Towards a Light Adressable Transducer Bacteriorhodopsin-based
1 El.B.A. Foundation, Via Giotto 2, Genova 16153 Italy
2 Polo Nazionale Bioelettronica - Parco Scientifico e Tecnologico
dell'Elba, Via Roma 28, 57030 Marciana (LI), Italy
3 Institute of Biophysics, University of Genova, Via Giotto 2, Genova 16153 Italy
This is a draft paper for a talk at theFifth Foresight Conference on Molecular Nanotechnology.
The final version has been submitted for publication in the special Conference issue of Nanotechnology.
This paper is available on the web at http://www.foresight.org/Conferences/MNT05/Papers/Nicolini1/index.html
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Highly oriented bacteriorhodopsin films were deposited by means of a specially
designed procedure of electric field assisted monolayer engineering. The
self-assembly of the monolayer at the air/water interface was controlled by the
increase of the surface pressure. The monolayers were transferred onto solid
substrates by Langmuir-Schaefer technique. Electrical measurements on the films
with a specially designed chamber confirmed the improved orientation of the
films. Possible application of the films in practical devices such as biosensor
transducer is discussed, keeping in mind features and inherent limitations when
compared with existing silicon transducers.
During last years the understanding of structure and function of several
biological systems has grown rapidly. Among them, the study of
Bacteriorhodopsin (BR) protein and the elucidation of its function as a light
driven proton pump represents one of the most interesting examples [1-3]. BR is
a light-transducing protein in the Purple Membrane (PM) of Halobacterium
Halobium. Its features allow one to identify and design several potential
bioelectronic applications aimed to interface, integrate or substitute the
silicon based microelectronics systems, as well as to develop molecular devices
. BR is a notable exception in respect with the usual biological molecules,
being mechanically robust, chemically and functionally stable in extreme
conditions, like high temperatures [5-7], which usually represents one of the
key parameters of working conditions. Furthermore, it possesses remarkable
photonic and photovoltaic properties which have been exploited for molecular
device constructions. For these reasons BR has been adopted as a building block
for a number of experimental prototypes, such as filters, photocells,
artificial photoreceptors, optical memories, image sensors and biosensors [3,
In addition, thin film technologies  allow the assembly of biological
materials in a 2D system, usually required for a device development. Among
these technologies, the Langmuir-Blodgett (LB) one [15-17] seems to be one of
the most promising, due to its ability to form molecular systems having an high
packing degree and a molecular order. Moreover, it has been possible to assess
that such a technique allows the fabrication of 2D protein closely packed
structures, showing an enhancement of some chemical-physical properties or an
induction of new properties commonly known for proteins in solution or even in
membranes [6, 18-20]. These properties include the long-term stability to
thermal and functional (photochemical) degradation.
Therefore, investigators have shown considerable interest in the adoption of
the Langmuir-Blodgett technique, or its modifications, to make molecular
electronic devices using, in particular, as an active component, a
light-transducing protein like BR. In fact, the ability of BR to form thin
films with excellent optical properties, and the intrinsic properties itself,
make it an outstanding candidate for use in optically coupled devices.
BR thin layers have been widely studied [21-25] as they perform bistability in
the optical absorbance and provide light-induced electron transport of protons
through the membrane. Furthermore, their extremely high thermal and temporal
stability allow to consider them also as sensitive elements for electrooptical
devices [26-25]. However, in order to use BR properties to provide photovoltage
and photocurrent, it is necessary to orient all the molecules in such a way
that all the proton pathways are oriented in the same direction. LB technique
in its usual version does not allow to realize it. When BR containing membrane
fragments are spread at the air/water interface, they orient themselves rather
randomly in such a way that proton pathway vector is oriented in opposite
directions in different fragments. Nevertheless, it is known a technique of
electrochemical sedimentation, which allows to deposit highly oriented BR
layers. However, the layers, deposited with this technique are rather thick and
not well controllable in thickness.
The aim of this work is then to modify the LB technique in order to obtain
highly oriented BR layers and to provide a suitable solution for the
development of a BR-based nanotransducer.
Materials and Methods
BR used in the work was from Sigma. Films were deposited on Langmuir trough
(MDT, Moscow, Russia). Water used in the work was purified by Milli-Q system
till the resistivity 18.2 M[Omega] cm. NaCl and KCl were also from Sigma.
Millipore filters with the hole diameter of 0.10 µ were used for the film
Film structure was studied with small-angle X-ray diffractometer with position
sensitive detector (AMUR-K) at the Elba Foundation laboratory in Genova and on
small-angle X-ray station at the Elettra Synchrotron in Trieste (Italy).
Langmuir-Blodgett (LB) technique allows to form a monolayer at the water
surface and to transfer it to the surface of supports. Formation of the BR
monolayer at the air/water interface, however, is not a trivial task, as it
exists in the form of membrane fragments. These fragments are rather
hydrophilic and can easily penetrate the subphase volume. In order to decrease
the solubility, the subphase usually contains a concentrated salt solution. It
was already shown the efficiency of the film deposition by this approach .
Nevertheless, it does not allow to orient the membrane fragments. As the
hydrophilic properties of the membrane sides are practically the same,
fragments are randomly oriented in opposite ways at the air/water interface.
Such film cannot thereby be useful to this work, as the proton pumping in the
transferred film will be compensated. On the other hand the technique of
electrochemical sedimentation is known to form rather thick BR films by
orienting them in the electric field.
Therefore, the following method was suggested and realized in this work (scheme
is shown in the Figure 1). 1.5 M solution of KCl or NaCl (the effect of
preventing BR solubility of these salts is practically the same) was used as a
subphase. Platinum electrode was placed in the subphase. Flat metal electrode,
with an area of about 70% of the open barrier through area, was placed at about
1.5 - 2 mm upper the subphase surface. Negative potential of 50-60 V was
applied to this electrode with respect to the platinum one. BR solution was
injected with syringe into the water subphase in dark conditions. The system
was left in the same conditions for electric field induced self-assembling of
the membrane fragments during 1 hour. After this, the monolayer was compressed
till 25 mN/m surface pressure and transferred onto the substrate (porous
membrane). The residual salt was washed with water. The water was removed with
the nitrogen jet.
Figure 1. Scheme of the electric field assisted BR monolayer
Photoresponse was studied with a home made working chamber. The filter with
deposited BR film was placed between two aqueous solutions (buffered with the
addition of KCl). It was fixed with a rubber ring to prevent the leakage of the
solutions from one section to the other. Photocurrent was measured between
these sections with 6517 electrometer (Keithley). Illumination was carried out
with a usual tungsten lamp through a fibre optic lightguide. The chamber was
placed into the metal box to be protected from electrical noise and light.
Light illumination resulted in the sharp increase of the current through the
membrane up to 1000 pA, while without illumination the noise current was of
about 10 pA. It is worth to mention that over up to twenty experiments carried
out over one year period with the same original BR preparation properly stored,
only about a half of the prepered samples allowed to register the above
mentioned photocurrent. Several experimental factors appear indeed to
negatively influence the resulting photocurrent, namely age of the BR, film
defects due to the porous nature of the supporting substrate and the
Results and Discussion
The dependence of the surface pressure upon the time with and without applied
electric field is shown in Figure 2. It is clearly visible, that electric field
improve strongly the ability of the membrane fragments to form a monolayer at
the water surface.
Figure 2. Dependence of the surface pressure of BR monolayer upon the time
in presence and absence of the electric field.
X-ray measurements of the deposited multilayers revealed practically the same
structure in films prepared with usual LB technique and electric field assisted
monolayer formation. Indeed, electric field only aligns the fragments at the
air/water interface, providing equal orientation of the proton pathways.
Layered structure in this case remains the same. X-ray curves (Fig. 3) from
both types of samples revealed Bragg reflection corresponding to the spacing of
46 , what is in a good correspondence with the membrane thickness.
Figure 3. X-ray pattern of films prepared with (dotted line) and
without (solid line) electric field assisted self assembling.
Furthermore, as previously shown (13, 18, 20, 21), with the LB technique the
heat stability of the BR mutilayer at 25 mN/m surface prssure apperas
significantly improved with respect to both the solution and the self-assembly
Table 1 Mean value of phototocurent observed in the system using porous
membranes covered with BR film deposited by ussual LB technique and electric
field assisted. A standard error of about 10 percent is observed over five
independent positive measurements.
on current [pA]
off current [pA]
field assisted monolayer formation
In order to control the degree of BR orientation photo induced current was
measured in the described measuring chamber. 1 monolayer of BR was deposited
onto the porous membrane. The results are summarized in the Table 1. It is
clear, that the photoresponce in the
case of electric field assisted monolayer formation is much higher with respect
to that after a normal LB deposition (in the last case the signal value is
comparable with the noise, indicating a mutually compensating orientation of
the membrane fragments in the film). The observed results allow to conclude,
that the suggested method of electrically assisted monolayer formation is
suitable for the formation of BR LB films, where the membrane fragments have
Figure 4 Molar ellipticity versus temperature for BR multilayers
prepered by LB technique (solid line) and by self-assembly (dotted line). The
corresponding value for BR in solution is also given (dotted-solid
As the electric field assisted monolayer formation at the air/water interface
turned out to provide highly oriented BR LB film formation, it appears possible
to suggest one new application of BR films as transducer. The principles of the
nanodevice realization are described below.
The scheme of the proposed device is presented in Figure 5. Porous membrane
with deposited BR film is separating two chambers with electrolytes. Light
fibre is attached to the X-Y mover, which allows to illuminate desirable parts
of the membrane. Illumination of the membrane part will result in the proton
pumping through it, carried out by BR. Therefore, a current between the
electrodes will appear. This current must depend upon several factors, such as
light intensity, pH of the electrolytes and gradient of the pH on the membrane.
One of the possible applications of the suggested device is mapping of 2D pH
distribution in the measuring chamber, which can result from the working of
enzymes, immobilized in this chamber. By scanning the light over the membrane
it will be possible to obtain the current proportional to the pH gradient in
the illuminated point and, maintaining the pH value fixed in the reference
chamber, it will be possible to calculate absolute pH values in different
points over the whole membrane surface. If different types of enzymes,
producing or consuming protons during their functioning, will be distributed
over the area closed to the membrane, the device will allow to determine the
presence of different substrates in the measured volume, performing, therefore,
a multienzymatic biosensing.
Figure 5. Schematic view of the measuring chamber used for the
experiment with the BR membrane.
Space resolution of the transducer, in principle, is extremely high. As each
BR molecule performs proton pumping, it will be comparable with the protein
size (about 2 nm). In practice, however, it will be limited by the technical
difficulty in focusing the light beam at such high resolution, but, in any
case, it will be more than in any existing transducer.
Comparison with the existing devices
Several silicon-based biosensors have been developed for various applications,
such as cell metabolism or immunoenzymatic activity determination. Due to its
performances and its structural simplicity one of most attractive transducers
based on a silicon heterostructure is the Light Addressable Potentiometric
Sensor (LAPS) [30,31]. It consist essentially of a silicon substrate coated, on
the front side, with an insulating layer. This layer is in direct contact with
the solution to be analyzed. The sensitive layer of the device consists of
Si3N4. A light source illuminates the rear or the front side of the transducer,
while an electronic circuit (namely a potentiostat) bias the structure. In its
main configuration, LAPS acts as a pH meter, able to detect a pH variation in a
microenvironment. In appropriate conditions a photocurrent flow through the
system and a pH variation is registered as a phocurrent displacement. Moreover,
it is very attractive the possibility to address different sensitive zones on
the chip, i.e. to perform a pH mapping  of the solution in the chamber,
which points out LAPS as the best candidate for comparison with the suggested
For a comparison purpose, the addressability of LAPS should be considered,
since it represents one of the most attractive features of both systems. LAPS
are usually addressed by means of light coming either directly from a light
emitting diode or through an optical fiber. The light impinging the silicon
substrate causes the generation of hole-elctron pairs, which diffuse in the
silicon bulk. Those pairs which reach the electric field present under the
oxide region are separated, and the minority carriers give rise to a
photocurrent, which is indeed the measuring signal. Since the carriers
diffusion is three dimensional, multiple light spots can interfere each
other ; in other words the hole-electron pairs generated by a spot can
diffuse into the region downstanding the adjacent spot, being then separated by
the electric field of that region . This fact, macroscopically, causes
interference between the photocurrents relative to different light excitation
sites. The spatial resolution of LAPS devices depends primarily on the minority
carriers diffusion lenght, defined as :
where D is the diffusion coefficient, and [tau] is the minority carriers
lifetime. The lifetime depends strongly on the silicon substrate [31, 32], and
is the major responsible of the above depicted effect. In practical cases, the
distance between light sources is related to the thickness of the silicon
substrate used. In fact the diffusion in the bulk is isotropic and we should
guarantee that hole electron pairs reach the space-charge region under the
oxide, that is diffuse, more or less, for a path equal to the silicon
Therefore the distance between two adjacent light spots should be larger that
two times the silicon thickness (referring to Figure 6 it must be W > 2
Hsi). One could think to build LAPS devices based on very thin silicon
substrates, but the counter effect is an increased fragility. In practice, one
can reach a limit spot distance of about 0.2 mm.
Figure 6. Schematic drawing of a LAPS devices addressed by multiple
Even if it is not easy to compare the suggested principle with already existing
devices, some of the characteristics for both of them are summarized in the
Table 2. Typical parameters and features of the silicon-based LAPS
transducer and suggested device.
|illumination light source
to the wafer thickness (0.2 mm in existing devices)
nm in principle but related to the minimal optical fiber diameter available
As it is obvious from the table, the suggested device is comparable and for
some aspects even better with respect to LAPS. These facts allow to conclude,
that the device could probably found applications in the biosensor field as
long as the existing problems could be overcomed. At this stage, however, many
difficulties still remain in the development of this type of devices with a
reproducibility equal to that of silicon-based ones. In addition, basic
problems such as the current detector, the membrane placement in real cells,
the correct addressability system and even the practical experimental setups
for biosensors should be solved.
Electric field assisted monolayer engineering at the air/water interface in a
Langmuir through as here introduced was proven to yield reproducable results.
Surface pressure measurements revealed indeed that membrane fragments in such a
film are quite more oriented with respect to usual LB technique deposition. At
the same time significant photoresponse appears present even if still rather
ectic and variabile.
With all due caution the attempt to construct a new usefull nanodevice based
on such elctric-field oriented LB film of bacteriorhodopsin appears thereby a
worthy undertaking, despite the numerous limitations and problems so far
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