TÜBİTAK Marmara Research Center, Chemical Technology Institute, TURKEY Selectivity of Amino Acid...

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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

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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

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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!

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• 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

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SerkanGÜRBÜZ

Dr. CemilDİZMAN

Dr. SerdarSEZER

Thanks…

• TÜBİTAK KAMAG 1007-110G085• Turkish National Police