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Published on January 15, 2009

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Surface Plasmon Resonance Based Fiber Optic Sensor For The Detection Of Bittering Component (Naringin) In Citrus Fruit Juice : Surface Plasmon Resonance Based Fiber Optic Sensor For The Detection Of Bittering Component (Naringin) In Citrus Fruit Juice By RAJAN JHA ICFO - Institut de Ciències Fotòniques Castelldefeles (Barcelona), Spain Slide 2: Fiber optic sensor: advantages Optical Technique for sensing Surface plasmon resonance (SPR) Biosensors: Fiber optic based SPR Detection of Naringin Conclusions OUTLINE Slide 3: Typical Fiber optic sensor Small in size,occupy less space and light in weight Electrical isolation and chemically inert On-line measurements and remote sensing Why Optical Fiber Sensor? Optical techniques used for sensing : Optical techniques used for sensing Evanescent field Fluorescence Interferometry Doppler’s effect Surface plasmon resonance (SPR) What are Surface Plasmons? : What are Surface Plasmons? Plasmon: The coherent oscillations of the free electrons are called plasmons At interface of metal and dielectric As confined to surface, these are called surface plasmon. Slide 6: Ksp ? Frequency Wavenumber K The Need Of Light Coupling The two curves never cross Direct light cannot excite surface plasmons. K0 = ?/c Slide 7: Excitation of Surface Plasmons: Kretschmann configuration Surface Plasmon Resonance : Surface Plasmon Resonance Resonance occurs at Measure qSP (different for different samples) em and w are known es can be calculated Slide 9: Fiber optic SPR sensors Prism is replaced by core of the multimode optical fiber Cladding is removed from a small portion and a metal layer is applied on the unclad portion of the fiber core Polychromatic light source is used A range of angles is launched The power transmitted through the fiber is detected Spectral interrogation mode of operation is considered Slide 10: Biosensors Allows detection of an analyte in sample matrix using selective reaction . The first biosensor was described by Clark and Lyons in 1962, for determination of glucose. The enzyme used was Glucose oxidase. Glucose + Oxygen +Water =Gluconic acid +hydrogen peroxide Slide 11: Reagent is immobilized over core of fiber. Analyte reacts with the reagent and changes the optical properties of the film and hence modulates the output signal Slide 12: Elements/ Reagent of Biosensors Enzymes Antibodies Nucleic acid These can attach to particular type of substrate Slide 13: Immobilization method in Biosensors These are processes by which enzymes/antibodies are attached to substrate. Adsorption Entrapment Cross-linking Covalent bonding Slide 14: Citrus fruit juice has faced problems in terms of bitterness and delayed bitterness, thereby affecting its consumer acceptability The bitterness in fruit juice is caused by naringin (4,5,7-trihydroxy flvanone-7-ramnoglucoside) To detect naringin, enzyme (Naringinase) is required Detection of Naringin Slide 15: Prunin+Rhamnose Prunin bitterness is about (60-70 %) less than that of naringin** Enzyme Naringinase is looked as a potential agent to reduce the naringin content and hence the bitterness in fruit juice The debittering component is the enzymes itself * M.Ono, J . Ferment. Technol, 59(4), 387-389 (1980) ** R.M.Horowitz, “Biochemistry of Phenolic compounds”, Academic press, New York, 545 (1964) * Fabrication of SPR based sensor for detection of naringin : Fabrication of SPR based sensor for detection of naringin Steps involved in sensor fabrication Deposition of silver on core of optical fiber Immobilization of enzyme on silver coated core of fiber Assay of enzyme Experimental execution and analysis of SPR based sensor Deposition of silver on fiber core using vapour deposition : Deposition of silver on fiber core using vapour deposition Silver was deposited on the core of a plastic clad silica (PCS) fiber of 600 ?m core diameter by vapor deposition technique Before coating with a silver film, the fiber cladding (1 cm length) was removed from fiber of about 20 cm length. After cleaning with acetone, it was kept in a vacuum chamber till deposition was achieved Enzyme immobilization : Enzyme immobilization Naringinase was immobilized on silver coated fiber using gel entrapment technique Polyacrylamide polymer was prepared by copolymerisation of acrylamide with N, N’-Methylenebisacrylamide Enzymes Naringinase (Fiber coated with enzymes) along with 30% acrylamide, 10% ammonium persulphate (APS) and tetramethyl-ethylenediamine (Temed) were mixed to catalyze the reaction Assay of Naringinase* : Assay of Naringinase* Immobilized fiber was dipped in a reaction mixture of 4ml of 0.125% naringin dissolved in 0.02 M acetate buffer (pH 4.5) Later fiber was removed and amount of naringin left was estimated, treating the remaining reaction mixture with diethylene glycol *M.Ono et al, J . Ferment. Technol, 55(5), 493-500 (1977) Experimental Setup : Experimental Setup Calibration curve of the sensor : Calibration curve of the sensor Performance parameters* : Performance parameters* dlres * S. A. Zynio et al., Sensors 2, 62-70, (2002). * A K Sharma and B D Gupta., Optics Comm. 245, 159-169, (2005). Slide 23: Sensitivity improves with naringin concentration Sensitivity of sensor Slide 24: As the concentration of naringin increases, the width of the response curve increases and hence SNR decreases SNR of sensor Slide 25: Concentration comparison with other existing method * E.Reichart et.al, Anal.Chim. Acta (1998) 179-187 Conclusions : Conclusions Fabrication and characterization of SPR based fiber optic sensor for detection of naringin is possible SPR wavelength increases with concentration of the naringin The sensitivity increases with an increase in the concentration of naringin and SNR decreases The sensors probe is re-usable Slide 27: Thank You Slide 28: Ksp ? Frequency Wavenumber K The Need Of Light Coupling The two curves never cross Direct light cannot excite surface plasmons. K0 = ?/c Slide 29: A fraction of light momentum transfers to Surface Plasmons and the resonance occurs at Kev = Ksp. Slide 30: A fraction of light momentum transfers to Surface Plasmons and the resonance occurs at Kev = Ksp. Metal Specification : Metal Specification Silver: Exhibits a very sharp SPR curve. Hence, a large detection accuracy (i.e., SNR). A poor chemical stability. Gold: Demonstrates a larger shift in resonance parameter. Hence, a better sensitivity. A reasonable chemical stability. Slide 32: Signal-to-noise ratio (SNR) is defined as inversely proportional to the SPR curve width Sensitivity is defined as the change in resonance wavelength per unit RIU change Slide 33: Advantages of Fiber Optic Biosensors Consume less analyte and so less expensive. Small in size and so more durable. Greater geometry versatility. No reference electrode is required as in electrochemistry. No electrical interference Resistant to nuclear and ionizing radiations. Naringin Estimation * : Naringin Estimation * To 1 ml of naringin solution 5 ml of 90% DEG (diethylene glycol) was added and incubated at 27?C for 10 minutes. 0.1 ml 4N NaOH was added and optical density was measured at 420nm * D.W.Davis, Anal. Chem. 19, 476, (1947) Slide 35: Specifications of chemicals Source of enzyme is Aspergillus niger* Acetate buffer= 13.6 gm of sodium acetate in 500 ml 0f water and pH was made to 4.5 with acetic acid Molecular weight of substrate (naringin) = 580 Enzyme concentration (stock solution) = 2mg/ml *Puri et.al, Critical Reviews in Biotechnology (1996), 144-155 Deposition of silver on fiber core using vapour deposition : Deposition of silver on fiber core using vapour deposition Silver was deposited on the core of a plastic clad silica (PCS) fiber of 600 ?m core diameter by vapor deposition technique Before coating with a silver film, the fiber cladding (1 cm length) was removed from fiber of about 20 cm length. After cleaning with acetone, it was kept in a vacuum chamber till deposition was achieved Slide 37: Specifications of chemicals Phosphate buffer=KH2PO4 (39 ml ) and K2HPO4 (61 ml) for pH=7 Molecular weight of substrate = 289.18 Molecular weight of pesticide = 350.59 Molecular weight of enzyme (from electric eel)= 260000 Enzyme concentration (stock solution)= 1.5mg/ml. Acetylcholine esterase : Acetylcholine esterase Acetylcholine +water=acetate+choline The enzyme is bound to cellular membranes of excitable tissue (synaptic junction, endoplasmic reticulum, etc.) and is believed to be associated with nerve impulse conduction AChE is also found in red blood cells. Slide 39: The enzyme Acetylcholine esterase (AChE) is bound to cellular membranes of excitable tissue (synaptic junction, endoplasmic reticulum, etc.) in our brain The neurotransmitter acetylcholine breaks into its constituents, acetate and choline in the presence of AChE Many pesticides and insecticides are potent inhibitor of acetylcholine esterase Acetylcholinesterase enzyme Acetylcholine esterase : Acetylcholine esterase An enzyme, found in the synaptic clefts of cholinergic synapses, that cleaves the neurotransmitter acetylcholine into its constituents, acetate and choline, thus limiting the size and duration of the postsynaptic potential. Many nerve gases and insecticides are potent acetylcholine esterase inhibitors, and thus prolong the timecourse of postsynaptic potentials. Chlorpyriphos : Chlorpyriphos Chlorpyriphos is an Organophosphorous insecticide possessing broad spectrum contact insecticidal properties for application on crops like cotton, chilly, rice, sorghun, soyabean, sugarcane, groundnut, vegetables, ornamentals and flowers; as a soil insecticide used for commercially important plantation crops like Citrus, Mango and Grapevine and it also finds application in the preservation of wood and timber Slide 42: Before taking measurements for other pesticide concentrations the surface was reactivated The surface was reactivated by using a mixture of substrate and 2-pyridine aldoxime methoiodide (PAM) The irreversible inhibition of AChE by organophosphate compound is accompanied by the formation of covalent phosphorus- enzyme linkage at the active site Reactivation of probe Slide 44: 1/SPR wavelength (nm)-1 1/Substrate concentration (mM)-1 Variation of inverse of SPR wavelength with the inverse of concentration of substrate for a fixed pesticide concentration: (?) 0.001 mM, (?) 0.01 mM, (?) 0.1 mM. N-layer model : N-layer model Slide 46: and Amplitude reflection coefficient (rp) Reflectance (Rp) Characteristic matrix of the combined structure Slide 47: Values of parameters used for simulation The value of different sellemier’s coeeficients Slide 48: As the Teflon thickness increases the curve shifts towards the lower value of resonant wavelength LRSP spectrum for metal thickness=40 nm Slide 49: Variation of resonance wavelength with refractive index follows a non linear behaviour Slope of the curve is much steeper when Teflon was employed

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