Enzymatic Cascade Reactions in Microchannels:
Monolayer Patterning & Reversible Embedding of DNA-Enzyme Hybrids
TuHa Vong,1,2 Jurjen ter Maat,1 Remco Regeling,1
Jan van Hest,2 Teris van Beek,1 Han Zuilhof1
1
Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB
Wageningen, The Netherlands. 2 Institute of Molecules and Materials, Radboud University
Nijmegen, P.O. Box 9010 6500 GL Nijmegen, The Netherlands.
E-mail: Han.Zuilhof@wur.nl
INTRODUCTION:
Glass microchips for lab-on-a-chip purposes are gaining rapid interest given the possibilities of
microfluidics-based diagnostics and microreactors. Since such chips are relatively expensive, it is
desirable to have a system that can be reused or changed according to one's needs. Typically,
biofunctional materials have a shorter active lifetime than the glass substrates. Therefore the goal of
this project is:
a) to provide local and reversible biofunctionalization of glass micro-channels, and
b) to use this approach to induce enzymatic cascade reactions within a single microchannel, via the
use of DNA-linked enzyme hybrids, that can be positioned and removed via DNA
hybridization/dehybridization.
RESULTS & DISCUSSION:
We have developed a novel site-specific surface modification method (patterning by UV
irradiation).1 This is a nice alternative for silane-based chemistry, as it is very mild,
circumvents the use of corrosive chemicals, is highly flexible with regard to the allowed
functional groups, and allows constructive photopatterning as shown by a variety of
scanning probe & optical microscopy techniques.
CF3 CF3 CF3
O
O
O
CF3
OH
OH
OH
Si
Si
Si
O
O
O
O
O
O
h
+
O
O
O
O
O
O
O
O
O
Si
Si
Si
O
O
O
O
O
O
O
Figure 1: Mild photochemical attachment of a functional monolayer onto glass surfaces.
This method is applied to attach a functional linker on the inside wall of a glass microchannel
(typical inner diameter: 10 – 100 m; Figure 2). The available linker is then used to covalently
couple an amino-terminated oligo-DNA. This thus yields a general platform for the reversible
binding of a complementary ssDNA-conjugate strand (e.g. DNA-enzyme).2
Figure 2: Local and covalent attachment of oligoDNA within a microchannel provides a general
supramolecular platform for the reversible functionalization with (bio-)hybrids.
Such a reversible attachment is especially attractive in the case of components that have an active
lifetime that is significantly shorter than that of the glass chip, such as is the case for many enzymes.
Therefore DNA-enzyme constructs were prepared, subsequently attached within the microchannel,
and their activity optimized.
Next, an enzymatic cascade reaction was performed within the microchannel, by local attachment of
DNA-hybrids of CalB and Horse Radish Peroxidase. It should be noted that after the initial local
prefunctionalization with two different oligo-DNA strands flowing the microchannel with a solution
containing a mixture of CalB and HRP allows the CalB and HRP to find their own position via
specific DNA hybridization. Finally, the cascade reaction was shown to work, and the dependence
on flow rate, length of functional moieties and specifics of the initial attachment reaction
investigated.
Fig. 3: Local positioning of DNA-hybrids of CalB and Horse Radish Peroxidase allows an enzymatic
cascade reaction within the microchannel.
Current work aims at increasing the reversibility of the system. After a start of 60% retaining
activity after 3 recycling steps,2 in mid-june 2010 we have improved this to >90% after 10 recycling
steps. We aim to fine-tune this even further, by a variation of especially dehybridization conditions.
REFERENCES:
1) J. ter Maat, R. Regeling, M. Yang, M.N. Mullings, S.F. Bent and H. Zuilhof - Photochemical
Covalent Attachment of Alkene-Derived Monolayers onto Hydroxyl-Terminated Silica Langmuir
2009, 25, 11592.
2) T.H. Vong, J. ter Maat, T.A. van Beek, B. van Lagen, M. Giesbers, J.C.M. van Hest and H.
Zuilhof - Site-Specific Immobilization of DNA in Glass Microchannels via Photolithography
Langmuir 2009, 25, 13952.
ACKNOWLEDGEMENTS: The authors thank NWO-ACTS (PoaC project 053.65.002) and
NanoNed (project WMM.6975), both funded by the Dutch Ministry of Economic Affairs,
for financial support.