Research Article
Open Access
Development of High Sensitivity and Cost Effective
Technique for the Surface Plasmon Resonance
Instrument
M Rajavelan1, S Ananthi2*, and K Padmanabhan3
1Department of Network Systems & Information Technology, Guindy Campus, University of Madras, Chennai, India
2Associate Professor & Head-in-Charge, Department of Network Systems & Information Technology, Guindy Campus, University of Madras, India
3Emeritus Professor (Hony), Alagappa College of Technology, Chennai, India
2Associate Professor & Head-in-Charge, Department of Network Systems & Information Technology, Guindy Campus, University of Madras, India
3Emeritus Professor (Hony), Alagappa College of Technology, Chennai, India
*Corresponding author: S Ananthi, Associate Professor & Head-in-Charge, Department of Network Systems & Information Technology, Guindy Campus,
University of Madras, Chennai -600 025, India, Tel: +044 22202767;E-mail: @
Received: November 01, 2016; Accepted: November 20, 2016; Published: December 10, 2016
Citation: Rajavelan M, Ananthi S, Padmanabhan K (2016) Development of High Sensitivity and Cost Effective Technique for the
Surface Plasmon Resonance Instrument. SOJ Mater Sci Eng 4(4): 1-5.DOI: http://dx.doi.org/10.15226/sojmse.2016.00139
AbstractTop
Surface Plasmon Resonance (SPR) is prevalent in the field of biotechnology
presently because of its label free detection of bio molecular
reactions. The SPR instrument finds its place in pharmacology and
drug research. SPR sensitivity is also remarkable by enabling the detection
of molecules in reactions and the affinity among the groups.
The theory behind the technique is well understood and attempt has
been made to improve the existing technique. The instrument involves
the motorized drive for the glass prism to vary the angle and
the source of laser using a solar panel held against the opposite prism
face, thereby catching the beam wherever it moves along that surface.
The use of gold layer in the SPR instrument is the vital component
that causes the SPR dip. Due to frequent use of test samples over the
gold layer, the gold vanishes from the glass plate. The gradual degradation
of gold layer requires frequent change of the gold slide. To
solve this problem, a thin layer of lacquer coating is applied over the
gold layer. But then, multiple peaks are generated than the single SPR
peak. Rather than using the angle change due to Refractive Index (R.I)
variations in the analyses sample, a new method of using these multiple
peaks aims to integrate the multiple reflections and find their
sum. This peak sum value changes in accordance with the R.I of the
substance. An embedded controller (microcontroller) is used to take
the signal samples and integrate them during the angle scan and to
display the peak sum in each and every scan. The peak sum value is
shown on an LCD interfaced to the microcontroller. An USB interface
to the computer is also developed. The program using Visual Basic
displays the SPR curve peaks and the related parameters on the computer
screen. This enables further analysis with the laptop computer.
The signals from such multiple reflections can be enhanced by an increased
power laser source. This increases the sensitivity of the new
technique. Thus, in this improvement to the conventional SPR instrument,
we get two advantages: One is that the expensive replacement
costs of the gold slide are absent. Second is the fact that more sensitive
results can be obtained by amplification of the optical signals. In the
conventional method based on sensitive angle variations, we cannot
get any amplification effect.
Keywords: Surface Plasmon Resonance; Gold Coated Glass Plate- Senor Chip; Laser Beam; Solar Cell and Multiple Reflections
Keywords: Surface Plasmon Resonance; Gold Coated Glass Plate- Senor Chip; Laser Beam; Solar Cell and Multiple Reflections
Introduction
Certain important aspects on the design and operation of the
SPR instrument are discussed in this paper. The SPR is a sensitive
method for optical Refractive index change based analytical
techniques and finds importance in bimolecular work, drug
synthesis, protein to protein affinity and fine measurements of
Nano-material properties [1, 2]. The instrument is a complex
combination of optical, electromechanical, and electronic and
microprocessor based devices [3, 4]. It requires only very
minute quantities of the substances analyzed. Currently available
instruments are very sophisticated and expensive.
It is felt necessary to design and develop a moderately simple enough instrument which can serve the above purposes with some additional features incorporated in it. In every aspect of the fabrication of the instrument, special attention was given to choose parts, materials and circuitry to minimize cost and boost sensitivity. The several improvements that have been experimented and studied include the optical parts [5], the electromechanical components and assembly, the detector and amplification of the signals, the processing and display of the signals as well as the choice of a suitable microcontroller to handle the operation of the entire instrument. The development of suitable software for a laptop to be associated with the set up was also an additional involvement.
To illustrate the various aspects of its design, the figure 1 gives an essence of the technique [6]. Its main component is a right angled BK7 glass prism. The laser source of red radiation with a suitably polarized light to fall on the broad face of the prism at varying angles of incidence is the next important optical component and assembly. The detection of the reflected light signal and transuding the same to a voltage is the next one. The angulations signal processing and evaluation are the aspects to be dealt with by the embedded controller [7, 8].
It is felt necessary to design and develop a moderately simple enough instrument which can serve the above purposes with some additional features incorporated in it. In every aspect of the fabrication of the instrument, special attention was given to choose parts, materials and circuitry to minimize cost and boost sensitivity. The several improvements that have been experimented and studied include the optical parts [5], the electromechanical components and assembly, the detector and amplification of the signals, the processing and display of the signals as well as the choice of a suitable microcontroller to handle the operation of the entire instrument. The development of suitable software for a laptop to be associated with the set up was also an additional involvement.
To illustrate the various aspects of its design, the figure 1 gives an essence of the technique [6]. Its main component is a right angled BK7 glass prism. The laser source of red radiation with a suitably polarized light to fall on the broad face of the prism at varying angles of incidence is the next important optical component and assembly. The detection of the reflected light signal and transuding the same to a voltage is the next one. The angulations signal processing and evaluation are the aspects to be dealt with by the embedded controller [7, 8].
Methods
Geometrical alignment for optimal ray positioning
In many of the designs, the angle of incidence of laser beam
is varied over a range of angles. In studying kinetics of fast bio
reactions through Refractive Index (R.I) changes, if we cannot
find the R.I. at a specific position on the liquid film, it will amount
to finding only an average change in R.I. over the length of the
film. Usually, the film is made to move slowly by the capillary
injection pump.
Therefore a small distance movement will amount to a time over which considerable reaction change might have taken place. Therefore, the reaction kinetics curve for a fast reaction with peak will flatten out in the measurement if the point does not coincide at the same position of the hypotenuse at all angles. So the accuracy in measuring the molecular sample will deviate by a large amount. In the (figure 2) it is shown that when the turning beams carrying the laser rotates with the centre of rotation at the table centre (where the prism is kept), for varying angles, the point of incidence of the laser moves along the hypotenuse of the prism-1, to 2 to 3. That will make the reading taken of the reflected light correspond to different positions on the hypotenuse and therefore from different points on the solution film that passes over the same. That may not find the R.I. at a specific point on the film of solution. That means we have to ensure that the point of laser light falling on the prism remains steady as the prism is turned in angle to vary the angle of incidence. Generally, the instruments designed do not seem to be aware of this critical problem of spot movement over the slide as the angle of incidence is varied. This is illustrated in figure 2 where at three angular values, the spot falls on different spots on the gold slide. Thus it has been found that some importance should be bestowed on the optical geometry design for SPR used for fast reaction detection.
Therefore a small distance movement will amount to a time over which considerable reaction change might have taken place. Therefore, the reaction kinetics curve for a fast reaction with peak will flatten out in the measurement if the point does not coincide at the same position of the hypotenuse at all angles. So the accuracy in measuring the molecular sample will deviate by a large amount. In the (figure 2) it is shown that when the turning beams carrying the laser rotates with the centre of rotation at the table centre (where the prism is kept), for varying angles, the point of incidence of the laser moves along the hypotenuse of the prism-1, to 2 to 3. That will make the reading taken of the reflected light correspond to different positions on the hypotenuse and therefore from different points on the solution film that passes over the same. That may not find the R.I. at a specific point on the film of solution. That means we have to ensure that the point of laser light falling on the prism remains steady as the prism is turned in angle to vary the angle of incidence. Generally, the instruments designed do not seem to be aware of this critical problem of spot movement over the slide as the angle of incidence is varied. This is illustrated in figure 2 where at three angular values, the spot falls on different spots on the gold slide. Thus it has been found that some importance should be bestowed on the optical geometry design for SPR used for fast reaction detection.
Solution to optical geometry problem of SPR setup
Suppose C is the Centre of rotation of the rod which rotates
over an angle range carrying the laser beam. Then, if all rays
should converge exactly at the centre of the prism hypotenuse,
then, what is the relative position of the prism w.r.t. the point C?
The solution to this problem is an optical Geometry problem. This
problem was solved using a computer program. That program
found out the optimal position of the rotating centre of the turn
table. So in order to increase the accuracy in the measurement of
molecular samples, it should be made that laser beam converges
at the centre of hypotenuse of the prism. The rotation centre is
the point where the green lines meet within the prism.
We have observed that in order to make the rays meet at one and the same point on the glass gold plate, it is required to determine the rotation centre to hypotenuse centre offset distance in the x and y directions. For this purpose, a computer program has been developed and the result of the program is just concisely shown in the figure 3, indicating the method of estimating these offset distances. The prism on the turn table is thus positioned accordingly.
We have observed that in order to make the rays meet at one and the same point on the glass gold plate, it is required to determine the rotation centre to hypotenuse centre offset distance in the x and y directions. For this purpose, a computer program has been developed and the result of the program is just concisely shown in the figure 3, indicating the method of estimating these offset distances. The prism on the turn table is thus positioned accordingly.
Graphical method for positioning the optimal centre of
rotation
By using a Graphics program, starting from point P, rays
are drawn using graphics line draw Commands. Rays as shown
in Red color are these internal rays. Each red ray coming down
meets the left small side of the prism. At this point, applying
Snell’s law, the ray on the air side is drawn. For each ray, there is
a ray coming to the prism from the light source (laser) (figure 4).
Now, extending each ray further to the right, there is a meeting
point of all such rays. That point is marked as C. This is the Centre
of rotation for the prism to turn. Keeping the laser beam fixed, if
Figure 1: The schematic of the Surfacce Plasmon resonance based
measurement system
Figure 2: When the turning beam varying the laser rotates with the centre
of rotation at the table centre (where the prism is kept), for varying
angles, the point of incidence of the laser moves along the hypotenuse
of the prism-1, to 2 to 3.
Figure 3: The Centre of Rotation is marked inside the Prism for two
different prisms.
the prism is turned over these angles, then the rays will all meet
at the point P.? Thus, a method has now been found to meet the
requirement that the light spot on the hypotenuse face stays at
one and the same point for all angles of incidence.
At all angles, the laser beam hits at the centre of hypotenuse of the prism.
At all angles, the laser beam hits at the centre of hypotenuse of the prism.
Experimental set-up
In the design of the rotation of the motor carrying the laser,
the stepper motor has to be driven by a suitable gear box of a
good ratio. A 30:1 ratio with a 200 steps per rotation stepper
motor gives an angular change of 360/(200x30) =0.06 degree
per step. By suitably using gear ratios, this can be enhanced to
1/100° at the most.
The drive for the stepper motor is arranged by a four wire interface from an embedded controller and a suitable four transistor driver circuit shown in the (figure 5).
Thus four different types of SPR experimental setups have been developed for a satisfactory result. These are as under:
Thus four different types of SPR experimental setups have been developed for a satisfactory result. These are as under:
SPR design setup II: Rotating prism and fixed laser source,
SPR design setup III: Rotating prism and detector with fixed laser source and goniometry -2 for detector
SPR design setup IV: Modified experimental setup II (Rotating prism with geared stepper motor and fixed laser)
The SPR design setup IV is well designed as the final SPR instrument in which certain reasonable features are embedded. It has sensitivity and good angular resolution. (Figure 6) illustrates the same.
The SPR reflected signal dip is constant and only angle changes with Specimen R.I. (figure 7). The dip in the curve of reflected light intensity happens at an angle θ Plasmon and this angle is characteristic of the sample kept adjacent to the gold film or metal. Actually, this angle is dependent on the R.I. values of prism, the gold and the sample. But when the R.I. of the sample changes, the dip magnitude does not change but only the angle at which the dip occurs changes.
The drive for the stepper motor is arranged by a four wire interface from an embedded controller and a suitable four transistor driver circuit shown in the (figure 5).
Thus four different types of SPR experimental setups have been developed for a satisfactory result. These are as under:
Thus four different types of SPR experimental setups have been developed for a satisfactory result. These are as under:
SPR design setup II: Rotating prism and fixed laser source,
SPR design setup III: Rotating prism and detector with fixed laser source and goniometry -2 for detector
SPR design setup IV: Modified experimental setup II (Rotating prism with geared stepper motor and fixed laser)
The SPR design setup IV is well designed as the final SPR instrument in which certain reasonable features are embedded. It has sensitivity and good angular resolution. (Figure 6) illustrates the same.
The SPR reflected signal dip is constant and only angle changes with Specimen R.I. (figure 7). The dip in the curve of reflected light intensity happens at an angle θ Plasmon and this angle is characteristic of the sample kept adjacent to the gold film or metal. Actually, this angle is dependent on the R.I. values of prism, the gold and the sample. But when the R.I. of the sample changes, the dip magnitude does not change but only the angle at which the dip occurs changes.
The gold coated specimen glass plate and other sanitized biochips:
There are two types of gold coated glass plates.
1. Pure gold coated over the glass plate using Sputter coater
2. Gold coated over the glass plate with TiO2.
There are two types of gold coated glass plates.
1. Pure gold coated over the glass plate using Sputter coater
2. Gold coated over the glass plate with TiO2.
Gold layer is very important to generate the surface Plasmon,
so it is necessary to coat the gold layer over the surface of the
glass plate and place it over the hypotenuse of the prism without
any air gap. The simplest and economical way of coating gold
layer over the glass plate is by using a Sputter Coater with a gold
anode 50nm of pure gold layer is coated for our purpose and
several such slides are prepared and stored in a vacuum chamber.
Sputter coaters are available with thickness monitoring facility.
Figure 4: Showing the convergence of rays internal (C) in the prism
while the actual point P is one and the same for all angles. Prism table
rotation centre will therefore be Point C
Figure 5: ULN 2003A driver IC for stepper motor.
Figure 6: The prism on the turn table driven by gear from stepper motor.
Figure 7: For substance R.I. values 1.33 and 1.34, the angle change is
from 66 to 64 degrees. The amplitude of the dip is not varying much.
The pure gold coated over the glass plate may be washed off
quickly within a few sample tests. This may be overcome to some
extent by using an adhesive layer like copper or titanium that
is coated over the glass plate before the gold is plated. The gold
with Titanium Oxide coated over the glass plate is available from
vendors dealing with SPR instruments.
When it is required to study bimolecular compound interactions, it is necessary to apply a suitable ligand coating on the gold surface. BiaCore Co. sells sensitized gold plates with Dextran, CM5 and some other chemicals. Preparation of gold plated slides with such sensitized coatings can be prepared by the analyst as and when needed.
The main running cost of the instrument lies in the expensive gold coated slide. In using the solar cell, the two cells are connected in series electrically. Each solar cell has a positive (+) and a negative (-) terminal. The two are joined and the two flat cells are kept adjacent to the second reflecting face of the prism. For He-Ne Laser, when the light incidence is such that the ray is totally reflected internally, the light beam directly impinges on the solar cell. The point of impingement may change as the incidence angle changes due to prism turning on the round table. But the ray is not missed for any angle. The D.C. signal voltage that is got is about 0.5V then. When there is surface Plasmon resonance at the particular angle, the light diminishes in intensity. Then, the voltage drops to anywhere between 0.3 to 0.4 V. Thus, with the Helium Neon laser of 10 mW, we can get about 100-200 mV change in signal. This signal is to be amplified. For the 50mW diode laser, we get 5V for full ray impingement and about 3-4 V for SPR condition. Thus, the voltage change we get is about 1 to 2 V, quite a sizeable value.
When it is required to study bimolecular compound interactions, it is necessary to apply a suitable ligand coating on the gold surface. BiaCore Co. sells sensitized gold plates with Dextran, CM5 and some other chemicals. Preparation of gold plated slides with such sensitized coatings can be prepared by the analyst as and when needed.
The main running cost of the instrument lies in the expensive gold coated slide. In using the solar cell, the two cells are connected in series electrically. Each solar cell has a positive (+) and a negative (-) terminal. The two are joined and the two flat cells are kept adjacent to the second reflecting face of the prism. For He-Ne Laser, when the light incidence is such that the ray is totally reflected internally, the light beam directly impinges on the solar cell. The point of impingement may change as the incidence angle changes due to prism turning on the round table. But the ray is not missed for any angle. The D.C. signal voltage that is got is about 0.5V then. When there is surface Plasmon resonance at the particular angle, the light diminishes in intensity. Then, the voltage drops to anywhere between 0.3 to 0.4 V. Thus, with the Helium Neon laser of 10 mW, we can get about 100-200 mV change in signal. This signal is to be amplified. For the 50mW diode laser, we get 5V for full ray impingement and about 3-4 V for SPR condition. Thus, the voltage change we get is about 1 to 2 V, quite a sizeable value.
Flow cell for analytes design
In general, the flow cell is a small compartment like
arrangement used to contain the samples to be tested. It is placed
over the prism surface after the gold coated glass plate in contact
with prism. The gold coating will be towards the flow cell. The
leading SPR instrument developers have used different types of
flow cells for different applications. But the most frequently used
type of flow cell is PDMS (Poly-DiMethyl-Siloxane) flow cell of
volumes of 1μl, 5μl, 100 μl and they are used as standard flow
cells in general. Also the SPR instrument developers made use of
custom flow. A suitable via media design of a flow cell which was
used for the set up constructed.
The design with the embedded controller
A PIC 18F2550 microcontroller based embedded board has
been developed and it is programmed in such a way that it supplies
the pulses for the stepper motor movement and acquires the data
on its ADC channel. The data is transferred to the computer and
plotted as the SPR signal in a picture window. The computer has
Visual basic software developed for the purpose of operating the
instrument. All software, both for the firmware of the PIC 2550 as
well as the Visual Basic based computer program was developed.
Hence the operation is made user friendly from the computer.
The PIC2550 communicates with the computer through the USB
port.
By feeding the number of steps to be turned on the computer, and by clicking start icon in the screen, the stepper motor starts, turns and the fixed laser source shines over the surface of the prism, gold layer and through the sample. After completion of the number of steps of rotation the stepper motor stops. Now, by clicking the read icon on the laptop, the reflected signal measured by the ADC channel is stored in the PIC microcontroller and transmits to the computer via the USB interface. Then, the data is plotted for the values corresponding to the SPR dip. The data is stored as a file in a folder. By loading this file in Mat lab, it has been possible to make further data analysis. The figure shows the real time plot of SPR in the laptop from the SPR instrument prototype.
By feeding the number of steps to be turned on the computer, and by clicking start icon in the screen, the stepper motor starts, turns and the fixed laser source shines over the surface of the prism, gold layer and through the sample. After completion of the number of steps of rotation the stepper motor stops. Now, by clicking the read icon on the laptop, the reflected signal measured by the ADC channel is stored in the PIC microcontroller and transmits to the computer via the USB interface. Then, the data is plotted for the values corresponding to the SPR dip. The data is stored as a file in a folder. By loading this file in Mat lab, it has been possible to make further data analysis. The figure shows the real time plot of SPR in the laptop from the SPR instrument prototype.
Results
The picture of the typical fabricated instrument with all
the features is shown in figure 9. The table below illustrates
the details of the optical and other components shown in the
table 1. With respect to the refractive index of material the SPR
dip changes. The figure 8a and b shows the generated surface
Plasmon resonance is measured at CRO and plotted in laptop
computer screen through microcontroller and visual basic GUI.
The figure 8c shows the SPR dip for two different dielectric
samples and change in dip occurs. The change in SPR dip is the
amount of binding of ligand and analyte sample measured.
Conclusion
A portable, low cost and high sensitivity SPR instrument is
constructed and developed for bio molecular interaction studies
and assessment applications. This will include the building up a
typical instrument, its hardware, associated software programs
as well as the computer interface. The associated techniques and
algorithms for interpretation of results. Optical ray positioning
for steady and accurate measurement of bio molecules is made
for increasing the sensitivity in the measurement of samples. The
higher cost Photo diode array is replaced by low cost, high sensitive
solar cell. This considerably reduced the making cost and
running cost of the instrument. Thus, sensitivity and resolution
are quite good when compared to commercial SPR instrument.
Thus, this SPR technique has been experimentally verified in our
investigations.
Figure 8: Shows a). SPR Dip in CRO, b). SPR Dip in the laptop screen, c).
SPR curve for two different samples
Figure 9: Shows Photograph of developed Surface Plasmon Resonance
instrument.
Table 1: Components used for the surface plasmon resonance.
Materials |
Thickness, nm |
Refractive index, n |
Absorption Coefficient, k |
BK7 Prism |
0 |
1.714 |
0 |
Matching oil |
1000000 |
1.714 |
0 |
Glass plate |
5000000 |
1.714 |
0 |
Gold layer |
50 |
0.1995 |
3.6425 |
Water(as sample) |
0 |
1.33 & 1.35 |
0 |
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