Download A Modified Surface Plasmon Resonance Instrument

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.