title: Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.
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. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary.
authors.forename: Adam
authors.initials: A
authors.lastname: Kecskemeti
authors.affiliation: Department of Biomaterials and Prosthetic Dentistry, University of Debrecen, Nagyerdei krt. 98., Debrecen, 4032, Hungary.
authors.forename: Jozsef
authors.initials: J
authors.lastname: Bako
authors.affiliation: Department of Experimental Physics, University of Debrecen, Bem ter 18./a, Debrecen, 4026, Hungary.
authors.forename: Istvan
authors.initials: I
authors.lastname: Csarnovics
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary.
authors.forename: Eva
authors.initials: E
authors.lastname: Csosz
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., Debrecen, 4032, Hungary. gaspar@science.unideb.hu.
authors.forename: Attila
authors.initials: A
authors.lastname: Gaspar
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004867:Equipment Design; D057927:Hydrophobic and Hydrophilic Interactions; D056656:Lab-On-A-Chip Devices; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013552:Swine; D014357:Trypsin / Q000737:chemistry*
chemical_list: D004129:Dimethylpolysiloxanes; D004800:Enzymes, Immobilized; D010455:Peptides; D012710:Serum Albumin, Bovine; C013830:baysilon; D014357:Trypsin
keywords: Digestion; Enzyme reactor; Immobilization; Microfluidic device; Peptide mapping; Trypsin