FIBER OPTIC EVANESCENT WAVE BIOSENSORdspace.cusat.ac.in/jspui/bitstream/123456789/10134/1/FIBER...
Transcript of FIBER OPTIC EVANESCENT WAVE BIOSENSORdspace.cusat.ac.in/jspui/bitstream/123456789/10134/1/FIBER...
FIBER OPTIC EVANESCENT WAVE BIOSENSOR
Project report submitted in partial fulfillment of the requirement of the award of
degree
MASTER OF TECHNOLOGY
IN
OPTOELECTRONICS AND LASER TECHNOLOGY
Submitted by
Smrithi.V
Register No. 95713009
Under the guidance of
Dr. P. Radhakrishnan,
International School of Photonics
Cochin University of Science & Technology
Cochin‐ 682022
ACKNOWLEDGEMENT
With deep sense of gratitude, I express my heartfelt thanks to
Dr. P. Radhakrishnan, Professor, ISP for the guidance, motivation, support and
encouragement given throughout my project work. I express my sincere gratitude to Dr.
M. Kailasnath, Director, ISP for the help rendered . I also express my sincere thanks to
Dr. V.P.N Nampoori Emeritus Professor, ISP for his help. I am thankful to all the
research scholars of ISP especially Mr. Bobby Mathews. C , Ms. Roopa Venkataraj and
Sister Rosmin for their constant support and help. I extend my sincere thanks to the
teaching and non‐ teaching staff of ISP for all the help and assistance. I would like to
remember my friends who helped me and supported me. I am extremely grateful to my
family who were a constant source of encouragement. Last, but most important of all, I
thank Almighty God.
Smrithi.V
ABSTRACT
Biosensors are analytical devices that can detect chemical or biological species or a
microorganism. A biosensor utilizes a biological recognition element that senses the presence
of an analyte ie; the species to be detected and creates a physical or chemical response that is
converted by a transducer to a signal. Biosensors can be used in clinical diagnostics, drug
development, environmental monitoring air, water, soil and food quality control.
Fiber Optic Biosensors (FOBS) are optical fiber derived devices which use optical field to
measure biological species. Because of their chemical inertness, their compatibility to a wide
range of surface modification, the potential for remote sensing, efficiency, accuracy, low cost,
and the ready availability of inexpensive lasers and photodetectors, FOBS are promising
alternatives to traditional methods for biomolecule measurements. One reliable and sensitive
optical method is evanescent sensing. A sensor based on evanescent field absorption relies on
the interaction of a target substance with the evanescent field adjacent to the fibre core.
Removing the cladding from a portion of an optical fibre permits the evanescent field to
interact with the substances in‐which the fibre is immersed. The objective of this project work
is to develop a Fiber Optic Biosensor based on evanescent wave to detect the microorganisms
such as Yeast molecule and to evaluate their activity in the presence of Curcumin and Neera.
CONTENTS
1. Introduction 1
1.1Optical Fiber 2
1.2 Fiber Optic Sensor 4
1.2.1 Classification of Fiber‐Optic Sensors 5
1.3 Biosensors 5
1.3.1 Introduction 5
1.3.2 Principles of Optical Biosensors 7
1.3.3 Optical Transduction 8
1.3.4 Immobilization of Biorecognition elements 9
1.4 Fiber Optic Biosensors 11
1.4.1 Different types of FOBS 12
1.5 Evanescent Wave Fiber Optic Biosensors 13
1.6 Yeast 171.7 Curcumin 181.8Neera 18
2. Measurement of absorption spectrum of yeast in curcumin 19
3. Measurement of absorption spectrum of yeast in neera 30
4. Measurement of absorption spectrum of curcumin in neera 38
5. Measurement of absorption spectrum of yeast in curcumin and neera 48
6. Conclusions 61
References 62
1
CHAPTER 1
INTRODUCTION
The field of biosensors has emerged as a topic of great interest because of the great
need in medical diagnostics and, more recently, the worldwide concern of the threat of
chemical and bioterrorism. The constant health danger posed by new strands of microbial
organisms and spread of infectious diseases is another concern requiring biosensing for
detecting and identifying them rapidly. Conventional laboratory methods for the detection of
microorganisms and biological toxins in food, water, and human specimens are often time
consuming, require extensive training in microbiology and give delayed results. Various rapid
methods have also been attempted. These methods, while rapid, require sophisticated,
expensive, non portable equipment, thus limiting their usefulness as real‐world detection
systems. These sensitivities also are often limited. Optical biosensors utilize optical techniques
to detect and identify chemical or biological species. They offer a number of advantages such as
the ability for principally remote sensing with high selectivity and specificity and the ability to
use unique biorecognition schemes.
A biosensor is an analytical device that combines a biological sensing element with a
transducer to produce a signal proportional to the analyte concentration. This signal can result
from a change in protons concentration, release or uptake of gases, light emission, absorption
and so forth, brought about by the metabolism of the target compound by the biological
recognition element. The transducer converts this biological signal into a measurable response
such as current, potential or absorption of light through electrochemical or optical means,
which can be further amplified, processed and stored for later analysis. Fiber Optic Biosensors
(FOBS) use optical fibers as the transduction element, and rely exclusively on optical
transduction mechanisms for detecting target biomolecules. Evanescent wave FOBS are
biosensors that utilize evanescent wave detection techniques. Electromagnetic waves
propagate within an optical fiber by total internal reflection at the exposed surface. Light
propagating through an optical fiber consists of two components: the guided field in the core
and the exponentially decaying evanescent field in the cladding. In evanescent wave FOBS the
cladding of a fiber is reduced or removed, the evanescent wave can interact with the
surroundings. Thus evanescent wave FOBS can identify such target analytes in minutes directly
from complex matrix samples, significantly improving the detection sensitivity, selectivity, and
speed.
The detection of chemical and biological agents is a key problem in environment
protection and food monitoring. Traditional laboratory methods can accurately detect the
chemical and biological agents. But the need for expensive devices, special operators, and also
long time for detection limit their wide applications. Thus, it is an urgent demand to develop a
simple, rapid, economical, portable and accurate detection device based on biological agent.
2
This project aims to investigate the properties of evanescent waves and to explore their novel
applications as sensing devices for detecting Yeast molecules, because the rapid detection and
identification of Yeast molecules are necessary for the assessment of their beneficial and
harmful roles in the production and spoilage of foods respectively
Fiber‐optic communication systems are light wave systems that employ optical fibers for
information transmission. Such systems have been deployed worldwide since 1980 and have
indeed revolutionized the technology behind telecommunications. Indeed, the light wave
technology, together with microelectronics, is believed to be a major factor in the advent of the
“information age.”
1.1 OPTICAL FIBER
An optical fiber is a dielectric waveguide that operates at optical frequencies. An optical
fiber is cylindrical in form consisting of the core, the cladding and the buffer. The basic structure
is shown in figure 1.1.
Fig 1.1 Basic structure of an optical fiber
The core is a cylindrical rod of dielectric material and is generally made of glass. Light
propagates mainly along the core of the fiber. The cladding layer is made of a dielectric material
with an index of refraction less than that of the core material. The cladding is usually made of
glass or plastic. The cladding reduces scattering loss that results from dielectric discontinuities
at the core surface, it adds mechanical strength to the fiber, and it protects the core from
absorbing surface contaminants with which it could come in contact. The coating or buffer is a
layer of material used to protect an optical fiber from physical damage. The material used for a
buffer is a type of plastic. The buffer is elastic in nature and prevent abrasions.
The light propagates through the fiber by total internal reflection. The angle at which
total internal reflection occurs is called the critical angle of incidence. At any angle of incidence,
3
greater than the critical angle, light is totally reflected back into the glass medium. The critical
angle of incidence is determined by Snell’s Law.
Figure 1.2 Total Internal Reflection in an Optical Fiber
Optical fibers are divided into two groups called single mode and multimode. Single
mode fiber is optical fiber that is designed for the transmission of a single ray or mode of light
as a carrier and is used for long‐distance signal transmission. Single mode fiber has a much
smaller core than multimode fiber. Multimode fiber is optical fiber that is designed to carry
multiple light rays or modes concurrently, each at a slightly different reflection angle within the
optical fiber core. Multimode fiber transmission is used for relatively short distances because
the modes tend to disperse over longer lengths (this is called modal dispersion).For longer
distances, single mode fiber sometimes called monomode) fiber is used. In classifying the index
of refraction profile, we differentiate between step index and graded index. Step index fibers
have a constant index profile over the whole cross section. Graded index fibers have a
nonlinear, rotationally symmetric index profile, which falls off from the center of the fiber
outwards. Figure 1.3 shows the different types of optical fibers
19
Chapter 2
MEASUREMENT OF ABSORPTION SPECTRUM OF YEAST IN CURCUMIN
This section deals with the determination of the absorption spectrum of
(a) Curcumin in ethanol,
(b) Yeast and Curcumin dissolved in ethanol
using Jasco V‐570 UV/ Visible/ NIR Spectrophotometer and Ocean Optics Spectrometer.
2.1. Measurement of absorption spectrum of Curcumin in ethanol and different
concentrations 0.2, 0.4, 0.6 and 0.8 gms of yeast in curcumin using
Spectrophotometer.
Preparation of the sample
(i) Curcumin
40 ml of ethanol is taken in a beaker. 10‐4 molar curcumin is weighed and
dissolved well in ethanol.
(ii) Yeast in Curcumin
Take four 100ml beakers and label them as 0.2, 0.4, 0.6 and 0.8 gms. Add
20 ml of sterilized water into each of these beakers. Then weigh 0.2, 0.4, 0.6
and 0.8 gms of Yeast using a weighing balance and dissolved well into the
sterilized water. Divide the curcumin solution into four equals parts and pour
into the four beakers containing yeast extract.
Experimental procedure
Absorption spectrum of the samples are taken using UV Visible NIR Spectrophotometer.
Graph showing absorption spectrum for curcumin and different concentrations of Yeast
in curcumin are shown in figures below.
20
(a)
(b)
(c)
21
(d)
(e)
Fig 2.1 Absorption spectrum for curcumin and different concentrations of yeast in curcumin.
Variation in the wavelength of peak 3 for different concentrations of yeast is plotted below.
0.2 0.3 0.4 0.5 0.6 0.7 0.8
425
426
427
428
429
Wav
elen
gth (nm
)
Concentration of yeast (gms)
Conc Vs wavelength
Fig 2.2 Graph showing the variation in wavelength of peak 3 for different concentrations of
yeast in the presence of constant amount of curcumin
22
Conclusions
1. Absorption spectrum was well defined at lower concentrations.
2. As the concentration of yeast in curcumin is increased peak 3 showed a blue shift in
wavelength.
3. The experiment was repeated for lower concentrations 0.0005, 0.001, 0.005, 0.01, 0.05,
0.1 and 0.2 gms and for higher concentrations 1, 3 and 5 gms of yeast. The above
result was repeated for lower concentrations. For higher concentrations structure
of the absorption spectrum changes with the addition of yeast.
2.2 Measurement of evanescent wave absorption spectrum of Curcumin in ethanol
and different concentrations of yeast in curcumin.
Absorption spectrum is obtained using evanescent wave sensor and ocean optics
spectrometer. Experimental layout is as follows. Here the variation in intensity of output light is
determined for different concentrations of yeast in curcumin.
Equipments required are
(i) White light LED source
(ii) Sensing cell : Made of glass, 15 cm long, 2.5 cm wide
(iii) Sensing fiber: Multimode, 400 μm core diameter, 430 μm cladding diameter
(iii) Ocean optics spectrometer: HR 4000, responsive from 200‐
1100 nm.
Preparation of Sensing Fiber
Take Plastic Clad Silica fiber of length 30 cm. The ends of the fiber should be polished for
maximum coupling of light from source to fiber and also from fiber to detector. A small portion
of the fiber is removed from both the ends of the fiber. These ends are then cut with a
diamond cutter. Hand polishing is done by drawing figure "8" patterns on a polishing sheet.
After determining the desired sensor length, it is marked at the middle portion of the fiber. The
sheath as well as the cladding of the marked portion is then removed using a razor blade. The
remaining cladding is removed by dipping that portion in acetone.
Preparation of Sensing Cell
The sensing cell is made from cylindrical glass tube of length 15cm and of diameter 2.5 cm.
The two ends of the tubes are closed and a hole is made at each ends through which the fiber
is passed.
23
Preparation of the sample
(i) Curcumin
40 ml of ethanol is taken in a beaker. 10‐4 molar curcumin is weighed and
dissolved well in ethanol.
(ii) Yeast in Curcumin
Take seven 100ml beakers and label them as 0.0005, 0.001, 0.005, 0. 01, 0.05,
0.1 and 0.2 gms. Add 20 ml of sterilized water into each of these beakers. Then
weigh 0.0005, 0.001, 0.005, 0. 01, 0.05, 0.1 and 0.2 gms of Yeast using a
weighing balance and dissolved well into the sterilized water. Divide the
curcumin solution into seven equals parts and pour into the seven beakers
containing yeast extract.
Experimental Set Up
24
Fig 2.3 Experimental set up for Evanescent wave biosensor
Experimental procedure
Fix the components required for the sensing on the optical bench. Switch on the white
light source. Align the components in such a way that light coming out from the fiber falls on
the ocean optics spectrometer. Take the spectrum of the cell. Then pour the different
concentrations of sample into the glass cell one by one and note the corresponding intensity of
output light in terms of wavelength. Ensure that each time before adding the new
concentration of sample into the glass cell, the sensing cell must be cleaned using the
sterilized water.
25
Absorption spectrum of source, cell, curcumin and different concentrations of yeast in
curcumin are shown below . The first peak is obtained at 453 nm.
Second peak is obtained at 545 nm.
(a)
Second peak is at 549 nm
(b)
26
Second peak for curcumin is at 555 nm.
(c)
200 400 600 800 1000 1200600
800
1000
1200
1400
1600Yeast 0.0005 gm + Curcumin
Intens
ity
Wavelength
Wavelength of the second peak is at 591 nm.
(d)
200 400 600 800 1000 1200700
800
900
1000
1100
1200
1300Yeast 0.001 gm + Curcumin
Intensity
Wavelength
Wavelength of the second peak is at 601 nm.
(e)
200 400 600 800 1000 1200700
800
900
1000
1100
1200
Yeast 0.005 gm + Curcumin
Intensity
Wavelength
27
Wavelength of the second peak is at 596 nm.
(f)
200 400 600 800 1000 1200700
800
900
1000
1100
1200
1300Yeast 0.01 gm + Curcumin
Intensity
Wavelength
Wavelength of the second peak is at 593 nm.
(g)
200 400 600 800 1000 1200700
800
900
1000
1100
1200
1300Yeast 0.05 gm + Curcumin
Intensity
Wavelength
Wavelength of the second peak is at 545 nm.
(h)
200 400 600 800 1000 1200
800
1000
1200
1400
1600
Intensity
Wavelength
Yeast 0.1 gm + Curcumin
Wavelength of the second peak is at 545 nm.
28
(i)
200 400 600 800 1000 1200600
800
1000
1200
1400
1600
1800Yeast 0.2 gm + Curcumin
Intensity
Wavelength
Wavelength of the second peak is at 545 nm.
(j)
Fig 2.4 Evanescent wave absorption spectrum for different concentrations of yeast in
curcumin
Graph showing the variation in relative intensity and wavelength of the second peak
with concentration of yeast are shown below.
0.00 0.05 0.10 0.15 0.201.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0Conc. Vs Relative intensity
Rela
tive in
tens
ity
Conc. oc yeast in gms
(a)
29
0.00 0.05 0.10 0.15 0.20
800
1000
1200
1400
1600
Inte
nsi
ty o
f firs
t pea
k
Conc. of yeast in gms
Y+C
(b)
0.00 0.05 0.10 0.15 0.20540
550
560
570
580
590
600
610
Wave
leng
th (
nm
)
Concentration of Yeast (gms)
Conc. Vs Wavelength
(c)
Fig 2.5 Graph showing the variation in (a) relative intensity , (b) Intensity of peak 1 and (c)
wavelength of peak 2 for different concentrations of yeast
Conclusions
1. It was observed that when yeast was added to curcumin,
there was a red shift in wavelength for peak 2 .
2. When concentration of yeast was increased further there is a
blue shift followed by saturation.
3. In the presence of curcumin, the first peak gets suppressed
especially at lower concentrations of yeast.
30
4. Thus yeast can be detected in the presence of curcumin
especially at low concentrations .
30
Chapter 3
MEASUREMENT OF ABSORPTION SPECTRUM OF YEAST IN NEERA
This section deals with the determination of the absorption spectrum of Yeast
dissolved in sterilized water in neera using Jasco V‐570 UV/ Visible/ NIR Spectrophotometer
and Ocean Optics Spectrometer.
3.1 Measurement of absorption spectrum of different concentrations 0.1, 0.5, 1
and 2 gms of yeast in neera.
Preparation of the sample
Yeast in neera
Take four 100ml beakers and label them as 0.1, 0.5, 1 and 2 gms. Add 20 ml of sterilized water
into each of these beakers. Then weigh 0.1, 0.5, 1 and 2 gms of Yeast using a weighing
balance and dissolve well into the sterilized water. Add 30 ml neera into these beakers and stir
well.
Experimental procedure
Absorption spectrum of the samples are taken using UV Visible NIR Spectrophotometer.
Graph showing absorption spectrum for neera, 0.1 gm concentration of Yeast and
different concentrations of yeast in neera are shown in figures below.
200 400 600 800 1000 1200
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Abso
rbanc
e (A
U)
Wavelength (nm)
Neera
(a)
31
(b)
Downward peak is observed at 298 nm and upward peak at 360 nm.
(c)
Upward peak is observed at 338 nm.
(d)
32
Upward peak is observed at 332 nm.
(e)
Upward peak is observed at 328 nm.
(f)
Fig 3.1 Absorption spectrum for neera, yeast and yeast in neera
Graph showing the variation in wavelength of the absorption peak with concentration of
yeast in neera is shown below.
33
-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2325
330
335
340
345
350
355
360
365
Wav
eleng
th (nm
)
Concentration of yeast (gms)
Y+N
Fig 3.2 Graph showing variation in wavelength for the peak with concentration of yeast
Conclusions
1. Peak absorption spectrum of neera is at 238 nm.
2. Addition of yeast shifts the absorption maximum of yeast from around 220 nm to
360 nm at low concentration.
3. With increase in concentration of yeast, the peak shifts to blue side.
4. In the presence of neera, there is a switch over from negative values of absorption to
positive values at lower concentrations of yeast.
5. This enables the measurement of concentration of yeast in the presence of neera.
3.2 Measurement of evanescent wave absorption spectrum of different
concentrations of yeast in neera.
Evanescent wave absorption spectrum is obtained using evanescent wave sensor and ocean
optics spectrometer. Experimental layout and set up is explained in section II of chapter 2.
Here the variation in intensity of output light is determined for different concentrations of
yeast in neera.
Evanescent wave absorption spectrum of of source, cell, neera and different
concentrations of yeast in neera are given below. For all cases first peak is obtained at 453 nm.
34
Second peak is obtained at 545 nm.
(a)
Second peak is at 549 nm
(b)
Wavelength of second peak is 556 nm.
(c)
35
Peak is obtained at 569 nm.
(d)
Second Peak is obtained 568 nm.
(e)
Peak obtained at and 568 nm.
(f)
36
Peaks is obtained at 568 nm.
(g)
Fig 3.3 Evanescent wave absorption spectrum for different concentrations of yeast in neera
Variation in wavelength of second peak with increase in the concentration of yeast is
shown below.
0.0 0.5 1.0 1.5 2.0
568.0
568.2
568.4
568.6
568.8
569.0Conc. Vs Wavelength
Wa
vele
ngth
(n
m)
Conc. of yeast in gms
Fig 3.4 Concentration of yeast Vs wavelength of second peak
37
Conclusions
1. In the presence of neera with increase in concentration of yeast, the relative intensity
increases.
2. There is no appreciable change in the evanescent wave absorption spectrum of yeast in
neera at higher concentrations. The first peak was immersed in noise.
3. Absorption spectrum gives a better signature regarding the measurement of yeast in the
presence of neera.
4. Experiment was performed for higher concentrations of yeast (upto 20 gms). But there
was not much variation in the output and hence has not been presented here.
38
Chapter 4
MEASUREMENT OF EVANESCENT WAVE ABSORPTION SPECTRUM OF CURCUMIN IN
NEERA
Absorption spectrum is obtained using evanescent wave sensor and ocean optics
spectrometer. Experimental layout and set up is explained in section II of chapter 2. Here the
variation in intensity of output light is determined for different concentrations of curcumin in
neera.
4.1Measurement of Evanescent wave absorption spectrum of 10 ‐3 molar
curcumin in neera.
Preparation of the sample
Curcumin in neera
Take four 100ml beakers and label them as 0.25*10 ‐3molar curcumin,
0.33*10 ‐3 molar, 0.5*10 ‐3 molar curcumin and 10 ‐3 molar curcumin,. Take 80ml,
60ml, 40ml and 20ml ethanol in the above beakers. Weigh 10 ‐3 molar curcumin using a
weighing balance and add into the beakers and dissolve well in ethanol. Add 30 ml
neera into these beakers and stir well.
Graph showing evanescent wave absorption spectrum for neera, different
concentrations of curcumin in neera are shown in figure below. The first peak was at
453 nm for all the cases.
(a)
The second peak is at 545 nm.
39
(b)
Second peak is observed at 547 nm.
(c)
The second peak is at 555 nm.
(d)
Second peak is observed at 553 nm.
40
(e)
Second peak is observed at 549 nm.
c
(f)
Second peak is observed at 545 nm.
(g)
Second peak is observed at 545 nm.
Fig 4.1 Evanescent wave absorption spectrum for different concentrations of curcumin
in neera.
41
Variation in the intensity of peak 1 and wavelength of peak 2 for different concentrations
of curcumin are plotted below.
0.25*10-3 0.33*10-3 0.5*10-3 10-3
1000
1500
2000
2500
3000
3500
C+N
Inte
nsity
(A
U)
Molar conc of curcumin
(a)
0.25*10-3 0.33*10-3 0.5*10-3 10-3544
546
548
550
552
554
Wave
length
(nm
)
Molar concentration of curcumin
C+N
(b)
Fig 4.2 Graphs showing (a) Concentration of curcumin Vs intensity of peak 1 & (b)
Concentration of curcumin Vs wavelength of peak 2 for 0.25 10‐3 to 10‐3 Molar
curcumin in the presence of neera.
42
4.2 Measurement of evanescent wave absorption spectrum of 10 ‐6, 10 ‐5,
10 ‐4molar curcumin in neera.
Preparation of the sample
Curcumin in neera
Take three 100ml beakers and label them as 10 ‐6molar curcumin,
10 ‐5 molar curcumin and 10 ‐4 molar curcumin. Take 40 ml ethanol in the
above beakers. Weigh 10 ‐6 molar curcumin, 10 ‐5 molar curcumin and 10 ‐4
molar curcumin using a weighing balance and add into the respective beakers
and dissolve well in ethanol. Add 10 ml neera into these beakers and stir well.
Add 10 ml ethanol to the above three samples to obtain the next sample to
obtain 0.8 molar concentrations. 10 ml ethanol is again added to obtain the 0.67
molar concentrations.
Graph showing evanescent wave absorption spectrum for neera, different
concentrations of curcumin in neera are shown in figure below. The first peak was at
453 nm for all the cases.
(a)
Second peak is observed at 560 nm.
43
(b)
Second peak is observed at 555 nm.
(c)
Second peak is observed at 555 nm.
(d)
Second peak is observed at 557 nm.
44
(e)
Second peak is observed at 560 nm.
(f)
Second peak is observed at 551 nm.
(g)
Second peak is observed at 552 nm.
45
(h)
Second peak is observed at 558 nm.
(i)
Second peak is observed at 549 nm.
(j)
Second peak is observed at 553 nm.
46
(k)
Second peak is observed at 557 nm.
Fig 4.3 Evanescent wave absorption spectrum for different concentrations of curcumin
in neera.
0.67*10-6 0.8*10-6 10-6 0.67*10-4 0.8*10-4 10-43600
3800
4000
4200
4400
4600
C+N
Inte
nsity
(AU)
Molar conc. of Curcumin
(a)
47
0.67*10-6 0.8*10-6 10-6 0.8*10-5 10-5 0.8*10-4 10-4548
550
552
554
556
558
560
C+N
Wave
length
(nm
)
Molar conc. of Curcumin
(b)
Fig 4.4 Graphs showing (a) Concentration of curcumin Vs intensity of peak 1 & (b)
Concentration of curcumin Vs wavelength of peak 2 in the presence of neera.
Conclusions
1. When neera is added to curcumin there was a blue shift in wavelength for the
second peak when the concentration of curcumin was increased.
2. Also the amplitude of the first peak decreased with the concentration of curcumin.
3. The device performs in a linear fashion at lower concentrations and shows
saturation at higher concentrations.
4. Hence neera can be used to measure the concentration of curcumin.
48
Chapter 5
MEASUREMENT OF ABSORPTION SPECTRUM OF YEAST IN CURCUMIN AND NEERA
This section deals with the determination of the absorption spectrum of Yeast dissolved in
sterilized water in neera and curcumin using Jasco V‐570 UV/ Visible/ NIR Spectrophotometer
and Ocean Optics Spectrometer‐HR 4000 ( 200‐1100 nm).
5.1 Measurement of absorption spectrum of different concentrations 0.001, 0.01, 0.1
and 1 gm Yeast in curcumin and neera.
Preparation of the sample
(i) Curcumin
40 ml of ethanol is taken in a beaker. 10‐4 molar curcumin is weighed and
dissolved well in ethanol.
(ii) Yeast in Curcumin and Neera
Take four 100ml beakers and label them as 0.001, 0.01, 0.1 and 1 gms. Add 20
ml of sterilized water into each of these beakers. Then weigh 0.001, 0.01, 0.1 and
1 gms of Yeast using a weighing balance and dissolved well into the sterilized
water. Divide the curcumin solution into four equal parts and pour into the four
beakers containing yeast extract. Add 30 ml of neera into four beakers and stir
well.
Experimental procedure
Absorption spectrum of the samples are taken using UV Visible NIR Spectrophotometer.
Graph showing absorption spectrum for curcumin and different concentrations of Yeast
in curcumin are shown in figures below.
(a)
49
200 400 600 800 1000 1200
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Abs
orba
nce (AU)
Wavelength (nm)
Neera
(b)
(c)
(d)
50
(e)
(f)
(g)
51
(h)
(i)
(j)
Fig 5.1 Absorption spectrum for yeast in curcumin and neera
52
Graph showing the variation in wavelength of the absorption peak with concentration of
yeast in neera and curcumin is shown below.
0.0 0.2 0.4 0.6 0.8 1.0
340
360
380
400
420
440
Wave
leng
th o
f pea
k (n
m)
Conc. of yeaast in gms
YNC
Fig 5.2 Absorption peak Vs concentration of yeast
Conclusions
1. With increase in concentration of yeast, the peak shifts to blue side.
2. This enables the measurement of concentration of yeast in the presence of neera and
curcumin.
5.2 Measurement of evanescent wave absorption spectrum of different
concentrations of yeast in curcumin and neera.
Absorption spectrum is obtained using evanescent wave sensor and ocean optics
spectrometer. Experimental layout and set up is explained in section II of chapter 2. Here the
variation in intensity of output light is determined for different concentrations of yeast in
curcumin and neera. Preparation of the sample is explained in section I.
Absorption spectrum using ocean optics spectrometer is shown below.
53
Second peak is obtained at 545 nm.
(a)
Second peak is at 549 nm
(b)
Wavelength of second peak is 556 nm.
(c)
54
Peak is obtained at 573 nm.
(d)
Second Peak is obtained 572 nm.
(e)
Peak obtained at and 569 nm.
(f)
55
Peak obtained at and 569 nm.
(g)
Fig 5.3 Evanescent wave absorption spectrum of yeast in neera and curcumin
Variation in wavelength of second peak with increase in the concentration of yeast is shown
below.
0.0 0.2 0.4 0.6 0.8 1.0568
569
570
571
572
573YNC
Wave
leng
th (nm
)
Concentration of yeast (gms)
Fig 5.4 Concentration of yeast Vs wavelength of the second peak
56
Conclusions
1. First peak was suppressed when neera and curcumin are added to yeast.
2. For lower concentrations of yeast, there is blue shift in wavelength of the second
peak .
3. For higher concentrations wavelength saturates so sensitivity is low.
4. This enables the measurement of lower concentration of yeast in the presence of
neera and curcumin.
5.3 Measurement of absorption spectrum of different concentrations of
curcumin in Yeast and neera.
Absorption spectrum is obtained using evanescent wave sensor and ocean optics
spectrometer. Experimental layout and set up is explained in section II of chapter 2.
Here the variation in intensity of output light is determined for different
concentrations of yeast in curcumin and neera.
Preparation of the sample
a) Curcumin in neera
Take four 100ml beakers and label them as 10 ‐4, 5* 10 ‐4, 10 ‐3 and 5* 10 ‐3
molar .Take 40ml ethanol into each of these beakers. Then weigh 10 ‐4, 5* 10 ‐4,
10 ‐3 and 5* 10 ‐3 molar curcumin using a weighing balance and add into the
beakers and dissolve well into the ethanol.
b) Yeast in neera
Take a 100ml beaker and add 20 ml of sterilized water into it. Then weigh 0.01 gm
of Yeast using a weighing balance and dissolve well into the sterilized water. Add 30ml
neera into this beaker and stir well. Now the solution is divided into four and poured
into the four beakers containing curcumin.
Evanescent wave absorption spectrum of the samples are determined using Ocean Optics
Spectrometer
Graph showing evanescent wave absorption spectrum for neera, different
concentrations of curcumin in neera and yeast are shown in figure below.
57
Second peak is at 547 nm
(a)
Wavelength of second peak is 556 nm.
(b)
Wavelength of second peak is 545 nm.
(c)
58
Peak is obtained at 557 nm.
(d)
Second Peak is obtained 552 nm.
(e)
Peak obtained at and 560 nm.
(f)
59
Peaks is obtained at 576 nm.
(g)
Peaks is obtained at 585 nm.
(h)
Fig 5.5 Evanescent wave absorption spectrum of curcumin in yeast and neera
Variation in wavelength of second peak with increase in the concentration of curcumin is
shown below.
60
10-4 5*10-4 10-3 5*10-3
550
555
560
565
570
575
580
585
Wave
length
(nm
)
Molar Concentration of curcumin
Y+N+C
Fig 5.6 Concentration of Curcumin Vs wavelength of second peak
Conclusions
1. There is complete elimination of first peak in the presence of neera and yeast.
2. Signal strength is comparatively high and noise is also absent.
3. When concentration of curcumin was increased, spectrum shows red shift which is a
concentration related feature.
4. This enables the detection of curcumin in the presence of neera and yeast .
Comparison
In Evanescent wave absorption
1. When concentration of curcumin in the presence of yeast and neera is increased, there
was red shift in wavelength of peak 2 from 552 nm to 585 nm.
2. When concentration of yeast in the presence of curcumin and neera was increased,
there was blue shift in wavelength of peak 2 from 573 nm to 569nm.
61
Chapter 6
CONCLUSIONS
This project started with the detection of yeast in the presence of curcumin. First
absorption spectrum for different concentrations of yeast in curcumin was studied.
Absorption spectrum was well defined at lower concentrations. As the concentration of
yeast in curcumin is increased peak 3 showed a blue shift in wavelength. Then the spectral
analysis of the samples was done using evanescent wave sensor and ocean optics
spectrometer. It was observed that as the concentration of yeast was increased there was a
small red shift in wavelength for peak 2 initially . When yeast concentration was increased
further there was a blue shift and subsequent saturation. Also the first peak gets
suppressed at lower concentrations of yeast.Thus yeast can be detected in the presence of
curcumin. For the next studies neera was added to yeast. With increase in concentration of
yeast, the second peak shifts to blue side. There was a switch over from negative values of
absorption to positive values of absorption at lower concentrations of yeast in the
absorption spectrum. This enables the measurement of concentration of yeast in the
presence of neera. There was no appreciable change in the evanescent wave absorption
spectrum of yeast in neera at higher concentrations. Next reaction between neera and
curcumin was studied using ocean optics spectrometer . When neera is added to curcumin
there was a blue shift in wavelength for the second peak when the concentration of
curcumin was increased. Also the amplitude of the first peak decreased with the
concentration of curcumin unlike in the case of reaction between curcumin and yeast where
there was an increase in the amplitude of the first peak. Hence neera can be used to
measure the concentration of curcumin. For the final studies yeast in the presence of
curcumin and neera was taken. Evanescent wave absorption spectrum for different
concentrations of yeast in curcumin and neera are taken using ocean optics spectrometer.
First peak was suppressed due to the presence of curcumin and neera. Second peak has a
blue shift and saturation, as concentration of yeast was increased. This enables the
measurement of concentration of yeast in the presence of neera and curcumin. Evanescent
wave absorption spectrum for different concentrations of curcumin in the presence of yeast
and neera was studied next. Here also the first peak was suppressed. As the concentration
of curcumin was increased, there was a red shift in wavelength. This enables the detection
of curcumin in the presence of neera and yeast . From this studies I conclude that even
small concentrations of yeast and curcumin can be detected in the presence of neera .
62
REFERENCES
[1] B.D.Gupta, “ Fiber Optic Sensors: Principles and Applications”, 3rd Edition, 2006, ( New India
Publishing Agency).
[2] Paras N Prasad, “ Introduction to Biophotonics”, 4th Edition, 2003, (published by John. Wile
and sons, Inc).
[3] Otto S Wolfbeis, “ Fiber optic chemical sensors and Biosensors”, Analytical Chemistry, 2004
Vol. 76, pages 3269‐3284.
[4] Mehrab Mehrvar and Chris Bis, “Fiber Optic Biosensors‐Trends and Advances”, Analytical
Sciences, 2000, Vol. 16, pages 677‐692.
[5] Angela Leung, P. Mohana Shankar, Raj Mutharasan, “A Review of Fiber Optic Biosensors”,
Sensors and Actuators B Vol. 125, 2007 pages 688‐703.
[6] S. F. D’Souza, “Microbial Biosensors”, Biosensors and Bioelectronics Vol 16, 2001
pages 337‐353.
[7] Chunhui Dai, Seokheun Choi, “Technology and Applications of Microbial Biosensors”, Open
Journal of Applied Biosensor, 2013 Vol 2, pages 83‐93.
[8] Yu Lei, Wilfred Chen, Ashok Mulchandani, “Microbial Biosensors”, Analytica Chimica Acta
2006, pages 200‐210
[9] Daniel V. Lim, “ Detection of Microorganisms and Toxins with Evanescent Wave Fiber Optic
Biosensors”, Proceedings of the IEEE, 2003, Vol 91, pages 902‐907.
[10] Maria Espinosa Bosch, Antonio Jesus Ruiz Sanchez, Fuensanta Sanchez Rojas, Catalina
Bosch Ojeda, “Recent Development in Optical Fiber Biosensors”, Sensors, 2007, Vol. 7,
pages 797‐859.
[11] Miso Park, Shen‐Long Tsai and Wilfred Chen, “Microbial Biosensors: Engineered
Microorganisms as the Sensing Machinery”, Sensors, 2013, Vol. 13, pages 5777‐5795.
63