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Fluorescence and Absorption
Spectroscopy of Bio-molecules
Ivanna Eusebe, René Cardona and
Katherine Gil
Mentor: Ms. Cheng-Hui Liu
Steady State Laser Spectroscopy Lab
City College of New York
High School Summer Research Program
Abstract
We have looked at the fluorescence and absorption
of the four bases of deoxyribonucleic acid (DNA),
and three amino acids (Phenylalanine, Tryptophan,
and Tyrosine). The purpose of this project was to
learn about the fundamental science behind disease
detection by using fluorescence and absorption.
This experiment can be used to find malignant and
benign forms of tumors and other cancerous
diseases.
Fluorescence
When a substance absorbs
radiation, atoms in the
substance are excited to a
higher energy level. The
atoms then emit light of
lower energy (longer
wavelength) and relax to the
ground state. The remainder
of the energy is released in
the form of heat.
Absorption
Absorption is the process in which a substance
absorbs or gathers incoming light. Light that is
not absorbed is either reflected or transmitted.
electron
E2 > E 1
http://hyperphysics.phy-astr.gsu.edu/hbase/hframe.html
Bio-molecules Investigated Table#1
Name
Deoxyadenosine
(A)
Molecular Formula
Molecular Weight
C10H13N5O3
251.24
Deoxycytidine
(C)
227.22
C9H13N3O4
Deoxyguanosine
(G)
C10H13N5O4
Thymine
(T)
C10H14N2O5
242.23
Tryptophan
C11H12N2O2
204.23
Tyrosine
C9H11NO3
181.19
Phenylalanine
C9H11NO2
165.19
595.2
http://www.nature.com/nbt/journal/v19/n4/thumbs/nbt0401_360_F1.gif
Molecular Structure
Procedures
1) The solution was inserted into a vial by:
a. measuring a certain amount of water (varying from 1mL to
4mL)
b. Then the DNA bases were mixed with powder and water
2) The solution was inserted into the vial and then the
narrowband filter (optional) was inserted into the
fluorescence meter.
3) Tests were run for the fluorescence and the
absorption.
4) The results were made into graphs using origin 5.0.
This is a picture of the C-Scan LDS-3
Fluorescence meter we used to
measure the fluorescence of the biomolecules.
This is a picture of the Perkin-Elmer Lambda
9 UV/VIS/NIS Spectrometer we used to
measure the absorption of the bio-molecules.
The differences between the two machines (not including the fact
that one measures fluorescence and the other measures absorption) is
that they collect data in different ways. For example if we wanted to
see the absorption we would run a background check to make sure
that the viles in which we put the bio-molecules would properly
allow light to travel through them. Whereas in fluorescence we do
not have to go through the previous procedure.
Experimental Results
Normalized Absorption Spectral Intensity of Amino Acids of
-3
Phenylalanine, Tyrosine and Tryptophan Solution in Concentration 10 M
Normalized Absorbance
1.0
293
273
256
Tryptophan
0.8
0.6
Tyrosine
0.4
Phenylalanine
0.2
0.0
240
260
280
300
320
Wavelength (nm)
340
360
FIG.1
Normalized Emission Spectral Intensity of Phenylalanine,
-3
Tyrosine and Tryptophan Solution of Concentration 10 M
Normalized Emission Intensity
1.0
287
305
357
0.8
Tryptophane
0.6
Tyrosine
0.4
Phenylalanine
0.2
0.0
260
280
300
320
340
360
380
400
Wavelength (nm)
420
440
460
FIG.2
FIG. 2
480
Normalized Flourescence Spectral Intensity of Phenylalanine, Tryptophan
and Tyrosine in Powder, Excitation Wavelengths at 240nm and 265nm
Normalized Intensity
1.0
295
305.5
335
0.8
Tryptophan
0.6
Tyrosine
0.4
Phenylalanine
0.2
0.0
260
280
300
320
340
360
380
400
Wavelengths (nm)
420
440
460
FIG.3
480
Electronic Absorption Spectra of Deoxynucleotides-Monophosphorate
(dAMP, dCMP, dGMP, TMP) in Water
1.4
Deoxyadonesine
1.2
Absorbance
Thymidine
1.0
Deoxycytidine
0.8
Deoxyguanosine
0.6
0.4
0.2
0.0
220
240
260
280
Wavelength (nm)
300
FIG. 4
320
Emission Spectra of Deoxynucleotides-Monophosphorate
(dAMP, dCMP, dGMP, TMP) in Water
( excitation WL 260 nm)
Emission Intensity (a.u.)
100
Adenine
80
Cytosine
60
Guanine
40
Thymine
20
0
300
320
340
360
380
400
420
Wavelength (nm)
440
460
480
FIG. 5
500
Emission Spectra of Deoxynucleotides-monophosphorate
(dAMP, dCMP, dGMP, TMP) in Powder
Emission Intensity (a.u.)
140
( excitation WL 240 nm)
312
120
Cytosine
100
Adenine
80
358
Guanine
346
60
Thymine
40
329.5
20
0
300
320
340
360
380
400
420
440
460
Wavelength (nm)
FIG. 6
Peak Importance
The peak is important to our research because the peak tells us at
what wavelength the bio-molecules fluoresce. Knowing at what
wavelength it fluoresces is important because then we have a
control sample to compare and contrast whether a tissue is
cancerous or not. If the tissue is cancerous, we can determine the
intensity or severity of the it.
Future Work
•
Our future goal is to learn about the Stokes Shift.
• We also would like to conduct experiments that would hopefully
lead to better and faster detection of cancerous diseases.
Conclusion
• We learned how to find the fluorescence and
absorption of different materials.
• We learned how to use many different kinds of
spectrophotometers (and spectrometers).
• We also learned how this experiment is
beneficial to today’s society and how it is
associated with cancer detection.
Discussion
After doing the entire experiment and looking over
the graphs we can see the differences in each of the
Amino Acids and DNA bases excitation and emission
wavelength. Our graphs show all the bio-molecules at
a normalized fluorescence and absorption spectra. We
can also see that when the bio-molecules are a
powder it shows a scattered curve, because it is
harder for the light to go through the powder. In a
solution the light can easily travel through it because
the solution is a liquid.
References
1.
2.
3.
4.
5.
R.R. Alfano, and Yuanlong Yang, “Stokes Shift Emission Spectroscopy of human tissue and
key Biomolecules” IEEE Journal of Selected Topics In Quantum Electronics. Vol. 9, No. 2,
2003
D. Onidas, D. Markovitsi,*S. Marguet, A. Sharonov, and T. Gustavsson*, “Fluorescence
Properties of DNA Nucleosides and Nucleotides: A Refined Steady-State and Femtosecond
Investigation” J. Phys. Chem. B 2002, 106, 11367-11374
http://en.wikipedia.org/wiki/Absorption_%28optics%29
http://en.wikipedia.org/wiki/Fluorescence
http://images.google.com/imgres?imgurl=http://www.stanford.edu/group/hopes/basics/dna/
Acknowledgements
•
Mentor: Ms. Cheng-Hui Liu
•
Doctor Sat
•
Professor R.R. Alfano
•
Dr. Peter Gross
•
Ms. Charlene Lee
•
Harlem Children Society
•
Mr. Gonzalez
•
Dr. Manuel Zevallos
•
NASA COSI