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

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 MS3.7 SALDI MS1.3 LDI MS0.644 HPLC MS0.003

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 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 EE Ground State Lowest Excited Electronic States Virtual States 2 Vibrational Energy States 1 0 Stokes λ>λ 0 Anti-Stokes λ<λ λ0λ0 λ0λ0

Surface-Enhanced Raman Scattering (SERS) Mechanism Enhancement of local electromagnetic field at a surface of metal. »EF=> x10 6 Chemical contribution due to the charge transfer between metal and sample molecule. EF => x10 2 metal Molecule Plasmons 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 AgNO 3 (90 mg) (3x) H 2 O distilled (0.5 L) 1% C 6 H 5 Na 3 O 7 (10 ml) AgNO 3 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 PretreatmentDry 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 μL 10 min to dry => 100 μL

Effects of Drying Temperature

pH Effects in the SERS GSH Detection

Optimized Parameters ParametersOptimized value Dropped sample volume60 μL Drying temperature100 o C Citrate buffer concentration10 mM Reduction time15 min pH4.0

SERS Glutathione Calibration concentration (μm) Raman Intensity at 660 cm -1 (Arb. Unit )

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 A v 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-(CH 2 ) 6 -TCCTATCCACCTATCC-3`  probe 2: 5`-HS-(CH 2 ) 6 -TTTTTTTTGGATAGGTGGTACGA-3`  Three Electrode System  Gold electrode, saturated calomel and platinum auxiliary electrode  [Ru(NH 3 ) 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 = x r = , 3 σ = 0.4 pM

Determination of GSH in Fetal Serum SamplesGSH concentration detected (mM) Standard Concentration (mM) Relative error (%)

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 Hours1 – 100 pMSelective Heat-induced SERS 50 nMMinutes 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 ?