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Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device

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Abstract

Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15–70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors.

Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

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References

  1. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci U S A. 1993;90:5011–5.

    Article  CAS  Google Scholar 

  2. Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001;19:242–7.

    Article  CAS  Google Scholar 

  3. Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nat Biotechnol. 2003;21:255–61.

    Article  CAS  Google Scholar 

  4. Stoll DR, Carr PW. Fast, Comprehensive two-dimensional HPLC separation of tryptic peptides based on high-temperature HPLC. J Am Chem Soc. 2005;127:5034–5.

    Article  CAS  Google Scholar 

  5. Ye M, Hu S, Schoenherr RM, Dovichi NJ. On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. Electrophoresis. 2004;25:1319–26.

    Article  CAS  Google Scholar 

  6. Zeisbergerova M, Adamkova A, Glatz Z. Integration of on-line protein digestion by trypsin in CZE by means of electrophoretically mediated microanalysis. Electrophoresis. 2009;30:2378–84.

    Article  CAS  Google Scholar 

  7. Bonneil E, Mercier M, Waldron KC. Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis. Anal Chim Acta. 2000;404:29–45.

    Article  CAS  Google Scholar 

  8. Cobb KA, Novotny M. High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography, using immobilized trypsin for protein digestion. Anal Chem. 1989;61:2226–31.

    Article  CAS  Google Scholar 

  9. Huang P, Wu J-T, Lubman DM. Separation of tryptic digests using a modified buffer in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer. Anal Chem. 1998;70:3003–8.

    Article  CAS  Google Scholar 

  10. Ekström S, Önnerfjord P, Nilsson J, Bengtsson M, Laurell T, Marko-Varga G. Integrated microanalytical technology enabling rapid and automated protein identification. Anal Chem. 2000;72:286–93.

    Article  Google Scholar 

  11. Feng S, Ye M, Jiang X, Jin W, Zou H. Coupling the immobilized trypsin microreactor of monolithic capillary with μRPLC−MS/MS for shotgun proteome analysis. J Proteome Res. 2006;5:422–8.

    Article  CAS  Google Scholar 

  12. Liu S, Bao H, Zhang L, Chen G. Efficient proteolysis strategies based on microchip bioreactors. J Proteomics. 2013;82:1–13.

    Article  CAS  Google Scholar 

  13. Cheng G, Chen P, Wang Z-G, Sui X-J, Zhang J-L, Ni J-Z. Immobilization of trypsin onto multifunctional meso-/macroporous core-shell microspheres: a new platform for rapid enzymatic digestion. Anal Chim Acta. 2014;812:65–73.

    Article  CAS  Google Scholar 

  14. Cheng G, Zheng S-Y. Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion. Sci Rep. 2014;4:6947.

    Article  CAS  Google Scholar 

  15. Sassolas A, Blum LJ, Leca-Bouvier BD. Immobilization strategies to develop enzymatic biosensors. Biotechnol Adv. 2012;30:489–511.

    Article  CAS  Google Scholar 

  16. Yamaguchi H, Honda T, Miyazaki M. Application of enzyme-immobilization technique for microflow reactor. J Flow Chem. 2016;6:13–7.

    Article  CAS  Google Scholar 

  17. Hajba L, Guttman A. Continuous-flow biochemical reactors: Biocatalysis, bioconversion, and bioanalytical applications utilizing immobilized microfluidic enzyme reactors. J Flow Chem. 2016;6:8–12.

    Article  CAS  Google Scholar 

  18. Rodrigues RC, Ortiz C, Berenguer-Murcia Á, Torres R, Fernández-Lafuente R. Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev. 2013;42:6290–307.

    Article  CAS  Google Scholar 

  19. Gao J, Xu J, Locascio LE, Lee CS. Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal Chem. 2001;73:2648–55.

    Article  CAS  Google Scholar 

  20. Cooper JW, Chen J, Li Y, Lee CS. Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry. Anal Chem. 2003;75:1067–74.

    Article  CAS  Google Scholar 

  21. Xu F, Wang W-H, Tan Y-J, Bruening ML. Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion. Anal Chem. 2010;82:10045–51.

    Article  CAS  Google Scholar 

  22. Ma J, Liang Z, Qiao X, Deng Q, Tao D, Zhang L, et al. Organic–inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity. Anal Chem. 2008;80:2949–56.

    Article  CAS  Google Scholar 

  23. Calleri E, Temporini C, Perani E, Stella C, Rudaz S, Lubda D, et al. Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins. J Chromatogr A. 2004;1045:99–109.

    Article  CAS  Google Scholar 

  24. Krenkova J, Kleparnik K, Foret F. Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries. J Chromatogr A. 2007;1159:110–8.

    Article  CAS  Google Scholar 

  25. Sun L, Li Y, Yang P, Zhu G, Dovichi NJ. High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres. J Chromatogr A. 2012;1220:68–74.

    Article  CAS  Google Scholar 

  26. Amankwa LN, Kuhr WG. Trypsin-modified-fused-silica capillary microreactor for peptide mapping by capillary zone electrophoresis. Anal Chem. 1992;64:1610–3.

    Article  CAS  Google Scholar 

  27. Nouaimi M, Möschel K, Bisswanger H. Immobilization of trypsin on polyester fleece via different spacers. Enzyme Microb Tech. 2001;29:567–74.

    Article  CAS  Google Scholar 

  28. Kim D, Herr AE. Protein immobilization techniques for microfluidic assays. Biomicrofluidics. 2013;7:041501–47.

    Article  Google Scholar 

  29. Liu Y, Lu H, Zhong W, Song P, Kong J, Yang P, et al. Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification. Anal Chem. 2006;78:801–8.

    Article  CAS  Google Scholar 

  30. Yamaguchi H, Miyazaki M. Enzyme-immobilized reactors for rapid and efficient sample preparation in MS-based proteomic studies. Proteomics. 2013;13:457–66.

    Article  CAS  Google Scholar 

  31. Cheng G, Hao S-J, Yu X, Zheng S-Y. Nanostructured microfluidic digestion system for rapid high-performance proteolysis. Lab Chip. 2015;15:650–4.

    Article  CAS  Google Scholar 

  32. Brivio M, Fokkens RH, Verboom W, Reinhoudt DN, Tas NR, Goedbloed M, et al. Integrated microfluidic system enabling (bio)chemical reactions with on-line MALDI-TOF mass spectrometry. Anal Chem. 2002;74:3972–6.

    Article  CAS  Google Scholar 

  33. Schoenherr RM, Ye M, Vannatta M, Dovichi NJ. CE-microreactor-CE-MS/MS for protein analysis. Anal Chem. 2007;79:2230–8.

    Article  CAS  Google Scholar 

  34. Pal R, Yang M, Lin R, Johnson BN, Srivastava N, Razzacki SZ, et al. An integrated microfluidic device for influenza and other genetic analyses. Lab Chip. 2005;5:1024–32.

    Article  CAS  Google Scholar 

  35. Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE. Soft lithography in biology and biochemistry. Annu Rev Biomed Eng. 2001;3:335–73.

    Article  CAS  Google Scholar 

  36. Gaspar A, Gomez FA. Application of surface plasmon resonance spectroscopy for adsorption studies of different types of components on poly(dimethylsiloxane). Anal Chim Acta. 2013;777:72–7.

    Article  CAS  Google Scholar 

  37. Rabe M, Verdes D, Seeger S. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci. 2011;162:87–106.

    Article  CAS  Google Scholar 

  38. Wu H, Zhai J, Tian Y, Lu H, Wang X, Jia W, et al. Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance. Lab Chip. 2004;4:588–97.

    Article  CAS  Google Scholar 

  39. Liu Y, Zhong W, Meng S, Kong J, Lu H, Yang P, et al. Assembly-controlled biocompatible interface on a microchip: strategy to highly efficient proteolysis. Chem Eur J. 2006;12:6585–91.

    Article  CAS  Google Scholar 

  40. Li Y, Xu X, Deng C, Yang P, Zhang X. Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis. J Proteome R. 2007;6:3849–55.

    Article  CAS  Google Scholar 

  41. Liu Y, Xue Y, Ji J, Chen X, Kong J, Yang P, et al. Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis. Mol Cell Proteomics. 2007;6:1428–36.

    Article  CAS  Google Scholar 

  42. Huang Y, Shan W, Liu B, Liu Y, Zhang Y, Zhao Y, et al. Zeolite nanoparticle modified microchip reactor for efficient protein digestion. Lab Chip. 2006;6:534–9.

    Article  CAS  Google Scholar 

  43. Peterson DS, Rohr T, Svec F, Frechet JMJ. Enzymatic microreactor-on-a-Chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem. 2002;74:4081–8.

    Article  CAS  Google Scholar 

  44. Peterson DS, Rohr T, Svec F, Frechet JMJ. High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers. J Proteome R. 2002;1:563–8.

    Article  CAS  Google Scholar 

  45. Boscaini E, Alexander ML, Prazeller P, Mark TD. Investigation of fundamental physical properties of a polydimethylsiloxane (PDMS) membrane using a proton transfer reaction mass spectrometer (PTRMS). Int Mass Spectrom. 2004;239:179–86.

    Article  CAS  Google Scholar 

  46. Pawliszyn J. Solid phase microextraction: theory and practice. Wiley-VCH; 1997.

  47. Yang Y, Hawthorne SB, Miller DJ, Liu Y, Lee ML. Adsorption versus Absorption of polychlorinated biphenyls onto solid-phase microextraction coatings. Anal Chem. 1998;70:1866–9.

    Article  CAS  Google Scholar 

  48. Shurmer B, Pawliszyn J. Determination of distribution constants between a liquid polymeric coating and water by a solid-phase microextraction technique with a flow-through standard water system. Anal Chem. 2000;72:3660–4.

    Article  CAS  Google Scholar 

  49. Ostuni E, Chen CS, Ingber DE, Whitesides GM. Selective deposition of proteins and cells in arrays of microwells. Langmuir. 2001;17:2828–34.

    Article  CAS  Google Scholar 

  50. Ron I, Sepunaru L, Itzhakov S, Belenkova T, Friedman N, Pecht I, et al. Proteins as electronic materials: electron transport through solid-state protein monolayer junctions. J Am Chem Soc. 2010;132:4131–40.

    Article  CAS  Google Scholar 

  51. Chumbimuni-Torres KY, Coronado RE, Mfuh AM, Castro-Guerrero C, Silva MF, Negrete GR, et al. Adsorption of proteins to thin-films of PDMS and its effect on the adhesion of human endothelial cells. RSC Adv. 2011;1:706–14.

    Article  CAS  Google Scholar 

  52. Secundo F. Conformational changes of enzymes upon immobilisation. Chem Soc Rev. 2013;42:6250–61.

    Article  CAS  Google Scholar 

  53. Rabe M, Verdes D, Seeger S. Surface-induced spreading phenomenon of protein clusters. Soft Matter. 2009;5:1039–47.

    Article  CAS  Google Scholar 

  54. Szollosi GJ, Derenyi I, Voros J. Reversible mesoscopic model of protein adsorption: From equilibrium to dynamics. J Phys Stat Mech Appl. 2004;343:359–75.

    Article  Google Scholar 

  55. Wilkins MR, Lindskog I, Gasteiger E, Bairoch A, Sanchez J-C, Hochstrasser DF, et al. Detailed peptide characterization using PEPTIDEMASS—a World-Wide-Web-accessible tool. Electrophoresis. 1997;18:403–8.

    Article  CAS  Google Scholar 

  56. Proc JL, Kuzyk MA, Hardie DB, Yang J, Smith DS, Jackson AM, et al. A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J Proteome R. 2010;9:5422–37.

    Article  CAS  Google Scholar 

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Acknowledgements

The research was supported by the EU and co-financed by the European Regional Development Fund under the project GINOP-2.3.2-15-2016-00008, GINOP-2.3.3-15-2016-00004, and NTP-EFÖ-P-15-0003 project. The authors also acknowledge the financial support provided to this project by the National Research, Development and Innovation Office, Hungary (K111932). We would like to thank Prof. Dr. Csaba Hegedűs for measurement possibilities in the Biomaterials Research Lab on the Faculty of Dentistry.

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Authors and Affiliations

  1. Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary

    Adam Kecskemeti & Attila Gaspar

  2. Department of Biomaterials and Prosthetic Dentistry, University of Debrecen, Nagyerdei krt. 98., Debrecen, 4032, Hungary

    Jozsef Bako

  3. Department of Experimental Physics, University of Debrecen, Bem ter 18./a, Debrecen, 4026, Hungary

    Istvan Csarnovics

  4. Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary

    Eva Csosz

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  1. Adam Kecskemeti
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Correspondence to Attila Gaspar.

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Kecskemeti, A., Bako, J., Csarnovics, I. et al. Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device. Anal Bioanal Chem 409, 3573–3585 (2017). https://doi.org/10.1007/s00216-017-0295-9

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  • DOI: https://doi.org/10.1007/s00216-017-0295-9

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