Fluorescence and Absorption Spectroscopy of Bio-molecules

Ivanna Eusebe, René Cardona and Katherine Gil

Mentor: Ms. Cheng-Hui Liu

Steady State Laser Spectroscopy LabCity College of New York

High School Summer Research Program

CNNanoPhotonics CenterCN

NanoPhotonics Center

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.

E2 > E1

electron

http://hyperphysics.phy-astr.gsu.edu/hbase/hframe.html

Bio-molecules Investigated Table#1

Name Molecular Formula Molecular Weight Molecular Structure

  Deoxyadenosine  (A)

  C10H13N5O3

  251.24

  Deoxycytidine (C)

  C9H13N3O4

  227.22 

  Deoxyguanosine (G)

  C10H13N5O4

  595.2  

  Thymine (T)

  C10H14N2O5

  242.23

  Tryptophan

   C11H12N2O2

   204.23

  Tyrosine

   C9H11NO3

   181.19

 Phenylalanine

  C9H11NO2

  165.19

http://www.nature.com/nbt/journal/v19/n4/thumbs/nbt0401_360_F1.gif

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 Perkin-Elmer Lambda 9 UV/VIS/NIS Spectrometer we used to measure the absorption of the bio-molecules.

This is a picture of the C-Scan LDS-3 Fluorescence meter we used to measure the fluorescence 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

240 260 280 300 320 340 3600.0

0.2

0.4

0.6

0.8

1.0 293273256

Phenylalanine

Tryptophan

Tyrosine

Normalized Absorption Spectral Intensity of Amino Acids of

Phenylalanine, Tyrosine and Tryptophan Solution in Concentration 10-3M

FIG.1

Norm

aliz

ed A

bso

rban

ce

Wavelength (nm)

FIG. 2FIG. 2

260 280 300 320 340 360 380 400 420 440 460 480

0.0

0.2

0.4

0.6

0.8

1.0

Normalized Emission Spectral Intensity of Phenylalanine,

Tyrosine and Tryptophan Solution of Concentration 10-3M

Phenylalanine

Tyrosine

Tryptophane

FIG.2

357305287

Norm

aliz

ed E

mis

sion Inte

nsi

ty

Wavelength (nm)

260 280 300 320 340 360 380 400 420 440 460 480

0.0

0.2

0.4

0.6

0.8

1.0 295

Phenylalanine

FIG.3

305.5 335

No

rma

lize

d In

ten

sity

Wavelengths (nm)

Tryptophan

Tyrosine

Normalized Flourescence Spectral Intensity of Phenylalanine, Tryptophan and Tyrosine in Powder, Excitation Wavelengths at 240nm and 265nm

220 240 260 280 300 3200.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

FIG. 4

Thymidine

Deoxyguanosine

Deoxycytidine

Deoxyadonesine

Electronic Absorption Spectra of Deoxynucleotides-Monophosphorate (dAMP, dCMP, dGMP, TMP) in Water

Ab

sorb

an

ce

Wavelength (nm)

300 320 340 360 380 400 420 440 460 480 500

0

20

40

60

80

100

Thymine

Guanine

Cytosine

Adenine

FIG. 5

Emission Spectra of Deoxynucleotides-Monophosphorate (dAMP, dCMP, dGMP, TMP) in Water

Em

issi

on I

nten

sity

(a.

u.)

Wavelength (nm)

( excitation WL 260 nm)

300 320 340 360 380 400 420 440 4600

20

40

60

80

100

120

140

Thymine

Guanine

Adenine

Cytosine

329.5

346

358

312

FIG. 6

Emission Spectra of Deoxynucleotides-monophosphorate (dAMP, dCMP, dGMP, TMP) in Powder

Em

issi

on I

nte

nsi

ty (

a.u

.)

Wavelength (nm)

( excitation WL 240 nm)

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.

References1. 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

2. 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

3. http://en.wikipedia.org/wiki/Absorption_%28optics%29 4. http://en.wikipedia.org/wiki/Fluorescence5. 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