Determination of Temporal and Spatial Concentration Gradients in Hydrogel Beads Using Multiphoton Microscopy Techniques

Abstract

Multiphoton microscopy is a promising technique to detect spatially and temporally resolved concentration gradients of chemical compounds, e.g., reactants in hydrogel-encapsulated biocatalysts. In contrast to current techniques, the improved spatial and temporal resolution of this method in data acquisition and its ability to measure hydrogel beads facilitates the identification of various kinetic phenomena. To our knowledge, multiphoton microscopy is used here for the first time to examine diffusion, mass transfer, and reaction in immobilized hydrogel systems. In a first step, the phenomena of diffusion and diffusion-coupled mass transfer through the phase interface are investigated in the bead center. Finally, the complete system--consisting of diffusion, mass transfer, and enzymatic reaction--is observed by measuring concentration gradients along the bead radius with temporal and spatial resolution. This metrology enables a subsequent mechanistic model identification, which in turn leads to an enhanced knowledge of reaction kinetics and supports the design of biotechnological processes. This task was only possible due to excellent spatial (25 microm) and temporal (5 s) resolution and the accuracy (+/-1%) achieved by using a multiphoton microscopy setup.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…A pH drop resulted in this case from the oxidation of d-glucose into d-gluconic acid. Fluorescence lifetime provides advanced measurement capabilities [178,179,186], eliminating signal distortion in dependence of the scanning depth as a known critical problem of intensity-based measurements in CLSM [193]. Internal pH changes at spatial resolution have been monitored in hydrogels and PEG microparticles using fluorescence lifetime microscopy techniques [166,186].…”

“…High spatial and temporal resolution is obtained enabling the simultaneous identification and monitoring of multiple events in immobilized biocatalysts. Diffusion and enzymatic reaction are resolved by elucidation of local concentration gradients through the catalytic particle [178,179].…”

The Microenvironment in Immobilized Enzymes: Methods of Characterization and Its Role in Determining Enzyme Performance

“…Unfortunately, lifetime and referenced measurements in CLSM depend on high-cost instrumentation that cannot be adapted to real-life reactor configurations and has limited throughput capacity. The application of multiphoton laser scanning microscopy [73,74] addresses some of the known limitations of measurements in CLSM. Multiphoton microscopy has been used to determine concentration gradients in hydrogel-encapsulated biocatalysts [73,74].…”

“…The application of multiphoton laser scanning microscopy [73,74] addresses some of the known limitations of measurements in CLSM. Multiphoton microscopy has been used to determine concentration gradients in hydrogel-encapsulated biocatalysts [73,74]. High spatial and temporal resolution is obtained, thus enabling multiple conversion events in immobilized biocatalysts to be monitored simultaneously.…”

Single‐Particle Studies to Advance the Characterization of Heterogeneous Biocatalysts

“…A pH drop resulted in this case from the oxidation of d ‐glucose into d ‐gluconic acid . The application of fluorescence lifetime or multiphoton laser scanning microscopy solve some of the known limitations of measurements in CLSM . Consequently, these techniques have been applied to the spatial resolution of internal pH inside hydrogels particles (1.5 mm) where the events of substrate diffusion and enzymatic reactions were resolved by elucidating the local concentration gradients through the catalytic particle .…”