Tutte e tre le prove risultano compatibili con i dati di letteratura.

BIOSENSORS FOR CELL SIGNALLING

J.M. Cooper

Over the last 10 years, research and development in bioelectronics has led to new techniques for using materials normally associated with the electronics industry in order to study biological phenomena. Perhaps the most striking example has been the development of biosensors. In order to fabricate these small diagnostic devices, it is necessary to modify a transducer surface (e.g. an electrode or a waveguide) with a biological molecule (e.g. an enzyme or an antibody) such that any degree of molecular recognition between the biological molecule and a sensed species will be communicated as a signal.

An example is shown in this figure which is a device made of 200 nm gold ring electrodes fabricated on a silicon substrate, indicating the potential use of nanotechnology for bioelectronics (see for example Cell Signalling). In the test pattern shown, the central gold circle acts as a model cell and can be used to electrochemically generate radicals.

Several projects are being pursued, including enzyme immobilisation in conducting polymers on ultra-microelectrodes, surface plasmon resonance sensing, microelectronic protein patterning, surface biocompatibility treatments, and the development of integrated electrochemical and optical immunosensors. Nanotechnology is particularly suited to this area since the dimensions of fabricated structures and of biological molecules are of the same order of magnitude; arrays of nanoelectrodes have been developed in the group and have been used to examine the molecular basis for electron transport processes between redox proteins and metal surfaces .

One example is the study of Electron transport processes between proteins and electrodes which has provided an elegant model for the modification of electrodes in order to investigate bio-electrochemical reactions at electrode surfaces. The process is therefore of fundamental interest in the design of a new generation of biosensors with faster response times.

The molecular organisation between enzymes and their substrates requires an understanding of their interactions in terms of charge-matching, spatial fitting and hydrogen bonding, and accounts for a mechanism by which electron transport between proteins can be observed at modified metals. In particular, self assembling charged monolayers at gold surfaces have been used at Glasgow to provide a template for proteins. Once "docked" in the correct molecular orientation, the molecules can be immobilised and studied using electroanalytical techniques to measure a variety of energetic and kinetic characteristics.

Research in this area has also investigated the use of conducting polymers which can be electrochemically deposited from solution onto electrodes. These polymers have diode-like characteristics in that they are conducting in their oxidised state and insulating in their reduced state. It has been found that biological molecules can be incorporated into these conducting polymers during the polymerisation process to provide a possible mechanism for localising proteins at a microelectrode. If the correct polymers are used, there is the possibility of creating a bioelectronic interface by providing molecular wires between the electrode and the biomolecular material.

Novel polymers are being synthesised within the Department of Chemistry, based upon polypyrrole and polythiophene which contain analogues of naturally occurring amino acids. These materials are capable of mimicking the enzyme's natural biological environment, and will ultimately lead to the development of 'solid state' bioelectronic interfaces. Faster biosensors will result, ultimately leading to the fabrication of a new generation of molecular sensors.

The combination of integrated silicon technology, semi-conductor fabrication processes and bio-electrochemistry has made it possible to develop highly specific, sensitive, and accurate micro-biosensors. Such sensors consist of biological material immobilised at a metal electrode and are capable of converting a biological signal into a quantitative response. Research is now in progress in the development of sub-µm sized electrodes that can be positioned in close proximity to individual cells in order to measure their responses to drugs and hormones.

A fundamental programme of research in Glasgow aims to investigate the role of the superoxide in cell biology. Superoxide has previously been shown to participate in a wide range of pathological processes such as arthritis and heart disease. It has also been demonstrated that cells produce this chemical naturally and that it has a benign role, possibly as a messenger. Until recently, it has not been possible to measure superoxide directly, and its biological effect has only been inferred by measuring (presumed) by-products of its activity. Research in collaboration with the University of Newcastle upon Tyne has resulted in the development of a specific biosensor which can attribute superoxide production to particular cellular systems.

Arrays of these sensors (see picture) (<1 µm) are being fabricated using electron beam lithography in the Department's Ultra-Small Structures Laboratories, and are being modified with biological material in order to measure highly specific signals that are produced by cells as part of the human antibody defence mechanism. By collaborating with a hospital clinical laboratory, the role of the immune system in rheumatoid arthritis is being addressed. The research is being carried out in parallel with projects involving dielectrophoresis (see later), in the hope that it will be possible to move individual cells within electric fields into an array of microbiosensors. This project is funded by the Medical Engineering and Sensors Committee of EPSRC, and illustrates the impact that biosensors have had on the research techniques available to the biomedical sciences.

A single latex bead with a diameter of 6 µm trapped using dielectrophoresis within an array of ring electrodes. The bead is a good physical model for a cell for these preliminary experiments.

There are two complementary collaborations on cell signalling. Work with the Wellcome Institute of Surgery, University of Glasgow, is underway to measure glutamate within the cortex of the brain during traumatic injury and involves problems of biocompatibility when implanting a sensor during neurosurgery. It is hoped to use information obtained to test the efficacy of new drugs to limit brain damage in post traumatic injury. In addition, a joint programme with the Department of Cell Biology, University of Liverpool, and funded by the Wellcome Foundation, involves measuring markers for heart disease in cultured heart cells to monitor the affect of new drugs.

PACS. 05.40.+j - Fluctuation phenomena, random processes and Brownian motion.
PACS. 82.70.-y - Disperse systems.
PACS. 87.80.+s - Biophysical instrumentation and techniques.

In recent years, electrorotation has developed in the field of biology as a technique for characterization of single cell dielectric properties. Applications to colloidal particles are scarce, although the method provides information on the electrical structure of the particle's interior. The method explores the frequency-dependent polarizability difference of the particles and the suspension medium by using rotating fields in the frequency range of 1 kHz to 200 MHz to induce individual particle rotation. To allow interpretation of the electrorotation spectra measured with light scattering techniques, the theoretical autocorrelation function of light scattered from rotating particles of cylindrical symmetry has been calculated. All particles were assumed to possess a single scattering site and the rotation axes were assumed to be parallel. An appropriate expression for the rotational diffusion around the longitudinal angle was also derived. Diffusion around the azimuthal angle could be neglected. The theoretical result consists of a well structured autocorrelation function. The theoretical limits of particle size and rotation speed that allow detection of electrorotation were explored. Experimental autocorrelation functions from electrorotation of human red blood cells could be explained theoretically when reasonable parameters for the cells' properties were assumed.

When a particle is placed in a non-uniform rotating a.c. electric field a dipole moment is induced and a torque applied, causing the particle to spin. The induced torque is dependant upon the applied voltage/frequency and the relative dielectric properties of the particle and its suspending medium. This phenomenon is known as ELECTROROTATION.

Electrorotation is exploited in combination with antibody technology to give the ELECTROROTATION ASSAY (ERA). In this a known particle is complexed with an analyte causing a change in the dielectric properties of the constituents. This dielectric shift can be detected as a change in the rotational characteristics in an ERA measurement cell. The selectivity of the assay can be controlled by the choice of binding agents used on the known particle. In short,
this assay enables the rapid and accurate detection of analytes in aqueous solution and is sensitive enough to detect analytes ranging from whole cells to molecular species.

The electrorotation assay is a technique which uses the phenomenon of electrorotation in combination with antibody technology to enable the rapid and accurate detection of analytes in aqueous solutions [1]. In the assay an analyte is bound to a known particle causing a change in the dielectric properties of the analyte-bead complex over the properties of just the bead. This change in the dielectric properties is subsequently detected using the electrorotation technique described above. Selectivity can be controlled by the choice of binding agents used on the known particle. This assay method can be used with different analytes ranging from whole cells to molecular species.

The graph above shows the electrorotational spectra of polystyrene beads after 3 different treatments. The first trace shows the response of an uncoated bead and can be described by three distinct regions. At low frequencies a slow rotation rate is observed which decreases with increasing electric field frequency. A region where the beads are almost stationary is then displayed from around 2kHz to 20kHz before the rotation rate increases to a large peak at approximately 600kHz. The shape of this spectrum is typical for most polystyrene particles and is used as a control for detecting changes in particle rotation as the Electrorotation Assay is performed. The remaining traces in figure 4 show the change in the rotation profile of beads as a result of applying a coating to the surface of the bead. In these cases the beads have been treated with different antibody/protein complexes. The effect of the coatings is to alter the dielectric properties of the bead-coating complex over the properties of the beads alone and these, in turn, are reflected in the rotation spectra of the treated beads. The beads after coating now exhibit rotational responses which have increased rotation rates at low frequencies and a change in the magnitude and frequency of the high frequency peak in rotation. By comparing the electrorotational spectra of complexed beads with uncoated beads it is possible to determine the presence of particular proteins within a mixed sample.

This work has been carried out in collaboration with Genera Technologies Ltd. Cambridge, UK

Cell-Virus Interaction

Rotating AC electric fields (electrorotation) are used to characterise the dielectric properties of individual cells and to monitor physiological changes in the properties of cells.

The speed of rotation of a cell varies with the applied frequency of the AC field and is in the region of a few revolutions per second. By modelling a cell as a series of concentric spheres (or shells) it is possible to obtain the dielectric parameters of the cell from the variation of the rotation rate with applied frequency.

Electrorotation is used to study the dielectric properties of individual Baby Hamster Kidney Fibroblasts (BHK(C-13)). These cells are being studied to probe the electrophysiology of viral infection using the Herpes Simplex Virus, type 1.

The variation in the electrical parameters of a cell has been compared with the known biophysical life-cycle of the virus, allowing for the first time a real time study of viral infection of a single cell.

Shown below is the rotation spectrum for a typical HSV infected fibroblast as a function of applied frequency.

A number of infected and non-infected cells have been studied and their average dielectric properties found to be different. This implies that it is theoretically possible to separate infected and non-infected BHK cells.

Based upon these differences, techniques for the separation of infected and non-infected cells BHK(C-13) cells are being developed.

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Ringraziamo il Prof. Bonincontro per la sua disponibilità , il tecnico Rocco del laboratorio di elettronica (senza il quale non avremmo potuto realizzare la nostra terza prova), il laureando Sergio Ferrari per i preziosi suggerimenti e naturalmente la Prof.ssa Congiu che ci ha consentito di portare avanti l'evoluzione dell'esperimento di E.R. con generatore esterno.

Speriamo che quanto finora scritto possa essere d'aiuto in futuro a chi sarà interessato a sviluppare l'argomento elettrorotazione e il relativo apparato di misura.