A Spring in Performance: Silica Nanosprings Boost Enzyme Immobilization in Microfluidic Channels

Abstract

Enzyme microreactors are important tools of miniaturized analytics and have promising applications in continuous biomanufacturing. A fundamental problem of their design is that plain microchannels without extensive static internals, or packings, offer limited exposed surface area for immobilizing the enzyme. To boost the immobilization in a manner broadly applicable to enzymes, we coated borosilicate microchannels with silica nanosprings and attached the enzyme, sucrose phosphorylase, via a silica-binding module genetically fused to it. We showed with confocal fluorescence microscopy that the enzyme was able to penetrate the ∼70 μm-thick nanospring layer and became distributed uniformly in it. Compared with the plain surface, the activity of immobilized enzyme was enhanced 4.5-fold upon surface coating with nanosprings and further increased up to 10-fold by modifying the surface of the nanosprings with sulfonate groups. Operational stability during continuous-flow biocatalytic synthesis of α-glucose 1-phosphate was improved by a factor of 11 when the microreactor coated with nanosprings was used. More than 85% of the initial conversion rate was retained after 840 reactor cycles performed with a single loading of enzyme. By varying the substrate flow rate, the microreactor performance was conveniently switched between steady states of quantitative product yield (50 mM) and optimum productivity (19 mM min) at a lower product yield of 40%. Surface coating with silica nanosprings thus extends the possibilities for enzyme immobilization in microchannels. It effectively boosts the biocatalytic function of a microstructured reactor limited otherwise by the solid surface available for immobilizing the enzyme.

Demystifying the Flow: Biocatalytic Reaction Intensification in Microstructured Enzyme Reactors

“…Fluidic Connect Pro Resealable flowcell 4515 holder, Teflon tubings and connection parts were from Micronit. Using procedures from our recent paper, the microchannel surface was derivatized with a layer of silica nanosprings to enhance the effective surface area for enzyme immobilization. Liquid flow was delivered from the syringe pump.…”

“…The amount of enzyme activity immobilized on the surface ( A imm ) was determined from activity measurements in solution. It was calculated as the difference between the total enzyme activity offered and the activity remaining in solution after the immobilization . Alternatively, A imm was determined by measuring the enzyme activity eluted from the channel surface .…”

The Promises and the Challenges of Biotransformations in Microflow

“…High surface‐to‐volume ratio of microchannel reactors offers the possibility to obtain relatively high enzyme loads of biocatalysts attached to the surfaces. It also enables attachment of a single layer of cells (Figure a), oriented single‐layer immobilization of enzymes fused with tags (Figure e), and creation of bacterial biofilms (Figure b), which together with short diffusional paths lead to excellent accessibility of a biocatalyst for the substrate and thereby high biocatalyst productivity.…”

“…Increased biocatalyst loads in microreactors could be obtained either by immobilization in surface‐attached thin layers of hydrogels (Figure c), on surface‐integrated nanostructures such as nanosprings (Figure g), or by the use of moving unsinkable graphene sheets, where biocatalyst reuse is provided by centrifugation of the outflow fluid . Further introduction of nanomaterials including biomimetic structures in microreactors, together with the genetic introduction of tags in bacterial cells and enzymes, offers the possibility for more specific immobilization and spatial organization of biocatalysts at specific sites, especially using DNA nanotechnology as a programmable tool for engineering multienzyme catalysis .…”

Enzyme immobilization on photopatterned temperature‐response poly (N‐isopropylacrylamide) for microfluidic biocatalysis

“…Photopolymerization conditions represent a critically important efficiency parameter for the immobilized enzyme microchip applied to continuous flow synthesis, which affects the behaviors of temperature-response PNIPAAm grafted on glass surface to absorb/release enzymes. 29 The effects of the crosslinker MBAAm mass in the polymerization solution, polymerization time under UV light and adsorption temperature were examined to increase the efficiency of enzyme immobilization. Due to the unique temperature-responsive characteristics of PNIPAAm for enzyme adsorption and desorption, as well as the convenient photopatterning technology, a novel and promising microchip was developed for further enzyme immobilization.…”