A new dual-spectroscopic strategy for the direct detection of

May 29, 2019 - ... detection of aristolochic acid I (AAI) is performed by surface-enhanced Raman spectroscopy (SERS), and its bioproduct, aristololact...
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A new dual-spectroscopic strategy for the direct detection of aristolochic acids in blood and tissue Lei Ouyang, Qian Zhang, Guina Ma, Lihua Zhu, Youqin Wang, Zhilin Chen, Yuling Wang, and Lei Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00442 • Publication Date (Web): 29 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Analytical Chemistry

A new dual-spectroscopic strategy for the direct detection of aristolochic acids in blood and tissue Lei Ouyang,a, b, † Qian Zhang,c, † Guina Ma,d Lihua Zhu,a, b,* Youqin Wang,e Zhilin Chen,c Yuling Wang,f Lei Zhaoc, * School of Chemistry and Chemical Engineering, Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Huazhong University of Science and Technology, Wuhan 430074, China. b Shenzhen Institute of Huazhong University of Science and Technology, Shenzhen, 518000, China c Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China d Radiology Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China e Department of Pediatric, Renmin Hospital, Hubei University of Medicine, Shiyan, 442000, China f Department of Molecular Sciences, Macquarie University, NSW 2109, Australia a

ABSTRACT: Aristolochic acids (AAs) contained in herbal plants are implicated in multiple organ injuries and have a high mutational burden in upper tract urothelial cancers. The currently available techniques for monitoring AAs include LC (liquid chromatography) and LC/MS (mass spectrometry), but the application of these approaches are limited due to the complex sample preparation and derivatization steps. Therefore, there is an urgent need to develop efficient methods for identifying and quantifying AAs. Here, we present a new dual-spectroscopic approach for the direct detection of AAs from blood and tissue samples; the detection of aristolochic acid I (AAI) is performed by surface-enhanced Raman spectroscopy (SERS), and its bioproduct, aristololactam (AAT), is detected by fluorescence spectroscopy based on their distinctive spectral response. Furthermore, a graphene assisted enrichment coupled with a magnetic retrieval strategy was developed to enhance SERS sensitivity toward AAI. Our method was successfully applied to directly determine both AAI and AAT from blood, liver and kidney of rats. The potential for real-world application was demonstrated by continuously monitoring AAI and AAT in rat blood and tissues after AAI feeding. The results showed that AAI was gradually metabolized to AAT and transported to different organs. It was found that the metabolism of AAI took place in the kidney, but AAT residue was detected in both liver and kidney, which might be related to long-term toxicity and gene mutation. The proposed dual-spectroscopic strategy is applicable to long-term toxicology research and to the direct diagnosis of AAI-induced organ injury.

Introduction Aristolochic acids (AAs) are a group of herbal components present in genera Aristolochia and some other plants.1 Many of these plants are used as herbal medicines to treat tumors, rheumatism, and pneumonia.2, 3 However, epidemiological studies show that AAs exposure is associated with a high risk of nephrotoxicity and upper urinary tract urothelial cell carcinoma. The misuse of aristolochic acid I (AAI) containing herbs in weight loss drugs resulted in several hundred cases of kidney failure in Belgium in the early 1990s.4 Recently, their distinct mutational signatures in the genomes of different tumors have been reported all over the world, such as intrahepatic bile duct carcinomas in China5 and hepatocellular carcinomas in southeast Asian countries.6 Strikingly high somatic mutation rates, even exceeding smoking-associated lung cancer, were revealed.7-9 Despite detailed indirect in vitro cell toxicity research and genomic mutation analysis,10, 11 the direct detection of AAI in blood and tissues for in vivo monitoring of its metabolic processes and clinical diagnosis of AAs induced organ injury is still a challenge due to its poor UV response and structural similarity to its bioproducts.

The current detection strategy for AAI from herbal remedies and botanical products is to transform it into fluorescence active molecules, such as aristololactam (AAT) through derivatization,12, 13 or to utilize mass spectrometry (MS) after chromatographic separation. 14-17 However, derivative methods suffer from poor yield at trace concentrations and are not efficient for biological samples since AAT is a bioproduct of AAI. Until now, only limited MS-based methods have been reported for its direct detection in fluids such as blood and urine. Liu et al. reported a LC-ESI-MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) approach for the detection of AAI in rat plasma.18 Later, Gu et al. proposed an HPLC-Q-TOF-MS (high-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry) method for effective detection of four types of AAs in rat serum.15 Due to the high cost and highly specialized instruments, none of these methods has been applied to AAI detection in tissue samples. Furthermore, it is important to detect AAI in vivo not only to fundamentally understand the metabolic processes but also to

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enable clinic diagnosis. Currently, no clinical method is available for diagnosing AAI induced organ injury. We propose a dual-spectroscopic strategy to directly detect both AAI and AAT without any derivatization or chromatographic separation by exploiting their distinct spectral responses. Fluorescently active AAT is directly detected by fluorescence spectrometry after extraction, while AAI is determined by surface enhanced Raman scattering (SERS) with a graphene enrichment strategy. Given the complex biological matrix of real samples, a pretreatment approach based on (NH4)2SO4-ethanol aqueous two-phase extraction (ATPE) is proposed to extract AAI and AAT with high efficiency and easy coupling to the spectral detection process. Our method was successfully applied in the analysis of trace AAI and AAT in rat blood and tissue samples, with sensitivity at the subnanomolar level. The potential of our approach is further demonstrated by continuously monitoring these two analytes in in vivo models. Metabolic analysis indicates that transformation of AAI to AAT took place in the kidney. The longer lived AAT was observed in both kidney and liver, which could be relevant in long-term multiple organ toxicity and gene mutation studies. Materials and Methods Chemical reagents Ferric chloride (FeCl3), ferrous sulfate (FeSO4), silver nitrate (AgNO3), sodium borohydride (NaBH4), aqueous ammonia (25%), ammonium sulfate ((NH4)2SO4), ethanol and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3Aminopropyl-trimethoxysilane (APTMS, 97%) was obtained from Aladdin Industrial Corporation (Seattle, USA). Aristolochic acid I (AAI) and aristololactam (AAT) were purchased from Sigma-Aldrich Inc. (St. Louis, USA) All reagents were of analytical reagent grade and used without further purification. Material Characterization UV-vis absorption spectra were recorded on an Evolution 201 UV-vis spectrometer (Thermo Scientific, USA). Transmission electron microscope (TEM) and energy dispersive spectroscopy (EDS) mapping were obtained from an S-TWIN HRTEM (FEI, USA) and a Tecnai G220. Scanning electron microscope (SEM) images were obtained with a SU8000 FESEM (Hitachi, Japan). Computational details Theoretical calculations concerning the geometry optimization were performed by using the Gaussian09 software package. Optimization of the molecular structures was performed by density functional theory (DFT) with the Becke’s 3 parameters and the Lee-Yang-Parr’s nonlocal correlation functional (B3LYP). The basis sets for C, N, O and H were 6-3111 G (d); for silver atoms, the valence and core electrons were described by the pseudopotential LanL2DZ basis set. The solvent (ethanol) effect was addressed with the conductor-like polarized continuum model (CPCM). Absorption and emission spectra of AAT were calculated using time-dependent DFT (TD-DFT) with TD-M062X TZVP methodology, which describes the excited states in terms of all possible single excitations from occupied to virtual orbitals (ethanol was used as solvent). Synthesis of Fe3O4-Ag NPs as SERS substrate Fe3O4 NPs were synthesized by an ultrasonic assisted reverse coprecipitation method.19 The shell of Ag was formed on the Fe3O4 NPs with APTMS as a linker. The fabrication method is based on our former work.20 Briefly, Fe3O4 (50 mg) dissolved in ethanol was mixed with APTMS (200 L); after magnetic

separation and washing with ethanol to rinse off unmodified linker, the surface-modified particles were dispersed in 0.1 mol L-1 AgNO3 (20 mL). After stirring for 1 h, 20 mL of 0.2 mol L-1 NaBH4 was added dropwise into the solution with a burette at an addition rate at about one drop per second. After further stirring for 45 min. The particles were separated with a magnet, washed and finally redispersed in 20 mL water. In vivo model establishment and biochemical testing of real samples Blood and tissue samples were obtained from rats fed with AAI. Male adult SD rats (160-250 g) were purchased from the laboratory animal center of Tongji Medical College, Huazhong University of Science and Technology. Rats were maintained under standard laboratory conditions and fed a normal diet and water ad libitum. All study protocols were approved by the Institutional Animal Care and Use Committee at Tongji Medical College of Huazhong University of Science and Technology and followed internationally accepted principles and the Guidelines for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology. AAI was dissolved in saline to a concentration of 1 mg/mL and daily administered intraperitoneally at a dose of 50 mg/kg bodyweight. Then, the rats were sacrificed, with blood collected via abdominal aorta at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h and 48 h followed by cervical dislocation. For freshly frozen tissues, the whole liver and kidney were excised and stored at −80°C. The collected blood specimens were centrifuged at 3 000 g for 10 min, and the supernatant was stored at −80°C until further analysis. The kidney and liver function of the rats treated with AAI at different times were also tested to determine correlation with AAI and AAT levels detected in blood and tissues. Real sample preparation The tissue samples were weighed and homogenized with saline to form homogenates for extraction. Blood sample was directly used for extraction. The sample was first mixed with ethanol, then (NH4)2SO4 was added and the solution was vortexed. Kept still for 3 min for phase separation, the targets were extracted into the upper phase. The volumes of each phase were obtained according to the phase interface. The ethanol mass ratio of the total extraction solution was tested from 19% to 25% and the (NH4)2SO4 mass ratio from 16% to 24% to optimize extraction efficiency. For real blood and tissue samples, to fully separate the targets from the complex matrix, after extraction the solution was centrifuged, and the upper phase was used for further detection. Part of the upper phase was used for fluorescence, and another part (0.5 mL) was mixed with the Fe3O4-Ag dispersion (20 µL) and graphene (50 µL for 20 min). The magnetic particles were separated with a magnet and dipped onto the slide for SERS detection. For comparison, the same sample was analyzed via HPLC. Before HPLC detection, the extracted AAI was converted to AAT based on previous work.13 SERS, fluorescence and HPLC detection Raman and SERS spectra were collected on a DXR confocal Raman microscope equipped with a CCD detector (Thermo Scientific, USA). A laser at 532 nm was used as excitation source with a power of 3.0 mW, and exposure time was 1 s with 10 accumulations. Fluorescence spectra were obtained on an FP-6200 Spectrofluorometer (JASCO, Japan) for AAT detection. The excitation wavelength was 393 nm, and the emission spectra were recorded from 400 to 700 nm; the maximum emission wavelength was approximately 474 nm. Ten spectra were

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Analytical Chemistry collected for each sample and averaged for quantitation. HPLC analysis was performed using a 1260 LC system (Agilent, USA) coupled with a diode array detector (DAD) and fluorescence detector (FLD). A reversed-phase Spherisorb C18 column (150 mm × 4.6 mm i.d., 5 µm, Waters, USA) was used to separate the targets. Methanol and HAc (1%) were used as the mobile phase with a ratio of 30:70 (v/v). The flow rate was 1.0 mL min−1, the injection volume was 20 µL and the column temperature was maintained at 35°C. For detection with DAD, the detection wavelength was 320 nm. The excitation and emission wavelengths were 393 nm and 474 nm for FLD, respectively. Results and discussion Spectral characteristics of AAI and AAT AAI and AAT coexist in biological fluid and tissues, and they have very similar molecular structures (Figure 1a and 1b). Their core structures are similar, but the functional groups -COOH and NO2 in AAI are converted to a lactam ring in AAT (indicated with dashed circles in a and b). Such structural differences lead to their distinct spectral responses. As shown in Figure 1c, characteristic Raman peaks were detected for 5 ppm AAI by using plasmonic Fe3O4-Ag particles as the enhancing substrate. However, poor SERS response was noted for AAT, even at a concentration of 50 ppm. SERS enhancement is a local field effect that occurs when the molecules are close to the surface of the substrate (within several nanometers); the SERS response originates from interaction with the Ag surface. The absorption orientation was further studied by analysis of its SERS fingerprint. Compared to its Raman spectrum, obvious peak shifts were observed in the SERS spectrum of AAI. For example, the peaks at 1545 cm-1, 1346 cm-1 and 1334 cm-1 in the normal Raman spectrum have shifted to 1538 cm-1, 1317 cm-1 and 1293 cm-1 in the SERS spectrum. Based on their assignments (Table S1 in Supporting Information),21 these peaks are attributed to the -COOH and -NO2 groups. The shifts of other peaks are also linked to the close interaction of AAI to the Ag surface since they are assigned to the nearby benzene ring. Based on these results, the adsorption of AAI onto the Ag shell was attributed to -COOH and -NO2. Such intense interaction between Ag and -COOH/-NO2 has also been validated by DFT simulation, as shown in Figure 1d. The molecular orientation of AAI after being adsorbed onto the Ag surface clearly shows a strong interaction of -COOH/-NO2 and Ag atoms. The distance between closest oxygen atoms to the surface of Ag atoms was calculated to be only about 1.97-2.40 Å, which ensured the excellent enhancement of the Raman signals of AAI molecule. It should be mentioned that our coreshell structure was formed by using NaBH4 as the reducer without any other surfactants, and the clean surface of Ag shell also benefits to the close interaction. On the other hand, the large steric hindrance and the lack of such groups lead to weak adsorption of AAT. It should be mentioned that the presence or lack of -COOH and -NO2 groups also leads to the differing fluorescence responses. As shown in Figure 1e, despite similar UV responses, completely different fluorescence responses were obtained at the same concentration. Although AAI has polycyclic aromatics, no native fluorescence response was found. The presence of the two electron-withdrawing groups (-COOH and-NO2) restrain its molecular fluorescence. However, AAT exhibits intense fluorescence because the electron-withdrawing groups are

converted into a lactam ring.22 The main contribution to the excitation and emission of AAT was computed to occur between orbitals No. 76 HOMO and No. 77 LUMO. Their isodensity plots are presented in Figure 1f. Based on their unique spectral responses, it is feasible to determine AAI with SERS and determine AAT with fluorescence spectroscopy, respectively. Additional experiments were performed to confirm the spectral detection of individual AAI and AAT without interference. It was observed that the addition of AAI at concentrations from 0.05 ppm to 5 ppm exhibited no observable interferences to the fluorescence spectrum of 0.05 ppm AAT (Figure S1). Similar results were found for SERS detection of AAI; no characteristic peaks of AAT were detected when its concentration was as large as 50 ppm (with AAI at 0.5 ppm). These results indicated the lack of spectral interference, ensuring specific detection.

Figure 1 Chemical structures and spectral characteristics of AAI and AAT. Molecular structures of (a) AAI and (b) AAT. (c) SERS response of AAI and AAT on Fe3O4-Ag substrate compared to Raman spectra of their solids. (d) Optimized molecular orientation of AAI adsorbed on Ag NPs from DFT simulation. (e) UV-vis absorption spectra of AAI and AAT (10 ppm) and their fluorescence spectra (1 ppm) with excitation at 393 nm. (f) Isodensity plots of frontier molecular orbitals contributing to the fluorescence excitation and emission, computed from DFT simulations.

Working scheme for AAI and AAT detection Due to the complex coexisting components in biological samples, it is important to extract AAI and AAT from the matrix to eliminate interference. Thus, the (NH4)2SO4-ethanol aqueous two-phase extraction (ATPE) was applied to achieve this purpose. This strategy offers an alternative to traditional liquid-liquid extraction because it involves an environmentally friendly solvent, has lower cost and is easy to perform.23-26 It has been shown that because of the mild extraction solvent, it

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is possible to greatly reduce the loss of targets that results from the absorption and embedding during the precipitation of massive proteins.27, 28 As shown in Figure 2a, homogenized AAI- and AAT-containing samples were mixed with ethanol; then, a defined quantity of (NH4)2SO4 was added, inducing a two-phase separation: the upper phase was the ethanol surplus phase, and the lower phase was the salt surplus phase. The benzene rings in the structures of both AAI and AAT make them more soluble in ethanol than water, which would lead to their extraction into the ethanol surplus phase. AAI and AAT were extracted into the ethanol surplus phase after aqueous two-phase extraction. One portion of the upper phase was directly used for fluorescence detection of AAT, while the other portion was used for SERS detection. The mass fractions of (NH4)2SO4 (from 16% to 24%) and ethanol (from 19% to 25%) were adjusted according to the phase diagram.27 Mass fractions of (NH4)2SO4 and ethanol at 20% and 22% were chosen, which ensured that the extraction efficiencies for both AAI and AAT were higher than 95% at those mass fractions (Figure S2). Using the parameters optimized above, the extractions was performed, and their SERS and fluorescence spectra in both phases are shown in Figure 2b and c. Intense SERS and fluorescence responses were obtained from the upper phase, while no observable signals were found in the lower phase. The Raman peak at 970 cm-1 was attributed to the presence of (NH4)2SO4. These results have also been cross compared with HPLC: neither AAI nor AAT was detected in the lower phase, indicating excellent extraction efficiency for both AAI and AAT. It should be mentioned that, with our extraction strategy, no obvious interference from the biomatrix was evident in either SERS or fluorescence spectra after extraction. The blood and tissue samples from rats without AAI feeding were used as biomatrix, in which different concentrations of AAI (50 ppb 5 ppm) and AAT (5 ppb - 500 ppb) were added and extracted with our sample preparation strategy. The spectra of AAI and AAT in standard solutions and in tissue extracts were compared (Figure S3 a and b showed the spectra of 5 ppm AAI and 0.5 ppm AAT). Similar spectral responses were obtained, indicating high extraction efficiency and good selectivity for our spectral sensing method. Irrespective of the type of sample, the recovery of the added standard was always within the range of 90-105%. Possible interference from components that might be extracted into the ethanol surplus phase was tested by monitoring the upper phase via HPLC. As shown in Figure S3 c and d, clean chromatograms were obtained from the extracted solutions; no obvious interference components were detected by either the DAD or the FLD detectors, illustrating the efficacy of our strategy to purify the targets. To further test for interference from possible components such as benzene amino acids, histidine (His), tyrosine (Tyr) and phenylalanine (Phe) (500 ppm) were mixed with AAI solutions (5 ppm). After the extraction, SERS detection was applied to the upper phase. As shown in Figure S4, no interference was observed, even though the concentration of the amino acids was 100 times higher than that of AAI. The anti-interference capabilities of our method can be attributed to the selective extraction of hydrophobic AAI into the ethanol surplus phase, while the hydrophilic amino acids remain in the salt surplus phase (as shown in Figure S5a), and to the stronger SERS response of AAI, which has an extended conjugated aromatic plane when compared to the small amino acids. Compared to the strong SERS response

of 5 ppm AAI, the SERS activity of these molecules with concentration of 50 ppm were much weaker (Figure S5b).

Figure 2 (a) Proposed strategy for sample preparation and detection of AAI and AAT. Fluorescence (b) and SERS (c) spectra of solutions after extraction (both upper and lower phases). The same concentration (shown in legends) of AAI and AAT were added to the water phase before extraction. Quantitative detection of AAI and AAT The detection performance of our proposed method for both AAI and AAT was tested over a wide concentration range. Figure 3a shows fluorescence spectra of AAT at different concentrations. By using the maximum emission peak at 474 nm as a representative peak, a linear range of 5 ppb to 500 ppb was obtained for intensity versus concentration; the limit of detection (LOD) was ~1 ppb (~2.9×10-9 mol L−1). For detection of AAI, plasmonic Fe3O4-Ag core-shell particles were used in the present work as the SERS substrate due to ease of separation and strong SERS enhancement.20, 29 SERS spectra at different concentrations of AAI are shown in Figure 3c; one characteristic peak at 1314 cm-1 was chosen for evaluation. The intensity at this peak was plotted against concentration, as shown in Figure 3d. A linear correlation over the range of 200 ppb to 10 ppm was obtained, and the LOD was approximately 100 ppb (~2.9×10-7 mol L−1). For each concentration, ten spectra were obtained and averaged for quantitation. As shown in Figure S6, good repeatability was obtained, with an relative standard deviation (RSD) of approximately 10%. The enhancement factor (EF) of our substrate for AAI was calculated to be approximately 8.1×105 by comparison with Raman spectra of AAI solutions without SERS enhancement (Figure S7). We also applied HPLC, with both DAD and FLD detectors, after derivatization. As shown in Figure S8, the LOD for both AAT and AAI was approximately 0.2 ppm with DAD because of the weak UV response. After the derivatization, the sensitivity increased to 10 ppb. The SERS method proposed here provides better sensitivity compared to HPLC with DAD detector, the sensitivity of which is still not good enough for AAI detection in real samples, because its concentration may be reduced to ppb level after metabolism.

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Analytical Chemistry

Figure 3 (a) Fluorescence spectra of different concentrations of AAT. (b) Plot of fluorescence intensity at 474 nm versus concentration. (c) SERS spectra of AAI at different concentrations. (d) Plot of SERS intensity at 1314 cm-1 versus concentration. Further enhancing AAI sensitivity by graphene enrichment and magnetic retrieval The concentration of the extracted AAI from tissue sample is very low (