Taylor dispersion analysis, resonant mass measurement and bioactivity of pepsin-coated gold nanoparticles.
Immobilized enzyme reactors based on nanoparticulate carriers are becoming increasingly popular. A toolbox of methods is usually utilized for their characterization in order to be capable of assessing their suitability for the intended purpose. In this work, as a model system pepsin was conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process. The success of the immobilization procedure was monitored by Vis spectroscopy via shifts of the localized surface plasmon resonance (LSPR) band, size characterization by dynamic light scattering (DLS), and ζ-potential determinations by electrophoretic light scattering. DLS revealed a significantly different hydrodynamic diameter for unmodified GNPs and protein-coated GNPs. However, the hydrodynamic diameters of pepsin-coated GNPs obtained with various concentrations of pepsin in the coating solution were not significantly different. In contrast, Taylor dispersion analysis allowed measuring the slight differences in the hydrodynamic radius. It provided also information on the viscosity of GNP suspensions and diffusion coefficients for the various pepsin@GNP preparations. For the determination of the pepsin surface coverage on the GNPs results from indirect protein quantitation of non-immobilized pepsin by Lowry assay were compared to direct measurement of immobilized pepsin by resonant mass measurements. Reasonable agreement was found. Accurate information on enzyme coverage is of utmost importance for a representative comparison of the turnover numbers and catalytic efficiencies of nanoparticulate immobilized enzyme reactors, which is shown by the adsorptively immobilized pepsin@GNP as model system.
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Markus
M
Höldrich
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Siyao
S
Liu
Malvern Instruments, Herrenberg, Germany.
Markus
M
Epe
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Electronic address: Michael.laemmerhofer@uni-tuebingen.de.
Michael
M
Lämmerhofer
D000818:Animals; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D004800:Enzymes, Immobilized; D006046:Gold; D010434:Pepsin A
Heterogeneous biocatalyst; Immobilized enzyme reactor; Nanoparticle; Protein corona; Resonant mass measurement; Taylor dispersion analysis