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This document describes a biosensor for detecting acrylamide in starchy foods using hemoglobin-modified carbon nanofibers. It first discusses how acrylamide forms in heated foods and can bind to hemoglobin. The document then reviews literature on similar biosensors and identifies limitations. It proposes developing the biosensor by confirming acrylamide-hemoglobin adduct formation through UV-Vis and FTIR spectroscopy. The work plan involves optimizing hemoglobin attachment to carbon nanofibers and comparing free and attached hemoglobin to create calibration plots for acrylamide detection in homemade potato crisps. Current work focuses on verifying adduct formation spectrophotometrically before further sensor development.
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- 1. A Biosensor Based on Electrodes Modified with Carbon Nano Fibres/Hemoglobin for Detection of Acrylamide in Starch Rich Food Samples Prabhakaran.M 115078011 GUIDE:Dr.Meera.p
- 2. Introduction • Acrylamide found in heated food stuffs. • High levels of acrylamide found in potato crisps, French fries. • It cause DNA damage and neurological and reproductive effects. • Acrylamide can create adducts with hemoglobin.
- 3. Literature • Recognition layer: SWCNT/MWCNT with hemoglobin. • Analyte: Acrylamide. • Concentration of Hb:10mg/ml in 0.2 mol acetate buffer containing of 0.05 M Nacl • Immobilization temperature:4°c. • Acrylamide concentration:10-3 M to 10-11 M. • Elecrolyte solution:0.05 M Nacl in 0.2 M acetate buffer.
- 4. Defects in the literature • The redox peaks observed in CV are attributed to COOH groups of CNTs and not Hb. • Peak current Vs scan rate plot is actually non-linear indicating a diffusion controlled response but authors claim that it is surface controlled. • If the peaks were only due to CNTs , acrylamide binding to Hb should not alter the response.
- 5. Our concept Prepare potato crisps in the lab with • Different types of oil. • Different temperature. • Different size. • Aligned carbon nano fibres • If possible can develop both optical and electrochemical biosensors
- 6. Work Plan • Confirm acrylamide – Hb adduct formation. • Optimize Hb at carbon nano fibres to conjugate to obtain redox peaks of Hb. • Compare the conformation of free Hb with adsorbed Hb. • Also compare the acrylamide binding capacity of free Hb Vs adsorbed Hb. • Make calibration plot (Optical and Electro Chemical )with different concentration of acrylamide at constant concentration of Hb. • Check the sensor with potato crisps.
- 7. Current work • To verify Hb-acrylamide adduct formation using UV-visible spectrophotometer and FTIR
- 8. reference 1. A Voltammetric Biosensor Based on Glassy Carbon Electrodes Modified with Single-Walled Carbon Nanotubes/Hemoglobin for Detection of Acrylamide in Water Extracts from Potato Crisps Agnieszka Krajewska, Jerzy Radecki and Hanna Radecka * 2. Programmable self-assembly of carbon nanotubes assisted by reversible denaturation of a protein P Nithyasri, K Balaji , P Brindha and M Parthasarathy
- 9. Hypothesis • Acrylamide + Hb → Adduct formed by Michael addition of N-terminal of Hb with alkenyl group of acrylamide. • This adduct formation confirmed by UV visible spectroscopy and FTIR.
- 10. To verify Hb-acrylamide adduct formation • Prepare Hb stock solution and acrylamide stock solution in pH 7 buffer. • Take six samples of Hb and buffer and mix well. • Add different concentration of acrylamide stock solution to 5 samples bottles and first sample act as the control. • Sample 1=>0.3ml Hb+2.7ml buffer(control). • Sample 2=>3ml acrylamide + Hb stock. • Sample 3=>2ml acrylamide + Hb stock. • Sample 4=>1ml acrylamide + Hb stock. • Sample 5=>0.5ml acrylamide + Hb stock. • Sample 6=>0.1ml acrylamide + Hb stock.
- 11. UV-visible spectroscopy 1. Hb alone: • Blank=>buffer(pH7),Sample=>Hb solution. • Maximum wavelength at 406 nm.
- 12. 2.Hb + acrylamide: • Blank=>buffer, sample=>Hb + acrylamide.
- 13. • Maximum wave length at 406 nm. • There is no peak shift because prosthetic(Heme group) group of Hb is not involed in adduct formation. • The metal-containing prosthetic group deeply buried inside the protein cage so it will not react with acrylamide. • Due to above problem we cannot design optical sensors.
- 14. FTIR spectroscopy 1.For pure acrylamide:
- 15. • Wave number of carboxy group 1660 1/cm and 1610 1/cm(doublet) • Wave number of alkenyl group 1282-1430 1/cm(singlets)
- 16. 2.For Hb + acrylamide:(sample 2) Hemoglobin+Acrylamide Deep frozen lyophilized FTIR
- 18. • Wave number of carboxyl group 1648 1/cm and alkenyl group is absent. • c=c group reacted with Hb and shift in c=o band is due to break of resonance as c=c has become c-c. • This process indicates adduct formation. • This is further verified by comparing the FTIR spectrum of Hb + acrylamide with propanamide.
- 19. Next work plan • Record FTIR for pure acrylamide and pure Hb. • Cyclic voltammetry of Hb at carbon nano fibre Vs Hb-acrylamide at carbon nano fibre. (a).Effect of air,nitrogen,oxygen. (b).Effect of solution PH.