Immobilization of proteolytic enzymes on replica-molded thiol-ene micropillar reactors via thiol-gold interaction

Abstract

We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic α-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-01674-9) contains supplementary material, which is available to authorized users.

Microfluidic Analysis Techniques for Safety Assessment of Pharmaceutical Nano‐ and Microsystems

“…In principal, the PDMS microstructures can be replicated to any heat-or UV-curable polymer by casting the monomer solution onto the PDMS mold, crosslinking the cast polymer when in contact with PDMS, and detaching the new polymer replica from the PDMS mold. The lifetime of the PDMS mold is typically less than that of the cleanroom master molds, because PDMS tends to absorb the monomers of the cast solution, but it easily lasts for at least a handful of repeated replication cycles [18]. In comparison to direct replication of heat-or UV-curable polymers (other than PDMS) with the help of cleanroom masters (e.g., SU-8), the elasticity of PDMS plays a critical role in facilitating straightforward detachment of the mold and the replica from each other (after curing) without damaging the reproduced microstructures.…”

“…An exception are, e.g., UV-curable off-stoichiometric thiol-ene (OSTE) polymers, which -depending the bulk monomer composition and the applied crosslinking conditions -may be sealed by one another following a fairly simple lamination protocol [19,20]. In addition to straightforward sealing, the OSTE chemistry provides the possibility to adjust both the bulk properties and the surface chemistry (type and number of functional groups available for further chemical conjugation reactions) on the basis of the end-application needs [18,[20][21][22]. These possibilities are briefly reviewed in the context of relevant applications in the next sections.…”

“…Ordered micropillar arrays can be made with high precision from silicon by anisotropic etching [147,154] or from polymer photoresists by direct UV lithography [155]. The lithographically defined micropillar arrays may also act as master molds for replicating the structures into other UV and heat curable polymers or thermoplasts, as described in Section 3.1, although the achievable aspect ratios of replicated pillars may be limited compared with direct microfabrication techniques [18]. Although silicon (oxide) readily facilitates a multitude of surface reactions, especially the possibility to use replication techniques increases the freedom in material selection with a view to enzyme immobilization on micropillar surfaces.…”

“…For example, the OSTE polymers feature either free surface thiols or allyl groups, the density of which can be tuned by simply manipulating the molar ratio of the thiol and allyl monomers in the bulk composition [20]. Especially the free thiol groups of the OSTEs have been extensively employed in a variety of microfluidic immobilized enzyme reactors making use of, e.g., photopolymerization [142,160] or gold nanoparticle interaction with the free thiol residues on both the chip surface and in the protein structure [18]. Besides surface chemistry, attention should be paid on the possible nonspecific interactions between the polymers and the biomolecules to ensure that the catalytic activity is not lost because of protein fouling.…”

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“…On the other hand, this supreme absorptivity can actually be exploited for the direct immobilization of enzymes [13,61]. Besides PDMS, thiol-ene (TE) microchips are experiencing increasing interest [60,[62][63][64]. In addition to optical transparency and low price, TE chips offer improved solvent resistance and the possibility of tuning the surface chemistry by modulating the stoichiometry of the monomers.…”