FIBER OPTIC EVANESCENT WAVE BIOSENSOR

Transcription

1 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 Under the guidance of Dr. P. Radhakrishnan, International School of Photonics Cochin University of Science & Technology Cochin

2 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

3 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.

4 CONTENTS 1. Introduction Optical Fiber Fiber Optic Sensor Classification of Fiber Optic Sensors Biosensors Introduction Principles of Optical Biosensors Optical Transduction Immobilization of Biorecognition elements Fiber Optic Biosensors Different types of FOBS Evanescent Wave Fiber Optic Biosensors Yeast Curcumin Neera Measurement of absorption spectrum of yeast in curcumin Measurement of absorption spectrum of yeast in neera Measurement of absorption spectrum of curcumin in neera Measurement of absorption spectrum of yeast in curcumin and neera Conclusions 61 References 62

5 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. 1

6 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, 2

7 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 3

8 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 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) (ii) Curcumin 40 ml of ethanol is taken in a beaker molar curcumin is weighed and dissolved well in ethanol. 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. 19

9 (a) (b) (c) 20

10 (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. Conc Vs wavelength Wavelength (nm) Concentration of yeast (gms) 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 21

11 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 , 0.001, 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 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. 22

12 Preparation of the sample (i) Curcumin 40 ml of ethanol is taken in a beaker molar curcumin is weighed and dissolved well in ethanol. (ii) Yeast in Curcumin Take seven 100ml beakers and label them as , 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 , 0.001, 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 23

13 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. 24

14 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) 25

15 Second peak for curcumin is at 555 nm. (c) 1600 Yeast gm + Curcumin 1400 Intensity Wavelength Wavelength of the second peak is at 591 nm. (d) 1300 Yeast gm + Curcumin 1200 Intensity Wavelength Wavelength of the second peak is at 601 nm. (e) 1200 Yeast gm + Curcumin 1100 Intensity Wavelength 26

16 Wavelength of the second peak is at 596 nm. (f) 1300 Yeast 0.01 gm + Curcumin 1200 Intensity Wavelength Wavelength of the second peak is at 593 nm. (g) 1300 Yeast 0.05 gm + Curcumin Intensity Wavelength Wavelength of the second peak is at 545 nm. (h) 1600 Yeast 0.1 gm + Curcumin 1400 Intensity Wavelength Wavelength of the second peak is at 545 nm. 27

17 (i) 1800 Yeast 0.2 gm + Curcumin 1600 Intensity Wavelength Wavelength of the second peak is at 545 nm. Fig 2.4 Evanescent wave absorption spectrum for different concentrations of yeast in curcumin (j) Graph showing the variation in relative intensity and wavelength of the second peak with concentration of yeast are shown below Conc. Vs Relative intensity 1.8 Relative intensity Conc. oc yeast in gms (a) 28

18 Y+C 1600 Intensity of first peak Conc. of yeast in gms (b) Conc. Vs Wavelength Wavelength (nm) Concentration of Yeast (gms) (c) Fig 2.5 Graph showing the variation in (a) relative intensity, (b) Intensity of peak 1 and wavelength of peak 2 for different concentrations of yeast (c) Conclusions 1. It was observed that when yeast was added to curcumin, there was a red shift in wavelength for peak 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. 29

19 4. Thus yeast can be detected in the presence of curcumin especially at low concentrations. 30

20 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. Yeast in neera Preparation of the sample 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. Neera Absorbance (AU) Wavelength (nm) (a) 30

21 (b) Downward peak is observed at 298 nm and upward peak at 360 nm. (c) Upward peak is observed at 338 nm. (d) 31

22 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. 32

23 365 Y+N Wavelength (nm) Concentration of yeast (gms) 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. 33

24 Second peak is obtained at 545 nm. (a) Second peak is at 549 nm (b) Wavelength of second peak is 556 nm. (c) 34

25 Peak is obtained at 569 nm. (d) Second Peak is obtained 568 nm. (e) Peak obtained at and 568 nm. (f) 35

26 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 Conc. Vs Wavelength Wavelength (nm) Conc. of yeast in gms Fig 3.4 Concentration of yeast Vs wavelength of second peak 36

27 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. 37

28 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 3 molar 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. The second peak is at 545 nm. (a) 38

29 Second peak is observed at 547 nm. (b) The second peak is at 555 nm. (c) Second peak is observed at 553 nm. (d) 39

30 c Second peak is observed at 549 nm. (e) (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. 40

31 Variation in the intensity of peak 1 and wavelength of peak 2 for different concentrations of curcumin are plotted below C+N 3000 Intensity (AU) * * * Molar conc of curcumin (a) 554 C+N 552 Wavelength (nm) * * * Molar concentration of curcumin (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 to 10 3 Molar curcumin in the presence of neera. 41

32 4.2 Measurement of evanescent wave absorption spectrum of 10 6, 10 5, 10 4 molar curcumin in neera. Preparation of the sample Curcumin in neera Take three 100ml beakers and label them as 10 6 molar 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. Second peak is observed at 560 nm. (a) 42

33 Second peak is observed at 555 nm. (b) Second peak is observed at 555 nm. (c) Second peak is observed at 557 nm. (d) 43

34 Second peak is observed at 560 nm. (e) Second peak is observed at 551 nm. (f) Second peak is observed at 552 nm. (g) 44

35 Second peak is observed at 558 nm. (h) Second peak is observed at 549 nm. (i) Second peak is observed at 553 nm. (j) 45

36 Second peak is observed at 557 nm. (k) Fig 4.3 Evanescent wave absorption spectrum for different concentrations of curcumin in neera. C+N Intensity (AU) * * * * Molar conc. of Curcumin (a) 46

37 C+N Wavelength (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. 47

38 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 ( 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 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) 48

39 Neera Absorbance (AU) Wavelength (nm) (b) (c) (d) 49

40 (e) (f) (g) 50

41 (h) (i) (j) Fig 5.1 Absorption spectrum for yeast in curcumin and neera 51

42 Graph showing the variation in wavelength of the absorption peak with concentration of yeast in neera and curcumin is shown below. 440 YNC 420 Wavelength of peak (nm) Conc. of yeaast in gms 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. 52

43 Second peak is obtained at 545 nm. (a) Second peak is at 549 nm (b) Wavelength of second peak is 556 nm. (c) 53

44 Peak is obtained at 573 nm. (d) Second Peak is obtained 572 nm. (e) Peak obtained at and 569 nm. (f) 54

45 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. 573 YNC 572 Wavelength (nm) Concentration of yeast (gms) Fig 5.4 Concentration of yeast Vs wavelength of the second peak 55

46 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, concentrations of curcumin in neera and yeast are shown in figure below. different 56

47 Second peak is at 547 nm (a) Wavelength of second peak is 556 nm. (b) Wavelength of second peak is 545 nm. (c) 57

48 Peak is obtained at 557 nm. (d) Second Peak is obtained 552 nm. (e) Peak obtained at and 560 nm. (f) 58

49 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. 59

50 Y+N+C Wavelength (nm) * *10-3 Molar Concentration of curcumin 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. 60

51 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. 61

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