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Continuous On-Chip Fluorescence Labelling, Free-Flow Isoelectric Lab on a Chip View Article Online COMMUNICATION View Journal | View Issue Continuous on-chip fluorescence labelling, free- flow isoelectric focusing and marker-free Cite this: Lab Chip,2016,16,1565 isoelectric point determination of proteins and Received 13th January 2016, peptides Accepted 23rd March 2016 a a a b DOI: 10.1039/c6lc00055j Christin Herzog, Elisabeth Poehler, Andrea J. Peretzki, Sergey M. Borisov, Daniel Aigner,b Torsten Mayrb and Stefan Nagl*a www.rsc.org/loc We present a microfluidic platform that contains a micro flow tion or postprocessing are required that are carried out out- reactor for on-chip biomolecule labelling that is directly followed side chip platforms or only a small portion of the sample is by a separation bed for continuous free-flow electrophoresis and transferred to the next step. Creative Commons Attribution-NonCommercial 3.0 Unported Licence. has an integrated hydrogel-based near-infrared fluorescent pH A promising method in this respect is micro free-flow – sensor layer. Using this assembly, labelling of protein and peptide electrophoresis (μFFE)2 4 because of its ability to continu- mixtures, their separation via free-flow isoelectric focusing and the ously and preparatively separate and isolate biomolecules determination of the isoelectric point (pI) of the separated prod- from mixtures on a small scale. Its flow rates that are com- ucts via the integrated sensor layer could be carried out within patible with reactors and other microfluidic structures, and typically around 5 minutes. Spatially-resolved immobilization of its inherent compatibility with many matrices and buffers for fluidic and sensing structures was carried out via multistep photo- biomolecules allow them to stay in their native conformation. lithography. The assembly was characterized and optimized with FFE is often used for preseparation in advance of other pro- respect to their fluidic and pH sensing properties and applied in cedures.5 In recent years, miniaturized integrated FFE assem- This article is licensed under a the IEF of model proteins, peptides and a tryptic digest from blies have been presented by multiple groups in many – physalaemine. We have therefore realized continuous sample variants.6 17 preparation and preparative separation, analyte detection, process In some works, μFFE has been connected with other on- Open Access Article. Published on 24 March 2016. Downloaded 9/25/2021 2:20:08 AM. observation and analyte assignment capability based on pI on a or off-chip functionalities. Geiger et al. have coupled an off- single platform the size of a microscope slide. chip nano-LC column18 with chip-based FFE for the 2D sepa- ration of a tryptic digest, whereas Benz et al. have coupled Introduction μFFE with subsequent nanospray-MS for the mass analysis of organic separation products.19 Jezierski et al. have linked a Miniaturized lab procedures on microfluidic chips enjoyed good flow reactor with micro free-flow zone electrophoresis on a successandarenowwidelyappliedincertainareassuchasflow single chip for the NBD-labelling and successive separation reactors, PCR, electrophoresis or cytometry. A generation ago, of amino acids.20 the concept of a micro total analysis system (μTAS) was intro- In order to gain real-time information about a preparative duced,aimingattheintegrationofallrequiredlabprocedures or analytical process on a microfluidic chip, an integrated on one single miniaturized chip platform.1 sensing approach is needed. Many different methods have Much progress has been achieved in this area, but in par- been pursued, but in particular, for the spatially-resolved ob- ticular, for preparative multistep procedures to date, almost servation of small molecules and process parameters, optical all platforms still fall short of being a satisfactory and eco- fluorescent and luminescent chemical sensors are very favor- nomic solution for integration of all required laboratory able.21,22 They may be assembled from molecular probes in working stages. Often, additional periods for sample prepara- suitable polymer matrices and integrated into microfluidic chips typically as a thin layer covering the whole micro- a Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 channel network or only select areas.23 Leipzig, Germany. Fluorescent and luminescent pH sensors have been ap- E-mail: [email protected]; Web: http://research.uni-leipzig.de/nagl; plied in microfluidic chips mainly for cell culture and moni- Tel: +49 341 97 36066 b Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität toring of other bioprocesses and could be incorporated with a 24–32 Graz, Stremayrgasse 9/III, 8010 Graz, Austria variety of different microfabrication techniques. This journal is © The Royal Society of Chemistry 2016 Lab Chip,2016,16,1565–1572 | 1565 View Article Online Communication Lab on a Chip As with other variations of electrophoresis, one mode of type III (bovine milk), β-lactoglobulin B (bovine milk), miniaturized FFE is microfluidic free-flow isoelectric focusing ubiquitin (bovine erythrocytes), neurotensin, oxytocin, leucine- (μFFIEF), where compounds are separated in a pH gradient enkephalin, physalaemin and endothelin were purchased from – according to their isoelectric point (pI).33 37 IEF offers an al- Sigma-Aldrich (Steinheim, Germany). n-Heptane was bought ternative dimension for separation and the pI offers a means from Roth (Karlsruhe, Germany). Dimethyl sulfoxide (DMSO), for determination or assignment of a particular compound. trisIJhydroxymethyl)-aminomethane (TRIS), H3BO3,H3PO4, 38 The pI may be used for identification of biological materials. CH3COOH, NaOH and NaHCO3 were from Merck (Darmstadt, So far, this is mostly performed using off-line methods and Germany). The ampholyte mixture pH 4–7waspurchasedfrom therefore needs additional laboratory working steps.39,40 AppliChem (Darmstadt, Germany) and the activated labelling We introduced a method for rapid determination of the dye Atto 425-NHS from Atto-Tec (Siegen, Germany). The isoelectric point (pI) of proteins and other (bio)-molecules NIR-fluorescent pH probe N-(3-([N-(3-(methacryloylamino)- during FFIEF using a fluorescent pH sensor layer in a separa- propyl)amino]sulfonyl)-2,6-diisopropyl-phenyl)-N′-(4-([N-(3- tion bed.41 This assembly was able to determine the pI of (methacryloylamino)propyl)-amino]-sulfonyl)-2,6-diisopropyl- proteins and other compounds on-chip with good precision phenyl)-1-(4-methyl-1-piperazinyl)-6,7,12-trichloroperylene- and repeatability. Later, we extended this approach using 3,4,9,10-tetracarboxylic bisimide (a perylene bisimide deriv- inkjet printing of pH sensing matrices in select areas in a ative, abbreviated as PBI herein) was synthesized by D. microfluidic chip42 and another readout method based on Aigner according to ref. 45. fluorescence lifetime measurements.43 However, in all cases, Britton–Robinson buffers (BRB) were made from 10 mM a fluorescence labelling step was still required prior to the H3BO3,H3PO4 and CH3COOH, and the pH was adjusted by chip-based FFIEF procedure. titration with 1 M NaOH solution and monitored using a To circumvent the off-chip labelling step in IEF, we intro- Lab 850 pH meter (SI Analytics, Mainz, Germany). duced a semi-UV-transparent FFE platform along with a read- Microchip fabrication Creative Commons Attribution-NonCommercial 3.0 Unported Licence. out assembly that is capable of simultaneous monitoring of the intrinsic fluorescence of certain biomolecules when ex- Fabrication of microfluidic chips with an integrated optical cited in the deep UV and near-infrared (NIR) monitoring of a pH sensor layer was performed using multiple photo- pH sensor layer for pI determination.44 Therefore, unlabelled polymerization steps in a procedure related to and extended biomolecular mixtures could be separated and analysed. from that in ref. 41. Briefly, in the first step, the sensor layer However, this system was limited to biomolecules that show was fabricated between two glass slides, one was with acrylate sufficient UV fluorescence (mainly proteins and peptides with surface modification and one was untreated. We used a 60 μL multiple tryptophans or tyrosines), required an elaborate and mixture of 85.0% (w/w) acryloylmorpholine, 14.8% (w/w) expensive optical setup and was lower in sensitivity than a oligoethylene glycol (OEG-DA700), 0.02% (w/w) PBI as a pH This article is licensed under a detection based on label fluorescence. probe and 0.2% (w/w) photoinitiator. Photopolymerization Herein, we now demonstrate a generally applicable solu- was carried out using a 4″ FE5 Flood Exposure (Süss MicroTec, tion for integration of the working steps of analyte visualiza- Munich, Germany) device equipped with a Hg lamp (14 mW −2 Open Access Article. Published on 24 March 2016. Downloaded 9/25/2021 2:20:08 AM. tion, FFIEF separation, and determination of isoelectric cm at 365 nm) at an illumination time of 15 s. After the points. We describe a combined miniaturized chip platform exposure, the unacrylated glass slide was removed. the size of a microscope glass slide that is able to perform In the second step, the microfluidic structure was prepared continuous on-chip labelling of biomolecular samples and on top of the sensing layer. 100 μL of OEG-DA258 with 1% (w/w) mixtures, their free-flow isoelectric focusing, observation of 2,2-dimethoxy-2-phenylacetophenone was spread over the sens- the pH gradient and assignment of isoelectric points (pI) ing layer
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