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Towards a Light Adressable Transducer Bacteriorhodopsin-based

by

Claudio Nicolini1,2,3,Victor Erokhin1,
Sergio Paddeu1 and Marco Sartore2

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 the
Fifth Foresight Conference on Molecular Nanotechnology.
The final version has been submitted
for publication in the special Conference issue of
Nanotechnology.

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Abstract

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.

Introduction

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 [4]. 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, 9-14].

In addition, thin film technologies [15] 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 were used for the film deposition.

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).

Film formation

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 [29]. 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 formation.


Photoresponse measurements

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 experimental configuration.

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 (Figure 4).

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.

  light on current [pA] light off current [pA]
usual LB technique 15 10
electric field assisted monolayer formation 820 10

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 preferential orientation.

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 line).
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.

Device principle

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 [32] of the solution in the chamber, which points out LAPS as the best candidate for comparison with the suggested BR-based nanodevice.

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 [31]. 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 thickness.

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 light sources.
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.

Table 2. Typical parameters and features of the silicon-based LAPS transducer and suggested device.

  LAPS Suggested device
output signal current (A) current (nA)
illumination light source 940 nm 520 nm
spatial resolution according to the wafer thickness
(0.2 mm in existing devices)
2 nm in principle but related to
the minimal optical fiber diameter available
typical dimensions 8mm*8mm; 16mm*16mm no limits
flexibility rigid very flexible
reliability months/years month/years


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.

Conclusions

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 identified.

References

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