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

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

Research output: Contribution to journalArticleScientificpeer-review

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 alpha-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.

Original languageEnglish
JournalAnalytical and Bioanalytical Chemistry
Volume411
Issue number11
Pages (from-to)2339–2349
Number of pages11
ISSN1618-2650
DOIs
Publication statusPublished - Apr 2019
MoE publication typeA1 Journal article-refereed

Bibliographical note

Julkaistaan OA-artikkelina.

Fields of Science

  • CHEMISTRY
  • Enzyme immobilization
  • FABRICATION
  • Gold nanoparticles
  • INTERFACE
  • MICROCHIP ELECTROPHORESIS
  • Mass spectrometry
  • Microfluidics
  • Microreactors
  • PERFORMANCE
  • PROTEINS
  • SOFT-LITHOGRAPHY
  • TRYPSIN
  • Thiol-enes
  • 317 Pharmacy

Cite this

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title = "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 alpha-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.",
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author = "Sari T{\"a}hk{\"a} and Jawad Sarfraz and Lauri Urvas and Riccardo Provenzani and Wiedmer, {Susanne K.} and Jouko Peltonen and Ville Jokinen and Tiina Sikanen",
note = "Julkaistaan OA-artikkelina.",
year = "2019",
month = "4",
doi = "10.1007/s00216-019-01674-9",
language = "English",
volume = "411",
pages = "2339–2349",
journal = "Analytical and Bioanalytical Chemistry",
issn = "1618-2642",
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Immobilization of proteolytic enzymes on replica-molded thiol-ene micropillar reactors via thiol-gold interaction. / Tähkä, Sari; Sarfraz, Jawad; Urvas, Lauri ; Provenzani, Riccardo; Wiedmer, Susanne K.; Peltonen, Jouko; Jokinen, Ville; Sikanen, Tiina .

In: Analytical and Bioanalytical Chemistry, Vol. 411, No. 11, 04.2019, p. 2339–2349.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Immobilization of proteolytic enzymes on replica-molded thiol-ene micropillar reactors via thiol-gold interaction

AU - Tähkä, Sari

AU - Sarfraz, Jawad

AU - Urvas, Lauri

AU - Provenzani, Riccardo

AU - Wiedmer, Susanne K.

AU - Peltonen, Jouko

AU - Jokinen, Ville

AU - Sikanen, Tiina

N1 - Julkaistaan OA-artikkelina.

PY - 2019/4

Y1 - 2019/4

N2 - 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 alpha-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.

AB - 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 alpha-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.

KW - CHEMISTRY

KW - Enzyme immobilization

KW - FABRICATION

KW - Gold nanoparticles

KW - INTERFACE

KW - MICROCHIP ELECTROPHORESIS

KW - Mass spectrometry

KW - Microfluidics

KW - Microreactors

KW - PERFORMANCE

KW - PROTEINS

KW - SOFT-LITHOGRAPHY

KW - TRYPSIN

KW - Thiol-enes

KW - 317 Pharmacy

U2 - 10.1007/s00216-019-01674-9

DO - 10.1007/s00216-019-01674-9

M3 - Article

VL - 411

SP - 2339

EP - 2349

JO - Analytical and Bioanalytical Chemistry

JF - Analytical and Bioanalytical Chemistry

SN - 1618-2642

IS - 11

ER -