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Nanoliter-scale electromembrane extraction and enrichment in a microfluidic chip Frederik André Hansen, Drago Sticker, Jörg P. Kutter, Nickolaj Jacob Petersen, and Stig Pedersen-Bjergaard Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01936 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018
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Analytical Chemistry
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Nanoliter-scale electromembrane extraction and enrichment in a microfluidic chip
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Frederik A. Hansen1, Drago Sticker1, Jörg P. Kutter1, Nickolaj J. Petersen1, Stig PedersenBjergaard1,2,*
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Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark 2 School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway
Abstract This paper reports for the first time nanoliter-scale electromembrane extraction (nanoliter-scale EME) in a microfluidic device. Six basic drug substances (model analytes) were extracted from 70 µL samples of human whole blood, plasma, or urine, through a supported liquid membrane (SLM) of 2nitrophenyl octyl ether (NPOE) and into 6 nL of 10 mM formic acid as acceptor solution. A DC potential of 15 V was applied across the SLM, and served as driving force for the extraction. The cathode was located in the acceptor solution. Due to the small area of the SLM (0.06 mm2), the system provided soft extraction with recoveries < 1 % for the 70 µL samples. Due to the large sample-toacceptor volume ratio, analytes were enriched in the acceptor solution. Enrichment capacity was 6-7fold per minute, and after 60 minutes operation most of the model analytes were enriched by a factor of approximately 400. Because of the SLM and the direction of the applied electrical field, a substantial sample clean-up was obtained. The chips were based on thiol-ene polymers, and the soft lithography fabrication procedure and the materials were selected in such a way that future mass production should be feasible. The chip-to-chip variability was within 23 % RSD (and less than 10 % in most cases) with respect to extraction recovery. Our findings have verified that nanoliter-scale EME is highly feasible and provides reliable data, and for future studies the concept should be tested for applicability in connection with in vitro micro-physiological systems, organ-on-a-chip systems, and point-of-care diagnostics. These are potential areas where the combination of soft extraction and high enrichment from limited sample volumes is required for reliable analytical measurements.
Introduction In many branches of analytical chemistry, sample preparation is an essential part of the analytical workflow, particularly within the field of bioanalysis, where samples often contain a complex matrix. During the past decade, increasing attention has been drawn to miniaturization of sample preparation methods to reduce the volumes of samples and chemicals, and to bring down the associated costs. As part of the push towards miniaturization, many platforms of sample preparation techniques have been developed on microfluidic devices, often simply referred to as “chips”. Examples of this include liquid phase microextraction,1,2 solid phase microextraction3,4 and protein digestion.5,6 The first generation chips were typically fabricated in glass, and often involved laborious and/or costly fabrication, including etching with hazardous hydrofluoric acid. Therefore, polymeric materials are gaining increasing interest for the fabrication of chips, as they enable use of fabrication methods, such as soft lithography, which allow rapid prototyping and low-cost mass-production of identical chips.7-9 One such family of polymers are the thiol-ene-based materials.10 The click-chemistry type photo-initiated polymerization reaction of thiol- and ene-components is characterized by a very rapid curing at ambient temperature and pressure. Additionally, thiol-ene materials offer low shrinkage upon polymerization and high mechanical strength, which makes them attractive polymers for fabrication of microfluidic devices.
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Electromembrane extraction (EME) is another sample preparation technique that has been implemented on a chip. In EME,11 analyte ions are transferred across a supported liquid membrane (SLM) that separates the sample from an acceptor solution, assisted by an electric field. Selectivity of the extraction is gained from the choice of SLM solvent,12 and the polarity and magnitude of the electric field.11,13,14 In 2010, we presented the first example of on-chip EME.15 The chip was composed of a flat porous membrane bonded between two poly(methylmethacrylate) (PMMA) pieces, into which compartments of sample and acceptor solutions were cut. In the first chip, the acceptor solution volume was 7 µL and kept stagnant, but later chips were operated with flowing acceptor solution.16,17 Since then, other chips constructed in similar fashion have been presented for various applications.18-21
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Recently, we proposed the concept of “nano-EME”, where selected basic drugs were extracted from 200 µL acidified sample solution, through an SLM of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor solution.22 The acceptor solution was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor solution by capillary electrophoresis with UV-detection. Due to the large sample-to-acceptor volume ratio, this system provided soft extraction with recoveries < 1 %, and at the same time very high enrichment factors even from limited sample volumes. Thus, with loperamide as one example, enrichments factors exceeding 500 were obtained after 5 minutes of extraction from 200 µL sample. Such performance data are not possible to obtain by traditional extraction methods. However, the first technical set-up for nano-EME was fragile, the precision was poor, and the set-up was not suited for routine operation, commercialization, or mass production. In addition, use of the term “nano” was not in accordance with the general recommendation only to use nano for describing nanoscience.23
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Therefore, in the current contribution, nano-EME was termed nanoliter-scale EME and performed in chip format for the first time, to overcome some of the above mentioned challenges. The idea was to construct a robust thiol-ene-based chip device for nanoliter-scale EME, providing repeatable extractions, and with potential for mass production. Thiol-ene was chosen as the polymer platform, as the pre-polymerized liquid state of this material is ideal for integration of a porous membrane. The purpose of the current paper was (a) to investigate the feasibility of nanoliter-scale EME in chip format, (b) to investigate extraction performance, (c) to investigate reliability, and (d) to investigate compatibility with more complex biological fluids, such as urine and blood.
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We emphasize that the current paper is conceptual and focused solely on technical development and fundamental characterization of performance. The final analysis of the acceptor phases was performed off-chip by LC-ESI-MS. Although this involved dilution of the acceptor phase, the quantitative data obtained to support the concept were acquired with high specificity, accuracy, and reliability. However, work is in progress in our laboratory to add electrophoretic separation to the nanoliter-scale EME chip. This challenging research will integrate nanoliter-scale EME, separation, and detection into a complete micro-total analysis system. Although nanoliter-scale EME is in its infancy, we expect such systems to be important in the future. The reason for this is that nanoliter-scale EME (1) enables soft extraction from very small sample volumes, and (2) is compatible with complex biological samples. Soft extraction is important in the study of complex biological systems, in order not to disturb biochemical equilibria. Among other applications, we expect nanoliter-scale EME has potential in connection with in vitro micro-physiological systems, organ-on-a-chip systems, and pointof-care diagnostics.
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Analytical Chemistry
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Experimental section Chemicals and reagents
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Acetonitrile, formic acid, 2-nitrophenyl octyl ether (NPOE), rhodamine 6G, amitriptyline hydrochloride, pethidine hydrochloride, pentaerythritol tetrakis(3-mercaptopropionate), and 1,3,5triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione were purchased from Sigma-Aldrich (St. Louis, MO). Sylgard 184−poly(dimethylsiloxane) (PDMS) elastomer kit was obtained from Dow Corning (Midland, MI,USA). Methadone hydrochloride, cocaine hydrochloride, amphetamine sulfate, and methamphetamine hydrochloride were all obtained from Nordisk Droge og Kemikalie A/S (Copenhagen, Denmark). Stock solutions of methadone, amitriptyline, cocaine, pethidine, methamphetamine and amphetamine were prepared in methanol at a concentration of 3 mg/mL and stored at 4℃. The individual stock solutions were used to prepare a mixture stock solution in 10 mM formic acid (pH 2.9) at a concentration of 100 µg/mL of each drug. From this, working solutions at lower concentrations with negligible methanol content (
