Nitrogen and Sulfur Co-doped Carbon-Dot-Assisted Laser Desorption

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Nitrogen and Sulfur Co-doped Carbon Dots-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Imaging for Profiling Bisphenol S Distribution in Mouse Tissues Zian Lin, Jie Wu, Yongqiang Dong, Pei Si Xie, Yan Hao Zhang, and Zongwei Cai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02362 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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Analytical Chemistry

Nitrogen

and

Sulfur

Co-doped

Carbon

Dots-Assisted

Laser

Desorption/Ionization Time-of-Flight Mass Spectrometry Imaging for Profiling Bisphenol S Distribution in Mouse Tissues Zian Lin,†* Jie Wu,† Yongqiang Dong,† Peisi Xie,‡ Yanhao Zhang,‡ and Zongwei Cai†‡*

†. Ministry of Education Key Laboratory of Analytical Science for Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China ‡. Partner State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong, SAR, P. R. China

Corresponding author: Zian Lin; Zongwei Cai Postal address: Department of Chemistry, Hong Kong Baptist University Kowloon Tong, Hong Kong, SAR, P. R. China Fax: +852-34117348 E-mail: [email protected] (Z.A Lin); [email protected] (Z. Cai)

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ABSTRACT Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) is rapidly maturing as an innovative technique for spatial molecule (m/z > 1000 Da) profiling. However, direct identification of low-molecular-weight compounds (m/z < 600 Da) by MALDI-TOF MSI using conventional organic matrices remains a challenge due to the ionization suppression and serious matrix-related background interference. Furthermore, the heterogeneous cocrystallization that is inherent to organic matrices can degrade spatial resolution in MSI. Herein, we developed a negative ion surface-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (SALDI-TOF MSI) protocol to detect bisphenol S (BPS) and map its spatial distribution in the mouse tissues by applying nitrogen and sulfur co-doped carbon dots (N,S-co-doped CDs) as a new matrix through spraying. The SALDI-TOF MS and imaging parameters, such as matrix concentration, ionization mode and matrix deposition, were optimized to improve imaging performance. In comparison to organic matrices, the use of N,S-co-doped CDs in negative ion mode exhibited free matrix background interference, enhanced MS signal intensity, and provided high spatial resolution (acquired at ~50 µm) in the analysis of BPS, which allowed the sensitive detection of the target compound on the surface of tissue sections. Quantitative assessment was also made by spotting BPS standards directly on the tissue surface, and a good correlation between the color change and BPS concentrations was found. The corresponding detection limit as low as ~pmol level for BPS was observed with the direct visualization from MS images. Furthermore, the feasibility of the proposed

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SALDI-TOF MSI method was extended for in situ identification of exogenous BPS in the different tissues of mouse involving liver, kidney, spleen and heart for exposure and profiling its spatial localization at different administration times. In addition, the general applicability of the proposed method was also evaluated by SALDI-TOF MSI analysis of BPAF in tissues. These successful applications of SALDI-TOF MSI not only demonstrated its promising potential as an alternative to MALDI-TOF MSI in profiling small molecules in tissue sections, but also provided tremendous insight into the assessment of BPS exposure.

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Introduction Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) offers an innovative tool for direct analysis of tissue sections and provides the capability for simultaneously monitoring the spatial distribution of various compounds such as proteins, peptides, lipids, drugs and metabolites at the molecular level.1,2 Since the first introduction by Caprioli in 1997,3 the utility of MALDI-TOF MSI has been widely demonstrated in a variety of fields, with particular emphasis on biomedical applications like drug distribution and biomarker discovery.4-6 Typically, the MALDI-TOF MSI workflow involves several steps of tissue cryosectioning, matrix deposition, data acquisition, and image construction. In this workflow, the choice and application of matrix to the surface of tissue sections is regarded as the most important step for obtaining sensitive MS signal response of molecules with good homogeneity, reproducibility, and high spatial resolution.7,8 However, severe background interference caused by traditional organic matrices such

as

α-cyano-4-hydroxycinnamic

acid

(CHCA),

sinapinic

acid

(SA),

and

2,5-dihydroxybenzoic acid (DHB) would strongly suppress the ionization and mask the detection of small molecules in low mass region (m/z < 600 Da), thus limiting their applications to the analysis of some endogenous/exogenous small molecules, such as pharmaceutical drugs and metabolites.9-11 To address this issue, two major strategies have been proposed. One is to synthesize or search for new organic matrices as alternative, which can shift matrix ion clusters and fragment peaks away from low mass region or generate less background interference.7,12,13

4


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However, different MALDI matrices exhibit different ionization properties that is rather difficult to provide sufficient ionization of the target compounds.13 Furthermore, the formation of sweet spots through heterogeneous crystallization, and the delocalization and diffusion of analytes in tissue sections during organic matrix desposition are unavoidable.14 The other one is to exploit new nanomaterials as LDI substrates,15,16 instead of organic matrices. This approach is quite popular and often defined as surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS).17 In the past few years, different types of nanostructual matrices including porous silicon,18 metal- and metal oxide-based nanoparticles,19 carbon-based nanomaterials,20 and metal-organic frameworks21 have been experimented to be effective LDI substrates for small molecules. Recently, there has been an increasing interest in the development of nanomaterials-based SALDI-TOF MSI for the sake of enhanced chemical selectivity, good reproducibility, high spatial fidelity and resolution compared to MALDI-TOF MSI. In 2007, Northen et al.22 first introduced a semitransparent nanostructure-initiator mass spectrometry (NIMS) for imaging of biomolecules in tissue sample. Since then, the SALDI-TOF MS approaches that substitute the nanomatrices for organic matrices, including silicon,23 gold,24 silver,25 titanium oxide nanoparticles,26 colloidal graphite,27 graphene,28 and thin films of nanostructured indium tin oxide,11 have been developed for imaging and identification of small molecules in tissue sections with low chemical background. Despite the progress that have been made in SALDI-TOF MSI, the exploitation of new nanomatrices with easy preparation, high ionization efficiency, and excellent stability and dispersibility that are compatible for MSI

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are highly desirable. Carbon dots (CDs) are a newly emerged class of carbon-rich nanoparticles with diameters less than 10 nm.29 Due to the excellent optical and electronic transport properties, good biocompatibility, and chemical stability, CDs are deemed to be an ideal LDI substrate for the analysis of small molecules.30 More recently, the nitrogen-doped CDs (N-doped CDs)31 and nitrogen and sulfur-co-doped CDs (N,S-co-doped CDs)32 have been successively exploited

as nanomatrices for negative ion SALDI-TOF MS of

hydroxypolycyclic aromatic hydrocarbons and metabolites, respectively. Nevertheless, so far the advantages of N,S-co-doped CDs applied as nanomatrices for SALDI-TOF MSI have not been demonstrated yet. Bisphenol A (BPA) is well-known endocrine-disrupting chemical (EDC). Many evidences have showed that BPA contributed to a variety of adverse health effects.33 Use of BPA in consumer products has been prohibited around the world. As a result, many BPA analogs, including bisphenol B (BPB), bisphenol C (BPC), bisphenol S (BPS) and bisphenol AF (BPAF), are commonly used as BPA alternative. Nevertheless, limited studies to date have suggested that the analogs showed similar toxic and estrogenic effects to BPA, provoking many unpredictable adverse health effects.34,35 For instance, exposure to BPS can be linked to obesity and hepatic steatosis.36 Although analytical methods have been developed for in vitro BPS determination,37 few in vivo studies, especially for in situ identification and monitoring its spatial distribution in different tissues and organs, have been published to date.38

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In this present work, N,S-co-doped CDs were synthesized via a facile one-step hydrothermal treatment and served as a nanomatrix for SALDI-TOF MSI. To the best of our knowledge, this is the first example on applying the N,S-co-doped CDs-based SALDI-TOF MSI to demonstrate the localization of BPS in tissue sections for exposure. The as-prepared N,S-co-doped CDs were characterized by different techniques and compared with traditional organic matrices (e.g. CHCA, DHB) for the analysis of BPS from standard bisphenol mixture and the mouse tissue sections in both ion modes. Potential of BPS quantification on the tissue surface was evaluated by SALDI-TOF MSI analysis. In addition, the practical applications of the N,S-co-doped CDs-based SALDI-TOF MSI for in situ identification and mapping the spatial distribution of BPS in mouse kidney and liver tissue sections after exposure were demonstrated.

EXPERIMENTAL SECTION Synthesis of N,S-Co-doped CDs. The N,S-co-doped CDs were prepared by thermal treatment according to the previous work with some modification.39 In brief, CA (2.10 g) and L-cysteine (0.605 g) were dissolved in 3 mL water and heated at 70 ℃ for 24 h. Subsequently, the obtained syrup-like mixtures were transferred to stainless-steel autoclave, and then heated to 220 ℃ for 3 h with a heating rate of 10 ℃/min. Finally, the obtained products were dialyzed against deionized water using a dialysis bag (the cutoff molecular weight: 1 kDa) for one week, followed by drying in vacuum at room temperature. As a control, the pure CDs were also prepared according to the same method by using CA (2.10 g) as the single carbon source.

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Solution Preparation. Matrix solution of CHCA or DHB with a concentration of 7 mg/mL containing 0.2% formic acid in water/ACN (3:7 v/v) was prepared for MALDI-TOF MSI analysis (10 mg/mL CHCA or DHB for MALDI-TOF MS analysis). Different concentrations of the N,S-co-doped CDs in the range of 0.5-2.5 mg/mL were prepared with methanol/water (7:3 v/v) and applied to SALDI-TOF MS and imaging analysis. All bisphenols were individually dissolved in ACN at a concentration of 10 mM as stock solutions and stored at 4 ℃ for use. BPS Administration and Tissue Preparation. All animal experimental procedures were approved by the animal ethics committee of Hong Kong Baptist University. 6-8 weeks of BALB/c mice were obtained from Chinese University of Hong Kong and were housed in sterile individually ventilated cages. The mice were treated by gastric infusion with BPS dissolved in olive oil at a dosage of 50 mg/kg body weight. The mice were anesthetized and then decapitated at 1h and 6 h after BPS exposure, respectively. The control experiments were also performed after gastric perfusion with olive oil according to the same procedure. The different tissues involving kidney, liver, spleen and heart were immediately dissected from the mice body and frozen in liquid nitrogen, and then stored at -80 ℃ for subsequent tissue preparation. All tissues were sliced to a thickness of 14 µm using a CryoStar NX70 cryostat (Thermo Fisher Scientific Inc, Germany) and thaw-mounted on ITO-coated glass slides. All tissues were dried in a vacuum desiccator for approximately 2 h before the matrix application. Matrix Coating. For imaging analysis, the matrices were sprayed by using an automatic

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sprayer (ImagePrep, Bruker Daltonics) through an optimized method. In brief, the sectioned tissues were sprayed with 7 mg/mL CHCA or 0.75 mg/mL N,S-co-doped CDs solution. More detailed spraying parameters were listed as follow: deposition for 60 cycles, 1.5 s of spraying at 45% power with 30% modulation, 15 s of incubation, and 120 s of drying. MALDI-TOF MS and SALDI-TOF MS Analysis. For MALDI-TOF MS or SALDI-TOF MS analysis, 1 µL of the organic matrix or N,S-co-doped CDs solution was firstly pipetted onto the stainless steel sample plate, and air-dried, followed by addition of 1 µL analyte solution. MALDI-TOF MSI and SALDI-TOF MSI Analysis. All MS and MSI experiments were performed on a rapifleX MALDI Tissuetyper (Bruker Daltonics, Germany) equipped with a smartbeam 3D laser at a repetition rate of up to 10 kHz. Mass spectra were acquired at a mass range of m/z 100-600 both in positive- and negative reflector ion modes by averaging signal from 1,000 shots at 3.0 × 2810 volts detector gain and 60% laser power. The mass resolution of this system for somatostatin 28 (m/z 3147.47) in reflector mode is higher than 40,000 and the mass accuracy for peptide mixture is lower than 5 ppm with external calibration. The other parameters were fixed during the whole experiments, including a reflector voltage of 20.84 kV, a lens voltage of 11.00 kV, an ion source voltage of 20 kV, a pulsed ion extraction time of 100 ns and the matrix suppression up to 100 Da. The data collection on the rapifleX was at a spatial resolution of 50 µm, and 500 laser shots were summed per raster position. The calibration of the instrument was performed with the organic matrices (CHCA and DHB) in positive and negative ion modes.

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MSI Data Processing. The obtained MS data were subsequently processed and analyzed by FlexImaging 5.0 software (Bruker Daltonics, Germany). Total ion count (TIC) was used to normalize the data. Regions of interest (ROI) were manually defined in the analysis software using both the optical image with the MSI data image. Masses were selected with variable mass-selection window width (which corresponds to a mass ratio of ±0.025%).

RESULTS AND DISCUSSION Characterization of N,S-Co-doped CDs. The N,S-co-doped CDs were synthesized via thermal treatment as described elsewhere.39 Representative TEM image (Fig.1A) demonstrated that the average size of the obtained N,S-co-doped CDs was ~7 nm with good monodispersity. High-resolution TEM images (Fig.1B) showed the high crystallinity of the N,S-co-doped CDs, in which the lattice spacing of 0.21 nm was well in agreement with the in-plane lattice spacing of graphene (100 facet), demonstrating the potential of the N,S-co-doped CDs in laser absorption and energy transfer. The atomic force microscopy (AFM) image (Fig.1C) revealed that the height of the N,S-co-doped CDs was less than 0.6 nm. Obviously, the ultrafine N,S-co-doped CDs made it possible for matrix spraying by using automated spraying device. Fourier transform infrared (FTIR) spectra provided a direct proof for the synthetic process. As presented in Fig.1(D), the typical absorption bands at around 1655 cm-1 and 1710 cm-1 was assigned to C=C and C=O stretching, respectively. Moreover, the peaks of O-H and COO- groups at 3210 cm-1 and 1550 cm-1, and the peaks of C-O, C-N and C-S group at around 1186 cm-1 were observed, indicating the successful preparation of the N,S-co-doped CDs. X-ray photoelectron spectroscopy (XPS) results

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(Fig.1(E)) also confirmed that the N,S-co-doped CDs were mainly composed of carbon, sulfur, and oxygen. In particular, the peak of S(2p) in the spectrum revealed the existence of C-S-C units indicating the incorporation of sulphur element into the N,S-co-doped CDs.39 As shown in Fig.1(F) and its inset, the bulk N,S-co-doped CDs were well dispersed in methanol/H2O and its solution exhibited a strong and wide UV absorption at ~339 nm, which matched well with the emission wavelength of the irradiation source (laser, 337 nm). It was suggested that the N,S-co-doped CDs could effectively absorb UV laser energy and transfer the energy to analyte.

SALDI-TOF MS for Bisphenol S. Our previous work40 has demonstrated that none of bisphenols except BPS cloud be detected with MALDI-TOF MS in both positive- and negative ion modes. As shown in Fig.S1 (Supporting Information), there were only BPS-related ions ([M+Na]+ at m/z 272.936), but no other analytes (BPA, BPB, BPC, and BPAF) were detected with CHCA matrix in positive ion mode, indicating that MALDI-TOF MS was ineffective for bisphenols. Using the N,S-co-doped CDs as LDI substrates, however, multiple bisphenols-related positive ions except for BPAF, assigned to the [M+Na]+, [M+2Na]+, [M+Na-H]+, [M+2Na-H]+, and [M+3Na-2H]+ ions, were obtained in positive ion spectrum (Fig.2A). Taking BPS as an example, the ion peaks at m/z 273.035, 295.015, and 316.993 were accordingly attributed to [BPS+Na]+, [BPS+2Na-H]+, and [BPS+3Na-2H]+ ions

(Fig.S2A,

Supporting

Information).

Nevertheless,

the

complicated

multi-alkali metal ion adducts in positive ion spectrum often made it rather difficult for identification and interpretation. Different from positive ion mode, negative ion mode

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showed a clean background for detection of the five bisphenols with enhanced signal responses. As presented in Fig.2B and Fig.S2B (Supporting Information), the deprotonated [M-H]- ions of the five bisphenols at m/z 227.181, 241.302, 249.105, 255.221, and 335.126, and the fragment ions of BPA ([M-CH4-H]- at m/z 211.142) and BPAF ([M-CHF3-H]- at m/z 265.125) were all observed in negative ion spectrum. As a control, the pure CDs were also used as LDI substrate for analyzing the five bisphenols by negative ion SALDI-TOF MS. However, there were only BPS ([M-H]- at m/z 249.078) and BPAF ([M-H]- at m/z 335.118 and [M-CHF3-H]- at m/z 265.101) peaks observed (Fig.S3, Supporting Information) with low signal responses. The results indicated that the N,S-co-doped CDs have better adaptability and practicability for BPs than the pure CDs and traditional organic matrices in negative ion mode. The concentration of the N,S-co-doped CDs adopted as LDI substrate played an important role in producing the MS signal of BPs in SALDI-TOF MS. As a result, the effect of the N,S-co-doped CDs concentrations from 0.5 to 2.5 mg/mL was further investigated in negative ion mode using BPS as a model compound. As shown in Fig.S4 (Supporting Information), the signal-to-noise (S/N) ratios of BPS at m/z 249.091 rapidly enhanced from 63 to 139 with the increase of the N,S-co-doped CDs concentrations from 0.5 mg/mL to 0.75 mg/mL. However, continuous increase of the N,S-co-doped CDs concentrations from 0.75 mg/mL to 2.5 mg/mL did not improve the signal response of BPS, where the S/N ratios of BPS gradually decreased from 139 to 56. Meanwhile, the background noise originating from the N,S-co-doped CDs also increased accordingly. Based on the above results, the

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concentration of the N,S-co-doped CDs with 0.75 mg/mL was chosen as the best for further studies. Fig.S5(A) (Supporting Information) presented the quantitative analysis of BPS standard with different concentrations. It was observed that the S/N ratios of BPS decreased from 546 to 39 with the decrease of BPS concentration from 100 µg/mL to 5 µg/mL. Furthermore, a good linear relationship was obtained between the S/N ratios of BPS and its concentration in the range of 5-100 µg/mL, and the regression equation was y=5.3776X+6.8507 with a high correlation coefficient (r2) of 0.9986 (Fig.S5(B), Supporting Information). Notably, the deprotonated ion of BPS could still be detected with the S/N ratio of 11.3, even in the case of 1 µg/mL (equivalent to 4 pmol), indicating the high ionization efficiency of the N,S-co-doped CDs as LDI substrate in SALDI-TOF MS. In addition, the reproducibility was also evaluated by analyzing BPS and the results (data not shown) demonstrated that the relative standard deviation (RSDs) for shot-to-shot and sample-to-sample assays were 5.6% (n=15) and 10.2% (n=15), much better than those obtained with MALDI-TOF MS. The good reproducibility was mainly attributed to the formation of homogeneous cocrystallization between the N,S-co-doped CDs and BPS, thus resulting in the elimination of sweet spot effect that often encountered in MALDI-TOF MS. Optimization of Matrix Deposition. The good performance of the N,S-co-doped CDs encouraged us to explore the possibility for SALDI-TOF MSI applications, in which the N,S-co-doped CDs were employed as like as organic matrices for spraying on the surface of tissue sections through automatic sprayer. Careful matrix deposition is a critical requirement

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to obtain a homogenous coverage of the N,S-co-doped CDs on the tissue surface and produce even ionization across the entire sample area. Herein, the coverage of the N,S-co-doped CDs on the mouse tissues was investigated and the result was shown in Fig.3. It was clearly observed by an optical microscope that the coverage density of the N,S-co-doped CDs on the tissue surface gradually enhanced with the increase of spraying time from 0 to 180 min. It should be noted that the N,S-co-doped CDs had entirely covered and distributed homogeneously on the surface of tissue over 120 min. In this case, the sufficient matrix deposition was very favorable for obtaining stable ion signals, meanwhile avoiding analyte delocalization that was encountered in spraying organic matrices. SALDI-TOF MSI for Bisphenol S. Because of its distinct histological features, the efficacy of the proposed N,S-co-doped CDs-based SALDI approach for MSI was further evaluated by using tissue sections, where 1µL of 100 µg/mL BPS standard solution was spotted on the kidney tissue section before matrix spraying. Fig.4(A1) showed histological image of the kidney tissue section and Fig.4(A2) presented the positive MS profile obtained from the kidney tissue section by MALDI-TOF MS using CHCA matrix. As demonstrated, the background peaks of CHCA matrix dominated the spectrum in the range of m/z 100-600 Da, and strongly suppressed the signal of BPS, where only [M+Na]+ ion of BPS was detected with a fairly low response. The characterized fragment ion of CHCA at m/z 184.1028 was extracted and its MS image was constructed. It was seen from Fig.4(A3) that the ion signal intensities were uneven across the tissue area, suggesting that CHCA was insufficient extraction and inhomogeneous cocrystallization with tissue. Although the [M+Na]+ ion of

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BPS was mapped, Fig.4(A4) did not show well-defined areas of the BPS spot as depicted in Fig.4(A1). Obviously, BPS delocalization with poor spatial resolution (Fig.4(A5-6)) was mainly attributed to the fact that liquid spraying of CHCA thoroughly wetted all surfaces of the tissue section, and thus resulting in local tissue dissolution and analyte diffusion. In contrast, negative ion mass spectrum from the kidney tissue analyzed following N,S-co-doped CDs deposition (Fig.4(B1)) exhibited the exclusive [M-H]- ion of BPS at m/z 249.0863 with a high signal response (Fig.4(B2)), firmly confirming that the N,S-co-doped CDs is capable of probing BPS in the tissue sections without background interference. Fig.4(B3) showed a selection of background ion image (e.g, m/z 124.0841) that highlighted the relative uniform coverage of the N,S-co-doped CDs on the tissue surface. Interestingly, BPS distribution on the tissue surface (Fig.4(B4)) was highly consistent with the spiked spot of histological image (Fig.4(B1)). As shown in Fig.4(B5-6), High spatial resolution images without analyte delocalization were obtained by merging two images of background and BPS ions, showing the potential of the N,S-co-doped CDs as matrix in MSI analysis. Fig.5(A) presented a dynamic range for potential quantification generated with the deprotonated BPS ion by SALDI-TOF MSI. The curve was plotted against six different amounts of BPS (1-100 ng), where BPS standards (1 µL) with different concentrations were micropipetted on liver tissue sections and negative ion SALDI-TOF MSI was performed on the spots. Each spot of BPS standards on the tissues was selected as region of interest and TIC was used to normalize the data. It was seen from Fig.5(A) that a good linear relationship between the intensities of the deprotonated BPS ion and its concentration on the blank tissues

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in a wide dynamic range and a high r2 value of 0.9988 were achieved with SALDI-TOF MSI. Moreover, the ion images of BPS were easily visualized with high spatial resolution by naked eyes as illustrated in Fig.5(B), where different amount of BPS were individually micropipetted on the surface of liver tissues. Interestingly, the color changes of BPS on the spots matched well with its corresponding concentration. It was estimated conservatively that the detection limit as low as ~ 20 pmol for BPS (the spot of 5 ng BPS) was obtained by direct observation from MS images. The results were relatively consistent with those obtained in SALDI-TOF MS, indicating the potential of BPS quantitation in tissue samples by the N,S-co-doped CDs-assisted SALDI-TOF MSI. SALDI-TOF MSI of Bisphenol S in the Mouse Tissues after Exposure. To further explore the potential applications of the N,S-co-doped CDs-assisted SALDI-TOF MSI in real samples, a mouse model of oral exposure to BPS was established, in which the same body weight dosages and two different administration times (1h and 6h, respectively) were studied according to the previous work.41 Fig.6(A) showed the negative ion MS profile and the SALDI-TOF MS images of the experimental and control groups with or without BPS exposure for 1h. It was observed from the experimental group that the deprotonated ion of BPS at m/z 248.7856 was identified with high signal response and the extracted MS image showed that BPS were mainly localized in the region of renal artery. However, no signal of BPS was probed in the control kidney tissue, which caused the failure to profile the spatial distribution of BPS in the same region. In situ analysis of the kidney tissues was performed after exposure to BPS for 6 h. As expected, BPS was distinctly identified in the experimental

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group and the negative ion MS profile was displayed in Fig.6(B). It was worthy to note that BPS was mainly distributed in the cortex region. The above finding was well consistent with the metabolic model of kidney. In addition, some unknown low-molecular-weight compounds (e.g. m/z=240, 255, 279, 283) were also detected after BPS administration for 6h and their corresponding ions were mapped with high spatial resolution (Fig.6(B)), although they needed to be further identified by LC-MS/MS. Fig.7(A-B) presented the MS images of BPS and the other unknown compounds in liver tissue sections at two different administration times. After BPS administration for 1 h, high concentration of BPS was unambiguously detected in liver tissue and its extracted BPS ion at m/z 249.02 showed a dark green in the MS image that was conspicuous against background (Fig.7(A)). Similar result was also obtained after BPS administration for 6h. As illustrated in Fig.7(B), many unknown compounds including m/z 255, 279, 283, 382, 394 and 514, more than BPS were probed simultaneously and these selected ions were successfully mapped with high spatial resolution. Besides, the spleen and heart tissues after BPS exposure were also analysed by SALDI-TOF MSI and the results (Fig.S6 and Fig.S7, Supporting Information) showed that the deprotonated BPS ion was not detected regardless of 1h and 6h exposure. In addition, the general applicability of the proposed method was further evaluated by SALDI-TOF MSI analysis of BPAF exposure in kidney and liver tissues. As presented in Fig.S8 and Fig.S9 (Supporting Information), although the deprotonated ion of BPAF was not sensitively probed, the fragment ion of BPAF ([M-CHF3-H]- at m/z 265.121) was clearly identified in the kidney and liver tissues. The corresponding spatial distribution of BPAF was

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similar to BPS at the same administration times. The aforementioned results firmly confirmed the practicability of the N,S-co-doped CDs-assisted SALDI-TOF MSI for BPS in real tissue samples.

CONCLUSIONS In summary, the N,S-co-doped CDs were successfully developed as a new matrix for negative ion SALDI-TOF MSI analysis of BPS in mouse tissue sections. The N,S-co-doped CDs exhibited the superior performance than organic matrices (e.g. CHCA, DHB) and pure CDs in background interference, sensitivity, selectivity, homogeneity and reproducibility. In addition, the potential of this matrix applied for the quantitative analysis of BPS in tissue samples was showed with a good linear relationship. In addition, in situ sensitive identification of BPS in the mouse and kidney liver tissues for exposure and profiling its distribution was achieved with high spatial resolution. The study of the distribution of BPS in different tissue samples can provide meaningful biological information and critical insight into the assessment of BPS exposure. It is expected that the proof-of-concept work can be certainly useful for N,S-co-doped CDs-assisted SALDI-TOF MSI analysis of other small molecules in biological tissue samples. ACKNOWLEDGEMENT This work was supported by the National Key Research and Development Program of China (2017YFC1600500), the National Natural Science Foundation of China (21675025, 21777010 and 91543202), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), the Natural Science Foundation of Fujian Province

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(2018J01683), and the Program for New Century Excellent Talents in Fujian Province University.

ASSOCIATED CONTENT Supporting Information Experimental details and additional data as noted in text. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Captions Figure 1. (A-B) HRTEM image of the N,S-co-doped CDs; (C) AFM image of the N,S-co-doped CDs; (D) FTIR spectrum of the N,S-co-doped CDs; (E) XPS spectrum of the N,S-co-doped CDs; and (F) UV-visible spectrum of the N,S-co-doped CDs (the inset is photograph of bulk N,S-co-doped CDs and its suspension). Figure 2. Mass spectra of five bisphenol mixture by the N,S-co-doped CDs-assisted SALDI-TOF MS (A) positive ion mode; and (B) negative ion mode; The concentration of each analyte was 20 µg/mL. Laser intensity: 60%. Figure 3. The effect of spraying times on the coverage of N,S-co-doped CDs on tissue surface. (A) Without spraying; (B) 30 min; (C) 60 min; (D) 90 min; (E) 120 min; and (F) 180 min. The concentration of the N,S-co-doped CDs was 0.75 mg/mL. Figure 4. (A) MALDI-TOF MSI and (B) SALDI-TOF MSI of the kidney tissue sections spiked with 100 µg/mL BPS. (A1 and B1) Histological images of the kidney tissues spiked with BPS before matrix spraying; (A2 and B2) Mass spectra of the kidney tissues obtained with positive ion MALDI-TOF MS and negative ion SALDI-TOF MS, respectively; (A3 and B3) MALDI image and SALDI image of the background ions; (A4 and B4) MALDI image and SALDI image of BPS; (A5 and B5) Merged images; (A6 and B6) Multi-color images of BPS obtained with positive ion MALDI-TOF MS and negative ion SALDI-TOF MS, respectively. Figure 5. Quantitative analysis of BPS in liver tissue sections by the N,S-co-doped CDs-assisted SALDI-TOF MSI in negative ion mode. (A) Calibration curve for BPS

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standards micropipetted on a liver tissue sections; (B) Histological images of the liver tissues spiked with different amount of BPS before N,S-co-doped CD spraying and their corresponding MS images. Figure 6. In situ identification and imaging of BPS in the mouse kidney tissues for exposure by the N,S-co-doped CDs-assisted SALDI-TOF MSI. (A) Exposure to BPS for 1h; (B) Exposure to BPS for 6h; Figure 7. In situ identification and imaging of BPS in the mouse liver tissues for exposure by the N,S-co-doped CDs-assisted SALDI-TOF MSI. (A) Exposure to BPS for 1h; (B) Exposure to BPS for 6h;

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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For TOC only

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Nitrogen and Sulfur Co-doped Carbon-Dot-Assisted Laser Desorption

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