LCD-based detection of enzymatic action

Article abstract of DOI:10.1039/b514048j

By incorporating an ester-containing substrate in a self-assembled alignment layer for liquid crystal cells, the presence of a lipase (CALB) can be directly detected through its enzymatic action on the alignment layer, without the need for fluorescent lab

Full text of DOI:10.1039/b514048j

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LCD-based detection of enzymatic action{
Johan Hoogboom,{* Kelly Velonia,§ Theo Rasing, Alan E. Rowan and Roeland J. M. Nolte
Received (in Cambridge, UK) 4th October 2005, Accepted 2nd November 2005
First published as an Advance Article on the web 24th November 2005
DOI: 10.1039/b514048j
of the layer. When used in an LC cell, this was clearly visible by the
By incorporating an ester-containing substrate in a self-
assembled alignment layer for liquid crystal cells, the presence
of a lipase (CALB) can be directly detected through its
enzymatic action on the alignment layer, without the need for
fluorescent labelling or enzyme assays.
absence of uniform liquid crystal alignment.
The substrate-containing alignment layer was constructed by
immersing a cleaned ITO-plate into a 3 wt% solution of 2 and
c-aminopropyltriethoxysilane (APTES) in toluene in a 9 : 1 molar
ratio." After 1 h, the plate was removed from the solution, washed
with acetonitrile and baked at 120 uC for 10 min to complete the
covalent bonding of the siloxanes to the surface. When these
surfaces were used in liquid crystal cells,11I polarising microscopy
showed that the siloxane layers were capable of inducing uniform
alignment of the common nematogen n-pentyl-4,49-cyanobiphenyl,
5CB (Fig. 1a).
Because of the strong interactions that exist between neighbouring
liquid crystal domains on a surface, any change in their surface
ordering is transmitted into the liquid crystal bulk, thereby
amplifying a small disturbance by several orders of magnitude.1–11
This qualifies liquid crystals as molecular magnifying glasses,
bringing (bio)reactions which occur on nanoscales into the realm
of the naked eye, without the need for additional processing.1–11
Recently, we described a procedure for the preparation of self-
assembled alignment layers for liquid crystal alignment purposes,
which makes use of parallel nanometre-sized grooves that are
present on commonly used indium–tin–oxide (ITO) surfaces.12
The size of these grooves is amplified over several orders of
magnitude by the anisotropic grafting of oligosiloxanes, which
spontaneously form in situ, onto the ITO surface, resulting in
liquid crystal alignment over millimetre-sized areas. Here we
expand on this procedure by incorporating siloxane 2 (for its
synthesis see Scheme 1), which contains a hydrolysable group, into
an alignment layer. When this layer is exposed to the lipase B from
Candida antarctica (CALB), the subsequent partial hydrolysis of
the layer results in a local disturbance of the alignment capabilities
Prior to LC cell construction, this alignment layer was incubated
with a solution of 0.3 mM CALB in a 20 mM phosphate buffer
pH 8.0, for 15 h in ambient conditions, after which it was washed
with water, iso-propanol and acetone to remove any residual
water, preventing dewetting of 5CB. When such plates were used
in the construction of a liquid crystal cell, polarising microscopy
showed that the cell contained areas of circa 100 mm, in which the
liquid crystal alignment had been disturbed (Fig. 1b). In addition,
Fourier-Transform Infra-Red experiments indicated the presence
of free acid groups on the parts of the surface which had been
exposed to the enzyme. NMR experiments also showed that
CALB was capable of hydrolysing 2 in solution. The patchy
nature of enzymatic activity is comparable to burst kinetics
observed before by Bjørnholm et al. for the action of a lipase on
substrate-containing Langmuir–Blodgett-monolayers.13,14 Blank
experiments, in which substrate-containing alignment layers were
incubated for 15 h with a 20 mM phosphate buffer pH 8.0, showed
Scheme 1 Synthesis of 2.
Institute for Molecules and Materials, Radboud University Nijmegen,
Toernooiveld 1, 6525ED, Nijmegen, The Netherlands.
E-mail: J.Hoogboom@science.ru.nl; Fax: +31 24 3652929;
Tel: +31 24 3652143
{ Electronic supplementary information (ESI) available: Synthesis &
molecular modeling. See DOI: 10.1039/b514048j
{ Present address: Massachusetts Institute of Technology, 77
Massachusetts Avenue, Bldg 18-143, Cambridge, MA 02139, USA,
hoogboom@mit.edu
§ Present address: Department of Organic Chemistry, University of
Geneva, Sciences II, 30 Quai Ernest Ansermet, CH-1211, Geneva 4,
Switzerland.
Fig. 1 Polarising micrographs of parallel liquid crystal cells with: (a) an
alignment layer of 2 and APTES (see text), polarisers at 0u; (b) the same
alignment layer, after exposure to CALB, polarisers at 45u. Images are 600
6 400 mm.
434 | Chem. Commun., 2006, 434–435
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Scheme 2 Schematic depiction of possible extreme differences in local liquid crystal orientation, arising from polarity differences upon hydrolysis of the
alignment layer, resulting in a reorientation of the mesogen near the boundary of enzymatic action and interfering with uniform liquid crystal alignment.
surface.12 To tune this rate, other siloxanes with different kinetics, such as
that a liquid crystal cell constructed from such plates contained no
APTES, were added.
non-aligned regions larger than 10 mm, ruling out background
I LC cells were prepared by using one sample plate and an unrubbed
hydrolysis. Scanning Electron Microscopy experiments indicated
that the initial surface ordering, i.e. the groove direction dictated
by the siloxane backbone of the alignment layer,12 was not affected
by the enzymatic action.
counter plate which was spin-coated with Polyimide Pyralin PI2555 (HD
Microsystems) at 5000 rpm for 20 s and baked at 120 uC for 90 min. Mylar
spacers of 6 mm were used to separate the plates. ITO plates were cleaned
with ozone (100 l h21) for 3 h prior to use. LC cells were filled with 5CB in
the isotropic phase at 40 uC (TNI = 35.3 uC) and were cycled 36 between
20 uC and 40 uC to exclude any memory effects.
These results can be explained as follows. Upon hydrolysis, the
alignment layer undergoes a dramatic local change in polarity,
going from a benzyl group to an acid group wherever the enzyme
is active. This entails that the interaction between the alignment
layer and the nematogen shifts from that between a benzyl group
and the apolar parts of 5CB, to one between an acid group and the
polar part of 5CB. This forces the mesogens near the surface,
especially near the boundary of enzymatic activity, to adopt a
different orientation (Scheme 2), disrupting uniform alignment.
Such changes in mesogen orientation caused by polarity
differences in the alignment layer are quite common and have
been reported before.15–19 In turn, this altered ordering of the
liquid crystal matrix near the surface is transferred into the
mesogenic bulk and amplified up to levels that can be easily
detected by the human eye.
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The method we present here offers a way to detect the presence
of a class of enzymes (e.g. lipases) by observing its action on a
substrate-containing alignment layer. The method does not require
additional labelling steps or enzymatic assays, can be easily
performed and gives clearly visible results. We are currently
investigating the use of different alignment layers for the detection
of different classes of enzymes, as well as determining a lower limit
on the detectable enzyme concentration in relation to reaction
time. We are also investigating the possibility of using this
technique to create surface patterns.
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Notes and references
" The formation of the alignment layer is very sensitive to the oligosiloxane
oligomerisation rate: the presence of the correct oligomer size at the right
time during layer formation, is crucial to the creation of an ordered
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Chem. Commun., 2006, 434–435 | 435