Transcript Detection of Glutathione By SERS and LSPR

Detection of Glutathione By HeatInduced 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.
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
• 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)
 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 (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
• Mass Spectrometry
LOD (µM)
MALD MS
3.7
SALDI MS
1.3
LDI MS
0.644
HPLC MS
0.003
• Fluorescence Spectroscopy
LOD = 16 nM
• Electrochemical detection
LOD = 10 nM
Difficulties in Detecting Glutathione
 Interference of complex compounds
 Sample preparation
 Derivatization
 Sensitivity
– e.g. enzymatic
 Poor Reproducibility
 Low enhancement factor – Raman detection
Development of a Heat-Induced SurfaceEnhanced
Raman Scattering Sensing Method for Rapid
Detection 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
• 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
Theory of Raman Spectroscopy
2
Lowest Excited
1
Electronic
States
0
Virtual
States
λ0
Ground
State
Stokes
λ>λ0
λ0
Anti-Stokes
λ<λ0
2 Vibrational
Energy
1
DE
States
0
Surface-Enhanced Raman Scattering (SERS)
Mechanism
• Enhancement of local electromagnetic field at a
surface of metal.
» EF=> x106
Incident light
SERS Signal
Plasmons
Molecule
metal
• Chemical contribution due to the charge transfer
between metal and sample molecule.
EF => x102
SERS Instrumentation
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
AgNO3
(90 mg)
(3x) H2O
distilled
(0.5 L)
AgNO3
Soln.
Hot plate
reduced
AgNP`s
colloidal
Soln
Ice bath
UV/vis spectrometer
Characteristics Silver Colloidal nanoparticles
10 x dilution
Characteristics Silver Colloidal Nanoparticles
Absorption intensity
Absorption maximum wavelength
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
No Pretreatment
Dry film method
Heat-Induced method
Blank Test
Effects of Silver Particle Size
(15 min)
(60 min)
Effects of the Amounts of Silver Colloids
5 min to dry => 60 μL
10 min to dry => 100 μL
Effects of Drying Temperature
pH Effects in the SERS GSH Detection
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
Raman Intensity at
660 cm -1 (Arb. Unit)
SERS Glutathione Calibration
concentration (μm)
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
Current supply
v
A
Counter
electrode
Reference
electrode
Working
electrode
Chronocoulometry (CC)
Anson plot
• Electrode Surface Area
• Diffusion Coefficients
• Concentration
• Adsorption
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
Ultrasensitive Detection of GSH
1
l GSH
) MCH
Ultrasensitive Detection of GSH
RuHex
GSH
AuNP
Quantitative Detection of GSH - Chronocoulometry
Anson plot
(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
Calibration Curve for GSH Concentration
y = 0.65809 + 0.00886x
r = 0.99634, 3σ = 0.4 pM
Determination of GSH in Fetal Serum
Samples GSH concentration
detected (mM)
1
0.092
2
1.80
3
4.30
Standard
Concentration (mM)
0.10
2.00
4.00
Relative
error (%)
8
10
7.5
Critique
 No real world samples detected
 Small dynamic range
 Takes many hours
Detection
Method
Detection Detection
limit
duration
Dynamic Selectivity
Range
Electrochemical
Sensing
0.4 pM
Hours
1 – 100 pM Selective
Heat-induced
SERS
50 nM
Minutes
100-800
nM
Selective
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
 Dr. Murray
 Murray Research Group
 Audience
Questions ?