Fluorescent Detection of Enzymatic Activity
Within a Liposome based Nano-Biosensor
1
Vicky Vamvakaki, Sofia Sotiropoulou, Didier Fournier , Nikos Chaniotakis
Laboratory of Analytical Chemistry, Department of Chemistry, University of Crete, 71 409 Iraklion, Crete, GREECE
[email protected] URL: www.analytical_chemistry.uoc.gr
1
Laboratoire d’Endomologie Appliquée, Université Paul Sabatier, 118 Route de Narbonne, 31062, Toulouse, Cedex 4, FRANCE
Aim of the project
Introduction
Encapsulation of enzymes in the microenvironment of
liposomes has proven to greatly improve enzyme
stabilization. Liposomes can effectively protect enzymes
from the aggression of external agents such as proteases1. In
addition, enzymes entrapped in liposomes are stabilized
against unfolding forces due to hydrophobic interactions
2
between the enzyme and the liposome membrane . Finally,
enzymes encapsulated in liposomes have proven to be fully
active even in very diluted samples in contrast to the free
3
enzyme in solution .
Apart from their stabilizing effect, liposomes have
physical properties that allow the use of optical detection
methods, when signal transduction is required.
The biocompatible microenvironment of the liposomes
along with the ability to control their physicochemical
properties, make them very appealing for a wide range of
applications4. The most widespread application of liposomes
is as carriers of functional substances for controlled release at
certain targets. However these attractive systems can also
find application in bioanalytical systems and especially in the
field of biosensors where enzyme stability is of great
importance and sensitive detection of the analyte is required.
5,6
Initial attempts to develop liposome-based biosensors have
been performed with glucose oxidase on screen printed
7
8
electrodes as well as on chitosan gel beads .
In this study a unique approach for the development of a
novel biosensor system based on liposomes and fluorescent
detection of the analyte is introduced. The inherently
unstable enzyme acetylcholinesterase from Drosophila
melanogaster is stabilized by encapsulation in liposomes.
The substrate partitioning into the liposome is facilitated by
the incorporation of porins within the liposome membrane.
The activity of the enzyme to substrate is monitored using a
pH sensitive fluorescent indicator. The nanobiosensors were
used in the detection of two organophosphorous pesticides,
dichlorvos and paraoxon which are AChE inhibitors.
Immobilization of the enzyme loaded liposome-based
nanobiosensor within an optimized sol-gel matrix is
examined in order to obtain a stand alone biosensor device.
Biosensor Design
Lipids
Enzyme
Fluorescent indicator
Encapsulation of
AChE in liposomes
Encapsulation of the pH sensitive
fluorescent indicator, pyranine
Porin
Insertion of the porin OmpF
in the liposome membrane to
allow substrate entrance
Substrate ATChCl
The enzymatic reaction lowers the pH value which is
correlated to substrate concentration
AChE
Acetylcholine + H2O
choline + acetic acid
Results
Sol Gel Nano-Biosensor
The encapsulation of the enzyme AChE and the fluorescent
indicator pyranine in liposomes was shown to be an efficient
process, producing nanosized biosensors. The response time of the
liposome sensor was fast (t<10min) (Fig. 1), the sensitivity for
substrate concentrations between 1.0 and 13.3 mM is calculated to
be 8.2x10-3 Abs/mM, while the detection limit is found less than 1.0
mM (Fig. 2).
Monitoring of the pesticides dichlorvos and paraoxon was
performed with the liposome based nanobiosensors and the
calibration curves are shown in Fig. 3 and 4 respectively. The
detection limit of the biosensor (calculated for 20% inhibition) for
dichlorvos was as low as 1.4x10-10 M. Under the same conditions,
the detection limit for paraoxon, was found 1.0x10-10 M.
The developed nanobiosensors were immobilized in a sol gel
matrix optimized for the specific case in order to obtain a stand
alone biosensor device that can be used continuously and for
successive measurements. The sensitivity of the resulting
biosensor to substrate was 7.5x10-3 Abs/mM (Fig. 5). Both
biosensor systems have similar sensitivity and response time, and
apparent Km values close to 5.5mM. This indicates that the
liposome nanobiosensors retain all their functionality and
enzymatic activity within the sol gel matrix.
6,1
6,0
I%
5,9
5,8
buffer
2.5 mM
5.0 mM
10.0 mM
13.3 mM
5,7
5,6
0
5
10
15
20
25
30
90
80
70
60
50
40
30
20
10
0
t (min)
6,00
90
5,98
80
5,96
70
5,94
60
5,92
8
9
10
- log [ dichlorvos ]
11
12
50
20
5,86
10
2
4
6
8
10
12
14
[ A T C h C l ] mM
Fig. 2 Calibration curve of the AChE/Liposome NanoBiosensor obtained at 10 minutes reaction time.
Experimental Setup
Encapsulation of acetylcholinesterase and pyranine in egg
phosphatidylcholine liposomes was performed following the lipid film's hydration
9
technique . Liposome diameter was estimated by dynamic light scattering to be
300(+/-4)nm. The non encapsulated enzyme was removed by adding 5mg/mL
pronase and incubating for 3h at room temperature. In all cases, AChE activity
10
was measured using the sensitive Ellman’s method (enzyme loading for each
measurement was 0.02pmol).
The fluorescence indicator pyranine is highly water soluble and has excitation
and emission wavelengths at 460nm and 513nm respectively.
Pesticides measrements were performed by incubating the nanobiosensors for
15min in each pesticide and monitoring the decrease of enzymatic activity.
The AChE/liposome biosensor with pyranine was mixed with a silica sol
(TMOS/water:5/1 in HCl) at a ratio of 2:1 and poured into cuvettes resulting in a
transparent sol gel film.
5,90
5,85
5,80
5,75
5,70
5,65
0
5
10
15
20
25
30
35
[ A T C h C l ] mM
Fig. 5 Calibration curve of the Sol Gel Nano-Biosensor
Conclusions
30
5,88
5,95
obtained at 10 minutes reaction time.
40
5,90
5,84
7
Fig. 3 Calibration curve of the AChE/Liposome NanoBiosensor with dichlorvos obtained at 30min reaction
time.
I%
Relative fluorescence
F i g . 1 R e l a t i v e F l u o re s c e n c e s i g n a l o f t h e
AChE/Liposome Nano-Biosensor with time, for different
ATChCl concentrations.
6
Relative fluorescence
Pesticide Measurements
Relative fluorescence
AChE/Liposome Nano-Biosensor
6
7
8
9
10
11
12
- log [ paraoxon ]
Fig. 4 Calibration curve of the AChE/Liposome NanoBiosensor with paraoxon obtained at 30min reaction time.
References
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A novel liposome-based nanobiosensor has been developed
using porin loaded liposomes containing the enzyme AChE and
the pH fluorescent indicator pyranine11.
Based on this
nanobiosensor system the monitoring of two organophosphorus
pesticides dichlorvos and paraoxon at nanomolar levels has been
achieved. The incorporation of the nanobiosensor into a sol gel
matrix provide an optically active stand alone biosensor with good
overall analytical characteristics, which is a very promissing
technology for the development of a novel class of detection
systems.
Acknowledgments
This work is being supported by the program “IRAKLITOS”
of the Greek Ministry of Education and the European Commission
Program “GANANO” (Contract No STREP505641-1).