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An Efficient and Versatile Pipette Microextraction Device Based on a Light-Heatable Sorbent Jianqiao Xu, Xiwen Liu, Qi Wang, Fuxin Wang, Zhoubing Huang, Dong-Yang Zhang, Zongwan Mao, Fang Zhu, and Gangfeng Ouyang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02345 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018
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Analytical Chemistry
An Efficient and Versatile Pipette Microextraction Device Based on a Light-Heatable Sorbent Jianqiao Xu,* Xiwen Liu, Qi Wang, Fuxin Wang, Zhoubing Huang, Dong-Yang Zhang, Zong-Wan Mao, Fang Zhu, Gangfeng Ouyang* KLGHEI of Environment and Energy Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, Guangdong 510275, China. ABSTRACT: Miniaturized sample pretreatment platforms have simplified analytical tasks in diverse disciplines. Herein, a novel pipette microextraction (PME) device is reported by making use of the photothermal property of a light-heatable sorbent (LHS) for the first time. Efficient and staining-free heating treatment of small volumes of liquids confined in the PME device is now enabled through light illumination. The light-induced heating treatment is capable of dramatically accelerating solvent elution rates, effectively unlocking bound toxin from its antibody, and rapidly quenching enzymatic activities, thus, provides PME with higher efficiencies, and enables its new applications in antibody-intermediated sampling of targeted toxin from stained food surfaces and powders, as well as in accurate revelation of enzymatic reaction kinetics. This study offers a new perspective of designing efficient and versatile microextraction platforms, and demonstrates their potential applications in different fields including public security, new drug development and environmental protection.
Miniaturization of analytical platforms have emerged as one of the major goals in analytical sciences.1-5 Miniature, low-cost and user-friendly analytical platforms can be applied both in laboratories and on site for different analytical tasks such as studying “omics“, inspecting environmental quality, and monitoring human health status.1-9 Miniaturization of chromatographic instruments and detection instruments is usually challenging without compromising their separation capacities and detection sensitivities. By contrast, sample pretreatment platforms commonly used for isolating and concentrating analytes from sample matrixes prior to instrumental analysis, are easier to be miniaturized and applied at any sites in need for their fair costs and satisfying performances.6,7 In this way, reliable instrumental analysis could be reserved in laboratories. Solid-phase microextraction (SPME) is a green and economical sample pretreatment technique, which exploits miniaturized devices based on solid phase extractive materials as integrated sample pretreatment platforms.6-9 The wide application of SPME in laboratories and on site greatly simplified sample pretreatment procedures in various fields ranging from isolating pollutants from environmental samples to capturing biomarkers from biofluids and biotissues.6-9 However, the principle of designing the extractive materials keeps unchanged over the years, which is mainly focused on obtaining high specific surface areas and high absorption/adsorption affinities through designing different microscopic structures and functional groups.10-12 These extractive materials leave the sample pretreatment procedures completely under passive modes. The extraction of analytes from sample matrixes, and the elution of analytes from extractive materials are solely driven by the spontaneouslyformed chemical potential gradients, with the mass transfer rates being limited by the diffusion of the analytes in boundary
layers and in solid phase extractive materials (Figure 1a). Few controllable interventions are available for favorably accelerating the mass transfer rates or modulating the distribution (mass flux directions) of the analytes to the receiving media, which could at least be beneficial for further improving efficiencies, and even promote new developments of miniaturized sample pretreatment platforms. In this study, we incorporated heating treatment, which is common in analysts’ toolkits for accelerating mass transfer rates and modulating distribution of analytes in different systems,13-15 to microextraction through an innovative way. We prepared a so-called light-heatable sorbent (LHS), and developed a novel pipette microextraction (PME) device by coating the LHS on the interior surfaces of pipette tips (Figure 1b). The PME device was easy to operate even for inexperienced operators. And the volumes of samples, derivatization solutions and elution solvents could be readily controlled by using a pipette (Figure 1c). More importantly, the LHS could produce heat under light illumination, which was successfully used to incorporate pre-extraction and postextraction heating treatment steps into PME (Figure 1c). Hence, rates of eluting the extracted analytes from the PME device were remarkably accelerated using a post-extraction heating step. Targeted harvest of toxin from food surfaces and food powders was realized by seamlessly coupling antibodybased micro-liquid extraction (µLE) with PME via a preextraction heating step. Besides, in vitro enzymatic reaction kinetics was accurately revealed by rapidly quenching enzymatic activities through a pre-extraction heating step to terminate the mass fluxes of the analytes towards being decomposed. Compared with the conventional heating methods using heat transfer media, light-induced heating could be more time- and energy-efficient,16 which could directly heat liquids inside the
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PME devices to required temperatures by adjusting light illumination time or intensity, and thus save extensive time and energy consumed on heating the cold heat transfer media or cooling the overheated heat transfer media to set temperatures. A time- and energy-efficient heating method is preferred especially when batteries are often the power supplies for on-site applications. In addition, light-induced heating could avoid staining of samples, solvents and the PME devices by heat transfer media. A light source is also more convenient than liquid heat transfer media to be transported to any sites in need.
Figure 1. (a) In the extraction step or elution step, the chemical potentials of the analytes decrease along the arrows, and the arrows also indicate the directions of the net mass fluxes. Diffusion in the extractive material and in the static boundary layers at the interfaces limits mass transfer rates. (b) Photograph of the PME devices and diagram of the vertical cross section of the coated part in the dashed box. (c) PME procedure. An optional derivatization step (not shown above) could be added following the extraction step when it is necessary. Two optional heating treatment steps could be adopted ahead of extraction to denature proteins, or after extraction to accelerate elution rates. After the elution step, the final solution was ejected to a vial waiting for instrumental analysis.
The LHS was prepared by coating magnetite (Fe3O4) microspheres with silica shells and subsequently grafting octadecyl groups on the silica shells (Figures S1 to S5 in the Supporting Information). Octadecyl group possessing a long flexible carbon chain exhibits fairly good affinities to a wide range of analytes. The magnetite cores were the heat sources of the LHS under light illumination owing to their photothermal properties.16 Instead of mechanically mixing magnetite microspheres and octadecyl-grafted solid silica microspheres, the integrated structure of embedding magnetite cores in octadecyl-grafted silica shells made full use of the surfaces of the magnetite microspheres, and provided higher specific surface areas by converting solid silica into silica shells. Besides, the superparamagnetic properties of the magnetite cores also facilitated the isolation of the
intermediate and final products from the reaction solutions during the synthesis of the LHS.17,18 After obtaining the LHS, the PME devices were prepared by coating the as-synthesized LHS on the interior surfaces of pipette tips using a watercurable glue (polyacrylonitrile dissolved in N,Ndimethylformamide) through a well-controlled procedure (Figure S6 in the Supporting Information). The LHS-based coatings were about 200 µm thick and possessed porous morphologies (Figure S7 in the Supporting Information). First, the PME devices were proven able to offer the full functions of other miniature sample pretreatment platforms including sampling, extraction, derivatization, washing and elution (Figure 1c).7 Under static extraction modes, the maximum extraction amounts from 30 µL of serum and urine were obtained within 5 min for most of the pharmaceuticals and steroid hormones (Figure S8 in the Supporting Information). For the steroid hormones, an additional hydroxylamine derivatization step consuming about 10 min was accomplished in the PME devices following the extraction step (Figure S9 in the Supporting Information). Even the extraction and derivatization steps were not fairly fast, a multichannel pipette could dramatically improve the efficiencies. When applying a pipette with twelve channels, the extraction step and the derivatization step would both take less than 1 min for each sample. High sensitivities (detection limits were calculated by defining the signal-to-noise ratios as three, they ranged from 0.04 to 3 ng mL-1, Table S1 in the Supporting Information) and wide linear ranges (Figure S10 in the Supporting Information) were achieved when the eluted analytes were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, the efficiency of PME was further improved, when the light-induced heating was utilized in the elution step (Figure 1c), to accelerate the rates of eluting the pharmaceuticals and the derivatization products of the steroid hormones from the PME devices. The elution steps previously took time comparable to the extraction steps, were accelerated by three to four times after light illumination (Figure S11 in the Supporting Information). The dramatically accelerated elution rates should be attributed to the elevated temperature (the elution solvents were heated to about 62 °C with no evaporation loss was observed, Figure S12 in the Supporting Information), which could provide energy to conquer the binding strength between the bound analytes and the sorbent, trigger convection within the confined solvents to diminish the boundary layers of the solvents, as well as accelerate the diffusion rates of the analytes in the LHS-based coatings and in the boundary layers of the solvents. Subsequently, based on the heat-susceptible binding between aflatoxin B1 (AFB1) and its monoclonal antibody (AFB1-mAb), AFB1-mAb-based µLE (AFB1-mAb-µLE) and PME were successfully bridged via a pre-extraction heating step, to develop a combined method for harvesting AFB1 from peanut kernel surfaces and powders. First, 30 µL of AFB1mAb solution was dropped on peanut kernel surface or into 5 mg of peanut kernel powder. Then, the AFB1-mAb solution was collected after 2 min using a PME device (Figures 2a and 2b), and immediately heated under light illumination for 1 min to unlock the bound AFB1 from AFB1-mAb (the aqueous liquids inside the PME devices could be heated to about 87 °C, Figure S13 in the Supporting Information). After cooling the heated AFB1-mAb solution back to room temperature, AFB1
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Analytical Chemistry in the solution was extracted by the LHS-based coating, the extractable AFB1 in the AFB1-mAb solution increased more than six times compared with the extractable AFB1 in the AFB1-mAb solution without heating treatment (Figure 2c), which indicated that the heating treatment step between µLE and PME effectively released the bound AFB1 from AFB1mAb, and facilitated the subsequent distribution of AFB1 to the LHS-based coating.
ratios as three, Table S1). Using other alternative antibodies, this method could be possibly extended to high sensitive analysis of other target analytes on solid surfaces or in solid powders, for example, trace amounts of illicit drugs stained on drug traffickers’ fingers or belongings.19,20 Thereafter, the PME devices were used to study in vitro enzymatic reactions by utilizing the light-induced self-heating effect of the LHS to rapidly quench enzymatic activities. Under light illumination for 1.5 min (the aqueous liquids inside the PME devices could be heated to about 94 °C, Figure S13), the activities of cytochrome c (cyt c) in the reaction solutions contained in the PME devices were successfully quenched (Figures S14 and S15 in the Supporting Information). After the reaction solutions inside the PME devices were subsequently cooled back to room temperature, the residual pharmaceuticals in the reaction solutions were extracted by the LHS-based coatings. The extracted pharmaceuticals were later eluted and determined to reveal the reaction kinetics (Figure 3a). The enzymatic reactions were also studied in parallel by adding methanol to the reaction solutions to quench the enzymatic activities (Figure 3b). The similar results obtained by PME and the conventional solvent quenching method indicated PME possessed satisfactory capacity to resolve the temporal concentrations through the short heat-quenching step. Compared with the solvent quenching method, PME could even use a washing step to eliminate the use of centrifugation and filtration setups for removing the denatured enzyme, and to remove salts to avoid their suppression on ionization efficiencies in mass spectrometry.
Figure 2. Collection of the AFB1-mAb solution from (a) peanut kernel surface or (b) peanut kernel powder by using the PME device. (c) After light illumination for 1 min, the extractable amounts of AFB1 from 30 µL of the AFB1-mAb solutions (AFB1-mAb 1.0 mg mL-1) inside the PME devices increased for over six times. (d) The extracted AFB1 from 5 mg of spiked peanut kernel powders (20 ng g-1) declined about fifty percent by using the µLE-PME combined method, when the AFB1-mAb solution (1.0 mg mL-1) was replaced by PBS solution. The error bars represent the standard deviations. All the data were obtained from three parallel experiments.
The µLE step enabled PME to harvest the nonvolatile toxin from solid surfaces and solid powders. However, when the AFB1-mAb solution was replaced by phosphate buffer saline (PBS) solution for µLE, the overall harvested AFB1 declined for about one half (Figure 2d). Being coupled with LCMS/MS, the AFB1-mAb-µLE-PME method achieved high sensitivities for the analysis of stained peanut kernel surfaces and powders (detection limits were 2.4 pg per spot and 0.4 ng g-1, they were also calculated by defining the signal-to-noise
Figure 3. The normalized residual amounts of the pharmaceuticals in the reaction solution (cyt c 1 µM, initial concentration of each pharmaceutical 200 ng mL-1, H2O2 450 µM) determined by using (a) PME or (b) solvent quenching method. The error bars represent the standard deviations. All the data were obtained from three parallel experiments.
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In the previous studies, enzymatic reactions were still undergoing during the microextraction procedures, the extraction steps must be confined in time durations short enough to resolve the temporal concentrations of the analytes, which however, sacrificed the sensitivity of microextractionbased analytical methods.21 In this study, the extraction time could be more flexible after the enzymatic activities were quenched and the mass fluxes of the analytes towards being decomposed were terminated. The PME device could also be used to study the enzymatic processes of other synthesized compounds for revealing their therapeutic or toxic effects, which is meaningful for developing new drugs or assessing the potential environmental effects of pollutants.22,23 In summary, this study developed a novel PME device as an efficient, versatile, as well as user-friendly sample pretreatment platform for biofluid analysis, food analysis and in vitro assay of enzymatic reactions. The incorporation of a LHS in the PME device enabled efficient and staining-free heating treatment in a miniaturized sample pretreatment platform with no need to consume time and energy on adjusting heat transfer media to set temperatures, or immerse the PME devices into heat transfer media. The light-induced heating effect of the LHS was successfully used to accelerate solvent elution rates, unlock bound toxin from its antibody, and quench enzymatic activities. The mass transfer of the analytes to the elution solvents and to the LHS-based coatings was facilitated, and the undesired mass fluxes of the analytes towards being decomposed were blocked. Therefore, higher efficiencies and new applications were achieved. All in all, this study provided a new perspective of designing novel miniature sample pretreatment platforms applicable in the analysis of liquid and solid samples with stable or changing analyte concentrations, thus could promote the applications of miniature sample pretreatment platforms in public security, new drug development, environmental protection and many other fields. It is notable that the magnetite core was reported to exhibit a broad absorbance over the ultraviolet-visible light region, however, its absorbance at shorter wavelength region was observed stronger.24 Thus, using light within ultraviolet to short visible light region might be beneficial for promoting the photothermal conversion efficiency in the future
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Chemicals, materials and equipments used in the experiments, preparation and characterization of the LHS and the PME device, light-induced heating, study of in vitro enzymatic reactions, instrumental analysis, Figures S1 to S15, and Table S1 (PDF)
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] (G.O.);
[email protected] (J.X.).
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT The authors acknowledge financial support from the projects of NNSF of China (21377172, 21477166, 21527813, 21677182 and 21737006), and the project of Jiangsu Key Laboratory of Vehicle Emission Control (OVEC039).
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Analytical Chemistry
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