Abstract
This paper describes a simple and efficient method for producing an on-chip enzyme immobilized monolith micro-reactor that integrates a microfluidic electrochemical cell for rapid characterization of enzymatic kinetics. The monolith was generated using a sol–gel method, followed by PEI functionalization and enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. Using the proposed immobilization strategy, a glucose oxidase (GOD) immobilized monolith micro-reactor has been produced with the controllable porosity that gives better enzyme kinetics compared to previously reported devices. This can be attributed to a favourable enzyme-substrate affinity in which more than 98% of the immobilized enzyme remains in an active conformation. The kinetic studies conducted have identified that a similar value of the k cat is obtained for immobilized GOD (13.4 s−1) and GOD free in solution (14 s−1) whilst the immobilized Michaelis constant K m(app) (7.2 mM) is ~4 times lower than GOD in solution (25 mM). In addition, the immobilized GOD exhibits increased stability, retaining at least 95% of the initial activity when stored of 30 days at 4°C, compared to only 60% for GOD in solution. Furthermore, the same enzyme immobilization strategy has been used for choline oxidase immobilization and similar kinetics to choline oxidase in solution were observed, once again indicating better maintenance of the enzyme conformation provided by the proposed method.
Similar content being viewed by others
References
Arai G, Noma T, Hayashi M, Yasumori I (1998) Electrochemical characteristics of d-amino acid oxidase immobilized in a conductive redox polymer. J Electroanal Chem 452:43–48
Deere J, Magner E, Wall JG, Hodnett BK (2002) Mechanistic and structural features of protein adsorption onto mesoporous silicates. J Phys Chem B 106:7340–7347
DeLouise LA, Miller BL (2005) Enzyme immobilization in porous silicon: quantitative analysis of the kinetic parameters for glutathione-s-transferases. Anal Chem 77:1950–1956
Demers N, Agostinelli E, Averill-Bates DA, Fortier G (2001) Immobilization of native and poly(ethylene glycol)-treated (‘PEGylated’) bovine serum amine oxidase into a biocompatible hydrogel. Biotechnol Appl Biochem 33:201–207
Fletcher PDI, Haswell SJ, Pombo-Villar E, Warrington BH, Watts P, Wong SYF, Zhang X (2002) Micro reactors: principles and applications in organic synthesis. Tetrahedron 58:4735–4757
Girelli AM, Mattei E (2005) Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B 819:3–16
Gleason NJ, Carbeck JD (2004) Measurement of enzyme kinetics using microscale steady-state kinetic analysis. Langmuir 20:6374–6381
Guo Z, Xu S, Lei Z, Zou H, Guo B (2003) Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 24:3633–3639
Han Y-J, Stucky GD, Butler A (1999) Mesoporous silicate sequestration and release of proteins. J Am Chem Soc 121:9897–9898
He P, Greenway G, Haswell SJ (2008) The on-line synthesis of enzyme functionalized silica nanoparticles in a micro-reactor using poly ethylenimine polymer and R5 peptide. Nanotechnology 19:315603
Hilder EF, Svec F, Fréchet JMJ (2002) Polymeric monolithic stationary phases for capillary electrochromatography. Electrophoresis 23:3934–3953
Holden MA, Jung S-Y, Cremer PS (2004) Patterning enzymes inside microfluidic channels via photoattachment chemistry. Anal Chem 76:1838–1843
Honda T, Miyazaki M, Nakamura H (2006) Facile preparation of an enzyme-immobilized microreactor using a cross-linking enzyme membrane on a microchannel surface. Adv Synth Catal 348:2163–2171
Kaku T, Karan HI, Okamoto Y (1994) Amperometric glucose sensors based on immobilized glucose oxidase-polyquinone system. Anal Chem 66:1231–1235
Kato M, Inuzuka K, Sakai-Kato K, Toyo’oka T (2005) Monolithic bioreactor immobilizing trypsin for high-throughput analysis. Anal Chem 77:1813–1818
Kawakami K, Sera Y, Sakai S, Ono T, Ijima H (2005) Development and characterization of a silica monolith immobilized enzyme micro-bioreactor. Ind Eng Chem Res 44:236–240
Kerby MB, Legge RS, Tripathi A (2006) Measurements of kinetic parameters in a microfluidic reactor. Anal Chem 78:8273–8280
Koh WG, Pishko M (2005) Immobilization of multi-enzyme microreactors inside microfluidic devices. Sens Actuators B 106:335–342
Krenková J, Foret F (2004) Immobilized microfluidic enzymatic reactors. Electrophoresis 25:3550–3563
Laarz E, Bergström L (2000) Dispersing WC–Co powders in aqueous media with polyethylenimine. Int J Refract Metals Hard Mater 18:281–286
Legido-Quigley C, Marlin ND, Melin V, Manz A, Smith NW (2003) Advances in capillary electrochromatography and micro-high performance liquid chromatography monolithic columns for separation science. Electrophoresis 24:917–944
Lei C, Shin Y, Liu J, Ackerman E (2002) Entrapping enzyme in a functionalized nanoporous support. J Am Chem Soc 124:11242–11243
Liu S, Sun Y (2007) Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol–gel membrane for glucose and ATP detections. Biosens Bioelectron 22:905–911
Luckarift HR, Spain JC, Naik RR, Stone MO (2004) Enzyme immobilization in a biomimetic silica support. Nat Biotechnol 22:211–213
Mao H, Yang T, Cremer PS (2002) Design and characterization of immobilized enzymes in microfluidic systems. Anal Chem 74:379–385
Mersal GAM, Bilitewski U (2005) Development of monolithic enzymatic reactors in glass microchips for the quantitative determination of enzyme substrates using the example of glucose determination via immobilized glucose oxidase. Electrophoresis 26:2303–2312
Murthy ASN, Sharma J (1998) Glucose oxidase bound to self-assembled monolayers of bis(4-pyridyl) disulfide at a gold electrode: amperometric determination of glucose. Anal Chim Acta 363:215–220
Nomura A, Shin S, Oulad MO, Kauffmann JM (2004) Preparation, characterization, and application of an enzyme-immobilized magnetic microreactor for flow injection analysis. Anal Chem 76:5498–5502
Ota S, Miyazaki S, Matsuoka H, Morisato K, Shintani Y, Nakanishi K (2007) High-throughput protein digestion by trypsin-immobilized monolithic silica with pipette-tip formula. J Biochem Biophys Methods 70:57–62
Park CB, Clark DS (2002) Sol–gel encapsulated enzyme arrays for high-throughput screening of biocatalytic activity. Biotechnol Bioeng 78:229–235
Park SS, Cho SI, Kim MS, Kim YK, Kim BG (2003) Integration of on-column immobilized enzyme reactor in microchip electrophoresis. Electrophoresis 24:200–206
Peterson DS (2005) Solid supports for micro analytical systems. Lab Chip 5:132–139
Peterson DS, Rohr T, Svec F, Fréchet JMJ (2002) Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem 74:4081–4088
Rogers MJ, Brandt KG (1971) Interaction of d-glucal with a niger glucose oxidase. Biochem 10:4624–4630
Sakai-Kato K, Kato M, Toyo’oka T (2003) Creation of an on-chip enzyme reactor by encapsulating trypsin in sol−gel on a plastic microchip. Anal Chem 75:388–393
Seong GH, Crooks RM (2002) Efficient mixing and reactions within microfluidic channels using microbead-supported catalysts. J Am Chem Soc 124:13360–13361
Seong GH, Heo J, Crooks RM (2003) Measurement of enzyme kinetics using a continuous-flow microfluidic system. Anal Chem 75:3161–3167
Thomsen MS, Pölt P, Nidetzky B (2007) Development of a microfluidic immobilized enzyme reactor. Chem Commun 252:7–2529
Tokeshi M, Kikutani Y, Hibara A, Sato K, Hisamoto H, Kitamori T (2003) Chemical processing on microchips for analysis, synthesis, and bioassay. Electrophoresis 24:3583–3594
Urban PL, Goodall DM, Bruce NC (2006) Enzymatic microreactors in chemical analysis and kinetic studies. Biotechnol Adv 24:42–57
Verpoorte E (2003) Beads and chips: new recipes for analysis. Lab Chip 3:60N–68N
Vidal J-C, Garcia E, Castillo J-R (1998) Electropolymerization of pyrrole and immobilization of glucose oxidase in a flow system: influence of the operating conditions on analytical performance. Biosens Bioelectron 13:371–382
Vodopivec M, Podgornik A, Berovič M, Štrancar A (2003) Characterization of CIM monoliths as enzyme reactors. J Chromatogr B 795:105–113
Wang B, Li B, Deng Q, Dong S (1998) Amperometric glucose biosensor based on sol–gel organic-inorganic hybrid material. Anal Chem 70:3170–3174
Wang C, Oleschuk R, Ouchen F, Li J, Thibault P, Harrison DJ (2000) Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface. Rapid Commun Mass Spectrom 14:1377–1383
Acknowledgments
We acknowledge the EU for funding through project NMP4-CT-2006-033254.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
He, P., Greenway, G. & Haswell, S.J. Development of enzyme immobilized monolith micro-reactors integrated with microfluidic electrochemical cell for the evaluation of enzyme kinetics. Microfluid Nanofluid 8, 565–573 (2010). https://doi.org/10.1007/s10404-009-0476-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10404-009-0476-8