wavelengths, 532, 633, and 785 nm, were used to record the SERS signals of MDA (100 µg/mL). As shown in Figure 9, when using 532 and 633 nm lasers, the characteristic Raman peaks of MDA could barely be observed due to visible laser thermal carbonization of the surface species. By contrast, excitation laser at 785 nm is a suitable option for the SERS experiment. It is well known that SERS intensities also depend on the excitation laser wavelength. Herein, three lasers with different wavelengths, 532, 633, and 785 nm, were used to record the SERS signals of MDA (100 μg/mL). As shown in Figure 9, when using 532 and 633 nm lasers, the characteristic Raman peaks of MDA could barely be observed due to visible laser thermal carbonization of the surface species. By contrast, excitation laser at 785 nm is a suitable option for the SERS experiment.

SERS Detection of MDA
By magnetically inducing sandwich-structure-based SERS sensing strategy, the quantitative detection performance of MDA was observed. As illustrated in Figure 10A, ultrasensitive detection of MDA with a minimum detection concentration of 1 ng/mL could be achieved. Figure 10B shows that a linear relationship between the denary logarithm of MDA concentrations in aqueous solutions and SERS intensities (I714 cm −1 ) could be obtained in the range from 5 to 10 5 ng/mL with a reasonable correlation coefficient (R 2 = 0.9750, and corresponding regression equation: y = 1486.253x − 507.167). The LOD value was estimated to be 0.0685 ng/mL according to the IUPAC standard method (Formula (1)): where RSD is the relative standard deviation of three replicates of the same experiment and BEC is the absolute value of the intercept between the linear regression equation and the x-axis. More recently, effective 1 October 2017, the Substance Abuse and Mental Health Services Administration (SAMHSA) established new testing criteria for MDA, for which the confirmation cutoff concentration is 500 ng/mL. Clearly, the LOD of the

SERS Detection of MDA
By magnetically inducing sandwich-structure-based SERS sensing strategy, the quantitative detection performance of MDA was observed. As illustrated in Figure 10A, ultrasensitive detection of MDA with a minimum detection concentration of 1 ng/mL could be achieved. Figure 10B shows that a linear relationship between the denary logarithm of MDA concentrations in aqueous solutions and SERS intensities (I 714 cm −1 ) could be obtained in the range from 5 to 10 5 ng/mL with a reasonable correlation coefficient (R 2 = 0.9750, and corresponding regression equation: y = 1486.253x − 507.167). The LOD value was estimated to be 0.0685 ng/mL according to the IUPAC standard method (Formula (1)): where RSD is the relative standard deviation of three replicates of the same experiment and BEC is the absolute value of the intercept between the linear regression equation and the x-axis. More recently, effective 1 October 2017, the Substance Abuse and Mental Health Services Administration (SAMHSA) established new testing criteria for MDA, for which the confirmation cutoff concentration is 500 ng/mL. Clearly, the LOD of the sandwich-structure-based SERS method is far below the required threshold. Therefore, the magnetically inducing sandwich-structure-based SERS sensing protocol is expected to be used during the initial period for inspecting or monitoring drug dependency.
Biosensors 2022, 12, x FOR PEER REVIEW 9 of 12 sandwich-structure-based SERS method is far below the required threshold. Therefore, the magnetically inducing sandwich-structure-based SERS sensing protocol is expected to be used during the initial period for inspecting or monitoring drug dependency. For actual application, human urine from a health volunteer was spiked with a required amount of MDA standard solution. The characteristic peaks of MDA (1 μg/mL) in human urine can obviously be detected by the sandwich-structure-based SERS protocol, as shown in Figure 11A. Checking interference from bioactive molecules coexisting in complex physiological urine is particularly crucial for detection of MDA. As demon- For actual application, human urine from a health volunteer was spiked with a required amount of MDA standard solution. The characteristic peaks of MDA (1 µg/mL) in human urine can obviously be detected by the sandwich-structure-based SERS protocol, as shown in Figure 11A. Checking interference from bioactive molecules coexisting in complex physiological urine is particularly crucial for detection of MDA. As demonstrated in Figure 11B, the corresponding characteristic Raman band of MDA at 714 cm −1 is free from the interference of nicotine, cholesterol, uric acid (UA), methamphetamine (MAMP), and amphetamine (AMP). Consequently, by applying the sandwich-structurebased SERS assay, MDA can be easily distinguished in urine, which is beneficial to clinical and forensic monitors. For actual application, human urine from a health volunteer was spiked with a required amount of MDA standard solution. The characteristic peaks of MDA (1 μg/mL) in human urine can obviously be detected by the sandwich-structure-based SERS protocol, as shown in Figure 11A. Checking interference from bioactive molecules coexisting in complex physiological urine is particularly crucial for detection of MDA. As demonstrated in Figure 11B, the corresponding characteristic Raman band of MDA at 714 cm −1 is free from the interference of nicotine, cholesterol, uric acid (UA), methamphetamine (MAMP), and amphetamine (AMP). Consequently, by applying the sandwich-structure-based SERS assay, MDA can be easily distinguished in urine, which is beneficial to clinical and forensic monitors. To validate the reliability of the SERS detection method, LC-MS as a standard method was used to detect MDA in the same urine sample. In Table 1, the good detection recoveries depict the acceptable reliability of the SERS method.  To validate the reliability of the SERS detection method, LC-MS as a standard method was used to detect MDA in the same urine sample. In Table 1, the good detection recoveries depict the acceptable reliability of the SERS method. Moreover, we compared the detection results with other methods reported in the literature. As shown in Table 2, the sandwich-structure-based SERS strategy has the highest sensitivity and a wide concentration dynamic linear range. In short, compared to other methods, our sandwich-structure-based SERS strategy shows superior sensitivity, which is crucial for detecting low drug concentrations in biosamples.

Conclusions
In summary, a magnetically inducing sandwich structure was proposed for development of an SERS sensing platform through optimal preparation of ITO/Au substrate and Fe 3 O 4 /Au magnetic sorbs. Integrating the stability and homogeneity of a two-dimensional substrate of ITO/Au, and magnetic enrichment of Fe 3 O 4 /Au with magnetically inducing SPR hotspots, the novel SERS strategy exhibited ultrasensitive detection of MDA and good Raman signal reproducibility. Based on SERS intensity at 714 cm −1 , the SERS detection of MDA presented a good linear relationship from 5 to 10 5 ng/mL with LOD at 0.0685 ng/mL. In the future, the sandwich-based SERS protocol provides the possibility for rapid, sensitive, and reliable on-site detection of MDA.