Detection of Glutathione By Heat- Induced Surface-Enhanced Raman Scattering (SERS) and...
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![Page 1: Detection of Glutathione By Heat- Induced Surface-Enhanced Raman Scattering (SERS) and Electrochemical Sensing Literature Seminar Thabiso Musapelo 03-01-10.](https://reader036.fdocuments.in/reader036/viewer/2022062407/56649d555503460f94a3221c/html5/thumbnails/1.jpg)
Detection of Glutathione By Heat-Induced Surface-Enhanced Raman
Scattering (SERS) and Electrochemical Sensing
Literature Seminar
Thabiso Musapelo03-01-10
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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 “
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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
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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)
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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
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Detection Methods for GSH
• Mass Spectrometry
• Fluorescence Spectroscopy
LOD = 16 nM
• Electrochemical detection
LOD = 10 nM
LOD (µM)
MALD MS 3.7
SALDI MS 1.3
LDI MS 0.644
HPLC MS 0.003
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Difficulties in Detecting Glutathione
Interference of complex compounds
Sample preparation
Derivatization Sensitivity
– e.g. enzymatic Poor Reproducibility
Low enhancement factor – Raman detection
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Development of a Heat-Induced Surface-Enhanced
Raman Scattering Sensing Method for RapidDetection of Glutathione in Aqueous Solutions
Genin Gary Huang, Xiao X. Han, Mohammad Kamal Hossain, and Yukihiro OzakiAnal. Chem. 2009, 81, 5881–5888
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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
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Theory of Raman Spectroscopy
E
Ground State
Lowest Excited
Electronic States
Virtual States
2 VibrationalEnergy States
1
0
Stokesλ>λ0
Anti-Stokesλ<λ0
2
1
0
λ0λ0
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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
metal
MoleculePlasmons
Incident light SERS Signal
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SERS Instrumentation
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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)
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Preparation of the Silver Nanoparticles Colloidal Solution
AgNO3
(90 mg)
(3x) H2O distilled (0.5 L)
1% C6 H
5 Na3 O
7
(10 ml)
AgNO3
Soln.
Ice bath
Hot plate
reduced AgNP`s colloidal Soln UV/vis spectrometer
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Characteristics Silver Colloidal nanoparticles
10 x dilution
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Characteristics Silver Colloidal Nanoparticles
Absorption intensity
Absorption maximum wavelength
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SERS with Different Pretreatments
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
GSH (10 μM) , Reduction 15 min ,pH 4.0
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SEM Images of GSH mixed with Silver Colloids
No Pretreatment Dry film method
Heat-Induced method Blank Test
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Effects of Silver Particle Size
(60 min)
(15 min)
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Effects of the Amounts of Silver Colloids
5 min to dry => 60 μL10 min to dry => 100 μL
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Effects of Drying Temperature
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pH Effects in the SERS GSH Detection
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Optimized Parameters
Parameters Optimized value
Dropped sample volume 60 μL
Drying temperature 100 o C
Citrate buffer concentration 10 mM
Reduction time 15 min
pH 4.0
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SERS Glutathione Calibration
concentration (μm)
Ram
an In
ten
sity
at
660
cm
-1
(Arb
. Un
it)
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Electrochemical Sensing Strategy for Ultrasensitive Detection of GSH by Using Two
Electrodes and Two Complementary Oligonucleotides
Peng Miaoa, Lei Liua, Yongjun Niea, Genxi LiBiosensors and Bioelectronics, 2009
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Three Electrode system
Av
Current supply
Working electrode
Reference electrode
Counter electrode
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Chronocoulometry (CC)
• Electrode Surface Area
• Diffusion Coefficients
• Concentration
• Adsorption
Anson plot
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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
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Ultrasensitive Detection of GSH
GSH l
1
) MCH
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Ultrasensitive Detection of GSH
GSH AuNP
RuHex
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Quantitative Detection of GSH - Chronocoulometry
(a) 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
Anson plot
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Calibration Curve for GSH Concentration
y = 0.65809 + 0.00886x r = 0.99634, 3σ = 0.4 pM
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Determination of GSH in Fetal Serum
Samples GSH concentration detected (mM)
StandardConcentration (mM)
Relative error (%)
1 0.092 0.10 82 1.80 2.00 103 4.30 4.00 7.5
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Critique
No real world samples detected Small dynamic range Takes many hours
Detection Method
Detection limit
Detection duration
Dynamic Range
Selectivity
Electrochemical Sensing
0.4 pM Hours 1 – 100 pM Selective
Heat-induced SERS
50 nM Minutes 100-800 nM
Selective
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ConclusionElectrochemical 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.
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Acknowledgments
Dr. Murray Murray Research Group Audience
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Questions ?