1. BME 4153-Independent project
Graphene oxide based fluorescence
resonance energy transfer biosensor
for breast cancer cells detection
HU RUIQI
11809482D
Supervisor: Dr. Mo Yang
April 2015
3. Background
• Cancer: the uncontrolled growth and spread of cells
(World Health Organization)
• Breast cancer: 3rd of top 10 cancers
(Hospital Authority, 2012)
• Severe threats & High mortality rate
• High significance and urgency for early diagnosis
4. Background
• The main cause of cancer deaths: metastasis
• leading cause of more than 90% of breast cancer
(Kanwar & Done, 2011)
• Circulating tumor cells (CTCs): main promoters of metastasis (Costa
et al., 2014)
• Released from the primary tumor into the bloodstream
• Detection of CTCs is actual purpose
• In this project: MCF-7 cells were used
5. Background
• Traditional method: Cancer screening tests
• Detect cancer before occurrence of symptoms
• Low accurate, low sensitive and not convenient
(Croswell et al., 2009)
• GO based biosensors were designed
• GO: nanomaterial
• Excellent optical and chemical properties
• High sensitivity and a significant part of biosensor
6. FRET phenomenon
• Fluorescence resonance energy transfer
• Photo excitation energy is transferred from a donor fluorophore to an accepter
molecule ( Morales-Narvaez and Merkoci, 2012)
• http://www.molecularbeacons.org/toto/Marras_energy_transfer.html
• Factors affecting on FRET:
• Distance
• Overlapping spectra
http://www.biomed.mtu.edu/StudentProjects/FRET.htm
In this project:
Donor: FAM/GQD
Acceptor: graphene oxide (GO)
7. Graphene oxide
• One-atom-thick nanostructure
• Has aromatic rings and binds with other molecules via π to π
stacking
• quenching capability
• In this project: GO acts as quencher
• http://www.graphenea.com/products/graphene-oxide
8. Aptamers
• Synthetic short sequences of DNA or RNA
• Bind to non-nucleic acid targets with high affinity and specificity
• More stable chemically and thermally
• A kind of Aptamer was selected: specifically recognize biomarker
on the MCF-7 cells and strongly bind with the cells
9. Objective
• To design and fabricate two types of GO based FRET biosensors
• FAM(fluorescent dye)-GO system
• GQD(fluorescent dye)-GO system
• To characterize the quenching efficiency of GO and the detection time of
detection for breast cancer cells
• To measure the sensitivity of the biosensor for breast cancer cells
12. Procedure:
• Step 1: Preparation
• FAM-aptamer was purchased from a company
• GQD-aptamer: conjugation of GQD with aptamers assisted by
EDC/NHS
• Step 2: Dilution
• Dilute GO into different concentrations
• Add GO & FAM/GQD-aptamer
into a container
Aptamer GQD GQD-Aptamer
13. Procedures
• Step 3: Obtaining FI & QE
• FAM-DNA: 465nm—520nm (Em:green)
• GQD-DNA: 360nm—465nm (Em:blue)
• Tecan Infinite F200 micro plate reader
• Step 4: Detection of MCF-7
• Add MCF-7 with different concentrations to determine detection limit
• http://www.news-medical.net/Infinite-200-PRO-NanoQuant-Microplate-Readers-from-Tecan
14. Signal Analysis
• Calculation of quenching efficiency
• F0’ =F0-background (PBS buffer)
• Fq’ =Fq-background
• QE (Quenching Efficiency) = (F0’-Fq’) / F0’
• Detection limit of MCF-7 were measured by plate reader
as well
16. FAM-GO system
• Overlapping spectra between GO and FAM-aptamerFRET occur
FAM-DNA emission and GO absorption spectra
0
0.2
0.4
0.6
0.8
1
200 300 400 500 600 700 800
NormalizedFAMEm&GOAbs(a.u.)
Wavelength (nm)
FAM-ssDNA Em
GO Abs
17. FAM-GO system
• Obtained directly from FI reading of plate
reader (data extract after 30 mins)
• High concentration low fluorescent intensity
0
5000
10000
15000
20000
25000
30000
35000
40000
0 50 100 150 200
Fluorescentintensity(RFU)
GO Concentration ug/ml
Fluorescent intensity of FAM-ssDNA with
different concentration of GO
Calculate QE based on data
90% QE 100 µg/ml of GO
18. FAM-GO system
• High concentration of
GO high restoration of
FI of FAM
• Detection limit: 103 /ml
• 0 to a few thousand CTCs
per 1-10 ml blood sample
and other biosensor: LOD
can go down to 102/ml
(Flores et al., 2010; Talasaz et al., 2009)
• FAM: organic molecule
not stable & detection limit is not
enough
• GQD: fluorescence
nanoparticle
• More stable0
500
1000
1500
2000
2500
3000
3500
4000
0 3 6 9 12 15 18 21 24 27 30 33
Fluorescentintensity(RFU)
Time/t
Restortation of fluorescent signal of various
concentration of MCF-7
8.3*10^4/ml
6.7*10^4/ml
8.3*10^3/ml
20. • Slight shift to right after conjugation with aptamers
• But FI increase dramatically after emitting UV light
0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600 700 800
NormalizedGQDEm&GQD—
ssDNAEm(a.u.)
Wavelength (nm)
GQD Em
GQD-ssDNA Em
GQD-GO system
0
2000
4000
6000
8000
10000
12000
14000
Fluorescentintensity(RFU)
GQD GQD-DNA
Fluorescent intensity of GQD and GQD-DNA
GQD-ssDNA emission and GQD emission absorption spectra
(Inset: GQD at the top and GQD-ssDNA at the bottom)
FI difference before/after conjugation
21. GQD-GO system
• UV-vis: GQD: 360nm DNA: 260nm
• GQD-ssDNA: two peaks shown to make sure successful conjugation
Normalized absorbance intensity of GQD, GQD-ssDNA and orginal DNA
260nm
360nm
22. • Overlapping spectra between GO and GQD-aptamerFRET occur
GQD-DNA emission and GO absorption spectra
0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600 700 800
NormalizedGQDEm&GOAbs(a.u.)
Wavelength (nm)
GO Abs
GQD-ssDNA Em
GQD-GO system
23. GQD-GO system
• Obtained directly from fluorescent intensity
reading of plate reader (data extract after 10
mins)
• High concentration low fluorescent intensity
0
500
1000
1500
2000
2500
0 500 1000 1500 2000
FluorescentIntensity(RFU)
GO Concentration (µg/ml)
Fluorescent Intensity with different conc.
GO
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 200 400 600 800 1000 1200 1400 1600
QuenchingEfficiency
GO Concentration (µg/ml)
Quenching efficiency with different conc.GO
Calculate QE based on data
90% QE 800 µg/ml of GO
25. GQD-GO system
• Conc. GO: 800 µg/ml
• 20 µl MCF-7: different concentration
• High concentration no restoration of FI
0
100
200
300
400
500
600
700
800
900
1000
0 0.5 1 1.5 2 2.5 3 3.5
Fluorescentintensity(RFU)
Time (hour)
Restoration of Fluorescent intensity after adding MCF-7 with
different conc.
9.0×104 /ml
6.8 ×104/ml
4.5 ×104/ml
2.3 ×104/ml
26. Possible reason
• FAM-GO system:
• Only aptamers are π to π
stacking with GO
• Fluorescent dye-FAM not bind
with GO
• However,
• GQD-GO system:
• GQD bind with GO via π to π
stacking
• Both aptamers & GQD bind with
GO
• much stronger binding than
aptamers with cells
• Solution:
Avoid binding between GQD and GO
27. Conclusion
• GO based FRET biosensors were designed
• FAM-GO FRET based essay:
• GO: 100 μg/ml to reach 90% QE
• Response time: 1 hour
• Detection of Limit: 103 /ml MCF-7
• GQD-GO FRET based essay:
• GO: 800 μg/ml to reach 90% QE
• Detection of MCF-7: not successful
• Reason: GQD can bind with GO via π to π stacking not only
aptamers
• Solution: passivation by PEG
28. Acknowledgement
• Special gratitude to supervisor: Dr. Mo Yang
• Appreciation on the research student SHI Jinyu
• Thanks for comment and advice from other supervisors
