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