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Detection of Glutathione By Heat-Induced Surface-Enhanced Raman Scattering (SERS) and Electrochemical Sensing. Literature Seminar. Thabiso Musapelo 03-01-10. Objective. To improve the simplicity, selectivity and sensitivity of Glutathione detection.

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slide1

Detection of Glutathione By Heat-Induced Surface-Enhanced Raman Scattering (SERS) and Electrochemical Sensing

Literature Seminar

Thabiso Musapelo

03-01-10

objective
Objective
  • To improve the simplicity, selectivity and sensitivity of Glutathione detection.

1.“Development of a Heat-Induced Surface-Enhanced Raman Scattering Sensing Method for Rapid Detection of Glutathione in Aqueous Solutions”

2. “Electrochemical Sensing Strategy for Ultrasensitive Detection of Glutathione (GSH) by Using Two Gold Electrodes and Two Complementary Oligonucleotides “

outline
Outline
  • Introduction
    • What is Glutathione ?
    • Surface enhanced Raman Scattering
    • Electrochemical Sensing
  • Results and discussion
    • Heat-induced Surface Enhanced Raman Scattering Method
    • Electrochemical Ultrasensitive Sensing Using modified Gold Electrode.

Critique/Comparison

Conclusion

glutathione gsh
Glutathione (GSH)
  • A tripeptide of glutamate, cysteine and glycine
    • (γ-L-glutamyl-L-cysteinylglycine; GSH) > 90 %
  • Has four different acid dissociation with the following pK`s :
  • 1. pK = 2.05 (glutamic acid) 2. pK = 3.40 (COOH, glycine)
  • 3. pK = 8.72 (-SH) 4. pK = 9.49 (amino group)
glutathione gsh1
Glutathione (GSH)
  • Most abundant reductive thiol in cells
  • Serves as an antioxidant for the cells.
  • Bioreductive reactions
  • enzyme activity maintenance
  • Amino acid transport
  • Abnormally low levels in Cervical cancer, Diabetes, liver diseases
  • Over expressed in tissues
    • Alzheimer, Parkinson`s diseases
detection methods for gsh
Detection Methods for GSH
  • Mass Spectrometry
  • Fluorescence Spectroscopy

LOD = 16 nM

  • Electrochemical detection

LOD = 10 nM

difficulties in detecting glutathione
Difficulties in Detecting Glutathione
  • Interference of complex compounds
  • Sample preparation
  • Derivatization
  • Sensitivity
    • e.g. enzymatic
  • Poor Reproducibility
  • Low enhancement factor – Raman detection
slide8
Development of a Heat-Induced Surface-EnhancedRaman Scattering Sensing Method for RapidDetection of Glutathione in Aqueous Solutions

Genin Gary Huang, Xiao X. Han, Mohammad Kamal Hossain, and Yukihiro Ozaki

Anal. Chem. 2009, 81, 5881–5888

raman effect
Raman Effect
  • Discovered in 1928 by Indian physicist C. V. Raman
  • Light inelastic scattering process; occurs at wavelengths that differ from that of incident light
  • Vibrational changes
slide10

Theory of Raman Spectroscopy

2

Lowest Excited

Electronic States

1

0

Virtual States

Stokes

λ>λ0

λ0

Anti-Stokes

λ<λ0

λ0

2

Ground State

Vibrational

Energy States

1

DE

0

surface enhanced raman scattering sers mechanism
Surface-Enhanced Raman Scattering (SERS) Mechanism
  • Enhancement of local electromagnetic field at a surface of metal.
          • EF=> x106
  • Chemical contribution due to the charge transfer between metal and sample molecule.

EF => x102

Incident light

SERS Signal

Molecule

Plasmons

metal

experimental
Experimental
  • Aluminum pan plates
  • 50 ml of 10 mM Citrate Buffer (pH = 4.0)
  • NIR laser (785 nm)
  • laser spot size (10 μm), Power (15 mW)
  • Exposure time (1 s)
  • Scanning Electron Microscopy (SEM)
preparation of the silver nanoparticles colloidal solution
Preparation of the Silver Nanoparticles Colloidal Solution

1% C6H5Na3O7(10 ml)

(3x) H2O

distilled

(0.5 L)

AgNO3

Soln.

AgNO3

(90 mg)

Hot plate

reduced

AgNP`s

colloidal

Soln

UV/vis spectrometer

Ice bath

characteristics silver colloidal nanoparticles1
Characteristics Silver Colloidal Nanoparticles
  • Absorption intensity
  • Absorption maximum wavelength
sers with different pretreatments
SERS with Different Pretreatments

GSH (10 μM) , Reduction 15 min ,

pH 4.0

a) Heat-Induced method (3 min) b) Dry film method (90 min) c) No Treatment d) Raman Spectrum 0.5 M GSH, no Ag Colloids e) Blank Test

sem images of gsh mixed with silver colloids
SEM Images of GSH mixed with Silver Colloids

No Pretreatment

Dry film method

Heat-Induced method

Blank Test

effects of the amounts of silver colloids
Effects of the Amounts of Silver Colloids

5 min to dry => 60 μL

10 min to dry => 100 μL

sers glutathione calibration
SERS Glutathione Calibration

Raman Intensity at

660 cm -1 (Arb. Unit)

concentration (μm)

slide25
Electrochemical Sensing Strategy for Ultrasensitive Detection of GSH by Using Two Electrodes and Two Complementary Oligonucleotides

Peng Miaoa, Lei Liua, Yongjun Niea, Genxi Li

Biosensors and Bioelectronics, 2009

three electrode system
Three Electrode system

Current supply

v

A

Counter

electrode

Working

electrode

Reference

electrode

chronocoulometry cc
Chronocoulometry (CC)

Anson plot

  • Electrode Surface Area
  • Diffusion Coefficients
  • Concentration
  • Adsorption
experimental1
Experimental
  • Electrochemical Analyzer, CHI660B (room temp.)
  • probe 1: 5`-HS-(CH2)6-TCCTATCCACCTATCC-3`
  • probe 2: 5`-HS-(CH2)6-TTTTTTTTGGATAGGTGGTACGA-3`
  • Three Electrode System
    • Gold electrode, saturated calomel and platinum auxiliary electrode
  • [Ru(NH3)6] 3+ used as electrochemical species
quantitative detection of gsh chronocoulometry
Quantitative Detection of GSH - Chronocoulometry

Anson plot

  • 0 pM, (b) 1 pM, (c) 10 pM, (d) 30 pM, (e) 50 pM,
  • (f) 80 pM, (g) 100 pM, (h) 200 pM, (i) 1000 pM
calibration curve for gsh concentration
Calibration Curve for GSH Concentration

y = 0.65809 + 0.00886x

r = 0.99634, 3σ = 0.4 pM

critique
Critique
  • No real world samples detected
  • Small dynamic range
  • Takes many hours
conclusion
Conclusion

Electrochemical Sensing

  • Relies on released DNA by GSH – Indirect method.
  • Amplification of Electrochemical signal by AuNPs.
  • Success in determination of GSH in fetal calf serum.

Heat-Induced SERS

  • Relies on heated GSH mix with Silver colloid solution.
  • With all the parameters optimized, it takes short detection time.
acknowledgments
Acknowledgments
  • Dr. Murray
  • Murray Research Group
  • Audience
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