Post on 26-Dec-2015
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
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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
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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
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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
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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.
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Acknowledgements
Prof. Dr. Pramuan Tangboriboonrat, Mahidol UniversityDr. Srung Smanmoo, BIOTEC
Faculty of Science, Mahidol UniversityBioresources Research Unit (BRU)