Detection of Glutathione By Heat-Induced Surface-Enhanced Raman

Scattering (SERS) and Electrochemical Sensing

Literature Seminar

Thabiso Musapelo03-01-10

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

• 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

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 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

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

E

Ground State

Lowest Excited

Electronic States

Virtual States

2 VibrationalEnergy States

1

0

Stokesλ>λ0

Anti-Stokesλ<λ0

2

1

0

λ0λ0

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

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)

1% C6 H

5 Na3 O

7

(10 ml)

AgNO3

Soln.

Ice bath

Hot plate

reduced AgNP`s colloidal Soln UV/vis spectrometer

Characteristics Silver Colloidal nanoparticles

10 x dilution

Characteristics Silver Colloidal Nanoparticles

Absorption intensity

Absorption maximum wavelength

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

SEM Images of GSH mixed with Silver Colloids

No Pretreatment Dry film method

Heat-Induced method Blank Test

Effects of Silver Particle Size

(60 min)

(15 min)

Effects of the Amounts of Silver Colloids

5 min to dry => 60 μL10 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

SERS Glutathione Calibration

concentration (μm)

Ram

an In

ten

sity

at

660

cm

-1

(Arb

. Un

it)

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

Three Electrode system

Av

Current supply

Working electrode

Reference electrode

Counter electrode

Chronocoulometry (CC)

• Electrode Surface Area

• Diffusion Coefficients

• Concentration

• Adsorption

Anson plot

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

GSH l

1

) MCH

Ultrasensitive Detection of GSH

GSH AuNP

RuHex

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

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)

StandardConcentration (mM)

Relative error (%)

1 0.092 0.10 82 1.80 2.00 103 4.30 4.00 7.5

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

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.

Acknowledgments

Dr. Murray Murray Research Group Audience

Questions ?