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A Modified Surface Plasmon Resonance Instrument with Improved Sensitivity S.Ananthi, University of Madras, Chennai. India; K.Padmanabhan, Anna University, Chennai, India; M.Rajavelan, University of Madras, Chennai, India Abstract: Today,advanced research in biotechnology involving biomolecular interaction studies, such as protein to protein interactions, utilises the Surface plasmon Resonance instrument for its label free and precise nature of measurement. In the current technique, the monochromatic p-polarised red light ray or beam, is totally reflected in a prism to analyte interface. With a gold layer at this interface, the resonance absorption of light causes the reflected light to dip in intensity at the specific angle of incident light beam, called the SPR dip angle. The analyte is kept above the gold layer and influences the dip angle very sensitively thereby enabling the dip angle measurement as a means of studying the analyte substance and bioreactions. Refractive index (R.I.) changes of value 10─4 is measurable by a change in angle at resonance to a precision of a milli-degree. There are several types of Biochips sold for use with the instrument. They are gold slides coated with Dextran and other chemicals which serve as biosensors to bind the biomolecules to the ligand. BiaCore Co. is one leading firm in this technology. We devised several modifications to the basic instrument to improve the sensitivity and also to suggest methods for reducing the running costs. The main point in the instrument currently is the change in dip angle with the analyte property variations. If n 1 ,n 2 and n 3 represent the complex refractive indices of the glass prism, the gold layer (50nm) and the analyte, the surface plasmon dip angle is a function θ s.p. = f(n 1 , n 2 , n 3 ). Since n 1 and n 2 are constant in a set up, the dip angle is just a function of n 3 of the analyte's R.I. The dip angle measurement is the basis of the current technique. The angle at dip alone changes and not the reflected light intensity. It was observed that angle variation and measurement cannot be made very accurately, either in the rotation based system or as in Biacore’s wide ray beam system. By our experiments and theoretical simulations with light refraction at multiple interfaces, we arrived at a modified gold slide to be used with the prism. A further coating of a lacquer was given over the gold film on the glass slide. The coat was 100 micron as a film. This material could be of a synthesised bio chemical so as to be specific to a particular bio-reaction. The flow chamber carrying the analyte also has at the other side, a mirror reflector. The simulation showed that because of this layer and return reflection, there are multiple reflections and hence there were noticed a number of dips in the experiments also. At the glass to gold, gold to coating and coating to analyte interfaces, light splits into refracted and reflected components. Each such component depends on the R.I. value n 3 . The resonance in the Gold layer occurs at different angles for these components. These dips vary in amplitude with the analyte R.I. The photo detected signal voltages of these several dips were summed up giving a value Φ n3 = Σ v n , where v n is the photo detected voltage values of the dips. By this, the measurement has been diverted from the angle to the amplitude of light reflected and transduced by the photo electric device. Since dips occur now severally at different angles, a large area reflecting light detector such as a solar cell was used to catch all these dips as the angle of incidence is varied. The dips could generate voltage drops in the 100-500 mV range. It was easily possible to get high resolution of the reflected Φ n3 by amplification of the voltage and subsequent Analog Digital conversion (ADC) with 12-bits resolution. Compared to the existing technique of sensitive angle assessment, in this modified multiple reflection method, by our ADC and pre-amplifier, much more sensitive results could be got. In the existing technique, enhancing the light source intensity is not contributive because it merely serves to fix the value of the minimum point or dip. But here, we can increase the sensitivity by a powerful light source since amplitude changes are used for detection. We had used a 100 mW red diode laser and it could sense the minor dips also occurring due to repeated reflections between the layers. The more the number of reflections, the better could be the sensitivity of the instrument. The figure given illustrates how the peaks change for a small change in analyte R.I. Giving a thin film coating over the gold does not wash off the gold after a few samples as in the existing procedure and hence the running cost is cut down. Fig.1. Showing multiple dips in light intensity and change due to analyte R.I.