SERS Biosensor for Endocrine Disruption Biomarker: Vitellogenin
Sachin Kumar Srivastava
Department of Electro-optic Engineering, &
Ilse Katz Institute for Nanoscale Science and Technology,
Ben-Gurion University of the Negev, Be’er Sheva, Israel
Mentor: Prof. Ibrahim Abdulhalim
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
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"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.
Indian Physicist: C. V. RAMAN
Nobel prize in Physics : 1930
Raman Spectroscopy
v3v2v1v0
Sto
kes
Ant
i-S
toke
sVirtual levels
Lowest Electronic State
Ground State
Rayleigh Scattering Raman Scattering
1 in 107 photons is scattered inelastically
Elastic
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Raman Scattering:
Rayleigh Scattering:
Inelastic
‘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
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Plasmonic Nanostructures
Incident light
Enhanced Raman Scattering
Adsorbed molecules
SERS Substrate
localp E
Nanosculptured Thin Films (nSTFs)
Biosensor
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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.
Sensor
Bio-sensor
Any device that uses specific biochemical reactions to detect chemical compounds
in biological samples.
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)
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Components of a Biosensor
/ Optical
Detector
Molecule imprinted gel matrix Chemical
interaction SPR/SERS etc
nm1<nm2
Wavelength
1. SENSITIVITY: Response per unit analyte numerical value.
2. DETECTION ACCURACY: How accurate the measurement is?
3. LIMIT OF DETECTION: What is the minimum numerical value of the parameter which can be sensed?
4. 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?
7. SHELF LIFE: What happens to the performance when kept for long time
8. SENSING VOLUME: Volume of analyte required for sensing
9. RANGE OF OPERATION: What is the working range of the sensor
Basic Characteristics of a Biosensor
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Endocrine System
http://www.cea1.com/anatomy-sistems/function-of-thymus-in-endocrine-system/attachment/function-of-thymus-in-endocrine-system/
Bond 1979, Biology of Fishes
General Functions of Hormones
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Metals(Pb, As, Hg, Ni, Cd)
Suspected Endocrine Disruptors
Industrial Chemicals(Bisphenol A)
Synthetic and Natural Hormones
(Phytoestrogens)
Pharmaceutical Drugs
(Corticosteroids)
Pesticides, Herbicides etc.
(DDT )
Personal Care Products(Phthalates,
Benzaldehyde)
Diabetes
Endocrine Disruptors
Growth inhibition
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
nSTF Fabrication
GLAD Technique
SEM Images
(20, 30, 40, 60 % porosity)
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Optimization of Performance
Maximum Enhancement @ 30% porosity
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
Sensor Chip Development
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From Laser To Spectrometer
SERS Probe
Extended view of SERS Probe
Anti-Vg Antibody
Vitellogenin
BSA
Sensor Chip
Experimental Setup
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Comparison of SERS Spectra@ Sensor vs 4-ATP
Small range effect (~ 2-3 nm)
Dieringer and van Duyne et al., Faradey Discuss., 2006, 132, 9-26
Negligible effect of further added molecules
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Sensor Response for Varying Vg Concentration
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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.
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Sensor Response Curve
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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
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Other Contributors/Co-authorsDr. Atef Shalabney, Laboratoire des Nanostructures, ISIS - Institut de Science et d'Ingénierie Supramoléculaires,Université de Strasbourg, 8 allée Gaspard Monge 67000-Strasbourg, Cedex
Dr. Isam Khalaila, The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben Gurion University, Beer Sheva-84105, Israel
Dr. Christoph Grüner and 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 AbdulhalimDepartment 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
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Thank You
Acknowledgements
1. Council of Higher Education of the Government of the State of Israel for PBC post-doctoral fellowship
2. Singapore National Research Foundation (CREATE programme)
3. German Excellence Initiative of the Deutsche Forschungs Gemeinschaft (DFG).
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I(l) >> IS >> IaS
1 >> 10-6 >> 10-9
Intensity Values at
~ 1500 cm-1
α0 - molecule equilibrium polarizabilityE0 - maximum electric fieldν0 & ν - excitation and vibrational frequency, respectivelyrmax - maximum vibrational amplitude’Modulation’ of linear optical polarizabiliy due to presence of internal vibrations
Dipole moment in a molecule
p E
Mechanism of Enhancement in SERS
Induced dipole moment in a molecule
localp E
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
Electromagnetic MechanismElectromagnetic MechanismElectromagnetic MechanismChemical Mechanism
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).
4localE
Enhancement Contribution: 102-103 Enhancement Contribution: 106-108
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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.
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
Endocrine System
Endocrine Disruptors
Chemical /Industrial waste, Natural reasons/factorsPreservatives, processed food, unbalanced metabolism Pesticides, etc.
Possible Factors
Detection of Vg is important to understand the estrogenic contamination of aquatic environment.
Representative of a kind of pollution in water/aquatic species