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

Sari Tähkä,Ville Jokinen,Susanne K Wiedmer,Tiina Sikanen,Riccardo Provenzani,Jouko Peltonen,Jawad Sarfraz,Lauri Urvas

doi:10.1007/s00216-019-01674-9

Sari Tähkä, Ville Jokinen + Show 6 more

Open Access

https://doi.org/10.1007/s00216-019-01674-9

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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

Highlights

Immobilization of proteolytic enzymes on solid support structures packed in capillary channels has gained considerable interest owing to its many benefits over soluble enzyme reactions [1, 2]
We examined the possibilities of exploiting the inherent high thiol density of the crosslinked offstoichiometric thiol-enes for efficient functionalization of the micropillar arrays with gold nanoparticles (GNPs)
We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs) by exploiting thiol-gold interaction

Summary

Introduction

Immobilization of proteolytic enzymes (e.g., trypsin, pepsin, and chymotrypsin) on solid support structures packed in capillary channels has gained considerable interest owing to its many benefits over soluble enzyme reactions [1, 2]. We make use of the native thiol-ene surface chemistry by preparing thiol-rich micropillar arrays and functionalizing them with gold nanoparticles (GNPs) via the well-characterized and strong thiol-gold interaction [21, 22]. A concentrated solution of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in PBS buffer was pumped through the micropillar array at a flow rate of 5 μL/min followed by quantitation of the reaction product, 2nitro-5-thiobenzoate (TNB), by UV absorbance (412 nm) using Varioskan LUX Multimode Microplate Reader (ThermoScientific, Vantaa, Finland). The sample solutions (20 μM bradykinin in 20 mM ammonium acetate, pH 8.2) were infused with a syringe pump at a constant flow rate of 2.5, 5, 10, 15, or 20 μL/min and the reactants were collected for MS analysis in appropriate volumes (typically 100–150 μL per fraction). The MS data was acquired over a mass range of m/z 100–2000 with a maximum accumulation time of 300 ms using Data Analysis 3.4

Results

Conclusions

Compliance with ethical standards