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

Outline

• Background

• Methodology

• Results & Discussion

• Conclusion

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

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

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

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/GQDAcceptor: graphene oxide (GO)

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

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

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

Methodology

• Principle

• Procedures

Principle

Fluorescent dye: FAM/GQD

Quencher: GO

Cells for detection: MCF

7

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

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

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

Results

• FAM-GO FRET based essay

• GQD-GO FRET based essay

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

No

rmal

ized

FA

M E

m&

GO

Ab

s (a

.u.)

Wavelength (nm)

FAM-ssDNA Em

GO Abs

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

Flu

ore

sce

nt

inte

nsi

ty (

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

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

Flu

ore

scen

t in

ten

sity

(R

FU

)

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

FAM-GO system

Whole process for quenching and restoring fluorescent signal

• Detection time: Around 1 hour

• 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

No

rmal

ize

d G

QD

Em

& G

QD—

ssD

NA

Em(a

.u.)

Wavelength (nm)

GQD Em

GQD-ssDNA Em

GQD-GO system

0

2000

4000

6000

8000

10000

12000

14000

Flu

ore

scen

t in

tensi

ty(R

FU

)

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

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

• 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

No

rmal

ized

GQ

D E

m&

GO

Ab

s (a

.u.)

Wavelength (nm)

GO Abs

GQD-ssDNA Em

GQD-GO system

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

Flu

ore

scen

t In

ten

sity

(R

FU

)

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

Qu

ench

ing

Eff

icie

ncy

GO Concentration (µg/ml)

Quenching efficiency with different conc.GO

Calculate QE based on data

90% QE 800 µg/ml of GO

GQD-GO system

Whole process for quenching fluorescent signal

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

Flu

ore

scen

t in

ten

sity

(R

FU

)

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

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

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

Acknowledgement

• Special gratitude to supervisor: Dr. Mo Yang

• Appreciation on the research student SHI Jinyu

• Thanks for comment and advice from other supervisors

Thank You!

Q&A