tÜbİtak marmara research center, chemical technology institute, turkey selectivity of amino acid...
TRANSCRIPT
TÜBİTAK
Marmara Research Center, Chemical Technology Institute, TURKEY
Selectivity of Amino Acid Reactive Reagents and a Magic Way of Development of
Latent Finger Marks
Dr. Melek Erol
28-30 September 2015Forensic Research 2015, Atlanta
TÜBİTAK
TÜBİTAK
What is in a fingerprint?
Eccrine secretion• Water (>98%)• Amino acids• Proteins• Urea• Uric acid• Lactic acid• Sugars• Inorganic ions (Na+, K+, Cl-, Ca2+, etc.)
Sebaceous secretion• Fatty acids, • Glycerides, • Wax esters, • Squalene • Sterol esters
L-Serine
L-Glycine
L-Alanine
L-Aspartic acid
Major amino acids present in fingerprints
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Amino Acid Reactive Reagents
Ninhydrin 2-hydroxy-1,4-naphthoquinone(Lawsone)
Genipin5-hydroxy-1,4-naphthoquinone(Juglone)
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Ninhydrin• Predominant reagent for visualization of fingermarks on porous
surfaces for criminal investigation.• First synthesized and discovered to react with amino acids in 1910
by Siegfried Ruhemann.• Forms a non-fluorescent purple product with amino acids.• Suggested for detection of latent fingermarks on porous surfaces in
mid-1950s by Oden and von Hofsten.
2-amino-1,3-indanedioneR.Jelly et al., Analytica Chimica Acta, 2009, 652, 128-142.
Strecker degradation
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Fresh
Fresh –UV(365 nm)
Ninhydrin – Amino Acid Spot Reactions at RT
L-Serine L-Glycine L-Alanine
2 hours -UV
2 hours
ALA-10
GLY-10
SER-10
SER-10
GLY-10
ALA-10
ALA-1
GLY-1
SER-1
SER-1
ALA-1
GLY-1
2 hours –UV(365 nm) 6 hours –UV(365 nm)
6 hours
SER-10
SER-10
GLY-10
GLY-10
ALA-10
ALA-10
ALA-1
GLY-1
SER-1
SER-1 GLY-1
ALA-1
L-Aspartic acid L-Histidine L-Cysteine
Filter paper used as the substrate due to its low photoluminescent properties.
1 mM Ninhydrin in 1:1.2:10
Ethanol:Ethyl acetate:
HFE7100
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80°C
80°C –UV(365 nm)
ALA-10
ALA-10
SER-10
SER-10
SER-1
SER-1
ALA-1
ALA-1
GLY-10
GLY-10
GLY-1
GLY-1
Ninhydrin–Amino Acid Spot Reactions at Different T’s
GLY-10
ALA-10
SER-10
GLY-1
SER-1
ALA-1
GLY-10
GLY1SER-10
SER-1
ALA-10
ALA-1
105°C –UV(365 nm)
105°CRT
RT –UV(365 nm)
ALA-10
GLY-10
SER-10
SER-10
GLY-10
ALA-10
ALA-1
SER-1
GLY-1
ALA-1
GLY-1
SER-1
150°C
150°C –UV(365 nm)
GLY-10
GLY-10
SER-10
SER-10
ALA-10
ALA-10
ALA-1
GLY-1
SER-1
ALA-1 SER-1
GLY-1
2 h reaction time at specified T’s
1 mM Ninhydrin in 1:1.2:10 Ethanol:Ethyl acetate: HFE7100
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Fluorescent Emission of Ninhydrin–Amino Acid Products
500 550 600 650 700 750 800
-1,0x106
-5,0x105
0,0
5,0x105
1,0x106
Fluor
esce
nce I
nten
sity
nm
L-Cysteine L-Glycine L-Histidine L-Aspartik L-Alanine L-Serine
Ninhydrin-Amino Acid Spots 80C
500 550 600 650 700 750 800-2,50x105
0,00
2,50x105
5,00x105
7,50x105
1,00x106
Fluor
esce
nce I
nten
sity
nm
L-Cysteine L-Glycine L-Histidine L-Aspartic L-Alanine L-Serine
Ninhydrin-Amino Acid Spots 150C
500 550 600 650 700 750 800-1,0x106
-5,0x105
0,0
5,0x105
1,0x106
Fluor
esec
ence
Inte
nsity
nm
80C 150C
Ninhydrin-Aspartic Acid Reaction
λex. = 505 nm
HORIBA Jobin Yvon FluoroLog - TCSPC
Spectrofluorometer
At 150C Aspartic acid > Glycine > Histidine > Serine > Alanine > Cysteine
• Cysteine forms fluorescent
product right after treatment even
at RT!
• Histidine forms light purple color
right after treatment!
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Lawsone• Responsible molecule for the dying of henna.• Forms a fluorescent brownish product with amino acids without
further treatment.• Maximum luminescence around 600 nm with λex. of 530 nm.
R.Jelly et al., Analytica Chimica Acta, 2009, 652, 128-142.
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RT
RT –UV(365 nm) 80°C –UV(365 nm)
80°C
Lawsone–Amino Acid Spot Reactions at Different T’s
2 h reaction time at specified T’s
GLY1
GLY-10SER-10
ALA-10
GLY-10 SER-10
ALA-10
105°C
105°C –UV(365 nm)
GLY-1SER-1
ALA-1
SER-1 GLY-1
ALA-1
150 °C –UV(365 nm)
150°C
ALA-10
ALA-10
SER-10
SER-10
GLY-10
GLY-10
SER-1ALA-1
GLY-1
ALA-1SER-1
GLY-1
1 mM Lawsone in 1:20:20 DMF:Acetone:HFE7100
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550 600 650 700 750 8000,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
L-Histidine L-Cysteine L-Glycine L-Aspartic L-Alanine L-Serine
Lawsone-Amino Acid Spots 80C
550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
L-Histidine L-Cysteine L-Glycine L-Aspartic L-Alanine L-Serine
Lawsone-Amino Acid Spots 150C
550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
80C 150C
Lawsone-Cysteine Reaction
Fluorescent Emission of Lawsone–Amino Acid Products
At 150C Cysteine > Serine > Glycine > Aspartic acid Alanine > Histidine
• Slight fluorescence occurence
at 80C!
• Most densely color observed
with Histidine!
λex. = 530 nm
HORIBA Jobin Yvon FluoroLog - TCSPC
Spectrofluorometer
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Juglone• A dye for cloth and inks, and a coloring agent for foods and cosmetics.• Suggested for its potential fingermark detection on porous surfaces.• Maximum luminescence around 597 nm with λex. of 530 nm.
RT
150°C –UV(365 nm) 80°C –UV(365 nm) RT –UV(365 nm)
150°C 80°C
GLY-10
SER-10
ALA-10
GLY-10
GLY-10
GLY-1
GLY-1
SER-10
SER-10
SER-1
SER-1
ALA-10
ALA-10
ALA-1
ALA-1
GLY-10
SER-10
ALA-10
105°C
105°C –UV(365 nm)
SER-10ALA-10
GLY-1
SER-1 ALA-1
GLY-10
GLY-1
SER-1ALA-1
GLY-1
SER-1
ALA-1
ALA-1
SER-1 GLY-1
ALA-10
SER-10GLY-10
2 h reaction time at specified T’s
1 mM Juglone in 1:20:20 DMF:Acetone:HFE7100
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550 600 650 700 750 800-5,0x105
0,0
5,0x105
1,0x106
1,5x106
2,0x106
2,5x106
Fluor
esce
nce I
nten
sity
nm
L-Histidine L-Cysteine L-Glycine L-Aspartic L-Alanine L-Serine
Juglone-Amino Acid Spots 80C
550 600 650 700 750 800-1,0x106
-5,0x105
0,0
5,0x105
1,0x106
1,5x106
2,0x106
2,5x106
Fluor
esce
nce I
nten
sity
nm
L-Histidine L-Cysteine L-Glycine L-Aspartic L-Alanine L-Serine
Juglone-Amino Acid Spots 150C
550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
2,5x106
Fluor
esce
nce I
nten
sity
nm
80C 150C
Juglone-Serine Reaction
Fluorescent Emission of Juglone–Amino Acid Products
At 150C Serine > Histidine > Alanine > Glysine Aspartic acid > Cysteine
• Cysteine forms highly
fluorescent product at RT and it
diminishes with increasing T!
λex. = 530 nm
HORIBA Jobin Yvon FluoroLog - TCSPC
Spectrofluorometer
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Genipin• Extracted from plants of Gardenia jasminoides Ellis and Genipa
Americana.• Used as a traditional Chinese medicine, food and fabric colourants and
as skin dyes, natural protein cross-linker, has low cytotoxicity.
R.Jelly et al., Analytica Chimica Acta, 2009, 652, 128-142.
• Forms a fluorescent blue product with amino acids without further treatment.
• Maximum luminescence around 613 nm with λex. of 555 nm.
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Genipin–Amino Acid Spot Reactions at Different T’s
2 h reaction time at specified T’s
80°C 150°C 105°C
150°C –UV(365 nm) 105°C –UV(365 nm) 80°C –UV(365 nm)
ALA10
GLY10
ALA-10
GLY-10SER-10
SER-10
SER-1
GLY-10
GLY-1ALA-10
ALA-1
ALA-10
GLY-10
GLY-1SER-1
ALA-1
SER-10
ALA-10GLY-10
GLY-10SER-10
ALA-10
RT
RT- UV(365 nm)
SER-10
ALA-1SER-1
GLY-1
SER-1
ALA-1
GLY-1
GLY-1SER-1
ALA-1
SER-10ALA-10
GLY-10
ALA-1
GLY-1
SER-1
1 mM Genipin in 1:1.2:10 Ethanol:Ethyl acetate: HFE7100
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550 600 650 700 750 800
0
1x106
2x106
3x106
4x106
Fluor
esce
nce I
nten
sity
nm
L-Histidine L-Cysteine L-Glycine L-Aspartic L-Alanine L-Serine
Genipin-Amino Acid Spots 80C
550 600 650 700 750 800
0
1x106
2x106
3x106
4x106
Fluor
esce
nt In
tens
ity
nm
L-Cysteine L-Glycine L-Histidine L-Aspartic L-Alanine L-Serine
Genipin-Amino Acid Spots 150C
550 600 650 700 750 800
0
1x106
2x106
3x106
4x106
Fluor
esce
nce I
nten
sity
nm
80C 150C
Genipin-Aspartic Acid Reaction
Fluorescent Emission of Genipin–Amino Acid Products
At 150C Aspartic acid > Glycine > Histidine > Alanine > Serine >> Cysteine
• Strongest fluorescence
observed between genipin and
aspartic acid at 150°C!
λex. = 555 nm
HORIBA Jobin Yvon FluoroLog - TCSPC
Spectrofluorometer
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Comparison of Fluorescence of Active Reagents at 150°C
Ninhydrin Genipin 2-methoxy-1,4-naphthoquinone
Juglone Lawsone
Active reagent At 150C most reactive with
Fluorescence intensity
Genipin Aspartic acid 3.8 x 106
Juglone Serine 2.2 x 106
2-methoxy-1,4-naphthoquinone
Histidine 2.1 x 106
Lawsone Cysteine 2.0 x 106
1,2-naphthoquinone-4-sulfonate
Histidine 1.3 x 106
Ninhydrin Aspartic acid 7.7 x 105
365 nm, yellow filter
Active reagentFluorescence intensity with
Serine
Genipin 2.7 x 106
Juglone 2.2 x 106
2-methoxy-1,4-naphthoquinone
1.4 x 106
Lawsone 1.4 x 106
1,2-naphthoquinone-4-sulfonate
5 x 105
Ninhydrin 1.3 x 105
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Fluorescence of Amino Acids and Fingerprints due to Thermal Effects
365 nm250 300 350 400 450
0,0
2,0x105
4,0x105
6,0x105
8,0x105
1,0x106
Fluor
esce
nce I
nten
sity
nm
L-Alanine L-Aspartic L-Cysteine L-Glycine L-Histidine L-Serine
Amino acid spots at 80 C; ex= 250 nm
500 550 600 650 700 750 8000,0
2,0x105
4,0x105
6,0x105
8,0x105
1,0x106
Fluor
esce
nce I
nten
sity
nm
L-Alanine L-Aspartic L-Cysteine L-Glycine L-Histidine L-Serine
Amino acid spots at 80 C; ex= 505 nm
300 350 400 450
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
L-Alanine L-Aspartic L-Cysteine L-Glycine L-Histidine L-Serine
Amino acid spots at 150C; ex= 250 nm
500 550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
L-Alanine L-Aspartic L-Cysteine L-Glycine L-Histidine L-Serine
Amino acid spots at 150C; ex= 505 nm
550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
Fingerprint Genipin Fingerprint+Genipin
150C, ex=555 nm500 550 600 650 700 750 800
0,0
5,0x105
1,0x106
1,5x106
2,0x106
Fluor
esce
nce I
nten
sity
nm
Fingerprint Ninhydrin Fingerprint+Ninhydrin
150C, ex=505 nm
365 nm
530 nm, yellow filter
365 nm, UV filter
DFO heat
Fluorescence sensitizer
TÜBİTAK
How do fingermarks develop with thermal effect, without any chemical?
OOHO O
OH
OHO
HO OH
OH
OH
n
O OH
HO
OH
HO
Cel.O
H
R-NH2Amino acid
Amadori Product
O OH
HO
OH
Cel.
OHN R
O OH
OH
Cel.
OO
H
Rearranged Product
OH
O
HO
O
HO NH
R O
HO
O
O
OHH
Cel. Cel.O
HN
OHO
OH
HNR
R
O
HO
O
O
OHH
Cel.
A
A
+
O
N
HO
O
OH
N
R
R
OHOH
OH
OHO
N
HO
O
OH
N
R
R
O
OHMagic fluorescent
product!
TÜBİTAK
• All reagents gave fluorescence with amino acids and fingerprints at 150C.
• All naphthoquinone derivatives and genipin worked as fluorescence sensitizer.
• Highest fluorescence observed with genipin.
• Fluorescence of ninhidrin treated fingermarks at 150C mostly due to fluorescence of fingermark, ninhydrin not fluorescence sensitizer!
• Fluorescent product formation between cellulose surface and amino acids at high temperature!
Conclusions
TÜBİTAK
SerkanGÜRBÜZ
Dr. CemilDİZMAN
Dr. SerdarSEZER
Thanks…
• TÜBİTAK KAMAG 1007-110G085• Turkish National Police