Surface-Coated Probe Nanoelectrospray Ionization Mass

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Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for Analysis of Target Compounds in Individual Small Organisms Jiewei Deng,† Yunyun Yang,‡ Mingzhi Xu,† Xiaowei Wang,§ Li Lin,† Zhong-Ping Yao,∥ and Tiangang Luan*,† †

MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, China ‡ Guangdong Provincial Key Laboratory of Emergency Test for Dangerous Chemicals and Guangdong Provincial Public Laboratory of Analysis and Testing Technology, China National Analytical Center Guangzhou, 100 Xianlie Middle Road, Guangzhou 510070, China § Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, China ∥ State Key Laboratory for Chirosciences and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong S. A. R., China S Supporting Information *

ABSTRACT: Analysis of target compounds in individual small organisms is of significant importance for biological, environmental, medicinal, and toxicological investigation. In this study, we reported the development of a novel solid-phase microextraction (SPME) based ambient mass spectrometry (MS) method named surface-coated probe nanoelectrospray ionization (SCP-nanoESI)MS for analysis of target compounds in individual small organisms with sizes at micrometer-to-millimeter level. SCP-nanoESI-MS analysis involves three procedures: (1) modification of adsorbent at the surface of a fine metal probe to form a specially designed surface-coated SPME probe with probe-end diameter at severalmicrometer level, (2) application of the surface-coated SPME probe for enrichment of target analytes from individual small organisms, and (3) employment of a nanospray tip and some solvent to desorb the analytes and induce nanoESI for mass spectrometric analysis under ambient condition. A SCP-nanoESI-MS method for determination of the perfluorinated compounds (PFCs) in individual Daphnia magna was developed. The method showed satisfactory linearities for analysis of real Daphnia magna samples, with correlation coefficient values (R2) of 0.9984 and 0.9956 for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), respectively. The limits of detection were 0.02 and 0.03 ng/mL for PFOS and PFOA, respectively. By using the proposed method, the amount, bioaccumulation kinetics, and distribution of PFOS and PFOA in individual Daphnia magna were successfully investigated.

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rich, etc.13,14 Matrix assisted laser desorption ionization (MALDI) 15,16 and secondary ion mass spectrometry (SIMS)17,18 are the two emerging MS based biological analysis methods, but the vacuum operating conditions restrict their applications in the analysis of living organisms and cells. Ambient MS19,20 provides a straightforward strategy for direct analysis of individual organisms and single cells at ambient and open-air conditions. Masujima’s group developed the live single-cell video MS method for detection of cellular and subcellular molecules, by applying a nanospray tip for direct suction of a single cell for subsequent mass spectrometric analysis.21 Pan et al. developed the single-probe which could be

nalysis of target compounds in individual small organisms and even single cells is of great importance, and it can give us significant insight in microsize for biological, biomedical, environmental, and cell toxicological investigation. However, it is still a very challenging task for both biological and chemical scientists. Several analytical techniques including fluorescence,1−3 nuclear magnetic resonance (NMR) spectroscopy,4 Raman scattering,5,6 chromatography,7 electrophoresis,8 microfluidics,9 and mass spectrometry (MS),10−12 etc., have been developed for analysis of the chemicals and metabolites in organisms and cells. Fluorescence based methods have played a prominent role for many years because of its high sensitivity and selectivity, but this technique typically requires labeling analytes, making the process laborious and time-consuming.12 MS is rapidly becoming a powerful approach for biological and even single-cell analysis due to its outstanding properties of high sensitivity, excellent specificity, label-free, and information© XXXX American Chemical Society

Received: July 2, 2015 Accepted: September 11, 2015

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DOI: 10.1021/acs.analchem.5b03110 Anal. Chem. XXXX, XXX, XXX−XXX

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nutrients in aquatic ecosystems.36 The size of a Daphnia magna is at a several-millimeter level, and thus it is selected to demonstrate the SCP-nanoESI-MS method for analysis of target compounds in individual small organisms.

inserted into single eukaryotic cells to sample the intracellular compounds for real-time MS analysis.22 Vertes’ group applied laser ablation electrospray ionization (LAESI)-MS for in situ analysis of individual cells to investigate metabolic variations in cell populations.23,24 Further improvements were made to enable LAESI-MS for in situ cell-by-cell imaging25 and observation of subcellular metabolites in single cells.26 Zhang’s group has also reported the application of probe electrospray ionization (PESI)-MS for single-cell analysis, by utilizing a tungsten probe with a tip diameter of 1 μm for sampling single cells and separation of analytes, and various metabolites at cellular and subcellular levels were successfully detected.27 Recently, several solid-phase microextraction (SPME)28,29 based ambient ionization methods were developed to achieve direct analysis of target compounds in complex samples with high sensitivity and low matrix effect. A PESI method by coupling an SPME probe (a nanosized TiO2 modified stainless steel needle with diameter of ∼300 μm) to nanoelectrospray ionization (nanoESI)-MS was developed by Zhang’s group, for selective and sensitive analysis of phosphopeptides in complex biological samples.30 Surface-coated wooden-tip ESI31 and coated blade spray ionization32 were developed for rapid extraction of analytes from complicated samples and direct ionization for MS analysis under ambient condition. However, these SPME based ambient MS methods are difficult for analysis of individual small organisms and single cells, because the SPME probes were designed with tip diameters of hundreds of micrometers, which is unsuitable for microsampling small organisms with sample amounts at picoliter-to-microliter level. Here we report the development of a novel surface-coated probe nanoelectrospray ionization mass spectrometry (SCPnanoESI-MS) method and its application for highly sensitive analysis of target compounds in individual small organisms with sizes at the micrometer-to-millimeter level. SCP-nanoESI-MS is developed from PESI-MS with the modification of adsorbent on the surface of a fine-tungsten probe for enrichment of target analytes. Indeed, SCP-nanoESI is a SPME based ambient ionization method, and its principle is similar to that of the recently developed SPME probe-nanoESI,30 surface-coated wooden-tip ESI,31 and coated blade spray ionization32 techniques but with improvement in the design of SPME probe for sampling the target position of individual small organisms and even single cells. First, the surface-coated SPME probe was designed with a tip diameter of one to several micrometers to make it possible to insert into a precise position of a small organism/cell for enrichment of target analytes. Second, the adsorbent was applied at nanometer-thickness to ensure fast equilibrium of analytes between SPME coating and sample matrix. In addition, the nanometer-scale coating does not obviously influence the size of the surface-coated SPME probe. Experiments were performed to investigate the exposure and bioaccumulation of perfluorinated compounds (PFCs) toward individual Daphnia magna. PFCs are persistent organic pollutants that have been of wide concern by both health authorities and the public in recent years,33,34 and the two most prevalent PFCs, i.e., perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA),35 were selected for investigation in this study. Daphnia magna is a small organism widely used as an important indicator of aquatic ecosystem health as well as a model organism in ecotoxicology.36,37 Particularly, Daphnia magna is an important trophic element of aquatic food webs and plays a prominent role in transporting energy and

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EXPERIMENTAL SECTION Chemicals and Reagents. n-Octadecyldimethyl[3(trimethoxysilyl)propyl]ammonium chloride (50% in methanol) was purchased from Fluorochem Ltd. (Graphite Way, Hadfield SK13 IQH, Germany). Calcium hydride and N,Ndimethylformamide (DMF, purified through calcium hydride desiccation and vacuum distillation before use) were obtained from Sigma-Aldrich (St. Louis, MO). CaCl2·2H2O, KCl, K2Cr2O7, KMnO4, MgSO4·2H2O, NaHCO3, H2SO4, and dichloromethane were from Guangzhou Chemical Reagent Factory (Guangzhou, China). HPLC grade methanol and acetonitrile were supplied by Burdick & Jackson (Muskegon, MI). Pure water was purified by a Milli-Q water purification system (Milford, MA). Standard substances of PFOS and PFOA were purchased from Accustandard Co. Ltd. (New Haven, CT). 13C4-PFOS and 13C4-PFOA were from Wellington Laboratories (Guelph, ON, Canada). Preparation of Surface-Coated SPME Probe. RS-6065 tungsten microdissecting needles (length of 5 cm, tip diameter of ∼1 μm, purchased from Roboz Surgical Instrument Co. Inc., Gaithersburg, MD) were used as substrates to prepare surfacecoated SPME probes. The needles were first washed with methanol/water (1:1, v/v) to clean their surfaces. Then, they were soaked in a 30% (wt/wt) H2SO4 solution containing KMnO4 (30 mg/mL) and K2Cr2O7 (15 mg/mL), and heated with reflux at 90 °C for 5 days (d). Subsequently, the needles were washed with water and put into a 30% (wt/wt) NaOH solution refluxed at 90 °C for 5 d to hydroxylate their surfaces. Afterward, the surface hydroxylated tungsten needles were applied to modify the adsorbent by silanization using a way similar to that of our previous study.31 In brief, the needles were immersed into a 50 mL anhydrous DMF solution, followed by adding 2.5 mL of n-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, and then heated with reflux at 120 °C under nitrogen atmosphere for reaction of 12 h. The surface-coated SPME probes were obtained after the coated tungsten needles were washed and dried. Characterization of the Surface-Coated SPME Probe. An ESCA LAB 250 X-ray photoelectron spectroscopy (XPS) instrument (Thermo Fisher Scientific, San Jose, CA) was applied for the XPS experiments, a Quanta 400 FEG field emission scanning electron microscope (SEM) instrument (FEI, The Netherlands) was employed for the SEM analysis, and a dimension Fastscan Bio atomic force microscope (AFM) instrument (Bruker Daltonics, Bremen, Germany) was utilized to perform the AFM analysis. Cultivation of Daphnia magna. Daphnia magna were cultured in the artificial freshwater (prepared by adding 58.5 mg of CaCl2·2H2O, 24.7 mg of MgSO4·2H2O, 13.0 mg of NaHCO3, and 1.2 of mg KCl into 1 L of pure water, and vigorously aerated for 24 h to dissolve the chemicals and stabilize the medium36) at a temperature of 20 ± 1 °C under a 16:8-h light:dark photoperiod as recommended by the guideline of Organization for Economic Cooperation and Development for the testing of chemicals.38 The suspension of Scenedesmus subspicatus was utilized to feed the Daphnia magna twice a day. B

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Figure 1. Schematic diagrams for development of a SCP-nanoESI-MS method to determine PFCs in individual Daphnia magna. (a) A surface-coated SPME probe designed for enrichment of PFCs. (b) Microscopic images of sampling one individual Daphnia magna and its one egg cell. (c) Experimental setup of the SCP-nanoESI-MS analysis. One loaded SPME probe was inserted into a nanospray tip filled with 1 μL of desorption/spray solvent, and a high voltage of −3.0 kV was applied on the SPME probe for mass spectrometric analysis. (d) Analytical performance and mass spectrum for quantitative analysis of PFOS and PFOA in the body fluid of Daphnia magna with internal standard method.

inserted into its cell membrane. Thus, all the fluids of an egg cell were applied for microextraction. All the operations were observed using a Leica DM IL inverted microscope (Leica Microsystems, Inc., Bannockburn, IL). After sampling, the surface-coated SPME probe was removed for subsequent nanoESI-MS analysis using a way similar to solid probe assisted-nanoESI.40 A BG12-94-4-CE nanospray tip (New Objective Inc., MA) was employed for the experiments, and 1 μL of desorption/spray solvent was filled into the tip end by a microsyringe. The loaded surface-coated SPME probe was inserted into the nanospray tip for desorption of 30 s, and then construction of a SPME-nanoESI assembly with the nanospray tip. The SPME-nanoESI assembly was then controlled by a three-dimensional moving stage and placed pointing to the MS inlet, adjusting to a position of 5 mm away from the MS inlet. Then, a high voltage of −3.0 kV was applied on the surfacecoated SPME probe to induce nanoESI for mass spectrometric analysis. Mass Spectrometry. Mass spectra were acquired on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with negative ion detection mode. Accurate mass measurement was accomplished with a mass resolution of 60 000, and recorded in the range m/z 400−550. Xcalibur 2.2 software (Thermo Fisher Scientific, San Jose, CA) was utilized for experimental control and data acquisition. Quality Assurance and Control. Quality assurance and control procedures were performed routinely. For each time of analysis, a blank experiment (using the same analytical procedure but without extraction of Daphnia magna) was carried out to check the blank surface-coated SPME probe, desorption/spray solvent, and mass spectrometer conditions. No obvious signals of PFOS and PFOA from blank experiment were found. A control group of Daphnia magna (without exposure of PFCs) was also set to study for each time of experiment. The results demonstrated that PFOS and PFOA in control Daphnia magna were around 2% of those in exposed

Exposure of Daphnia magna toward PFCs. The exposure experiments were carried out in 250 mL polypropylene beakers. Juvenile Daphnia magna (6−24 h old) were exposed in a 200 mL aqueous solution with both PFOS and PFOA at a concentration of 10 ng/mL (which is an effective and safe concentration similar to that used in the previous bioaccumulation investigation studies36,37 and much lower than the 48 h-LC50 values, i.e., 37.36 mg/L for PFOS and 476.52 mg/L for PFOA39). The maximum amount of methanol (used as a carrier for PFOS and PFOA) was less than 0.1 mL/L in the final test medium. For investigation of individual difference of bioaccumulation, the exposure time was selected as 3 d and 28 Daphnia magna were tested. For bioaccumulation kinetic experiment, the sampling time points were selected at 0 h, 1 h, 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 5 d, 7 d, and 10 d, respectively. At each time point, 6 individual Daphnia magna were applied for analysis in parallel. To analyze the distribution, 6 individual Daphnia magna after 3 d exposure were analyzed. After the bioaccumulation experiments, the Daphnia magna were transferred from beaker by pipet to polystyrene culture dishes and rinsed with the artificial freshwater for five times, then dried by filter paper and further transferred to a clean microscope glass slide for sampling and extraction. Sampling and Detection. A three-dimensional manipulator (Fu-Li-Qian-Tian Optoelectronics Technology, Beijing, China), which possesses the smallest microstep size of 1 μm, was used for controlling one surface-coated SPME probe to precisely insert into a target position of Daphnia magna for sampling. The depth of the insertion to the Daphnia magna body was approximately 50 μm, and the sampling time was 60 s. For sampling the egg cells of Daphnia magna, another micromanipulator equipped with a RS-6065 needle (with a tip diameter of ∼1 μm) was used for microdissection to separate the egg cells from the body of Daphnia magna. In general, the egg cell was broken when a surface-coated SPME probe was C

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Figure 2. XPS spectra of the (a) tungsten needle and (b) surface-coated SPME probe.

Daphnia magna. To eliminate the interference of exposure medium, the exposed Daphnia magna was washed with artificial freshwater for five times before analysis. The amounts of PFOS and PFOA present in the washed artificial freshwater of the last time were measured no higher than 5% of those in exposed Daphnia magna. Sample carryover was also monitored, by a blank experiment after the analysis of an exposed Daphnia magna, and no evident carryover was observed. To perform an accurate quantitative calculation, a series of concentrations of PFOS and PFOA were spiked into blank body fluids of Daphnia magna to construct calibration curves. First, Daphnia magna were dried by filter paper and transferred to a 1 mL PP centrifuge tube. Then, the Daphnia magna were homogenized and centrifuged at 4000 rpm for 10 min, and the supernatants were obtained as the body fluids of Daphnia magna. A 10 μL portion of body fluid was spiked with 0.1 μL of PFOS and PFOA methanol/water (1:9, v/v) solution, forming a series of spiked samples with PFOS and PFOA at concentrations of 0.1, 0.5, 1, 5, and 10 ng/mL, respectively. Principal Component Analysis. Principal component analysis (PCA) was performed by SIMCA-P (11.5 Umetrics, Umea, Sweden). The concentration values of PFOS and PFOA were used as variables, and the samples were used for observations. The data sets were mean-centered scaled prior to modeling.

solvent to desorb the analytes and induce nanoESI for mass spectrometric analysis under ambient and open-air conditions. Previously reported techniques such as solid probe assistednanoESI40 and solvent vapor condensing PESI,42,43 etc., can be introduced in this step. Design of the Surface-Coated SPME Probe for Enrichment of PFCs in Individual Daphnia magna. A suitable solid substrate should be selected to prepare the surface-coated SPME probe. A mature Daphnia magna generally has a size of 3−5 mm, and its egg cells are usually at the sizes of 100−200 μm (Figure S-1 in the Supporting Information). Thus, the surface-coated SPME probe should be designed with tip-end of micrometer-scale to make it possible to insert into a precise position of a Daphnia magna for enrichment of analytes. In this study, a tungsten needle was selected as the substrate for modification of adsorbent to prepare a surface-coated SPME probe. The tungsten needle is very hard and has a tip-end diameter of ∼1 μm, which makes it easy to pierce into the skin/membrane of a small organism/cell and reach a precise target position for sampling. The diameters of the tungsten needle at the positions of 10, 50, and 100 μm away from tip-end are approximately 5, 15, and 30 μm, respectively. Thus, it can be inserted into a precise position of Daphnia magna with a rigorous depth for sampling and microextraction. Before functionalization of adsorbent, the surface of tungsten needles should be hydroxylated first. We applied a previously reported oxidant,43 which is 30% (wt/wt) H2SO4 solution containing 30 mg/mL of KMnO4 and 15 mg/mL of K2Cr2O7, to oxidize the tungsten needles surfaces, and then hydroxylate them using a 30% (wt/wt) NaOH solution. n-Octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride was selected as the adsorbent to prepare the surfacecoated SPME probe. Our previous study31 has confirmed that this adsorbent showed a desirable ability for direct extraction of PFCs from complex biological samples such as whole blood and milk, via the mechanisms of both reversed phase adsorption and ion exchange adsorption. The adsorbent was bonded to the hydroxyl groups on the tungsten needles surface via silanization using a method similar to that from our previous study (Figure S-2 in the Supporting Information).31 Characterization of the Surface-Coated SPME Probe. In the XPS spectrum of the tungsten needle surface (Figure 2a), only carbon-, oxygen-, and tungsten-related peaks were found. While in the XPS spectrum of the surface-coated SPME probe surface (Figure 2b), the carbon-, oxygen-, nitrogen-, silicon, and tungsten-related peaks were observed. The content

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RESULTS AND DISCUSSION Procedures for Development of a SCP-nanoESI-MS Method. Generally, the development of a SCP-nanoESI-MS method for analysis of target analytes in individual small organisms involves three procedures (Figure 1): First, one must select a suitable adsorbent and a modification method to develop a specially designed surface-coated SPME probe toward target analytes. The adsorbents are selected according to the characteristics of target compounds. Octyl (C8), octodecyl (C18), cation exchanger, and anion exchanger, etc., are the commonly used materials. The use of highly selective adsorbents such as aptamers for specific compounds is recommended. Attachment chemistries for covalent surface modification include silane, phosphonate, carboxylate, catechol, alkene/alkyne, and amine, etc.41 Silanization is one of the most widely used approaches, by which covalent linkages between substrates and adsorbents are rapidly formed. Second, one must control the surface-coated SPME probe to insert into a precise position of the small organism for enrichment of target analytes. This step is usually operated under microscopic observation. Third, one must employ a nanospray tip and some D

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Figure 3. SEM images from the tips of (a−c) original tungsten needle tip and (d−f) surface-coated SPME probe at magnifications of 200× , 1500× , and 6000× , respectively.

percentages of Si and N in the SPME probe surface are 4.23% and 2.60%, respectively. Deconvolution of the Si 2p peak displays the presence of both Si−O at 102.2 eV and Si-CH2 at 102.8 eV (Figure S-3a in the Supporting Information), and deconvolution of the N 1s peak reveals the presence of N−CH2 at 399.6 eV and N−CH3 at 402.2 eV (Figure S-3b in the Supporting Information). Thus, the XPS results demonstrated the successful functionalization of the n-octadecyldimethyl[3(trimethoxysilyl)propyl]ammonium chloride on the SPME probe surface. The SEM images showed the morphologic differences between the original tungsten needle and surface-coated SPME probe. Figure 3a−c revealed that the original tungsten needle tip-end diameter was ∼1 μm with smooth surface. After functionalization, the surface morphology of the surface-coated SPME probe looked quite different from the original tungsten needle surface, and became rough (Figure 3d−f). The tip-end diameter was measured to be ∼2 μm, which was sufficient for sampling the organs of Daphnia magna. AFM was applied to further investigate the surface morphology and thickness of the coating. The AFM height images showed that the surface of uncoated tungsten needle was relatively neat (Figure S-4a in the Supporting Information), while regular globules were observed on the surface of our developed SPME probe (Figure S-4b in the Supporting Information). The root-mean-square roughness, average roughness, and maximum roughness of the coated material were 5.44, 4.09, and 39.9 nm, respectively (Figure S-5 in the Supporting Information). The heights of the globules were measured in several dozens of nanometers, and the radii of the globules were measured at the level of hundreds of nanometers (Figure S-5 in the Supporting Information). Thus, the obtained coating of surface-coated SPME probe was at nanometer-level thickness, which does not influence the size of surface-coated SPME probe obviously. In addition, the nanometer-scale adsorbent facilitates the process of microextraction by enhancement of the mass transfer. Optimization of the Experimental Conditions. The experimental conditions including extraction time, type, and amount of desorption/spray solvent; desorption time; high

voltage; and distance between tip-end and MS inlet, etc., were optimized by analyzing the body fluids of Daphnia magna spiked with PFOS and PFOA (both of 10 ng/mL). The extraction equilibrium time was investigated first because the adsorbent of the surface-coated SPME probe was designed at the nanometer-scale to enhance the mass transfer and facilitate the microextraction process. A series of extraction periods including 5, 10, 20, 30, 60, 120, and 300 s were studied, and the result revealed that 60 s was sufficient for a dynamic equilibrium of the distribution of PFOS and PFOA between the coating and matrices (Figure S-6 in the Supporting Information). Subsequently, different desorption/spray solvents, i.e., methanol, acetonitrile, methanol/water (v/v = 1:1), acetonitrile/water (v/v = 1:1), and dichloromethane, were investigated. Methanol gave the most abundant and stable signals of PFOS and PFOA, and thus, it was selected as the desorption/ spray solvent for the analysis of PFOS and PFOA in Daphnia magna by SCP-nanoESI-MS. The amount of desorption/spray solvent was also tested. The increasing volume of desorption/ spray solvent increases the signal duration but decreases the signal intensity. When 1 μL of methanol was used, the signal duration time was more than 30 s, and the analysis sensitivity was enough for determination of real Daphnia magna samples. Different desorption times (i.e., 10, 20, 30, 45, 60, and 120 s) were studied. It was found that the signal intensities increased with the increasing desorption time, and the equilibrium of desorption was reached at 30 s. Different high voltage (i.e., −2.0, −2.5, −3.0, −3.5, −4.0, −4.5, and −5.0 kV) were tested, and the optimized value was selected as −3.0 kV. The distance between the tip-end of the nanospray tip and the MS inlet was optimized, and the optimum distance was selected as 5 mm. Extraction Properties and Quantitative Abilities. Previous study27 has demonstrated that a fine-tungsten needle could also be used as a SPME probe for sampling single plant cells and enrichment of metabolites for PESI-MS analysis, enhancing the signal intensity about 30-fold even without the adsorbents coated on the needle surface. To demonstrate the capacity of the surface-coated SPME probe for enrichment of PFCs, here we compared a fine-tungsten needle and a surfaceE

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calibration curve method, and 13C4-PFOS and 13C4-PFOA were used as the IS compounds of PFOS and PFOA, respectively. Both 13C4-PFOS and 13C4-PFOA were added into desorption/spray solvent with a concentration of 1 ng/mL. After SCP-nanoESI-MS analysis, satisfactory linearities were obtained for both PFOS and PFOA in the range 0.1−10 ng/ mL, with correlation coefficient values (R2) of 0.9984 and 0.9956, respectively. The limit of detection (LOD) and quantitation (LOQ) were 0.02 and 0.06 ng/mL for PFOS, and 0.03 and 0.09 ng/mL for PFOA, respectively. The method showed good repeatability with relative standard deviation (RSD) values of 5.8% and 8.6% for PFOS and PFOA, respectively, by using one probe for six replicate extractions of Daphnia magna body fluid spiked with 1 ng/mL of PFOS and PFOA. Probe-to-probe repeatability was also examined by analyzing six Daphnia magna body fluid samples spiked with 1 ng/mL of PFOA and PFOS using six different surface-coated SPME probes, with RSDs of 7.9% and 10.4% for PFOS and PFOA, respectively. The method also exhibited good accuracy, with recoveries in the range 90.4−117.6% for analyzing different concentrations (0.5 and 5 ng/mL) of spiked samples (Table 1). The coating stability was also investigated, and the results showed that the coating property of the SPME probe was stable, allowing it to be used over 50 times without measurable loss of performance. It was noted that every time it is reused, the surface-coated SPME probe should be washed with methanol and pure water. The method has been proven to be desirable for analyzing real Daphnia magna samples after PFOS and PFOA exposure. By sampling the body fluid in the abdomen of one Daphnia magna for analysis, significant signals of PFOS and PFOA were observed (Figure 4b). Similar signals were also found by sampling its tissue and egg cell for analysis (Figure S-7 in the Supporting Information). The body fluid and tissue of the Daphnia magna were extracted twice for replicated analysis, and similar signal intensities of PFOS and PFOA were obtained, which suggested that one extraction did not significantly affect the concentration of PFOS and PFOA in one Daphnia magna. Investigation of the Individual Difference of Bioaccumulation after Daphnia magna Exposed to PFCs. The developed SCP-nanoESI-MS method was first applied to investigate the individual difference of bioaccumulation after exposure to PFCs. There were 28 Daphnia magna tested, by analysis of the abdomen body fluids after 10 ng/mL of PFOS and PFOA exposure for 3 d. The measured concentrations of each Daphnia magna were summarized in Table S-1 (Supporting Information), and there were 3 Daphnia magna with bioaccumulation concentrations significantly higher (p < 0.01, One-Way ANOVA) than other 25 Daphnia magna. The obtained concentrations of PFOS and PFOA were applied for

coated SPME probe for analysis of one Daphnia magna (expose to 10 ng/mL of PFOS and PFOA for 3 d) at the same time, by inserting two probes into the abdomen of one Daphnia magna for extraction of the body fluid simultaneously, and then for nanoESI-MS analysis. Weak signals from PFOS and PFOA together with many other signals from tissue components were observed in the obtained PESI-MS spectrum (Figure 4a). While

Figure 4. Mass spectra obtained by analyzing the body fluid of one Daphnia magna exposed to 10 ng/mL of PFOS and PFOA for 3 d by using (a) a fine-tungsten needle and (b) a surface-coated SPME probe for sampling.

in the obtained SCP-nanoESI-MS spectrum (Figure 4b), significantly higher signals from PFOS and PFOA were observed, and the signals from tissue components were much lower and immersed in the background. The experimental results demonstrated that our developed surface-coated SPME probe showed high enrichment ability toward PFCs, and the amounts of PFOS and PFOA enriched by the SPME probe and desorbed into the spray solution for mass spectrometric analysis might be higher than those of tissue components. The experiments were repeated six times in parallel, by analyzing six different Daphnia magna, and similar results were obtained. The enrichment abilities for PFOS and PFOA by our developed surface-coated SPME probe were 33 ± 4- and 26 ± 3-fold higher than those by the fine-tungsten needle without surface modification. PFOS and PFOA were added into the body fluid of Daphnia magna to form a series of concentrations of matrix spiked solution to construct calibration curves. A quantitative calculation was performed using internal standard (IS)

Table 1. Validation of the Proposed Method for Analyzing PFOS and PFOA in the Body Fluid of Daphnia Magna repeatability (RSD, %, n = 6)b

recovery (mean ± SD, %, n = 3)

analyte

linear range (ng/mL)

regression eq

R2

LODa (ng/mL)

LOQa (ng/mL)

one probe

probe-to probe

0.5 ng/mL

5 ng/mL

PFOS PFOA

0.1−10 0.1−10

y = 0.3289x − 0.0053 y = 0.0641x + 0.0023

0.9984 0.9956

0.02 0.03

0.06 0.09

5.8 8.6

7.9 10.4

112.4 ± 25.3 117.6 ± 29.6

95.2 ± 12.3 90.4 ± 14.5

a

LOD and LOQ were determined as the concentrations producing signal-to-noise ratios of 3 and 10, respectively. bAnalysis of Daphnia magna body fluid spiked with 1 ng/mL of PFOS and PFOA. F

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Analytical Chemistry PCA, and the results demonstrated that 25 of the 28 Daphnia magna showed good bioaccumulative consistency, while 3 Daphnia magna (samples of nos. 5, 14, and 19) exhibited significantly different bioaccumulative properties (Figure 5), suggesting their individual differences of bioaccumulation.

Figure 5. PCA score plot of 28 investigated Daphnia magna exposed to 10 ng/mL of PFOS and PFOA for 3 d.

Investigation of the Bioaccumulation Kinetics of Daphnia magna Exposed to PFCs. The bioaccumulation kinetics of Daphnia magna exposed to PFOS and PFOA were successfully investigated by analyzing individual organisms with different exposure times. For each time point, six individual Daphnia magna were analyzed in parallel, by sampling the body fluid in the abdomen. As illustrated in Figure 6, both PFOS and PFOA concentrations exhibited a rapid increase during the first 3 d of the exposure phase followed by equilibrium afterward. The observed bioaccumulation kinetics is similar to that of a previous study36 using conventional methods obtained from a number of Daphnia magna and extensive pretreatment steps. PFOS was observed to show higher bioaccumulative ability than PFOA, which were explained by that compound with higher log Kow value (5.5 for PFOS) accumulating more quickly than the lower one (4.3 for PFOA).36 It should be noted that although similar trends of bioaccumulation kinetics were obtained, the SCP-nanoESI-MS method showed obvious advantages of being simple and fast as compared to the conventional methods, and variations of bioaccumulation concentrations among different Daphnia magna were readily observed. Investigation of the PFCs Distribution in Individual Daphnia magna. Sampling different parts of one Daphnia manga was easily achieved because the tip size of the SPME probe was much smaller than Daphnia magna‘s body and even its egg cell. Concentrations of PFOS and PFOA in different parts of Daphnia manga were investigated, and the obtained results are shown in Table S-2 (Supporting Information). The concentration order of PFOS and PFOA from high to low was abdomen, head, back, tail, and egg cell. It is interesting that both PFOS and PFOA could be detectable in the egg cells, and the measured concentrations were 10−20% of those in the abdomen, which suggested that the risks of bioaccumulation can be transferred from mother to offspring even with the existence of a protective barrier from the egg cell membrane.

Figure 6. Bioaccumulation kinetics of (a) PFOS and (b) PFOA obtained by analyzing individual Daphnia magna with different exposure times. (The different symbols represented the detected concentrations of Daphnia magna in different time points, and the black line was the average curve of the bioaccumulation trend of every six samples.)

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CONCLUSION In this study, we report the development of a novel surfacecoated SPME probe and its application for determination of target compounds in individual small organisms. With the modification of a C18-anion exchange adsorbent at the surface of a fine metal probe, a specially designed surface-coated SPME probe toward PFCs was developed. By using the SPME probe for microsampling of individual Daphnia magna, the bioaccumulation behaviors of PFCs were successfully investigated. The proposed methodology processes the advantages of simplicity, quantitativeness, and reproducibility, showing promising prospects in microscale studies of biology, toxicology, environment, medicine, and single cell, etc. The developments of surface-coated SPME probes with different highly efficient materials for further investigation of various chemicals at cellular and subcellular levels are ongoing in our laboratory.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03110. Additional information as noted in text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-20-84112958. E-mail: [email protected]. G

DOI: 10.1021/acs.analchem.5b03110 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry Author Contributions

(29) Deng, J.; Yang, Y.; Wang, X.; Luan, T. TrAC, Trends Anal. Chem. 2014, 55, 55−67. (30) Zhao, Y.; Gong, X.; Si, X.; Wei, Z.; Yang, C.; Zhang, S.; Zhang, X. Analyst 2015, 140, 2599−2602. (31) Deng, J.; Yang, Y.; Fang, L.; Lin, L.; Zhou, H.; Luan, T. Anal. Chem. 2014, 86, 11159−11166. (32) Gómez-Ríos, G. A.; Pawliszyn, J. Angew. Chem., Int. Ed. 2014, 53, 14503−14507. (33) Wang, T.; Wang, Y.; Liao, C.; Cai, Y.; Jiang, G. Environ. Sci. Technol. 2009, 43, 5171−5175. (34) Zushi, Y.; Hogarh, J. N.; Masunaga, S. Clean Technol. Environ. Policy 2012, 14, 9−20. (35) Zareitalabad, P.; Siemens, J.; Hamer, M.; Amelung, W. Chemosphere 2013, 91, 725−732. (36) Dai, Z.; Xia, X.; Guo, J.; Jiang, X. Chemosphere 2013, 90, 1589− 1596. (37) Xia, X.; Rabearisoa, A. H.; Jiang, X.; Dai, Z. Environ. Sci. Technol. 2013, 47, 10955−10963. (38) OECD. Guidlines for the Testing of Chemicals; Organization for Economic Co-Operation and Development: Paris, 2008. (39) Ji, K.; Kim, Y.; Oh, S.; Ahn, B.; Jo, H.; Choi, K. Environ. Toxicol. Chem. 2008, 27, 2159−2168. (40) Mandal, M. K.; Yoshimura, K.; Saha, S.; Ninomiya, S.; Rahman, M. O.; Yu, Z.; Chen, L. C.; Shida, Y.; Takeda, S.; Nonami, H.; Hiraoka, K. Analyst 2012, 137, 4658−4661. (41) Pujari, S. P.; Scheres, L.; Marcelis, A. T. M.; Zuilhof, H. Angew. Chem., Int. Ed. 2014, 53, 6322−6356. (42) Mandal, M. K.; Saha, S.; Yoshimura, K.; Shida, Y.; Takeda, S.; Nonami, H.; Hiraoka, K. Analyst 2013, 138, 1682−1688. (43) Yu, Z.; Chen, L. C.; Mandal, M. K.; Yoshimura, K.; Takeda, S.; Hiraoka, K. J. Am. Soc. Mass Spectrom. 2013, 24, 1612−1615.

J.D. and Y.Y. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 21277177, 21307167, 41473092, 21207121), Scientific and Technological Project of Guangdong Province (No. 2011B060100005). We also acknowledge the financial support of Joint Supervision Scheme with The Chinese Mainland, Taiwan, and Macao Universities of The Hong Kong Polytechnic University.

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DOI: 10.1021/acs.analchem.5b03110 Anal. Chem. XXXX, XXX, XXX−XXX