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Sep 29, 2014 - Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China. Anal. Chem. , 2014 ...
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Macrofluidic device for preparative concentration based on epitachophoresis Frantisek Foret, Vladimíra Datinská, Ivona Vorá#ová, Jakub Novotný, Pantea Gheibi, Jan Berka, and Yann Astier Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019
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Macrofluidic device for preparative concentration based on epitachophoresis František Foret ab*, Vladimíra Datinskáa,c, Ivona Vor Berkac, Yann Astierc
Jakub Novotnýa, Pantea Gheibic, Jan
Czech Academy of Sciences, Institute of Analytical Chemistry, (Brno, Czech Republic) Masaryk University (Brno, Czech Republic) c Roche Sequencing Solutions, Inc. (Pleasanton, USA)
ABSTRACT: We have developed a new separation device to concentrate and collect ions from several milliliter sample volumes to microliter fractions. Unlike most conventional platforms, this device has a circular architecture. The electrophoretic migration operates from the outer perimeter towards the center. Separations can be performed both in continuous (zone electrophoresis) and discontinuous (moving boundary) electrolyte systems. We use a discontinuous electrolyte system comprising of a leading and a terminating electrolyte to concentrate samples containing small organic anions and DNA fragment. The agarose gel stabilizes the boundary between the leading and terminating electrolytes. The milliliter volume sample is mixed with the terminating electrolyte and migrates through the gel towards the center. The concentrated total sample is collected in microliter fraction at the center. The potential for preparative concentration of DNA is demonstrated using a DNA ladder. Because zone migration accelerates as it moves towards the center, we named this method Epitachophoresis from the Greek word “ ”, meaning “acceleration”. To the best of our knowledge, this unique circular architecture has not been previously described.
Introduction Electrophoresis is one of the commonly used separation techniques in bioanalysis. Preparative electrophoresis is mainly practiced in the free flow electrophoresis mode1,2, , in a form of compartmentalized electrolyser3 or as a separate step after slab gel electrophoresis . Some attempts to use capillary electrophoresis for fraction collection have also been described5,6. In many electrophoresis protocols, a form of moving boundary electrophoresis is often used for sample focusing and concentration. For example, in disk electrophoresis, the protein sample is focused as it enters the SDS gel7, similarly, transient isotachophoresis can be applied for sample preconcentration in capillary zone electrophoresis8. Capillary isotachophoresis, where a steady state is reached during the sample migration in a discontinuous electrolyte system, was developed decades ago 9,10 especially for analyses of small inorganic and organic ions. Nucleic acid extraction from biological samples is a key factor for all molecular biology and genetics applications. Numerous methods have been developed and optimized for DNA and RNA extraction from blood, tissue, or other biospecimen11. With the recent adoption of liquid biopsy as a source of cell-free nucleic acids, additional demands on handling large (ml) sample volumes, as well as purity requirements, have arisen. In some applications, such as analysis of circulating tumor DNA (ctDNA) in plasma by nextgeneration sequencing or qPCR, quantitative recovery of every available genome copy, concentration of extracted DNA into small volumes, and separation of ctDNA from white blood cell derived genomic DNA, are of paramount importance for errorfree molecular diagnostic assays12,13.
Recently there has been a growing interest in developing isotachophoretic protocols for concentration and purification of DNA as an alternative to the solid phase extraction protocols of DNA preparation14. While some of the first published experiments described agarose filled tubes for isotachophoresis (ITP) concentration of DNA sample15,16, most of the recent papers focus on a microfluidic scale17-19. In our previous report, we have used a commercial capillary ITP instrument equipped with a conductivity detector and micropreparative valve for focusing and fraction collection of DNA samples20 loaded from buffer or saline solutions. Since the commercial instrument was originally designed mainly for analytical purposes, the maximum sample volume that could be loaded and focused was about 150 microliters. This limitation relates to the limited size of the separation capillary with an internal diameter of 0.8 mm. In ITP the separated/transported amount of the analyte (in moles) relates to the electric charge, which passes through the separation column during the analysis21. High separation charge can be achieved by using high electric current in large bore tubes; however, the resulting Joule heating often leads to bubble formation and boiling of the electrolyte solution. On the other hand, at low currents, the Joule heating is not a problem; although the analysis time may be exceedingly long. It is worth noting that ITP is much more current efficient than zone electrophoresis where most of the electricity is transported by the background electrolyte22. The heating problem can be partially addressed by using flat separation channels with better cooling and sample loading capacity23; however, none of the previously described devices can address focusing sample volumes in the several ml range.
We show how a circular geometry applied to the principle of ITP can resolve the large sample input problem. A DNA ladder is used to demonstrate separation of the nucleic acid fraction from 15 ml input into 200 µl volume resulting in up to 75-fold concentration with yields up to 80 %. The concentrated nucleic acid fraction is easily recovered by simple pipetting out the dialysis cup. The described method of DNA extraction presents advantages compared to traditionally used solid phase extraction, providing higher yield and lower sample losses from large input volumes. High mobility sample ions as well as large proteins with very low mobility separate from the DNA fragments. From the recently published techniques for DNA purification the SCODA based on a synchronous coefficient of drag alteration of DNA in pulsed-field gel electrophoresis is also promising24. Similarly, to the present report, the DNA fragments also migrate from the edge towards the center of a multiple electrode agarose filled device. The presented results show very good DNA extraction yields compared to standard techniques; however, it seems that the concentration of large volumes of salt containing DNA fragments below 20 kbp is challenging25. MATERIALS AND METHODS List of chemicals and material Buffer components L-histidine monohydrochloride monohydrate (99%), L-histidine (99%), Ntris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS; 99.5%) and tris-(hydroxymethyl)aminomethane (TRIS; 99.8%) are purchased from Sigma-Aldrich (USA). Agarose NEEO ultra-quality Roti®garose with low electroendosmosis was purchased from Carl Roth (Germany). Anionic dye Patent blue V sodium salt were from SigmaAldrich; red anionic dye 1,8-dihydroxy-2-(4sulfophenylazo)naphthalene-3,6-disulfonic acid trisodium salt (SPADNS) was from Lachema, Brno, Czech Republic. Low molecular weight dsDNA ladder labeled with Fluorescein (ten fragments from 75 base pairs - bp to 1622 bp) was purchased from Bio-Rad, USA and a non-labeled ladder (10 fragments from 50 bp to 766 bp) from New England BioLabs, USA. Nucleic acid fluorescence stain (SYBR™ Gold, in DMSO) used for visualization of DNA ladder during the analysis was purchased from Invitrogen, Carlsbad, CA, USA. Material used for the device Ertacetal® was purchased from Quadrant Plastic Composites GmbH, Lotte, Germany. The wire ring electrode was made from 0.5 mm diameter stainless steel wire (stainless steel 1.4301, Hobby Draty, Czech Republic). The second electrode was 0.4 mm x 20 mm Pt wire (SAFINA, Vestec, Czech Republic). Slide-A-Lyzer™ mini dialysis cups (2000 Da MWCO) were from Thermo Fisher Scientific, USA. Resistivity detector cell was constructed by using Pt wires (300 µm, SAFINA, Vestec, Czech Republic), 1 ml pipette tip (PZ HTL S.A., Warszawa, Poland) and epoxy resin (Bison International B.V., Goes, Netherlands). Device architecture and fabrication The device (Figure 1 A,B) was machined from Ertacetal®. The stainless steel wire ring electrode was secured on the edge of the circular separation compartment (radius of 55 mm). The second Pt-wire electrode was placed in the leading electrolyte reservoir on the corner of the device. The ring electrode was
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connected to the right banana type connector (Figure 1 B). The left banana connector was attached to the platinum wire electrode positioned in the leading electrode reservoir. The internal channel is 9 mm internal diameter and 20 cm long (Figure 1 A). The side openings were plugged by silicon septa. The central collection well with the diameter of 9 mm was drilled through the device and closed from the bottom by a moving Ertacetal® rod sealed by a rubber O-ring – not shown. For capture and collection of the analyte zone, a mini dialysis cup with a semipermeable membrane was used. The collection cup is cut to minimize the collection volume with a razor blade. The final collection volume is 200 µl. (Figure 1 B). The cup was placed in the central collection well for experiment. Experimental Procedure All experiments were run in electrolyte buffers systems. The leading electrolyte (LE) contained 100mM HCl-Histidine buffer at pH 6.25 and the terminating electrolyte (TE) contained 10mM TAPS titrated by TRIS to pH 8.30. The agarose stabilized LE disk was prepared in 20mM LE (HCl-Histidine; pH 6.25). All buffers were prepared in deionized water. The 0.3% agarose gel disk was prepared using 67 mg of low electroendosmosis agarose dissolved in 20 ml of 20mM LE buffer, heated and poured into a laboratory made plastic mold on a glass plate. The final gel had circular shape (74 mm diameter, 4 mm thickness with a 4 mm center hole). The device was set up for experiment. Mobile central piston rod was moved to a lower position and the device channel was filled with approximately 20 ml of 100mM LE buffer. The collection cup was placed in the central hole and also filled with 100mM LE. The polymerized LE gel disk was carefully transferred into the device center. A 75x1 mm round glass plate with a 4 mm center hole was placed on top of the gel. The sample (Table 1) solution in the 15 ml of the 10 mM TE buffer was applied by a serological pipette into the space between the gel disk and ring electrode. Power supply PowerPac 3000 (BioRad) was run at a constant power mode at 2 W (this corresponds approximately to 16 mA and 120 V at beginning of the analysis). The experiment took approximately 1 hour *Q 10 mA and 200 V at the end of analysis). After the focused sample zone entered the collection cup the electric current was turned off and the sample was pipetted out for analysis. The empty collection cup was lifted up by the moving rod and discarded. Table 1: Amount of analytes in experiments Sample
Red dye experiment
150 S of 0.1mM SPADNS
100 S of 0.1mM SPADNS, 100 S of 0.1mM Patent blue V 10 S of 10mM acetic acid
1 µg of DNA ladder
DNA analysis DNA concentration in the collected fraction was evaluated using Qubit fluorimeter (Invitrogen, Carlsbad, CA, USA) by using a high sensitivity dsDNA Qubit quantitation assay kit. The collected fractions were further analyzed using the chip CGE-LIF instrument Agilent 2100 Bioanalyzer (Agilent, Santa
Frantisek Foret: 0000-0002-0060-7350 ResearcherID: D-9495-2012 Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the Grant Agency of the Czech Republic, project P20612G014. Additional support was provided by Roche Sequencing Solutions (Pleasanton, USA) and by Institutional support RVO 68081715 of Institute of Analytical Chemistry, Czech Academy of Sciences in Brno, Czech Republic. The research was partly carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II.
REFERENCES 1 Krivankova, L.; Bocek, P. Continuous free flow electrophoresis. Electrophoresis, 1998, 19, 1064-1074 2 Spanik, I.; Vigh, G. Effect of feed zone width on product purity in preparative-scale, continuous free-flow isoelectric focusing separation of enantiomers. J. Chromatogr. A, 2002, 979, 123-129 3 Tomas, R.; Yan, L.; Krenkova, J.; Foret, F. Autofocusing and ESIMS analysis of protein digests in a miniaturized multicompartment electrolyzer. Electrophoresis 2007, 28, 2283–2290 4 Seelert, H.; Krause, F. Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels. Electrophoresis 2008, 29, 2617-2636 5 Müller, O.; Foret, F.; Karger, B.L. Design of a High-Precision Fraction Collector for Capillary Electrophoresis. Anal. Chem., 1995, 67, 2974-2980. 6 Luo, Z.F.; Zhou, H.M.; Jiang, H.; Ou, H.C.; Li, X.; Zhang, L.Y. Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection. Analyst, 2015, 140, 2664-2670. 7 Bendickson, L.; Nilsen-Hamilton, M. Western Blots, and Other Applications. In: Making and Using Antibodies: A Practical Handbook, 2nd Edition (Howard, G.C.; Kaser, M.R. Editors), CRC Press-Taylor & Francis Group, Boca Raton, FL, USA, 2014, pp. 275301 8 Foret, F.; Szoko, E.; Karger, B. L. On-Column Transient and Coupled Column Isotachophoretic Preconcentration of Protein Samples in Capillary Zone Electrophoresis. J. Chromatogr. 1992, 608, 3-12. 9 Everaerts, F. M.; Beckers, J. L.; Verheggen, P. E. M. Isotachophoresis: Theory, Instrumentation and Applications, Elsevier, Amterdam 1976. 10 # P.; Deml, M.; Gebauer, P.; Dolnik, V. Analytical isotachophoresis (B.J. Radola- Editor), VCH, Weinheim 1988.
Page 6 of 11
11 Thatcher, S.A. DNA/RNA preparation for molecular detection. Clin. Chem. 2015, 61, 89-99. 12 Heitzer, E.; Ulz, P.; Geigl, J.B. Circulating tumor DNA as a liquid biopsy for cancer. Clin. Chem. 2015, 61, 112-23. 13 Crowley, E.; Di Nicolantonio, F.; Loupakis, F.; Bardelli, A. Liquid biopsy: monitoring cancer-genetics in the blood, Nature Rev. Clin. Oncol. 2013, 10, 472–484. 14 Datinska, V.; Voracova, I.; Schlecht, U.; Berka, J.; Foret, F. Recent progress in nucleic acids isotachophoresis. J. Sep. Sci. 2018, 41, 236-247. 15 Kondratova, V. N.; Serd'uk, O. I.; Shelepov, V. P.; Lichtenstein, A. Concentration and isolation of DNA from biological fluids by agarose gel isotachophoresis. Biotechniques 2005, 39, 695-699. 16 Kondratova, V. N.; Botezatu, I. V.; Shelepov, V. P.; Lichtenstein, A. V. Tube gel isotachophoresis: A method for quantitative isolation of nucleic acids from diluted solutions. Anal. Biochem. 2011, 408, 304308. 17 Rogacs, A.; Marshall, L. A.; Santiago, J. G. Purification of nucleic acids using isotachophoresis. J. Chromatogr. A 2014, 1335, 105-120. 18 Ostromohov, N.; Schwartz, O.; Bercovici, M. Focused upon Hybridization: Rapid and High Sensitivity Detection of DNA Using lsotachophoresis and Peptide Nucleic Acid Probes. Anal Chem. 2015, 87, 9459-9466. 19 Martins, D.; Levicky, R.; Song, Y. A., Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip. Biosens. Bioelectron. 2015, 72, 87-94. 20 Datinska, V.; Voracova, I.; Berka, J.; Foret, F. Preparative concentration of nucleic acids fragments by capillary isotachophoretic analyzer. J. Chromatogr. A 2018, 1548, 100-103. 21 Bocek, P.; Deml, M.; Kaplanova, B.; Janak, J. Analytical isotachophoresis concept of separation capacity. J. Chromatogr. 1978, 160, 1-9. 22 Mikkers, F.E.P.; Everaerts, F.M.; Verheggen, T.P.E.M. Highperformance zone electrophoresis. J.Chromatogr. 1979, 169, 11-20. 23 Dolnik, V.; Deml, M.; Bocek, P. Large sample volume preseparation for trace analysis in isotachophoresis. J. Chromatogr. 1985. 320, 89-97. 24 Marziali, A; Pel, J.; Bizzotto, D.; Whitehead, L.A. Novel electrophoresis mechanism based on synchronous alternating drag perturbation. Electrophoresis 2005, 26, 82–90. 25 So, A.; Pel, J.; Rajan, S.; Marziali, A. Efficient genomic DNA extraction from low target concentration bacterial cultures using SCODA DNA extraction technology. Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5506 26 Stellwagen, N.C.; Gelfi, C.; Righetti P.G. The free solution mobility of DNA Biopolymers 1997, 42, 687-703. 27 Shintaku, H.; Nishikii, H.; Marshall, L. A.; Kotera, H.; Santiago, J. G. On-chip separation and analysis of RNA and DNA from single cells. Anal. Chem. 2014, 86, 1953-1957.Krivankova, L.; Bocek, P. Electrophoresis, 1998, 19, 1064-1074
Figure 3: Separation modes and detection. A Zone electrophoresis in continuous buffer system of red dye SPADNS and blue dye Patent Blue V. Constant 3 W electric power. Electrolyte: 10mM TAPS-TRIS pH 8.3. Gel: 0.3% agarose in 10mM TAPS-TRIS pH 8.3. Dyes were injected from water. B Discontinues buffered conditions with leading (HCl-HIS pH 6.25) and terminating electrolyte (TAPS-TRIS pH 8.3). Constant 2 W electric power.
Figure 4: Epitachophoretic extraction, concentration and recovery of a DNA ladder with fluorescence detection. A Absorbance spectra of covalently labeled DNA ladder before and after Epitachophoresis. B chip-CGE-LIF electropherograms of covalently labeled DNA ladder 1 µg/15 ml before Epitachophoresis. ACS Paragon Plus Environment