Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and

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Disposable MoS-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples Yaju Zhao, Minmin Tang, Qiaobo Liao, Zhoumin Li, Hui Li, Kai Xi, Li Tan, Mei Zhang, Danke Xu, and Hong-Yuan Chen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00051 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples Yaju Zhao,† Minmin Tang,† Qiaobo Liao,† Zhoumin Li,† Hui Li,*,† Kai Xi, † Li Tan, ‡ Mei Zhang, ‡ Danke Xu,*,† and Hong-Yuan Chen† †State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P.R. China

‡Jiangsu Institute for Food and Drug Control, Nanjing 210008, P.R. China Corresponding Authors *E-mail: [email protected]. Tel/Fax: (+) 00862583595835 *E-mail: [email protected].

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ABSTRACT: In this work, we demonstrate, for the first time, the development of a disposable MoS2-arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analysed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS2-arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analysed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuffs detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5−10 ng/mL (R2 >0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

KEYWORDS: MoS2, MALDI MS, chip, quantification, sulfonamides


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Nowadays, antibiotic residues in foods are of great concern to regulatory agencies and consumers, because the residual veterinary drugs may cause adverse health effects to humans.1 To ensure food safety, fast screening methods are necessary for routine and large-scale detection of these residues. Biosensors, especially affinity-based sensors, are innovative detection methods in the field of rapid antibiotic residues screening.2 Their main advantages are simplicity, short analysis time, good selectivity and sensitivity, and easy operation by relatively unskilled personnel.3 Optical, electrochemical, thermal or acoustic-based transducers are common signal transduction methods of the biosensors.4 Among them, enzyme-linked immunosorbent assay (ELISA)1 and lateral-flow immunoassays (LFIA)5 are the most widely used biosensor assays, which employ colorimetric detection to realize qualitative or quantitative analysis. However, the lack of molecular weight and structural information of targets may induce high rates of false positive results.6 Therefore, there is a dire need for a simple, rapid, and accurate antibiotic detection method. The establishment of a biosensing method using mass spectrometry (MS) as a signal transducer can promote accurate and quick antibiotic detection.7,8 MS detection can identify the analytes that are difficult to distinguish by the simple use of a bioreceptor, greatly improving the detection accuracy. For example, Wang et al. reported an antibody-graphene oxide nanoribbon conjugate to separate and enrich chloramphenicol in both river water and human serum samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is then used to identify the target analytes.7 Gan et al. also combined aptamer-functionalized SiO2@Au nanoshells with MALDI-TOF MS to enrich and analyse kanamycin from milk samples.8 Compared with conventional chromatography coupled with tandem MS methods for antibiotic detection, MALDI-TOF MS detection-based biosensing method is much simpler, and requires less time to complete. However, these enrichment systems only carried out a single molecule detection, and quantitative analysis strategy was not established.7-10 MALDI-TOF MS is a well-established analytical technique for high throughput and rapid analysis of large molecules. Recent exploration of new MALDI matrices has solved the matrix interferences in the low mass region (m/z < 500 Da) and inferior shot-to-shot reproducibility for small molecule analysis.11-15 Specifically, surface-enhanced laser


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desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) technique, with nanomaterial serves as both a MALDI matrix and an adsorbent, has shown great promise in practical applications due to the capacity of selectively enrichment of targets from complex samples.11,13,16 However, only a few analytes with aromatic groups have been reported to be practicable since their interaction mechanisms mainly rely on hydrophobic interaction and π–π stacking interaction.11,15,17,18 Or affinity ligand was conjugated onto nanomaterials as SELDI probes. This requires us to seek nanomaterials with conjugation sites and grope reaction conditions, which is not universally applicable. Moreover, two steps of commercial steel plate cleaning and matrix loading were required before every MALDI MS measurement, which is labor intensive, time-consuming, and not suitable for high throughput analysis. These challenges have greatly limited the use of MALDI-TOF MS in fast screening of antibiotics. Here we report the development of a disposable MoS2-arrayed MALDI MS chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of SAs in multiple complex samples (Figure 1). Our recent study has revealed that MoS2 as the MALDI matrix exhibited good performances in the characterization of small molecules.19 On the basis of previous results, we describe the specific preparation and use of MoS2-arrayed ITO glass slide as a disposable, inexpensive, and conductive sample plate for MALDI MS analysis on standard MALDI-TOF mass spectrometers. An enrichment and purification procedure, with antibody-functionalized immunomagnetic techniques, was used to improve the detection sensitivity and the anti-interference ability to the impurity substances contained in real complex samples. We also introduced the internal standard (IS)-first method to simplify the experimental process in MALDI-TOF MS quantitation. We demonstrated that the IS- contained MoS2-arrayed MALDI MS chip achieves both the establishment of standard calibration curves and high throughput analysis of multiple real food samples in a single assay.

EXPERIMENTAL SECTION Preparation of MoS2–arrayed MALDI MS chip. The MoS2 nanosheets were prepared by chemical exfoliation methods according to the previously reported procedure (Figure


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S1a).19 The details are given in Supporting Information. The MoS2 solution was prepared in water at a concentration of 0.5 mg/mL. For the formation of matrix array on a chip, we designed and obtained a sticker template with 96 arrayed well. The template has the same dimension as the ITO glass chip. The sticky paper was aligned and attached to an ITO glass slide surface. Subsequently, 1 µL of MoS2 solution was pipetted onto each well and dried under ambient condition to form a thin matrix layer. For the calibration curves, 1 µL of SDM-d6 (5 ng/mL) was predeposited onto the MoS2 array before sample loading. The template layer was then peeled from the ITO glass slide to form a MoS2–arrayed MALDI MS chip (Figure S1b). LDI-MS Analysis of SAs on MALDI MS chip. 1 µL of analyte solution including sulfacetamide (SA), sulfapyridine (SPY), sulfadiazine (SDZ), sulfamethoxazole (SMZ), sulfathiazole (STZ), sulfisoxazole (SIX), sulfamethazine (SM2), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfaquinoxaline (SQX), sulfadimethoxine (SDM), and sulfadimethoxine-d6 (SDM-d6) was pipetted on top of the matrix on the MALDI MS chip and air-dried. The chip was then immobilized on a target plate by using double-sided tape and subjected to LDI-MS analysis. Immunoaffinity Extraction and Enrichment of Target SAs. 500 µL of monoclonal antibody against SAs (SA-mAb) -functionalized MagBeads (denoted SA-mAb/MB) was washed with 1PBS, and incubated with 1 mL of sample solution with gentle shaking at room temperature for 20 min. Then, SAs-captured SA-mAb/MB were washed twice with water. Methanol (100 µL) was added to the SAs-captured SA-mAb/MB and the tube was vortexed for 10 s to elute the analytes. The elution step was repeated once. The collected sample eluate (200 µL in total) was evaporated to near-dryness under a gentle stream of N2 at 50 °C. The residue was reconstituted with 5 µL of deionized water for MALDI-TOF MS analysis. Establishment of Standard Calibration Curve for Quantitation of Target SAs. For multiplexed SAs quantitation, standard calibration curves were developed by a series of standard solutions composed of varying amounts of SMZ, SM2, SMM, SQX, and SDM (ranging from 0.5 to 10 ng/mL). The mixed SAs solutions were incubated with the SA-mAb/MB, and followed by the immunoaffinity enrichment process. For relative quantitation, 5 µL of the residue was pipetted onto the MALDI MS chip containing IS by steps and analyzed by MALDI-TOF MS. Real Sample Analysis. Both of the real samples (pork and egg) were kindly provided by the Jiangsu Academy of Agricultural Sciences (Nanjing, China). 0.5 g of the homogenized sample was weighed into centrifuge tube. For egg sample, 1 mL of acetonitrile was added and the mixture was shaken for 30 min at room temperature, followed by centrifuging at 8000 rpm for 5 min. The supernatant was collected, and the residue was extracted again with 1 mL


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of acetonitrile. For pork sample, the same procedure was applied except that the extraction reagent was replaced with 80% ethanol. Take 300 µL of the collected supernatant extracts, and evaporate to near-dryness. The residue was reconstituted with 1.5 mL with 1PBS and filtrated. 1 mL of the filtrate was then incubated with the SA-mAb/MB, and followed by the immunoaffinity enrichment process for MALDI-TOF MS analysis. MALDI-TOF MS analysis. MALDI-TOF MS measurements were performed on a 4800 Plus MALDI-TOF/TOF mass spectrometer (AB Sciex, U.S.A.) equipped with a pulsed Nd: YAG laser (355 nm wavelength) with negative reflection mode. For each spectrum, 50 shots from different positions of the target spot (automatic mode) were collected and analysed. The data processing was performed with the Data Explorer Software from AB Sciex. RESULTS AND DISCUSSION Characterization and Design of the MALDI MS Chip. MoS2 nanoflakes were synthesized by chemical Li-intercalation and exfoliation with n-butyllithium according to the previously reported methods (Figure S1a).19 MoS2 were exfoliated into monolayers by intercalation with lithium followed by reaction with water. The reaction between the water and the intercalated lithium forms hydrogen gas between the MoS2 layers. The expansion of hydrogen gas tends to separate the MoS2 layers to form exfoliated MoS2 nanosheets. The morphology and structure of the prepared MoS2 was analyzed using transmission electron microscope (TEM) (Figure S2a). The transparent and corrugated single-layered nanosheets could be observed. The lateral dimensions of the MoS2 sheet are in the 400-1500 nm range. The characterization results clearly indicated the successful exfoliation of bulk MoS2 into MoS2 nanoflakes. The nanosheet structure could provide a large surface area for analyte adsorption and allows the nanomaterial to attach to the ITO chip tightly, thus preventing the detachment of MoS2 and contamination under vacuum.20 The high resolution TEM (HRTEM) image (inset in Figure S2a) shows the typical hexagonal single crystal structure with a lattice spacing of 0.27 nm for the (100) Mo atoms. Furthermore, a strong absorption of the MoS2 nanosheets at the wavelength of laser used in MALDI (355 nm) enables it to serve as a matrix for absorbing laser energy and transferring energy to the analyte (Figure S2b). Inset in Figure S2b shows the photograph of MoS2 aqueous solution, exhibiting excellent water dispersibility of MoS2. We then fabricated a MoS2 matrix arrayed MALDI MS chip containing 96 spots of 1.9 mm diameter each. An optical image of the MALDI MS chip showed a 16 6 array with acceptable spot morphology and homogeneous distribution (Figure S2c), which was further confirmed by the photograph taken by the camera equipped on the MS instrument (Figure S2d). Scanning electron microscopy (SEM) image provided a more detailed information of the MoS2 matrix spot. As shown in Figure S2e, MoS2 are remarkably evenly distributed and


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densely covered on the array spot. The uniform distribution of the matrix made it possible for improving shot-to-shot reproducibility and the accuracy of signal distribution. The conditions of the coupling reaction for preparing the SA-mAb/MB were used according to the manufacturer’s instruction. The amount of immobilized SA-mAb on the surface of Carboxyl MagBeads is critical for their capture and enrichment capability. Thus, the supernatants after every immobilization process were collected and measured using BCA Protein Assay. According to the results, the average amount of SA-mAb coupled on the Carboxyl MagBeads was estimated to be 40.75 mg/g. Based on IgG (Mw ~ 150 kDa), about 0.27 nmol of SA-mAb were coupled onto 1 mg Carboxyl MagBeads. This measured amount helps to further estimate the capture of SAs, and guide the optimization of experimental conditions as well. The MoS2–arrayed MALDI MS chip allowed us to conduct quantitative analysis, recovery studies, and real foodstuffs analysis all in a single assay. The experiments were aligned as shown in Figure S2f. If there are more samples to be tested, an additional MALDI MS chip could be utilized. It is notable that this arrayed chip provides a high-throughput and rapid strategy for multiple sample analysis. MALDI-TOF MS analysis of SAs on matrix-arrayed ITO chip. To assess the performance of MoS2 matrix-arrayed MALDI MS chip, a variety of SAs dissolved in deionized water were analyzed. Previous study has demonstrated that MoS2 was a useful matrix for the facile analysis of amino acids, peptides, fatty acids and drugs in negative ionization mode by our research group.19 The negative ion spectrum was easier to interpret with only [M-H]- ion signal present. The edges and defects of MoS2 nanosheet can function as deprotonation sites for sample ionization and proton acceptance, thus facilitating the charging process in negative ion mode. On the basis of previous results, negative ion mode was chosen in this work. Figure 2a displayed representative spectra for the compounds SA (MW 214.24), SPY (MW 249.29), SDZ (MW 250.28), SMZ (MW 253.28), STZ (MW 255.32), SIX (MW 267.30), SM2 (MW 278.33), SMM (MW 280.33), SCP (MW 284.72), SQX (MW 300.37), SDM (MW 310.33), and SDM-d6 (MW 316.37) (inset in Figure 2a show their structures). All of the 12 SAs could be successfully detected with strong characteristic [M - H]- ion peaks and low background signals. Notably, SDM-d6 and SDM were also detected at m/z 297.9 and 294.9, due to the loss of CD3 and CH3. Then mixture of the above SAs was measured under the same condition with the matrix-arrayed ITO chip. Likewise, the twelve SAs were all detected with no signal overlapping (Figure 2b). The results validated that the matrix-arrayed


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ITO chip could detect a variety of small-molecular-weight SAs simultaneously and effectively. For the further investigation, three different sample plates were prepared by loading MoS2 matrix onto different substrates: stainless steel plate, ITO glass slide, and glass slide. The LDI-MS of SAs was next performed on the three plates under the same instrumental conditions (Figure 2c). SMZ, SM2, SMM, SQX, and SDM were selected as the model analytes. The mass peak intensities obtained from the three substrates are shown as bar graphs for comparison. The results showed that the ion intensities of the five SAs from the ITO glass slide were comparable to that obtained from commercial steel plate, indicating that MoS2-coated ITO glass chip is suitable for MALDI-MS. ITO coated glass slide has been used as a standard sample plate in MALDI-MS imaging due to its electrical conductivity and optical transparency.21 Compared to the commercially available 384 spot stainless steel plate, the ITO glass chip is much cheaper and avoids the cleaning steps of the sample plate. On the other hand, the ion intensities of the SAs desorbed from glass chip were more than twofold lower than those from ITO glass chip. The only difference between them lied in the ITO thin films deposited on glass substrates. This demonstrated the importance of ITO thin film in the enhancement of LDI efficiency owing to its electrical conductivity. In addition, we found bare ITO glass chips showed no activity for MALDI-TOF MS (Figure S3). These results clearly indicate that the MoS2 matrix layer is essential to the LDI process and the as-prepared MoS2-arrayed ITO chip can be a good alternative and disposable substrate for MALDI-TOF MS. To demonstrate our concept as depicted in Figure 1, proof-of-principle experiments were performed. SA-mAb/MB was prepared for immunoaffinity capture of target analyte in spiked standard samples and SDM was selected as the model analyte. We first analyzed a dilution series of SDM aqueous solutions to measure the detection sensitivity of the MoS2-arrayed ITO chip. Mass spectra of 5 µg/mL, 1 µg/mL, 500 ng/mL, 400 ng/mL, 300 ng/mL and 200 ng/mL of SDM are shown in Figure 3a. As the concentration of SDM decreased, the signal intensity reduced and no signal was obtained when the concentration was 200 ng/mL due to insufficient sensitivity. We then conducted the enrichment process using SA-mAb/MB. SDM dissolved in PBS was incubated with SA-mAb/BP to facilitate immunoaffinity extraction and


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enrichment process. The SDM captured on SA-mAb/MB was then collected by magnetic separation, eluted by methanol, reconstituted with water, and spotted onto the MoS2-arrayed MALDI MS chip for direct MALDI-TOF MS analysis. After enrichment with SA-mAb/MB, SDM down to 400 pg/mL could be detected with S/N ratio of 11.33 (Figure 3b). This detection limit after enrichment reached almost 3 orders of magnitude lower than that without enrichment, fully demonstrating the feasibility of the immunoaffinity enrichment strategy. Development of the IS Method for Quantitative Analysis. The homogeneous distribution of nanomaterials as MALDI matrices facilitated the quantitative application of MALDI MS.11,14 However, the quantitation accuracy could be affected in real samples when using intensity as a quantitative standard. The addition of isotopically-labelled molecules as an IS can minimize the error produced in a complicated system.22 Meanwhile, the use of IS can efficiently improve the accuracy of MALDI MS analysis to RSD < 10%.23,24 Thus, an IS SDM-D6 was used for the quantitative analysis of SAs in real samples. Typically, a mixed sample suspension containing the IS was prepared first, and the mixture was loaded onto the matrix prior to MALDI MS analysis. When preparing the sample solutions, at least 5 µL of the IS was needed for one sample. However, this sample mixing method becomes cumbersome and consumes a large amount of IS substance when dealing with multiple samples. Hence, the exploration of simple preparation approach with low cost is still necessary. In this study, we proposed a new sample preparation method with isotope IS deposited first and subsequently loaded with analytes for quantitative analysis. Figures S4a and S4b show a comparison of the MS signals of SDM (5 ng) and IS SDM-d6 (5 ng) with two different sample preparation procedures (mixed method and IS-first method). Deprotonated ions of SDM and SDM-d6 with similar signal intensities were obtained for both methods. This could be benefited from uniform distribution of MoS2 matrix in each spot and homogeneous spread of IS on the MoS2 matrix. Considering the convenience of sample preparation and IS cost savings, the IS-first method was more beneficial. To examine the stability of the IS covered MoS2-arrayed ITO chip, 1 µL of SDM (5 µg/mL) was analysed after the chip was stored with different periods. As time goes by, the signal intensities of SDM and SDM-d6 maintained at a consistent level, indicating both the IS and MoS2 matrix were stable under the storage


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conditions (Figure S5). Furthermore, the reproducibility was assessed and the results are shown in Figure S6. The shot-to-shot RSD (n = 15) at different locations on one sample spot was 3.57%, and the sample-to-sample RSD (n = 10) was 6.48%. This result demonstrated the feasibility of the IS-first method for quantitative analysis of analytes. To maximize the efficiency of SAs enrichment, experimental conditions were optimized by comparing the relative intensity of enriched SDM to IS. 1mL of SDM (10 ng/mL) sample was chosen as the test sample. In the beginning, the amount of SA-mAb/MB required was theoretically estimated. Assuming that the molar ratio of antibody to SDM is 1: 1, 10 ng of SDM requires a volume of about 12 µl of SA-mAb/MB. Considering the possible loss of activity of the antibody after conjugation to Carboxyl MagBeads, we optimized the amount of SA-mAb/MB ranging from 25 µL to 200 µL. As illustrated in Figure S4c, the enrichment efficiency at capture time of 30 min gradually increased when the amount of SA-mAb/MB increased from 25 µL to 100 µL and then remained almost constant at amount of SA-mAb/MB >100 µL. Therefore, 100 µL of SA-mAb/MB was chosen as the optimal amount. We then studied the effects of different adsorption time (5 min, 10 min, 20 min, 30 min, and 45 min). As shown in Figure S4d, the best capture efficiency was obtained at 20 min. Obviously, a longer incubation time reduced the extraction efficiency of target analytes. This phenomenon could be due to the fact that the room temperature and salt ion environment may affect the binding of antibodies and antigens at a longer time. Therefore, an adsorption time of 20 min was selected. The elution of captured analytes from antibodies with methanol is routinely performed for immunoaffinity column. Methanol can break the antigen−antibody interaction efficiently. According to the results reported in the literature, the desorption time had no obvious effect on the enrichment efficiency with methanol elution 25. Thus, a short elution time of 210 s was utilized in the following experiments. Multiplexed Quantitative Detection of SAs. Another important advantage of MALDI MS is that it can simultaneously identify multiple species of SAs within one run. The antibody against SAs can recognize and bind to a class of SAs, thus eliminating the need to optimize enrichment conditions for individual compounds. Moreover, simultaneous analysis of multiple components presents advantages of shorter analysis time, lower cost, and less sample usage. By taking advantage of innate ability of MALDI MS and antibody’s characteristic, we


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performed multiplex quantitative detection of five most commonly tested SAs. A series of standard SAs solutions composed of varying amounts of SMZ, SM2, SMM, SQX, and SDM were prepared with pure solvent 1PBS buffer and processed by the multiplexed immunoaffinity enrichment assay to establish standard calibration curves. Figure 4a shows representative MALDI-TOF mass spectra obtained from the SA-mAb/MB incubated with standard SAs mixtures ranging from 0.5 ng/mL to 10 ng/mL. As expected, all the five SAs dominantly appeared in the mass spectra. The pure solvent standard working curves using relative peak intensity displayed in Figure 4b-f show linear relationships with concentrations (R2 = 0.9917 for SMZ, R2 = 0.9937 for SM2, R2 = 0.9962 for SMM, R2 = 0.9903 for SQX and R2 = 0.9979 for SDM), which demonstrated satisfactory quantitative behavior of this immunoaffinity MALDI-TOF MS method. The limits of detection (LOD), based on signal–noise ratio ≥ (S/N), were in the range 0.043−0.236 ng/mL (0.86-4.73 µg/kg correspondingly in this work). According to the maximum residue limits (MRLs) established by EU Council Regulation EEC 2377/90, the total SAs in food should not exceed 100 µg/kg.26 The sensitivity of present method can satisfy the demands for routine analysis of SAs residues, and showed a competitive sensitivity compared to other previously reported mass spectrometry-based approaches (Table S1).27-30 Below the LOD, it is considered that the test sample to be negative and in line with safety requirements. The whole MALDI MS analysis procedure developed here took approximately 5 s per sample, whereas it takes more than 5 min to run a sample with traditional LC/MS instrument. Moreover, MALDI MS analysis could be easily operated by relatively unskilled personnel. LC-MS-based analytical methods usually suffer from matrix effects (ME) in food analyses which affect the analyte signal and accuracy of quantitative analyses. Thus, ME in the ESI source are usually estimated by the ratio of slopes of the matrix calibration working curve and the pure solvent standard curve (Rslope).31 However, to the best of our knowledge, the ME in MALDI ion source with nanomaterials as MALDI matrices has never been reported. To assess the accuracy of pure solvent standard working curve for quantitative analysis, we also evaluated ME using three representative types of matrix calibration standard working solutions. Before analysis of the SAs-spiked negative sample extraction solutions, 1PBS solution, blank pork extract, blank egg extract, and blank milk extract without any SAs were


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analysed as negative controls. No other peaks except the IS were observed in the low mass range (Figure S7), demonstrating the good specificity and high selectivity of the immunoaffinity magnetic separation in complex media. In the following, a series of concentrations of SDM spiked pork exact, SM2 spiked egg extract, and SMM spiked milk extract were analysed by our proposed method and three matrix calibration curves were obtained. The results turn out that very little matrix suppression was observed for the three SAs with Rslope ranging from 0.935 to 1.120 (Figure 5a-c), indicating negligible ME and pure solvent standard working curves were suitable for quantitative analysis.32,33 This phenomenon may be due to the following three reasons. Firstly, immunoaffinity purification provides the specificity to minimize or eliminate the matrix interferences in complex samples because of its selective extraction ability.25 Secondly, many nanomaterials-based matrices showed excellent salt tolerance in MALDI MS detection, indicating that small amount of salt from complex samples cannot affect the signal intensities of the targets.34,35 Thirdly, in the current method, we use relative quantification method rather than absolute quantification of peak intensities. Even small amounts of impurities may suppress analyte signals, this effect will still be offset by the introduction of IS. We then evaluated the recoveries of the five SAs in pork, egg, and milk samples. As shown in Table S2, at the spiked concentrations of 20, 50, and 100 µg/kg, the mean recoveries of the five analytes ranged from 74.6% to 109.5% with RSD values less than 14.7% (n=3). We also analysed five SAs spiked pork sample (50 µg/kg) without using SA-mAb/MNP for purification. SAs spiked pork sample was extracted by 80% ethanol, and 200 µL of the extract was evaporated to near-dryness under a gentle stream of N2. Then the residue was reconstituted with 5 µL of deionized water, and loaded onto the MALDI MS chip for direct MALDI-TOF MS analysis. Interestingly, no signal peak can be observed in the spectrum due to the complex sample matrices (Figure 5d). The impurities containing large amounts of fat and lipids covered the matrix substrate, inhibiting the signals of SAs and IS. We used one-step solvent extraction method, which is quick and easy operating. However, this method could bring some undesired substances, thus further purification process was needed. After purification and enrichment with SA-mAb/MNP, all of the five SAs could be detected (Figure 5e). This immunoaffinity enrichment method can not only preconcentrate the target


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compounds but also exclude matrix interferences from samples due to its specific affinity of SA-mAb. To verify the reusability of the SA-mAb/MB, five replicate measurements were performed on the same SA-mAb/MB. The regeneration was achieved by immediately washing with PBS after methanol elution and the results were shown in Figure 5f. Recoveries for SDM were satisfactory when SA-mAb/MB was regenerated after 4 continuous adsorption cycles. At the fifth analysis, recovery for SDM decreased slightly, which means the SA-mAb/MB assay could be recycled four times at least. This excellent reusability means this immunoaffinity magnetic separation method keeps great potentials to be applied in the enrichment of SAs, which would reduce the average cost of antibodies. Applications to Real Sample Analysis. Finally, we applied the proposed method to quantify and identify SAs in real food samples, which were collected from local markets. Figure 6a,b show typical MS spectra for the pork sample (No. 941) and egg sample (No. 833). Besides characteristic peaks of IS at m/z 297.9, and 314.9, we observed one strong peak in negative ion mode at m/z 276.9 in both samples. In particular, MS/MS spectra of m/z 276.9 were presented to provide additional structure informations (shown in Figure 6c, d). The fragments at m/z 106.2, 121.2, and 185.2 all confirmed the existence of SM2 in the samples.36 Then the quantification of target analytes was performed with the obtained pure solvent calibration standard curve. As listed in Table 1, SM2 were found at concentrations of 58.0 and 9.71 µg/kg for sample 941 and 833, respectively. The values measured by the proposed method are consistent with the data measured by HPLC-ESI-MS/MS. These results demonstrates that the developed method is competent and reliable for rapid identification and quantitative analysis of SAs in complex samples. Furthermore, due to the high throughput and simplicity of this method, we could finish analysis of 96 sample spots within 10 min in one single MoS2-arrayed MALDI MS chip. Thus, the disposable MoS2-arrayed MALDI MS chip coupled with immunoaffinity magnetic enrichment method provides a much more facile, selective, high-throughput, and sensitive tool for the detection of small-molecular-veterinary drug residues in food samples.

CONCLUSIONS


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This work proposes a disposable MoS2-arrayed MALDI MS chip combined with immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple SAs. The inexpensive disposable MALDI MS chip developed here has proven to be a good alternative to commercial steel plate for SAs analysis. Higher sensitivity was obtained by targeted extraction and enrichment using antibody-conjugated magnetic beads. By previously loading IS onto the MoS2 array, the MALDI-TOF MS quantitative detection of five SAs was performed. The calibration curves exhibited good linearity with correlation coefficients better than 0.990. This method was successfully applied to identify and quantify SAs in different types of real food samples, including pork and egg samples. This proposed strategy shows excellent analytical performance with good convenience, low-cost, high throughput, high sensitivity as well as acceptable precision. It could be anticipated that this novel MALDI MS chip could be coupled with miniaturized MALDI-TOF mass spectrometers as a biosensor instrument for rapid and high throughput screening of antibiotic residues in food. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supporting experimental details, supporting tables, supporting figures, and supporting references.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Tel/Fax: (+) 00862583595835 *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We acknowledge financial support of the National Natural Science Foundation of China (21775068, 21227009, 21475060, and 21405077), Natural Science Foundation of Jiangsu Province (BK20140591), and the National Science Fund for Creative Research Groups


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(21121091). REFERENCES (1) Jiang, W. X.; Wang, Z. H.; Beier, R. C.; Jiang, H. Y.; Wu, Y. N.; Shen, J. Z. Simultaneous Determination of 13 Fluoroquinolone and 22 Sulfonamide Residues in Milk by a Dual-Colorimetric Enzyme-Linked Immunosorbent Assay. Anal. Chem. 2013, 85, 1995-1999. (2) Gaudin, V. Advances in Biosensor Development for the Screening of Antibiotic Residues in Food Products of Animal Origin – A Comprehensive Review. Biosens. Bioelectron. 2017, 90, 363-377. (3) Chen, T.; Cheng, G.; Ahmed, S.; Wang, Y.; Wang, X.; Hao, H.; Yuan, Z. New Methodologies in Screening of Antibiotic Residues in Animal-Derived Foods: Biosensors. Talanta 2017, 175, 435-442. (4) Zamora-Gálvez, A.; Ait-Lahcen, A.; Mercante, L. A.; Morales-Narváez, E.; Amine, A.; Merkoçi, A. Molecularly Imprinted Polymer-Decorated Magnetite Nanoparticles for Selective Sulfonamide Detection. Anal. Chem. 2016, 88, 3578-3584. (5) Chen, Y. Q.; Chen, Q.; Han, M. M.; Liu, J. Y.; Zhao, P.; He, L. D.; Zhang, Y.; Niu, Y. M.; Yang, W. J.; Zhang, L. Y. Near-Infrared Fluorescence-Based Multiplex Lateral Flow Immunoassay for the Simultaneous Detection of Four Antibiotic Residue Families in Milk. Biosens. Bioelectron. 2016, 79, 430-434. (6) Han, S. J.; Zhou, T. J.; Yin, B. J.; He, P. L. A Sensitive and Semi-Quantitative Method for Determination of Multi-Drug Residues in Animal Body Fluids Using Multiplex Dipstick Immunoassay. Anal. Chim. Acta 2016, 927, 64-71. (7) Wang, J.; Cheng, M. T.; Zhang, Z.; Guo, L. Q.; Liu, Q.; Jiang, G. B. An Antibody-Graphene Oxide Nanoribbon Conjugate as a Surface Enhanced Laser Desorption/Ionization Probe with High Sensitivity and Selectivity. Chem. Commun. 2015, 51, 4619-4622. (8) Gan, J. R.; Wei, X.; Li, Y. X.; Wu, J.; Qian, K.; Liu, B. H. Designer SiO2@Au Nanoshells towards Sensitive and Selective Detection of Small Molecules in Laser Desorption Ionization Mass Spectrometry. Nanomedicine 2015, 11, 1715-1723. (9) Gulbakan, B.; Yasun, E.; Shukoor, M. I.; Zhu, Z.; You, M. X.; Tan, X. H.; Sanchez, H.; Powell, D. H.; Dai, H. J.; Tan, W. H. A Dual Platform for Selective Analyte Enrichment and Ionization in Mass Spectrometry Using Aptamer-Conjugated Graphene Oxide. J. Am. Chem. Soc. 2010, 132, 17408-17410. (10) Ma, R.; Lu, M.; Ding, L.; Ju, H.; Cai, Z. Surface-Assisted Laser Desorption/Ionization Mass Spectrometric Detection of Biomolecules by Using Functional Single-Walled Carbon Nanohorns as the Matrix. Chem. - Eur. J. 2013, 19, 102-108. (11) Wang, J.; Sun, J.; Wang, J.; Liu, H.; Xue, J.; Nie, Z. Hexagonal Boron Nitride Nanosheets as a Multifunctional Background-Free Matrix to Detect Small Molecules and Complicated Samples by MALDI Mass Spectrometry. Chem. Commun. 2017, 53, 8114-8117. (12) Kuo, T.-R.; Chen, Y.-C.; Wang, C.-I.; Shen, T.-H.; Wang, H.-Y.; Pan, X.-Y.; Wang, D.-Y.; Liou, C.-C.; Chang, Y.-H.; Chen, Y.-C.; Wu, Y.-H.; Liu, Y.-R.; Lin, Y.-H.; Hu, C.-C.; Chen, C.-C. Highly Oriented Langmuir-Blodgett Film of Silver Cuboctahedra as an Effective Matrix-Free Sample Plate for Surface-Assisted Laser Desorption/Ionization Mass Spectrometry. Nanoscale 2017, 9, 11119-11125.


ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(13) Huang, X.; Liu, Q.; Huang, X.; Nie, Z.; Ruan, T.; Du, Y.; Jiang, G. Fluorographene as a Mass Spectrometry Probe for High-Throughput Identification and Screening of Emerging Chemical Contaminants in Complex Samples. Anal. Chem. 2017, 89, 1307-1314. (14) Lu, W. J.; Li, Y.; Li, R. J.; Shuang, S. M.; Dong, C.; Cai, Z. W. Facile Synthesis of N-Doped Carbon Dots as a New Matrix for Detection of Hydroxy-Polycyclic Aromatic Hydrocarbons by Negative-Ion Matrix-Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry. ACS Appl. Mater. Interfaces 2016, 8, 12976-12984. (15) Xu, G. J.; Liu, S. J.; Peng, J. X.; Lv, W. P.; Wu, R. A. Facile Synthesis of Gold@Graphitized Mesoporous Silica Nanocomposite and Its Surface-Assisted Laser Desorption/lonization for Time-of-Flight Mass Spectroscopy. ACS Appl. Mater. Interfaces 2015, 7, 2032-2038. (16) Huang, X.; Liu, Q.; Fu, J.; Nie, Z.; Gao, K.; Jiang, G. Screening of Toxic Chemicals in a Single Drop of Human Whole Blood Using Ordered Mesoporous Carbon as a Mass Spectrometry Probe. Anal. Chem. 2016, 88, 4107-4113. (17) Ma, Y. R.; Zhang, X. L.; Zeng, T.; Cao, D.; Zhou, Z.; Li, W. H.; Niu, H. Y.; Cai, Y. Q. Polydopamine-Coated Magnetic Nanoparticles for Enrichment and Direct Detection of Small Molecule Pollutants Coupled with MALDI-TOF-MS. ACS Appl. Mater. Interfaces 2013, 5, 1024-1030. (18) Liu, Q.; Cheng, M. T.; Wang, J.; Jiang, G. B. Graphene Oxide Nanoribbons: Improved Synthesis and Application in MALDI Mass Spectrometry. Chem. - Eur. J. 2015, 21, 5594-5599. (19) Zhao, Y.; Deng, G.; Liu, X.; Sun, L.; Li, H.; Cheng, Q.; Xi, K.; Xu, D. MoS2/Ag Nanohybrid: A Novel Matrix with Synergistic Effect for Small Molecule Drugs Analysis by Negative-ion Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Anal. Chim. Acta 2016, 937, 87-95. (20) Dong, X. L.; Cheng, J. S.; Li, J. H.; Wang, Y. S. Graphene as a Novel Matrix for the Analysis of Small Molecules by MALDI-TOF MS. Anal. Chem. 2010, 82, 6208-6214. (21) Hu, J. J.; Liu, F.; Ju, H. X. MALDI-MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance. Angew. Chem. Int. Ed. 2016, 55, 6667-6670. (22) Wei, Y.; Li, S.; Wang, J.; Shu, C.; Liu, J. a.; Xiong, S.; Song, J.; Zhang, J.; Zhao, Z. Polystyrene Spheres-Assisted Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Quantitative Analysis of Plasma Lysophosphatidylcholines. Anal. Chem. 2013, 85, 4729-4734. (23) Hosu, I. S.; Sobaszek, M.; Ficek, M.; Bogdanowicz, R.; Drobecq, H.; Boussekey, L.; Barras, A.; Melnyk, O.; Boukherroub, R.; Coffinier, Y. Carbon nanowalls: a new versatile graphene based interface for the laser desorption/ionization-mass spectrometry detection of small compounds in real samples. Nanoscale 2017, 9, 9701-9715. (24) Chen, S.; Zheng, H.; Wang, J.; Hou, J.; He, Q.; Liu, H.; Xiong, C.; Kong, X.; Nie, Z. Carbon Nanodots As a Matrix for the Analysis of Low-Molecular-Weight Molecules in Both Positive- and Negative-Ion Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Quantification of Glucose and Uric Acid in Real Samples. Anal. Chem. 2013, 85, 6646-6652.


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(25) Xie, J.; Jiang, H.; Shen, J.; Peng, T.; Wang, J.; Yao, K.; Sun, S.; Shao, B.; Tang, J. Design of Multifunctional Nanostructure for Ultrafast Extraction and Purification of Aflatoxins in Foodstuffs. Anal. Chem. 2017, 89, 10556-10564. (26) Jesus Garcia-Galan, M.; Silvia Diaz-Cruz, M.; Barcelo, D. Combining Chemical Analysis and Ecotoxicity to Determine Environmental Exposure and to Assess Risk from Sulfonamides. Trends Anal. Chem. 2009, 28, 804-819. (27) Veach, B. T.; Mudalige, T. K.; Rye, P. RapidFire Mass Spectrometry with Enhanced Throughput as an Alternative to Liquid–Liquid Salt Assisted Extraction and LC/MS Analysis for Sulfonamides in Honey. Anal. Chem. 2017, 89, 3256-3260. (28) Zhu, W.-x.; Yang, J.-z.; Wang, Z.-x.; Wang, C.-j.; Liu, Y.-f.; Zhang, L. Rapid Determination of 88 Veterinary Drug Residues in Milk Using Automated TurborFlow Online Clean-Up Mode Coupled to Liquid Chromatography-Tandem Mass Spectrometry.Talanta 2016, 148, 401-411. (29) Malá, Z.; Gebauer, P.; Boček, P. New Methodology for Capillary Electrophoresis with ESI-MS Detection: Electrophoretic Focusing on Inverse Electromigration Dispersion Gradient. High-Sensitivity Analysis of Sulfonamides in Waters. Anal. Chim. Acta 2016, 935, 249-257. (30) Li, C.; Wang, Z. H.; Cao, X. Y.; Beier, R. C.; Zhang, S. X.; Ding, S. Y.; Li, X. W.; Shen, J. Z. Development of an Immunoaffinity Column Method Using Broad-Specificity Monoclonal Antibodies for Simultaneous Extraction and Cleanup of Quinolone and Sulfonamide Antibiotics in Animal Muscle Tissues. J. Chromatogr. A 2008, 1209, 1-9. (31) Li, R. J.; Wu, M. C. High-Throughput Determination of 30 Veterinary Drug Residues in Milk Powder by Dispersive Solid-Phase Extraction Coupled with Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry. Food Anal. Methods 2017, 10, 3753-3762. (32) Yang, P.; Chang, J. S.; Wong, J. W.; Zhang, K.; Krynitsky, A. J.; Bromirski, M.; Wang, J. Effect of Sample Dilution on Matrix Effects in Pesticide Analysis of Several Matrices by Liquid Chromatography High-Resolution Mass Spectrometry. J. Agric. Food Chem. 2015, 63, 5169-5177. (33) Hoff, R. B.; Rubensam, G.; Jank, L.; Barreto, F.; Peralba, M. D. R.; Pizzolato, T. M.; Diaz-Cruz, M. S.; Barcelo, D. Analytical Quality Assurance in Veterinary Drug Residue Analysis Methods: Matrix Effects Determination and Monitoring for Sulfonamides Analysis. Talanta 2015, 132, 443-450. (34) Lin, Z.; Zheng, J. N.; Lin, G.; Tang, Z.; Yang, X. Q.; Cai, Z. W. Negative Ion Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Analysis of Small Molecules Using Graphitic Carbon Nitride Nanosheet Matrix. Anal. Chem. 2015, 87, 8005-8012. (35) Kim, Y. K.; Na, H. K.; Kwack, S. J.; Ryoo, S. R.; Lee, Y.; Hong, S.; Hong, S.; Jeong, Y.; Min, D. H. Synergistic Effect of Graphene Oxide/MWCNT Films in Laser Desorption/Ionization Mass Spectrometry of Small Molecules and Tissue Imaging. ACS Nano 2011, 5, 4550-4561. (36) Geis-Asteggiante, L.; Nunez, A.; Lehotay, S. J.; Lightfield, A. R. Structural Characterization of Product Ions by Electrospray Ionization and Quadrupole Time-of-Flight Mass Spectrometry to Support Regulatory Analysis of Veterinary Drug Residues in Foods. Rapid Commun. Mass Spectrom. 2014, 28, 1061-1081.


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Table 1. Comparison of detection results by our method and concentrations determined by HPLC-ESI-MS/MS. sample

amount detected by

amount detected by

HPLC-ESI-MS/MS (µg/kg)

our method (µg/kg)

Pork (No. 941)

SM2 57.3 ± 0.6

SM2 58.0 ± 2.3

Egg (No. 833)

SM2 10.6 ± 0.3

SM2 9.71 ± 3.3


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FIGURES

Figure 1. Schematic representation of selective enrichment and quantitative analysis of SAs on disposable MoS2-arrayed MALDI MS chip combined with immunoaffinity enrichment method.


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Figure 2. (a) MALDI-MS spectra of various SAs obtained on MoS2-arrayed MALDI MS chip. The concentration of each analyte is 500 µg/mL. (b) MALDI-TOF mass spectrum of the mixture of the twelve SAs obtained on MoS2-arrayed MALDI MS chip. The concentration of each analyte is 50 µg/mL. (c) Comparison of mass peak intensities of five SAs with MoS2-arrayed commercial steel plate, MoS2-arrayed ITO glass chip, and MoS2-arrayed glass chip for MALDI-TOF MS. The concentration of each analyte is 50 µg/mL.


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Figure 3. MALDI-MS sensitivity assay. MALDI-MS spectra of SDM at different concentrations (a) without enrichment and (b) using SA-mAb/MB for enrichment.

Figure 4. Multiplex quantitative analysis of five SAs standard working solutions with enrichment using SA-mAb/MB. (a) Representative MALDI-TOF mass spectra of a serial dilution of SAs (0.5 ng/mL, 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, and 10 ng/mL). The standard calibration curves of (b) SMZ, (c) SM2, (d) SMM, (e) SQX, and (f) SDM. Throughout the experiments, MoS2-arrayed MALDI MS chips were previously covered with 5 ng SDM-d6 as an IS. The error bars represent the standard deviations of three independent measurements.


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Figure 5. Calibration curves of (a) SDM, (b) SM2, and (c) SMM obtained from pure solvent standard working solutions (red lines) and (a) pork, (b) egg, and (c) milk extract standard working solutions (blue lines). Representative MALDI-TOF mass spectra of five SAs spiked pork sample (50 µg/kg) (d) without and (e) with SA-mAb/MB enrichment. (f) Repetition of SDM capture and re-capture on the regenerated SA-mAb/MB: the intensity ratios of enriched SDM to SDM-d6 after 5 capture and regeneration cycle. Throughout the experiments, MoS2-arrayed MALDI MS chips were previously covered with 5 ng SDM-d6 as an IS.


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Figure 6. The detection and identification of SAs in real foodstuffs: (a) pork sample and (b) egg sample. (c-d) The structures of the detected SAs were further identified with corresponding MS/MS spectra. Throughout the experiments, MoS2-arrayed MALDI MS chips were previously covered with 5 ng SDM-d6 as an IS.


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TOC: Graphical Abstract


Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and

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