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SERS Biosensor for Endocrine Disruption Biomarker: Vitellogenin

SERS Biosensor for Endocrine Disruption Biomarker: Vitellogenin. Sachin Kumar Srivastava. Mentor: Prof. Ibrahim Abdulhalim. Department of Electro-optic Engineering , & Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Be’er Sheva , Israel.

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SERS Biosensor for Endocrine Disruption Biomarker: Vitellogenin

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  1. SERS Biosensor for Endocrine Disruption Biomarker: Vitellogenin Sachin Kumar Srivastava Mentor: Prof. Ibrahim Abdulhalim Department of Electro-optic Engineering, & Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Be’erSheva, Israel

  2. Outline • Introduction • Raman-/Surface Enhanced Raman- Spectroscopy (SERS) • Biosensor • Endocrine Disruption Compounds • Nano-Sculptured Thin Films (n-STFs): Optimization of performance • Fabrication : Steps involved in developing the sensor • Results: Sensing and other characterizations • Summary

  3. Raman Spectroscopy Indian Physicist: C. V. RAMAN "A small fraction of sunlight passing through a transparent medium is scattered in all directions, with a different wavelength than the incident radiation. These shifts depend upon the chemical structure of the molecules.“ "A new radiation", Indian J. Phys.,2(1928)387. Nobel prize in Physics : 1930 Raman Scattering Rayleigh Scattering Lowest Electronic State Virtual levels Anti-Stokes Stokes Rayleigh Scattering: Elastic Inelastic Raman Scattering: v3 v2 v1 v0 1 in 107 photons is scattered inelastically Ground State

  4. ‘Fingerprint’ of molecular bonds and crystalline structure • Very low cross-section: Applications in real life are hindered Steps of Raman development • Laser applications – Dramatically improved power of excitation and Raman signal • SERS effect – Enhanced intensities up to factors of 108-9 • Raman microscope – Decreased probe volume (light spot diameter below 1 μm) What is SERS? Large local field enhancement on rough metallic surfaces due to plasmonic and charge transfer enhancement mechanisms • Incident light • Enhanced Raman Scattering • Adsorbed molecules Nanosculptured Thin Films (nSTFs) • SERS Substrate

  5. Biosensor Sensor A device having the role of converting a change in one physical parameter into a change in magnitude of a second different parameter which can be measured more conveniently and perhaps more accurately. Bio-sensor A biosensor is a self-contained integrated device that is capable of providing specific quantitative or semi-quantative analytical information using a biological recognition element which is in direct soatial contact with a transduction element (IUPAC, 1996) Any device that uses specific biochemical reactions to detect chemical compounds in biological samples.

  6. Components of a Biosensor / Optical Molecule imprinted gel matrix Chemical interaction SPR/SERS etc Detector

  7. Basic Characteristics of a Biosensor SENSITIVITY: Response per unit analyte numerical value. DETECTION ACCURACY: How accurate the measurement is? LIMIT OF DETECTION:What is the minimum numerical value of the parameter which can be sensed? SELECTIVITY: Interference from other factors must be minimized for obtaining correct results 5. RESPONSE TIME: Time necessary for having about 95% of the response. 6. REUSABILITY: Can the sensor be used again and again? SHELF LIFE:What happens to the performance when kept for long time SENSING VOLUME:Volume of analyte required for sensing RANGE OF OPERATION: What is the working range of the sensor nm1<nm2 Wavelength

  8. Endocrine System General Functions of Hormones http://www.cea1.com/anatomy-sistems/function-of-thymus-in-endocrine-system/attachment/function-of-thymus-in-endocrine-system/ Bond 1979, Biology of Fishes

  9. Endocrine Disruptors Metals (Pb, As, Hg, Ni, Cd) Personal Care Products (Phthalates, Benzaldehyde) Industrial Chemicals (Bisphenol A) Suspected Endocrine Disruptors Diabetes Growth inhibition Pesticides, Herbicides etc. (DDT ) Synthetic and Natural Hormones (Phytoestrogens) • Vg : Egg yolk protein in aquatic females, excreted during sex • Elevated amounts: A sign of estrogenic contamination of aquatic environment. • Representative of a kind of • pollution in water/aquatic species Pharmaceutical Drugs (Corticosteroids)

  10. nSTF Fabrication GLAD Technique SEM Images (20, 30, 40, 60 % porosity)

  11. Optimization of Performance SERS from 4-Aminothiophenol (4-ATP) adsorbed at the n-STF • Highest SERS signal due to plasmons coupling • Mutual interaction between the EM fields of the localized surface plasmons (LSPs) at the neighbouring nanorods • Effective electromagnetic field of the LSPs in the voids • Field in voids decreases with increase in porosity • The number of adsorbed molecules increases, however. • Trade off between the enhancement due to rods and voids Maximum Enhancement @ 30% porosity

  12. Sensor Chip Development

  13. Experimental Setup From Laser To Spectrometer Anti-Vg Antibody Vitellogenin BSA • SERS Probe • Extended view of SERS Probe Sensor Chip

  14. Comparison of SERS Spectra@ Sensor vs 4-ATP Small range effect (~ 2-3 nm) Negligible effect of further added molecules Dieringer and van Duyne et al., Faradey Discuss., 2006, 132, 9-26

  15. Sensor Response for Varying Vg Concentration

  16. Control Experiments • Confirmation for the specificity of the sensor to vitellogenin. • Another similar protein called Fetuin was used for study. Reusability and Repeatability • A number of sensor chips were prepared and experiments were performed • over a period of three months at different days, times and ambient conditions • The sensor surface was regenerated by immersing in 50mM glycine buffer for • 2 minutes in between two sensing events. • Each chip was used at least three times for full sample concentration range to • ensure the repeatability and reusability.

  17. Sensor Response Curve

  18. Summary • A highly sensitive, fast Vg sensor chip utilizing SERS has been fabricated • SERS can be used for sensing of bigger molecules in non-direct mode of sensing • Vitellogenin concentrations as small as 5pg/ml can be detected • The sensor is specific to Vg only. • The repeatability, reusability and shelf life of the sensor are reasonably good For more detailed discussion of the presented results, please see SERS Biosensor Using Metallic Nano-Sculptured Thin Films for the Detection of Endocrine Disrupting Compound Biomarker Vitellogenin, SMALL (Wiley),2014. DOI: 10.1002/smll.201303218

  19. Other Contributors/Co-authors Dr. AtefShalabney, • Laboratoire des Nanostructures, ISIS - Institut de Science et d'IngénierieSupramoléculaires,Université de Strasbourg, 8 alléeGaspardMonge 67000-Strasbourg, Cedex Dr. IsamKhalaila, • The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben Gurion University, Beer Sheva-84105, Israel Dr. ChristophGrünerand Prof. Bernd Rauschenbach • Leibniz Institute of Surface Modification, Permoserstrasse 15, 04318 Leipzig and University Leipzig, Institute for Experimental Physics II, Linnéstr.5, 04307 Leipzig, Germany Prof. Ibrahim Abdulhalim Department of Electro optic Engineering and Ilse Katz Institute for Nanoscale Sciences and Technology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel & School of Materials Science and Engineering, Nanyang Technological University, 637722, Singapore

  20. Acknowledgements • Council of Higher Education of the Government of the State of Israel for PBC post-doctoral fellowship • Singapore National Research Foundation (CREATE programme) • German Excellence Initiative of the Deutsche ForschungsGemeinschaft (DFG). Thank You

  21. Dipole moment in a molecule α0 - molecule equilibrium polarizability E0 - maximum electric field ν0 & ν - excitation and vibrational frequency, respectively rmax - maximum vibrational amplitude ’Modulation’ of linear optical polarizabiliy due to presence of internal vibrations Intensity Values at ~ 1500 cm-1 I(l) >> IS >> IaS 1 >> 10-6 >>10-9

  22. Mechanism of Enhancement in SERS Chemical Mechanism Electromagnetic Mechanism Electromagnetic Mechanism Electromagnetic Mechanism Induced dipole moment in a molecule The molecular polarizability increases due to the formation of charge transfer (CT) metal-molecule complex. The CT band is in resonance with both the incident and scattered light. The local electric field experienced by the adsorbed molecule increases due to excitation of localized surface plasmons by both the incident and scattered photons • SERS depends on the local field to the fourth power ( ) in the vicinity of • an adsorbate molecule. • It is a small range effect (~ 2-3 nm). Enhancement Contribution: 102-103 Enhancement Contribution: 106-108

  23. Endocrine System Endocrine system is an ensemble of glands which secrete different types of hormones directly into the blood stream to maintain homeostasis. The endocrine system regulates many vital processes in a living organism, such as development, growth, reproduction and metabolism. Endocrine Disruptors Extrogeneous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and regulation of developmental processes Possible Factors • Chemical /Industrial waste, Natural reasons/factors • Preservatives, processed food, unbalanced metabolism Pesticides, etc. • Detection of Vg is important to understand the estrogenic contamination of aquatic environment. • Representative of a kind of pollution in water/aquatic species

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