G. PANAYOTOU. M.D. WATERFIELD AND P. END MACROMOLECULAR INTERACTIONS

Recent advances in optical biosensors allow biologists to monitor x

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Many problems in modern biology require the study of the formation of a complex between two or more macromolecules. The binding of an antibody to an antigen, a growth factor or hormone to its receptor, an enzyme to its substrate or a transcription factor to DNA are just a few examples from the variety of complex- forming processes that biologists study. The analysis of these interactions involves a number of different tech- niques, many of which are used in attempts to quantify binding affinities. As all the interactions are dynamic processes, the ability to measure precisely their kinetic parameters and determine how fast one molecule binds to and dissociates from another is very important for understanding the regulation of the biological processes involved.

Some recent advanlces in the development of biosen- sors are beginning to find widespread use in this field. Although a considerable amount of effort has been expended in the construction of different types of biosensor fll, it is the optical biosensors that show the

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most promise of becoming bench-top companions to researchers studying macromolecular interactions. We shall describe the basic principles of operation of these biosensors, using as examples two commercially avail- able instruments, one (Pharmacia BIAcore) based on surface plasmon resonance and the other (Artificial Sensing Instruments BIOS-l) using integrated optics incorporating waveguiding films.

Both types of biosensor rely on the immobilization of one of the interacting components on a sensor surface (either through direct coupling or through an adaptor molecule), while the second component is injected at a constant flow rate over that surface with the help of a ~uid-handling system that can be automated. Both instruments record the change in refractive index of the medium very close to this surface, which is directly pro- portional to the amount and molecular weight of the macromolecules bound at any time. Using this method- ology, it is not necessary either to label one of the interacting molecules or to separate the complexes

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Fig. I. ComparisoG’&he basic principles of operation of the two optical biosensors used to measure macromolecular interactions: BfAcore, based on surface plasmon resonance, and the integrated optics instrument BIOS-l, based on waveguiding.

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from free componems. The interaction can be observed as it takes place in. real-time by plotting the signal against time. The trick, of course, is to measure the refractive index changes accurately, and that has been the focus of intense research in developing the optical biosensors.

Both instruments employ the principle of total internal reflection. When monochromatic light travelling within a medium reaches the interface with another medium of lower refractive index, it is totally reflected, provided the angle of light i,s kept above a critical value. At a certain incident angle above the critical angle, a small electromagnetic field component of the light, called the evanescent wave, penetrates into the side of lower refractive index. The value of this incident angle is dependent on the ratio of the two refractive indices. As its name suggests, the evanescent wave decays expo- nentially with the ‘distance from the interface and its penetration is of the order of one wavelength. It is this electromagnetic field component that actually ‘probes’ the surface on which the interaction takes place and can provide the necessary information about the refrac- tive index changes. The way that this is achieved differs between the two types of optical biosensor (Fig. 1).

In the BIAcore instrument, monochromatic, plane- polarized light is emitted from a laser source as a wedge-shaped beam, thereby providing a fixed range of incident angles, all above the threshold for total internal reflection. The beam is aimed, through a prism, at a disposable sensor chip, which consists of a glass support, a thin (5Onm) layer of gold and a carboxylated dextran matrix onto which the capturing molecule is immobilized by any of several distinct immobilization chemistries [2]. When the light hits the interface between the gold layer and the matrix, the evanescent wave interacts with the cloud of electrons on the gold surface and resonance occurs, resulting in a quantum mechanical wave called a surface plasmon. This surface plasmon wave, therefore, takes up part of the energy of the incident light, and a ‘dip’ in the intensity of the reflected light will be observed for a particular incident angle. By placing a position-sensitive diode array on the reflected wedge of light, the angle at which surface plasmon resonance occurs can be determined t31. As the refractive index of the medium close to the sensor surface changes, so does the angle at which surface plasmon resonance occurs, and the resulting dip in light intensity is detected. Measurements are performed continuously and the angle differences are expressed in arbitrary resonance units, so that a real-time plot (or sensorgram) of the sensor response in resonance units versus time can be obtained.

As the BIAcare uses a fixed range of angles of inci- dence and a fi$ed detector, there is no need for moving components. A& alternative approach is taken by the BIOS-1 instrum$ht, where the range of incident light angles is provided by rotation of the sensor chip against a fixed-angle beam of light. This scanning procedure takes up to three seconds, but allows the measurement

of a larger range than is possible with the BIAcore. The major difference between the BIOS-l and the BIAcore is that the disposable sensor surface of the former does not contain a gold layer but is an inte- grated optical unit, comprising a wavegtiiding film of high refractive index (SiOz/TiO, or Ta,Os) on a glass or plastic support. Instead of a prism, the BIOS-I uses a sensor chip with an integrated sub-micron diffraction grating, which allows the diffracted light to propagate in the waveguide (Fig. 1).

The waveguiding film can trap light under conditions of total internal reflection. The incidence angle at which this can happen again depends on the refractive index of the surface layer in contact with the film. When light couples into the waveguide, it can be detected at the end of the sensor chip (see Fig. 1) [4l. Capturing mole- cules can be immobilized either by direct attachment to the waveguiding film (in the same way molecules are attached to an ELISA plate) or by covalent coupling to an adhesion layer (which can be done using a variety of chemistries). As interactions take place at the sensor surface, the refractive index changes and so does the angle at which the incoming light couples into the waveguide, again providing a real-time plot of the response.

The detection limit of optical biosensors depends on the sensitivity of the transducer element (be it a metal layer or waveguide). Integrated optics biosensors appear to be more sensitive than the surface plasmon resonance biosensors, as a sharp resonance signal can be obtained with the high refractive index waveguiding films [4]. On the other hand, the use of a very hydrophilic dextran layer on surface plasmon reso- nance sensor surfaces results in negligible non-specific binding, and the capturing molecules are well exposed to the incoming sample for analysis. A further differ- ence between the two instruments is the size of the flow cell. The small flow cell of the BIAcore (60nl) reduces the problem of diffusion-limited reaction rates to a minimum, and therefore improves the quality of the data, especially when kinetic parameters are evalu- ated. The BIOS-l, on the other hand, provides a range of larger flow cell sizes (10-lOOpl), which allows the application of larger volumes and samples such as viruses and even whole cells.

It is obvious that this technology can have wide-ranging applications in biological research (for an overview, see [51), and indeed more and more papers are appearing in international journals reporting data obtained in this way. A good example of a biological system in which biosensors have been particularly useful is signal trans- duction in response to growth factors. Starting from the cell surface, where a growth factor can interact with specific receptors 161, and moving into the cell, where a large number of signalling molecules containing SH2 domains form complexes with activated receptors, growth regulation involves a perplexing combination of protein-protein interactions. The specificity, relative

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Fig. 2. An example of thle kind of data obtained with the BlAcore instrument. Three superimposed sensorgrams are shown. A purified SH2 domain was injecteNd over an immobilized, tyrosine-phospho- rylated peptide, or a control, non-phosphorylated peptide. Dissociation of the SHZ--peptide complex was compared in buffer flow and upon injection of an excess of competing peptide. The arrows point to the beginning and the end of the protein injection.

affinities and speed of exchange of different interacting molecules are very important determinants of the sequence of events that transmit a signal from the outside of a cell to the nucleus (where, of course, protein-DNA interactions are prominent and require a similar analysis [71).

Figure 2 shows a representative example of the kind of data that can be obtained with a typical, high-affinity interaction. Injection of a recombinant SH2 domain over an immobilized, specific binding molecule (in this case, a tyrosine-phosphorylated peptide from the PDGF P-receptor) results iin a fast association phase, from which association rate constants can be determined. The subsequent dissociation of the bound domain from the surface can then be observed either in buffer flow or in the presence of a competing peptide in solution. It is clear from this figure that the ‘true’ dissociation rate is much faster than the apparent rate observed in buffer flow (where rebinding to the surface occurs), suggest- ing that this type of interaction is characterized by fast exchange [81. This may be relevant to the in vivo regu- lation of the signall.ing molecules, which transiently associate with and become activated by receptors. Bacterial chemotaxis is another interesting system where biosensor analysis has been used recently to study the dynamics of thk assembly of a quaternary signal transduction system. A previously uncharacter- ized interaction between a protein kinase and its sub- strate, which is a response regulator, was identified in

this way, and the dissociation of the complex was fol- lowed under conditions that allowed phosphorylation to occur on the sensor surface [91.

These exciting new advances show that the field of biosensor-based biological research is rapidly coming out of its infancy period, allowing data to be generated that would be difficult to obtain by other methods. Further improvements in the sensitivity of the method, immobilization chemistries and data analysis tech- niques will certainly increase the number of potential applications. Acknowledgements: We would like to thank K Tiefenthaler, P Marbach and MJ Fry for helpful comments on the manuscript.

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8. PANAYOTOU G, GISII G, END P, TRUONG 0, Gour I, DIIAKU R, FRY MJ, HILES I, PAWSON T, WATERPIIXD MD: Interactions between SH2 domains and tyrosine-phosphorylated platelet- derived growth factor B-receptor sequences: Analysis of kinetic parameters by a novel biosensor-based approach. Mel Cell Bioll993, 13:3567-3576.

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G. Panayotou, Ludwig Institute for Cancer Research, 91 Riding House Street, London WlP 8BT, UK. M.D. Waterfield, Ludwig Institute for Cancer Research and Department -of Biochemistry and Molecular Biology, University College, London WClE 6BT, UK. P. End, Department of Biopharmaceutics, Sandoz Pharma Ltd, CH-4002, Basle, Switzerland.