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Transcript of 1 Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand 2...
1Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand2Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC)Thailand Science Park, Klong luang, Pathumthani, 12120 Thailand
1
Selective and Sensitive Sensing of L-Aspatic Acid and L-Glutamic Acid
Employing Dansyl Functionalized Dextran
Weerachai Nasomphan1, Srung Smanmoo2 and Pramuan Tangboriboonrat1*
Advantages of L-Asp & L-Glu
Biosynthesis precursor
Amino acid metabolism
Human nutrition
Identifying diseases
Biological roles
2Okamoto H. et al. J Sep Sci 2005; 28: 522. Yen K. et al. Talanta 2007; 71: 2007.
H2N CH C
CH2
OH
O
CH2
C
OH
O
H2N CH C
CH2
OH
O
C
OH
O
(L-Asp) (L-Glu)
Acidic amino acid
High level in blood
Stroke Diabetic
L-Aspatic acid L-Glutamic acid
3
Disadvantages: Expensive Complicate Skillful Time consuming
STANDARD METHOD
FluorescenceChemosensor
Advantages: High selectivity* High sensitivity** Low cost Simple-to-use Naked-eye diagnostic tools
*,** depend on chemical property of sensor
UV-Vis HPLC ICP-MS
Standard Method Vs Chemosensor
4Ramon M. et al. Chem Rev 2003; 103: 11. Suslick KS. et al. Tetrahedron 2004; 60: 11133.
Signaling subunit
Binding subunit “Ionophore”
Amino Acid
Amino Acid
Fluorescence quenching/enhancement Color change
Interactions
• Acid-base interaction • Hydrogen bonding• Metal-ligand coordination• Van der Waals and physical interaction
Fluorophore
Design of Chemosensor
OC6H13C6H13O
C6H13O
OC6H13
OC6H13C6H13O
C6H13O OC6H13
n
Molecular Vs Polymeric Chemosensors
Molecular chemosensor
Advantages • High sensitivity • Solubility enhancement
Polymeric chemosensorConjugated system
Non-Conjugated system
Long Y. et al. Macromolecules 2009; 42: 6501.Malashikhin S. et al. J Am Chem Soc 2008; 130: 12546.
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OMe
Signal Enhancement
OOH
OH OHO
OOH
OH OHO
Dextran (D)
Non-Conjugated Polymer: Dextran Probe
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Acidic amino acids (AAs)
H2N CH C
CH2
OH
O
CH2
C
OH
O
H2N CH C
CH2
OH
O
C
OH
O
L-Asp L-Glu
Polymeric template: Dextran
(Water soluble template)
OOH
OH OHO
OOH
OH OHO
Oxidation
OHCOHC
O
OOHC
OHC
O
O
Dextran (D)
Oxidized Dextran
BindingUnit
O
O
O
O
N
OH OHOH
HN
S OO
N
O
O
O
O
N
OH OHOH
HN
S OO
N Dansyl Dextran Conjugate (DD)
Fluorophore
Signaling UnitS OO
HNNH2
N
Dansyl Hydrazine(Acidic Sensitive)
Interaction of Dextran Probe & Amino Acid
“ Fluorescent on ”
Hydrogen bondingCharge-Charge interaction
“ Fluorescent off ”
Internal Charge Transfer (ICT) Process
ICT
7Ohtake K. et al. J Control Release 2002; 82: 263.Yen K. et al. Talanta 2007; 71: 2007.
DD
H2N CH C
CH2
OH
O
CH2
C
O
O
Objective
To synthesize a polymeric chemosensor for L-Asp and L-Glubased on dansyl hydrazine dextran conjugate (DD) via fluorescence quenching
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O
O
O
O
N
OH OHOH
Dansyl Conjugated Dextran (DD)
HN
S OO
N
Non-Conjugated Polymer: DD
Synthetic scheme of dextran dansylhydrazine conjugate
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Molecular weight (kDa) = 2600Polydispersity index (Mw/Mn) = 1.33
OOH
OH OHO
OOH
OH OHO
NaIO4, 25 oC
Dextran (D)
O
O
O
O
N
OH OHOH
Dansyl Conjugated Dextran (DD)
S OO
HNNH2
N
HN
S OO
N
OHCOHC
O
OOHC
OHC
O
O
Oxidized Dextran
Dansyl Hydrazine
25 oC
OHCOHC
O
OOHC
OHC
O
O
Oxidized Dextran
Selectivity of DD
10
Fluorescence (FL) spectra of DD upon the addition of 20 amino acids
Conditions:
•[DD] = 5 × 10-6 mol L-1
•[Amino Acids] = 25 × 10-3 mol L-1
• 1% PBS buffer
• Ex = 275 nm.
(L-Asp) (L-Glu)DD
Fluorescence image
DD Sensitivity to L-Asp
FL change of DD in the presence of various concentrations of L-Asp
Conditions: • [DD] = 5 µM (1% PBS buffer)• [Amino Acids] = 2.5-25 mM
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Limit of detection (LOD)10 µM
DD + L-AspDD
DD Sensitivity to L-Glu
FL change of DD in the presence of various concentrations of L-Glu
Conditions: • [DD] = 5 µM (1% PBS buffer)• [Amino Acids] = 2.5-25 mM
12
Limit of detection (LOD)50 µM
DD + L-GluDD
Conclusions
The FL quenching of DD exhibited excellent degree of selectivity towards L-Asp and L-Glu.
A new FL sensor, Dansyl Hydrazine dextran conjugate (DD) was prepared to use as a selective polymeric chemosensor for amino acids.
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DD is highly soluble in water, it prompts the application for the intracellular sensing of L-Asp and L-Glu.
14
Acknowledgements
Prof. Dr. Pramuan Tangboriboonrat, Mahidol UniversityDr. Srung Smanmoo, BIOTEC
Faculty of Science, Mahidol UniversityBioresources Research Unit (BRU)