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We have previously shown that lipid bilayers containing specific recognition elements can be completely coated on the inside of PDMS microfluidic channels bound to glass supports

Design and characterization of immobilized enzymes in microfluidic systems.

ANALYTICAL CHEMISTRY, no. 2 (2002): 379-385

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摘要

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of ...更多

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简介
  • The authors report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes.
  • Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flowcontrolled dilution on-chip.
  • This allowed LineweaverBurk analysis to be performed from a single experiment with all the data collected simultaneously.
  • The presence of glucose was detected by two coupled steps employing immobilized avidinDconjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase
重点内容
  • We report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes
  • In the studies presented here, we developed a simple method for immobilizing biocatalysts on the walls of PDMS microfluidic channels for three specific goals
  • We have previously shown that lipid bilayers containing specific recognition elements can be completely coated on the inside of PDMS microfluidic channels bound to glass supports.[12]
  • The results indicated that the turnover rate for dephosphorylation was
方法
  • Design and Characterization of Immobilized

    Enzymes in Microfluidic Systems

    Hanbin Mao, Tinglu Yang, and Paul S.
  • The authors report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes.
  • Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flowcontrolled dilution on-chip.
  • This allowed LineweaverBurk analysis to be performed from a single experiment with all the data collected simultaneously.
  • The presence of glucose was detected by two coupled steps employing immobilized avidinDconjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase
结果
  • RESULTS AND DISCUSSION

    Immobilization and Characterization of Phosphatase Catalyst in Microcapillary Tubes.
  • The tube was incubated in the buffer overnight, as waiting several hours allowed BSA to fully reorganize at the interface
  • At this point, a solution of streptavidin-conjugated alkaline phosphatase (0.02 mg/mL) was made to flow into the tubes, incubated for 15 min, and washed out with the same pH 9.8 buffer.
  • It should be noted that even the 0.0 mol % bilayer gave a small residual response, which was probably due to a tiny amount of enzyme nonspecifically adsorbed to the underlying substrate
结论
  • In the experiments presented here, enzymes were immobilized in phospholipid bilayer-coated microcapillary tubes and microfluidic channels.
  • The turnover numbers for phosphatase immobilized inside microcapillary tubes and microfluidic channels were similar and represented a reasonable fraction of the corresponding bulk value.
  • A one-shot Lineweaver-Burk plot using arrayed microfluidic channels demonstrated the feasibility of using microchannels to obtain kinetic data rapidly with a high S/N ratio.
  • Serial enzyme reactions inside a microfluidic device were established to show the potential application of this general methodology to multistep chemical synthesis
总结
  • Introduction:

    The authors report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes.
  • Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flowcontrolled dilution on-chip.
  • This allowed LineweaverBurk analysis to be performed from a single experiment with all the data collected simultaneously.
  • The presence of glucose was detected by two coupled steps employing immobilized avidinDconjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase
  • Methods:

    Design and Characterization of Immobilized

    Enzymes in Microfluidic Systems

    Hanbin Mao, Tinglu Yang, and Paul S.
  • The authors report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes.
  • Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flowcontrolled dilution on-chip.
  • This allowed LineweaverBurk analysis to be performed from a single experiment with all the data collected simultaneously.
  • The presence of glucose was detected by two coupled steps employing immobilized avidinDconjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase
  • Results:

    RESULTS AND DISCUSSION

    Immobilization and Characterization of Phosphatase Catalyst in Microcapillary Tubes.
  • The tube was incubated in the buffer overnight, as waiting several hours allowed BSA to fully reorganize at the interface
  • At this point, a solution of streptavidin-conjugated alkaline phosphatase (0.02 mg/mL) was made to flow into the tubes, incubated for 15 min, and washed out with the same pH 9.8 buffer.
  • It should be noted that even the 0.0 mol % bilayer gave a small residual response, which was probably due to a tiny amount of enzyme nonspecifically adsorbed to the underlying substrate
  • Conclusion:

    In the experiments presented here, enzymes were immobilized in phospholipid bilayer-coated microcapillary tubes and microfluidic channels.
  • The turnover numbers for phosphatase immobilized inside microcapillary tubes and microfluidic channels were similar and represented a reasonable fraction of the corresponding bulk value.
  • A one-shot Lineweaver-Burk plot using arrayed microfluidic channels demonstrated the feasibility of using microchannels to obtain kinetic data rapidly with a high S/N ratio.
  • Serial enzyme reactions inside a microfluidic device were established to show the potential application of this general methodology to multistep chemical synthesis
基金
  • This work was supported by ARO (DAAD19-01-1-0346), an ONR-YIP Award (NOOO14-00-1-0664), the Texas Advanced Technology Program (Grant 010366-0181-1999), startup monies from Texas A&M University, a Nontenured Faculty Award from 3M Corp., and a Research Innovation Award from the Research Corporation of America (RI0437). Received for review July 20, 2001
引用论文
  • 380 Analytical Chemistry, Vol. 74, No. 2, January 15, 2002 the same for both systems within experimental error. Furthermore, the use of the PDMS system made data collection more than 1 order of magnitude more rapid than in the microcapillary platform, while possessing an improved signal-to-noise ratio. In the final section of this work, we immobilized glucose oxidase and horseradish peroxidase in series to create a simple glucose sensor that works in two steps.
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  • 382 Analytical Chemistry, Vol. 74, No. 2, January 15, 2002 enzymes. The first stems from the obstruction of active sites at the interface. Steric effects have often been observed for immobilized proteins when their binding pockets face toward the surface.24 As shown in Figure 1, the alkaline phosphatase used here had two active sites for dephosphorylation. Depending upon the packing density and protein orientation, some of these may not have been available for reaction. Furthermore, since the streptavidin-conjugated alkaline phosphatase was prepared by generic chemical cross-linking, the active sites on even neighboring alkaline phosphatases may have had different orientations relative to the underlying streptavidin. If these steric effects strongly blocked the substrate from getting to the most buried locations, this could have caused a significant reduction in turnover rate.
    Google ScholarFindings
  • 384 Analytical Chemistry, Vol. 74, No. 2, January 15, 2002
    Google ScholarFindings
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