A novel "pro-sensitizer" based sensing of enzymes using Tb(iii) luminescence in a hydrogel matrix

Article abstract of DOI:10.1039/c2cc30904a

Chemically synthesized "pro-sensitizers" release the sensitizer in the presence of lipase or β-glucosidase, triggering a significant luminescence response from a lanthanide based hydrogel. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc30904a

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COMMUNICATION
A novel ‘‘pro-sensitizer’’ based sensing of enzymes using Tb(III)
luminescence in a hydrogel matrixw
Sandip Bhowmika and Uday Maitra*ab
Received 8th February 2012, Accepted 13th March 2012
DOI: 10.1039/c2cc30904a
Chemically synthesized ‘‘pro-sensitizers’’ release the sensitizer
in the presence of lipase or b-glucosidase, triggering a significant
luminescence response from a lanthanide based hydrogel.
Recently we discovered a novel way to sensitize trivalent
lanthanide ions where the spatial proximity between the
sensitizer and the lanthanide ion was achieved in a cholate
gel matrix through self-assembly of commercially available
ingredients.8 We were thus able to eliminate the need for a
chelating ligand and use the gel matrix as a general platform to
carry out all sensitization and sensing studies. 2,3-Dihydroxy-
naphthalene (DHN) was shown to sensitize Tb3+ exclusively
in the gel medium (sensitization by DHN in a fluid medium was
negligible). We reasoned that the hydroxyl groups on DHN
can in fact be suitably functionalized to generate a pro-sensitizer
(which would not sensitize the lanthanide ion). The idea was to
incorporate the pro-sensitizer into the gel media which upon
the action of an enzyme would release the sensitizer and hence
make the gel luminescent (Scheme 1).
Optical probes which can provide direct information about
enzyme activities are in great demand as enzyme mediated
transformations are central to all biological processes. While
most enzyme assays are based on colorimetry, recent years
have witnessed increasing intent for developing analogous
fluorescent systems, mainly due to their higher intrinsic
sensitivity.1 A significant number of fluorescent probes have
been reported in the literature which can successfully sense
enzymes and other biomolecules, small organic molecules,
metal ions etc.2 Fluorescence based sensing of biomolecules
such as enzymes or in vivo measurements often pose a chal-
lenge due to interference from background fluorescence arising
either from other fluorogenic species or auto fluorescence from
cells.3 Time-delayed luminescence measurements provide a
possible solution to such problems as fluorescence from probes
with longer lifetimes can easily be separated from the short-
lived background. In this context, luminescent lanthanide
complexes are attractive choices because of their long life-
times, sharp emission bands and negligible interference from
the environment.4 We now report a novel, ‘‘pro-sensitizer’’
based sensing of enzymes using a gel-based Tb(III) lumines-
cence assay.
An obvious starting point was to test a diester of DHN and
cleave it with an esterase to release DHN (Scheme 2). A lipase
was chosen for this purpose, since lipases belong to a very well
known class of enzymes. A malfunctioning lipase metabolic
pathway is reflected in high blood cholesterol and triglyceride
levels and can lead to several cardiac diseases and diseases
related to liver such as hepatomegaly, liver fibrosis, splenomegaly
etc.9 A simple and efficient lipase assay system is thus of
Lanthanide complexes have been extensively used for
sensing metal ions, small organic molecules, and also in some
cases for enzyme assays. Such examples typically follow the
well-known strategy towards lanthanide sensitization by an
[enzyme substrate–antenna–ligand] complex of a lanthanide5,6
or by using a binary system of an appropriate chelator and a
modified sensitizer.7 However, for all these systems, chelation
of the lanthanide ion by a suitable multidentate ligand is
essential, usually requiring additional synthetic steps to obtain
the chelating ligand suitable for the job.
Scheme 1 A strategy towards the design of enzyme-induced lumines-
a Department of Organic Chemistry, Indian Institute of Science,
Bangalore 560 012, India. E-mail: maitra@orgchem.iisc.ernet.in;
Fax: +91-80-2360-0529; Tel: +91-80-2360-1968
b Also at: Chemical Biology Unit of the Jawaharlal Nehru Center for
Advanced Scientific Research, IISc Campus, Bangalore 560 012,
India
cent gel.
w Electronic supplementary information (ESI) available: Synthetic
procedures, details of gel preparation, microscopy. See DOI:
10.1039/c2cc30904a
Scheme 2 Free sensitizer generation due to lipase activity.
c
4624 Chem. Commun., 2012, 48, 4624–4626
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Fig. 1 Lipase (0.9 mg mLꢀ1) assay with 2 (33 mM) in a Tb : cholate
gel system (5 mM : 15 mM) at 298 K; (a) evolution of luminescence
spectra with time; (b) increase in intensity (l 544 nm) as a function of
time for native enzyme and comparison with denatured enzyme or
without any enzyme.
Fig. 2 b-Glucosidase (0.7 mg mLꢀ1) assay with 3 (0.37 mM) in a
Tb : cholate gel system (5 mM : 15 mM) at 298 K; (a) evolution of
luminescence spectra with time; (b) increase in intensity (l 544 nm) as a
function of time for native b-glucosidase and comparison with denatured
enzyme or without any enzyme.
considerable importance. The substrates for lipases are generally
hydrophobic in nature, which makes designing of an assay
system based on a fluorogenic probe in aqueous media non-
trivial. Interestingly, bile salts are critical for the activity and
efficiency of most classes of lipases.10 The hydrogel system
enjoys an advantage as the gel network primarily consists of
bile salt aggregates thus providing a near-natural environment
for the enzyme. Lipase from Candida rugosa sp. was used for
all the assays. The enzyme was dissolved in aqueous terbium
acetate, while the pro-sensitizer (2) was dissolved in sodium
cholate stock solution.
a key role in the carbohydrate metabolism, was chosen.11
b-(Mono)glucoside derivative of DHN (3) was synthesized
(see ESIw) to act as a pro-sensitizer (Scheme 3).
The luminescence studies showed similar enhancements in
intensity for the assay with the native enzyme as observed with
lipase (Fig. 2).
In order to unambiguously establish that it is indeed the
generation of free DHN that is responsible for the luminescence
The gel formed upon mild sonication of the mixture, whose
(time-delayed) luminescence was recorded as a function of
time (Fig. 1).
Compound 2 did not appreciably sensitize Tb3+ and only in
the presence of native lipase the luminescence intensity started
to increase. A remarkable enhancement in lanthanide emission
was observed that was clearly visible even with a hand-held
long-wave UV lamp (photo in Scheme 1). A control was done
using the denatured enzyme which did not show any enhanced
luminescence from the lanthanide ion, thereby proving that
the luminescence response was indeed a result of the enzyme
activity.z Higher analogues of DHN diesters (with propionate,
butyrate and hexanoate) were also studied with similar results.
In order to explore the generality of the sensing system with
a completely different type of enzyme, b-glucosidase, which
cleaves water-soluble b-glucosides to release sugars and plays
Fig. 3 Comparison of enzyme activity measured by luminescence
and HPLC studies for (a) lipase (33 mM of 2 and 0.9 mg mLꢀ1 enzyme
were used for both luminescence and HPLC measurements) and (b)
b-glucosidase (0.37 mM of 3 and 0.7 mg mLꢀ1 enzyme were used for
both luminescence and HPLC measurements).
Scheme 3 Free sensitizer generation due to b-glucosidase activity.
c
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enhancement, HPLC analysis for both the gel systems was
carried out to monitor the evolution of DHN with time (Fig. 3).
When the relative peak areas obtained from the HPLC
analysis were plotted against time, the trends matched quite
well with the luminescence enhancement, confirming that both
were indeed the result of the same process.
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In conclusion, we have developed a novel luminogenic
lanthanide based gel probe for inexpensive and rapid detection
of two completely different types of enzymes, lipase and
b-glucosidase. The sensing technique does not involve any
specialized multidentate chelating ligand and instead uses the
gel matrix as the general platform. To the best of our knowl-
edge this is the first report describing a luminescent gel system
used for enzyme sensing. The synthesis of our pro-sensitizers
is straightforward and has a unique advantage of being
adaptable to assay different enzymes. We believe that this
technique would provide an exciting alternate approach
towards the sensing of various enzymes and should enjoy
significant advantages over the other existing sensing techni-
ques because of its simplicity and generality. This strategy
should also allow one to rapidly test enzyme inhibition. We are
currently working on the design of other pro-sensitizers for
clinically important enzymes and fine tuning the assay system
to make it more sensitive.
We thank the Department of Science and Technology,
New Delhi, for support of this work through a J. C. Bose
Fellowship to UM. The Institute Nanoscience Initiative (INI)
is acknowledged for SEM and TEM facilities. We thank
Tumpa Gorai for help with some experiments. The Council
of Scientific and Industrial Research (CSIR) is thanked for the
award of a research fellowship to SB.
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Notes and references
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z (a) Moderate concentrations of enzymes were used for all the studies
and b-glucosidase (6.1 U mgꢀ1). (b) Both lipase and b-glucosidase
were denatured by heating a sealed container of enzyme stock solution
at 85 1C for 10 min.
as the intrinsic activities were less for both enzymes, lipase (2.9 U mgꢀ1
)
c
4626 Chem. Commun., 2012, 48, 4624–4626
This journal is The Royal Society of Chemistry 2012