Ceyhun Ekrem. Kirimli, PhD.

Advisors: Dr. Wan Y. Shih1

Dr. Wei-Heng Shih2

1

Circulating DNA• Existence of nucleic acids in blood is first

discovered in 1948 by Mandel and Métais1

• In 1994, association of circulating DNA with cancer was discovered.2,3

• Circulating DNA diagnostic and even prognostic association with different cancer types

• Total number of estimated deaths in US related to the cancer types which may be diagnosed using circulating DNA is 400.000.

1.Mandel P. CR Acad Sci Paris. 2.Sorenson Gd Fau - Pribish DM, Pribish Dm Fau - Valone FH, Valone Fh Fau - Memoli VA, Memoli Va Fau - Bzik DJ, Bzik Dj Fau - Yao SL, Yao SL. 3.Vasioukhin V Fau - Anker P, Anker P Fau - Maurice P, Maurice P Fau - Lyautey J, Lyautey J Fau - Lederrey C, Lederrey C Fau - Stroun M, Stroun M.

Adopted from http://www.inostics.com/?page_id=39

2

Trans-renal DNA

• It was later found out that low molecular weight DNA fragments can actually pass through kidneys

1)Image adopted from: Green C, Huggett JF, Talbot E, Mwaba P, Reither K, Zumla AI. 2009;9(8):505-11.2) Su YH, Wang M, Brenner DE, Ng A, Melkonyan H, Umansky S, Syngal S, Block TM, Journal of Molecular Diagnostics. 2004 May;6(2):101-7.

3

Why Tr-DNA?

• Non-invasive• Cleaner than most body fluids (almost no

proteins etc.)• Enrichment of low molecular weight DNA• Large Volume

4

Clinical Applications• Prenatal diagnostics

Trisomies, disomies, gender detection etc…

• Tumor detection & monitoring (almost all markers are applicable to cf-DNA is also applicable to tr-DNA)

• Transplantation monitoring >10 biopsies in first year after surgery

• Infectious Diseases

5

Objective• To develop a method that can be applied to detection of transrenal

DNA Mutation• With attomolar (10-18 M) detection sensitivity1

• With specificity enough to detect mutant DNA in a background with abundant Wild Type DNA

• Can be Multiplexed• Most clinical conditions require detections from multiple loci for diagnosis

• Time requirement• Labeling, isolation, amplification can be time consuming

6

2) Ying-Hsiu Su, Mengjun Wang,Dean E. Brenner, Alan Ng, Hovsep Melkonyan, Samuil Umansky, Sapna Syngal, and Timothy M. Block, Journal of Molecular Diagnostics, Vol. 6, No. 2, May 2004

1) Frank Caruso, Elke Rodda, and D. Neil Furlong, Anal. Chem. 1997, 69, 2043-2049

Piezoelectric Plate Sensor (PEPS)

MPS= 3-mercaptopropyl trimethoxysilane

1) Image adopted from,Wei Wu, Ceyhun Kirimli, Wei-Heng Shih, Wan Y. Shih, Real-time, Biosensors and Bioelectronics,2) Qing Zhu, Wan Y. Shih, and Wei-Heng Shih, Mechanism of flexural resonance frequency shift of a piezoelectric microcantilever sensor during humidity detection, Appl.

Phys. Lett. 92, 183505 (2008)

1

Schematic of PEPS

Micrograph of PEPS

Highly sensitive piezoelectric sensor due to piezoelectric Layer’s Young’s modulus change1 due to binding

Flexural resonance frequency shift was more than 300 times larger than could be accounted for by mass loading.2

7

Piezoelectric Plate Sensor (PEPS)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0-90

-75

-60

-45

-30 Width Extension ModeLength Extension Mode

Pha

se A

ngle

Frequency (MHz)1) Image adopted from,Wei Wu, Ceyhun Kirimli, Wei-Heng Shih, Wan Y. Shih, Real-time, Biosensors and Bioelectronics,2) Qing Zhu, Wan Y. Shih, and Wei-Heng Shih, Mechanism of flexural resonance frequency shift of a piezoelectric microcantilever sensor during humidity detection, Appl.

Phys. Lett. 92, 183505 (2008)

0 5 10 15 20 25 30

-2.0

-1.5

-1.0

-0.5

0.0

f/f

(x10

3)

Time (min)

Streptavidin

Impedance Spectrum of PEPS Detection Experiment

8

Accomplishments

1. Development of a signal processing algorithm to increase the sensitivity by reducing noise in the resonance spectrum to achieve 10-19 M LOD

2. Initial optimization of flow speed and temperature for in situ mutation detection using glass slides with fluorescent microspheres

3. Validation of specific mutation detection with reporter fluorescent microspheres

9

Accomplishments

4.In situ mutation detection with optimal temperature and flow speed with better than 1:250 MT/WT specificity in detecting both single mutation and double mutation

• Double\Single mutation

5.Development of in situ double-stranded target DNA mutation detection • Target DNA can be detected without the need for DNA isolation, concentration, and

amplification.

10

Sensitivity : Noise problem in data processing• Inability to detect low concentrations of analytes due to

noise in data.

Reason

1.Imprecise peak finding algorithm.2.Stationary window losing important data while peak shifts.

0 5 10 15 20 25 30

-2

-1

0

1

2

Freq

uenc

y C

hang

e (k

Hz)

Time (min)

[tDNA]=10-19M Control

11

More accurate peak finding algorithm

3.41 3.42 3.43-11.5

-11.0

-10.5

-10.0

-9.5

Pha

se A

ngle

Frequency (MHz)

Raw data Smoothed raw data Max. of raw data Max. of smoothed data

Window Size (kHz)3.41 3.42 3.43

-11.5

-11.0

-10.5

-10.0

-9.5

3.4195 3.4202 3.4209

-9.500

-9.498

-9.496

-9.494

Pha

se A

ngle

Frequency (MHz)

Pha

se A

ngle

Frequency (MHz)

Smoothed raw data Peak of fitted parabola Neighbourhood Fitted parabola

Polynomial fitting on data around the maximum of Raw Datacan be misleading

Solution: Polynomial fitting on data around maximum of smoothed raw data.

Statistical analysis on ≅35±10 polynomials per spectrum.

12

Moving window peak monitoring

3.43 3.44 3.45-34

-33

-32

-31

-30

-29

-28

Pha

se A

ngle

Frequency (MHz)

Scan @ t=0 min Scan @ t=30 min

3.40 3.41 3.42 3.43 3.44 3.45 3.46-29.4

-29.2

-29.0

-28.8

-28.6

-28.4

-28.2

-28.0

-27.8

Pha

se A

ngle

Frequency (MHz)

Scan @ t=0 min Scan @ t=30 min

Real peak position will not be lost Important data to determine the real peak position will remain in

the spectrum

13

Battery Powered Impedance Analyzer

14

Model Study: Specificity15

Effect of Laminar Flow and Temperature on Specificity

0 2 4 60

5

10

15

20

25

30

35

SM

T/SW

T

Flow Rate (ml/min)

Room Temp. 30oC 35oC

Increasing the temperature alone, SMT/SWT, 11 12 at 35C. With flow, SMT/SWT increased dramatically.

16

Effect of Laminar Flow and Temperature on Specificity

• SMT/SWT 24 at 4 ml/min > SMT/SWT at any flow rate at 35C,

• Optimal detection conditions MT occurred at 30C and a flow rate of 4 ml/min. (Hepatitis B Virus 1762T/1764A double mutation)

0 2 4 60

5

10

15

20

25

30

35

SM

T/SW

T

Flow Rate (ml/min)

RT 30oC 35oC

17

Validation: 2 color FRM hybridization Scheme

18

DNA Marker (Double Mutation)

19

Detection of Mixture of MT and WT tDNA

0 10 20 30 40 50 60-1.2-1.0-0.8-0.6-0.4-0.20.0

100 zM 1 aM 10 aM 100 aM

f/f

(10-3

)

Time (min)

tDNA FRM

0.15 0.30 0.45 0.60 0.750

10

20

30

40

50

60

70

10 aM

# FR

Ms

f/f (10-3)

1 aM

100 aM

100 zM

100 zM 1 aM

10 aM 100 aM

MT FRM

WT FRM

20

Switching from Double Mutation to Single Mutation

Problem : Melting Temperature (Tm) difference between perfect and mismatch decreases making it more difficult to distinguish MT and WT tDNA hybridizations.

30 40 500

25

50

75

100

tm1

% o

f Den

atur

ed D

NA

Temperature (oC)

Perfect Match Single Mismatch Double Mismatch

tm2

21

Locked Nucleic Acids

Locked Nucleic Acids increase the melting temperature difference between perfectly matching and mismatching target DNA sequences

DNA

22

Detection of Mixture of MT and WT tDNA

0 10 20 30 40 50 60

-1.8-1.5-1.2-0.9-0.6-0.30.0

f/f

(10-3

)

Time (min)

100 zM 1 aM 10 aM 100 aM

tDNAmix. FRM

0.0 0.2 0.4 0.6 0.8 1.0 1.20

15

30

45

60

75

90

# FR

Ms

f/f(10-3)

100 zM1 aM

10 aM

100 aM

MT FRM

WT FRM

100 zM 1 aM

10 aM 100 aM

23

Denaturation of dsDNA• Problem

• Most of tr-DNA is in double stranded form• PEPS detection depends on hybridization requiring ssDNA

• Solution

Flow Cell with

PEPS

Incubator

Water Bath

Boiled Sample

T1=95oCT2=21oC-63oC

L

Flow Cell with capture

DNA

24

Capture Probe

• Capture probes hybridize to complementary tDNA sequences

HeatssDNA

Capture DNAImmobilized on

Gold coated Glass Slides

Probe DNA immobilized

PEPS

Complementary tDNAtDNA

25

Detection of Double Stranded DNA

0 10 20 30 40 50 60

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

f/f

(10-3

)

Time (min)

100 zM 1 aM 10 aM 100 aM

MT tDNA FRM

0 10 20 30 40 50 60

-0.20

-0.15

-0.10

-0.05

0.00

f/f

(10-3

)Time (min)

10 aM 100 aM 10 fM 100 fM 1 pM

WT tDNA FRM

26

Detection of Cell Culture Extracted DNA

0 10 20 30 40 50 60

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

Time (min)

100 zM 1 aM 10 aM 100 aM

f/f

(10-3

)

tDNA FRM

0.0 0.2 0.4 0.6 0.80.0

0.3

0.6

0.9

1.2

1.5

1.8

f/f FR

M(1

0-3)

f/ftDNA

(10-3)

100zM1 aM

10 aM

100 aM

10-19 10-18 10-17 10-16

10

20

30

40

# FR

Ms

tDNA Concentration (M)

100 zM 1 aM

10 aM 100 aM

27

Conclusions• Peak Determination method developed allowed detection of <60

copies/ml tDNA hybridization by reducing the noise in the resonance spectrum

• Laminar flow and Temperature is optimized to increase the specificity to allow hybridization of 15 fold more FRMs after tDNA hybridization with a MTtDNA:WTtDNA 1:107

• By introducing flow, with minimal need to increase the temperature, specificity is maximized

• Detection of 60 copies/ml of single stranded Mutant tDNA is achieved using DNA probes on a background of 250 and 1000 fold more WT DNA in double/single mismatch mixture experiments• More importantly, unambigous confirmation of detection is achieved by 2 color FRM hybridization

method with a 80% MT FRMs in mixture experiments.

28

Conclusions• Fast cooling method is developed to detect ds-tDNA

• 87% of efficiency in detecting tDNA from ds-DNA is achieved using capture DNA

• Double stranded DNA of double/single mutations are detected with a limit of detection of 60 copies/ml.

• Cell line extracted DNA detected at 60 copies/ml LOD

• It has been shown that probes can be designed to allow for multiplexed detections of very similar tDNA fragments simultaneously with no cross hybridization.

29

Potentiostat Assisted FTIR

30

Potentiostat Assisted FTIR

31

Wave Number (cm-1)

1000 1500 2000 2500 3000 3500 4000

R/R

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

Experiment 4 Normalized Spectra

-200 mV

900 mV

-100 mV

32

DNA marker

33

Effect of Cooling Rate and Capture Probes on Recovery of ssDNA

87 % recovery with respect to ssDNA77% recovery without capture DNA

0 5 10 15 20 25 30

-0.6

-0.4

-0.2

0.0

Slow, Capture (-) Slow, Capture (+) Moderate, Capture (-) Fast, Capture (-) Moderate, Capture (+) Fast, Capture(+) ssDNA

f/f

(10-3

)

Time (min)Slow Moderate Fast

0

25

50

75

100

Capture DNA (+) Capture DNA (-)

(f/f

) dsD

NA/(

f/f) ss

DN

A

Cooling Rate

34

Detection of Mixture of MT and WT tDNA

MT FRM

WT FRM

0.0 0.2 0.4 0.6 0.80.00.30.60.91.21.51.82.1

100 aM

f/f FR

M(1

0-3)

f/ftDNA

(10-3)

100 zM

1 aM

10 aM

10-19 10-18 10-17 10-160

20

40

60

80

100

% #

MT

FRM

MT tDNA Concentration (M)

100 zM 1 aM

10 aM 100 aM

35

Detection of Double Stranded DNA

10-18 10-16 10-14 10-12

0.0

0.2

0.4

0.6

0.8

MT tDNA WT tDNA

f/f

(10-3

)

tDNA Concentration (M)

0

50

100

150

200

250

300

(f/f

) MT/(

f/f) W

TtDNA Concentration (M)

10-17 10-16

36

Standard method of Tr-DNA Detection• Polymerase Chain Reaction

PROS CONS

Unmatched in sensitivitySingle molecule/reaction

Requires right method of nucleic acid isolation

Loss of low molecular weight fragmentsPCR amplicon size

Potential PCR inhibition by co-isolated factors.

37

Detection of Mixture of MT and WT tDNA

100 zM 1 aM

10 aM 100 aM

MT FRM

WT FRM

0.0 0.1 0.2 0.3 0.4 0.5 0.6

0.2

0.4

0.6

0.8

1.0

1.2

f/f FR

M (1

0-3)

f/ftDNA

(10-3)

10-19 10-18 10-17 10-16 10-150

20

40

60

80

100

% #

MT

FRM

MT tDNA Concentration (M)

MT:WT = 1:250

38

Blocking of non-specific binding in urine

• Use of Bovine Serum Albumin (BSA) before detection• Non-specific binding was not observed only when PEPS is pre-

treated with at least 3% BSA before being immersed in urine

0 10 20 30 40 50 60-1.6

-1.2

-0.8

-0.4

0.0

Washing

No BSA 1% BSA 2% BSA 3% BSA

Freq

uenc

y S

hift

(kH

z)

Time (min)

Urine

• Urine contains urea, chloride, sodium, potassium, creatinine, very low amounts of peptides and proteins and other organic molecules

39

Other Platforms• No other platform on direct detection from urine.

 Method Sensitivity Disadvantages AdvantagesQuartz Crystal Microbalance .1 fM1,2

Low Sensitivity Time consuming (4

Hrs.)1,2

Low cost3

Surface Plasmon Resonance 1 fM4

Expensive, Low sensitivity5

Can be multiplexed,5

9.5 min-1.5 Hrs.6  

Carbon Nanotubes 35 fM7 Low Sensitivity Low cost8

Piezoelectric Microcantilevers 10pM9 Low Sensitivity Inexpensive

Atomic Force Microscopy Attomolar10

Expensive Equipment

High Sensitivity

Electrochemical Attomolar11Time consuming (4

Hrs.)6High Sensitivity

1) Liu T, Tang J, Jiang L (2002) Biochem Biophys Res Commun 295:14–16 2) Liu T, Tang J, Jiang L (2004) Biochem Biophys Res Commun 313:3–73) Sung-Rok Hong, Hyun-Do Jeong, Suhee Hong, Talanta 82 (2010) 899–903 4) D’Agata R, Corradini R, Grasso G, Marchelli R, Spoto G (2008) ChemBioChem 9:2067–20705) S. Paul P. Vadgama A.K. Ray, IET Nanobiotechnol., 2009, Vol. 3, Iss. 3, pp. 71–80 6) Laura Maria Zanoli, Roberta D’Agata, Giuseppe Spoto, Anal Bioanal Chem (2012) 402:1759–17717) S. Niu, M. Zhao, R. Ren, S. Zhang, J. Inorg. Biochem. 103 (2009) 43. 8) Alexander Star, Eugene Tu, Joseph Niemann, Jean-Christophe P. Gabriel, C. Steve Joiner, and Christian Valcke, PNAS January 24, 2006 vol. 103 no. 4 921–9269) Su M, Li SU, Dravid VP. Appl Phys Lett. 2003;82(20):3562-4. doi: Doi 10.1063/1.1576915. PubMed PMID: ISI:000182823300062. 10) Husale S, Persson HHJ, Sahin O. DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.11) Hu K, Lan D, Li X, Zhang S (2008) Anal Chem 80:9124–9130

40

• Attomolar (10-18M) level detection sensitivity• Real Time Detection of 1.6x10-18M target DNA sequences in

PBS by monitoring the first longitudinal extension mode (LEM) resonance frequency shift of the PEPS1

• Multiplexed Detections • Array PEPS are used for in situ, real-time, all-electrical

detection of Bacillus anthracis (BA) spores in an aqueous suspension using the first longitudinal extension mode of resonance.2

• Label-free detection in complex body fluids • Highly sensitive detection of HER2 extracellular domain in

the serum of breast cancer patients by piezoelectric microcantilevers.3

41

1) Wei Wu, Ceyhun Kirimli, Wei-Heng Shih, Wan Y. Shih, Real-time, Biosensors and Bioelectronics,2) McGovern JP, Shih WH, Rest RF, Purohit M, Mattiucci M, Pourrezaei K, Onaral B, Shih WY. The review of scientific instruments3) Loo L, Capobianco JA, Wu W, Gao X, Shih WY, Shih WH, Pourrezaei K, Robinson MK, Adams GP.

PEPS

Conclusions

• Detection of < 60 copies/ml is possible (SNR >3)

• SNR ratio increased at least 5 fold

0 5 10 15 20 25 30 35-2

-1

0

1

2

Freq

uenc

y S

hift

(kH

z)

Time (min)

Control 10-19MtDNA

0 5 10 15 20 25 30-0.10-0.08-0.06-0.04-0.020.000.02

Control 10-19M tDNA

Freq

uenc

y S

hift

(kH

z)

Time (min)

f=100 Hz

Single Parabola Peak Determination

10-18 10-16 10-14 10-12 10-10 10-8

100

101

102

SN

R

tDNA Concentration (M)

Peak Determination Single Parabola Raw

Threshold for Detection (SNR = 3)

42

Conclusions (Simulations)

0.3 0.6 0.9 1.2 1.5 1.8-2

0

2

4

6

8

10

12

|f s-

f a|/f a(%

)f

a/f(10-3)

50zM

0 5 10 15 20 25 30

3.456

3.459

3.462

3.465

Freq

uenc

y (M

Hz)

Time (min)

Peak positions determined by data analysis

Real peak positionsfReal

fcalculated

At most 12% error in the lowest concentration is estimated by simulations

43

Preliminaries• Multiplexed Simultaneous Detection of kRas 6 different

codon 12 mutations using Array PEPS

44

Probes and Tms

• Detections done at 63 oC• Tms calculated using salt adjusted values and nearest

neighbor algorithm for the mismatch types1,2

Mutation Tm (Mt, Perfect Match) oC Tm (WT, Mismatch), oC Difference, oC

GGTAGT 68 52.7 15.3GGTCGT 71 50.1 20.9GGTTGT 69 53.3 15.7GGTGAT 70 54.7 15.3GGTGCT 72 51.1 20.9GGTGTT 70 54.3 15.7

1) J. SantaLucia, Jr., Proceedings of the National Academy of Sciences of the United States of America, 1998, 95, 1460-1465.2) Yong You, Bernardo G. Moreira, Mark A. Behlke and Richard Owczarzy, Design of LNA probes that improve mismatch discrimination, Nucleic Acids Research, 2006,

Vol. 34, No. 8

45

Results

• No cross hybridization is observed at 63 oC, tDNA (10-15M) concentration

• Multiplexed single mismatch tDNA detection in urine at 10-15M

T1= Target DNA Complementary to Probe 1

46

Challenges • Urine contains urea, chloride, sodium, potassium, creatinine, very low amounts of peptides and proteins and other organic molecules

• Non-specific binding in urine decreases the sensitivity of any detection scheme in this complex environment

• Specificity enabling separation of single base difference in hybridization is required.

Probe DNATarget DNA

Non-specifically binding molecules, ions, proteins, etc…

1) Ying-Hsiu Su, Mengjun Wang,Dean E. Brenner, Alan Ng, Hovsep Melkonyan, Samuil Umansky, Sapna Syngal, and Timothy M. Block, Journal of Molecular Diagnostics, Vol. 6, No. 2, May 2004

47

Challenges• DNA in urine is double stranded

• Detection based on hybridization, requires single stranded DNA.• Denatured DNA can renature to form dsDNA

48