U.S. EPA 2004 Nanotechnology Science To Achieve Results (Star) Progress Review Workshop-Nanotechnology and the Environment II

Ultrasensitive Pathogen Quantification in Drinking Water Using High Piezoelectric PMN-PT Microcantilevers

200 µm100 µm

Wan Y Shih and Wei-Heng Shih

Dept Materials Science and Engineering

Drexel University

R829604August 18-20, 2004

E.coli 0157:H7

• E.coli 0157:H7 is the main cause of :– Foodborne illness

• Contaminated meat – improperly cooked beef• Lettuce• Unpasteurized milk and juice• Salami• Sprouts

– Waterborne illness• Creeks, rivers, streams, lakes, ground water,

portable water and swimming pools

E coli alone killed some 240 people in 2001!

Current pathogen detection technology

Sample water

filterWait for a day

Disadvantages:SlowNot real-time, in-situNot quantitative

Need: a sensor that is direct, sensitive, rapid, real-time, and in-situ

Dynamic Applications: Biosensors

nonpiezoelectric

piezoelectric

DESIGNDetection: DETECT MASS CHANGE

BY RESONANCE FREQUENCY SHIFTMeasurement: ELECTRICAL

-88

-86

-84

-82

-80

-78

18.8 19.0 19.2 19.4 19.6 19.8 20.0 20.2

f (KHz)

phas

e (d

eg)

in air0.04 mg0.09 mg

PHA

SE A

NG

LE (D

EG.)

FREQUENCY (kHz)

y = 2412.4xR2 = 0.9979

0

50

100

150

200

250

0 0.02 0.04 0.06 0.08 0.1

dm (mg)

df

(Hz)

FREQ

UEN

CY

SH

IFT(

Hz)

MASS CHANGE (mg)

Clamp

Impedance analyzer

PZT

Steel

Label freePortableRapid real-time detectionAirborne and in-liquid

Advantages:

Piezoelectric Microcantilever Biosensing

1 g/L Yeast Solution

-88

-87

-86

-85

-84

-83

-82

-81

16.5 17.0 17.5 18.0 18.5

f (KHz)

Phas

e (d

eg)

0 min10 min23 min35 min48 min61 min

0

50

100

150

0 20 40 60 80Time (min)

∆f

(Hz)

1 g/L

2 g/L2µm

Yeast Quantification

-87

-86

-85

-84

64 66 68 70 72

0 min

15 min

30 min

θ

frequency (kHz)0 10 20 30 40 50 60

0

200

400

600

800

1000

1200

∆f (

Hz)

time (min)

Protein Detection: Avidin

(a) (b)

0 10 20 30 400

50

100

150

200

250

300

−∆f (

Hz)

time (min)

Biotin-Avidin Binding Detection

Steel PZT

Clamp

0

100

200

300

400

500

0 10 20 3 0 4 0 50 60 70

time (min)−∆

f (H

z)

2µm

E coli Detection

Comparisons

Focusing Optics

Cantilever

Photodetector

Laser Diode

T. Thundat, E. A. Wachter, S. L. Sharp, and R. J. Warmack, Appl. Phys. Lett., 66, 1695 (1995).B. Ilic, D. Czaplewsli, H. G. Craighead, P. Neuzil, C. Campagnolo, and C. Batt, Appl. Phys. Lett., 77, 450 (2000).

Silicon microcantileverDisadvantages:

need a driver for actuation Need optical system for detectionQ factor ≈1 in water, in-water detection difficult Difficult for multiplexing

Silicon microcantilever Quartz Crystal Microbalance(QCM)Avidin binding on 3-mercatopropionic acid

0

200

400

600

800

1000

0 20 40 60 80

time (min)

∆f

(Hz) piezoelectric

cantilever

Cantilever of 0.7 mm long PZT with a 2.5 mm tip is already 16 times more sensitive in ∆f than the most common 5MHz QCM

Also difficult for multiplexing

QCM

QCM

2cm

Biophysical Journal,78, 487 (2000)

Mass Detection sensitivity

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛=≅

∆∆

ρρπν

~~

~12236.011

421

3

2 EwLM

fmf

hphsw

LPZT

steel ∆m

J. W. Yi, W. Y. Shih, W. –H. Shih, J. Appl. Phys., 91(3), 1680-1686 (2002).

1104

105

106

slope = -3

4mm wide, mode 14mm wide, mode 24mm wide, mode 32mm wide, mode 1

∆f/∆

m(ν

12 /

0.1 1 1010-3

10-4

10-5

10-6

10-7

10-8

10-9

10-10

10-11

10-12

without a tip with a tip Ilic et al. Thundat et al.

∆m

/∆f (

g/H

z)

L (mm)

At 100µm length, ∆m/∆f will approach 10-16 g/Hz !

Slope = -3

Objectives

To develop high piezoelectric microcantilever sensors for real-time, in situ ultrasensitive pathogen pathogen detection in water

Tasks:

To develop highly piezoelectric lead magnesium niobate-lead titante (PMN-PT) films for microcantilever miniaturization

To fabricate and characterize highly PMN-PT microcantileversensors

To use sub-millimeter PZT cantilevers for immediate pathogen detection development

Ecoli 057:H7 Detection and Releasein Ecoli Solution (100 mg/L)

0

100

200

300

400

500

0 10 20 30 40 50 60 70

time (min)

Attachment

PZT: 0.6 mm long, 2 mm wide, 127 µm thickstainless steel tip: 4 mm long, 2 mm wide, 50 µm thick

1 µm

In glycine solution (pH =2)

-400

-300

-200

-100

0

0 10 20 30 40

time (min)

∆fg

(Hz)

Release

Immobilization on glass, Ti, and Au

Gold

Glass

MPA in NHS+EDC

Antibody

Salmonella T.

PZT/Au-coated glass cantilever

Titanium

GOPTS

Antibody

Salmonella T.

PZT/Ti cantilever

Multiplexing

0 10 20 30 4012300

12400

12500

12600

12700

12800

Res

onan

ce F

requ

ency

(Hz)

T im e (m in)

Ab Immobilization Control (B lank)

GO

PTS

Con

trol

Antibody solution

Detection with a Control

-10 0 10 20 30 40 50 60 70-0.40

-0.35

-0.30

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

Pea

k D

evia

tion

(ft-f 0 in

kH

z)

Time (in minutes)

Ab Immobilization Control

-10 0 10 20 30 40 50 60 70

-0.25

-0.20

-0.15

-0.10

-0.05

0.00

Pea

k P

ositi

on C

hang

e (k

Hz)

Time (minutes)

-2500

-2000

-1500

-1000

-500

0

0 20 40 60 80

Time (Hz)

∆f (

Hz) EDC+NHS

∆f=100 HzAntibody

∆f=450 Hz

109 cells/mlSamonella T.

∆f=1500 Hz

Gold

Glass

MPA in NHS+EDCAntibody

Salmonella T.

PZT/Au-coated glass cantilever

Salmonella T

Salmonella detection on gold coated PZT/glass cantilever

0 5 10 15 20 25 30

-1600

-1400

-1200

-1000

-800

-600

-400

-200

0

1x104 cells/ml1x105 cells/ml

5x105 cells/ml

1x109 cells/ml

Res

onan

ce fr

eque

ncy

shift

(Hz)

Time (min)

109cfu/ml 105cfu/ml 104cfu/ml5x104cfu/ml

Salmonella detection on gold coated PZT/glass cantilever

104 105 106 107 108 1090

200

400

600

800

1000

1200

1400

1600

Res

onan

ce fr

eque

ncy

shift

(Hz)

Concentration (cells/ml)

Infection dosage

*Anal. Chem. 71, 3846 (1999), Anal Chem. 75, 5293 (2003)

Detection limit of ELISA,* optical based sensor*

0 5 10 15 200

20

40

60

80

100

120

140

MPA exposure to EDC+NHS

-∆f(H

z)

Time (min)

MPA exposure to EDC +NHS

Gold

Glass

MPA withNHS+EDC

10 MHz QCM*∆f=30 Hz

PZT/glass cantilever∆f=100 Hz

*Y. S. Fung* and Y. Y. Wong, Anal. Chem.2001, 73,5302-5309

0 5 10 15 20 25 300

100

200

300

400

500

Antibody im m obilization

-∆f(H

z)

T im e (m in)

Antibody immobilization detection

Gold

Glass

MPA with NHS+EDC

Antibody

10 MHz QCM*∆f=150 Hz PZT/glass cantilever

∆f=450 Hz*Y. S. Fung* and Y. Y. Wong, Anal. Chem.2001, 73,5302-5309

0 5 10 15 20 25 30

-350

-300

-250

-200

-150

-100

-50

0

Salmonella detection at 105 cells/ml

∆f(H

z)Time (min)

Salmonella T. at 105 cfu/ml

Salmonella detection at 105 cells/ml

Gold

Glass

MPA with NHS+EDC

Antibody

10 MHz QCM*∆f=40 Hz

PZT/glass cantilever∆f=300 Hz

*Y. S. Fung* and Y. Y. Wong, Anal. Chem.2001, 73,5302-5309

Summary for 0.4 mm long PZT/glass cantilever

Detection sensitivity: 3x10-11 g/Hz

C (cells/ml) 1x109 1x105 5x104 1x104

∆f 1400 Hz 300 Hz 180 Hz 90 Hz

∆m 42 ng 10 ng 6 ng 3 ng

Cellsneeded

3x105 4000 3000 1500

Vol. needed

4 µl 50 µl 70 µl 170 µl

Diffusiontype

Linear 3-d 3-d 3-d

Titanium

GOPTSAntibody

Salmonella T.

PZT/Ti cantileverBinding of

CSA-1

Antibody

to GOPTS

Binding of S.

Typhimurium

to Antibody

Brief Rinse in

BufferUn-Binding of S.

Typhimurium in

Glycine (pH =

2.5)

1.5 mm long PZT/Ti cantilever

Novel Coating Process for PMN-PT

Pb(Ac)2EG

Mg(OH)2 Nb2O5

500 600 700 800 900 1000

60

70

80

90

100

Soaking time: 2 hr

Bulk

den

sity

(%)

Sintering temperature (oC)20 25 30 35 40 45 50

20 µm thick PMN-PT tape

Inte

ns

ity

(a

.u.)

2θ (degree)

PMN-PT/Cu Microcantilevers

20 µmCu PMN-PT10 µm

Freestanding PMN-PT film

400 µm-40 -20 0 20 40

-40

-20

0

20

40

P (µ

C/c

m2 )

E (kV/cm)

1 cm

PMN-PT/Cu Microcantilevers

0 5 1 0 1 5 2 0 2 5 3 0

0

2 0

4 0

6 0

8 0

1 0 0

Tip

dis

pla

ce

me

nt

(µm

)

V o l t a g e ( v )

1 s t r u n ( 0 - 1 0 V ) 2 n d r u n ( 0 - 2 0 V ) 3 r d r u n ( 0 - 1 5 V ) 4 t h r u n ( 0 - 1 5 V ) 5 t h r u n ( 0 - 2 5 V ) 6 t h r u n ( 0 - 2 5 V ) *

( * : d e c r e a s e f r o m V m a x )

2 mm long PMN-PT/Cu cantilever

PMN-PTCuVDC

Laser displacement

meter

0 4 8 1 20

5 0 0

1 0 0 0

1 5 0 0

d3

1 (

10

-12m

/V)

E l e c t r i c f i e l d ( k V / c m )

1 s t r u n ( 0 - 1 0 V ) 2 n d r u n ( 0 - 1 5 V ) 3 r d r u n ( 0 - 1 5 V ) 4 t h r u n ( 0 - 1 5 V ) 5 t h r u n ( 0 - 2 5 V )

)tt(tEE)tt3t2t2(tEtE2tEtE

VL3hd

21121

212

22

122114

22

24

12

1231 +

++++⋅=

ZnO2 d31 < 10Bulk PMN-PT d31 ~ 250

PMN-PT single crystal cut in (100) d31 ~ 1500

R T

E

PMN-PT/Cu Microcantilevers

4 0 0 0 0 8 0 0 0 0 1 2 0 0 0 0

- 8 0

- 6 0

- 4 0

- 2 0

Ph

as

e A

ng

le (

de

gre

e)

F r e q u e n c y ( H z )

Peak 1

Peak 2

Peak 3

Q ~ 100

0.6 mm long PMN-PT/Cu cantileverPMN-PT 20 µm thick, Cu 5 µm thick

PMN-PTCuVAC

Humidity Sensing

2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3

6 9 3 0 0

6 9 3 5 0

6 9 4 0 0

6 9 4 5 0

6 9 5 0 0

6 9 5 5 0

6 9 6 0 0

6 9 6 5 0

2n

d P

ea

k F

req

ue

nc

y (

Hz

)

R e l a t i v e H u m i d i t y ( % )

b y d e s i c c a n t f l o w i n g H

2O / N

2 m i x t u r e

2nd peak

Slope=43 Hz/RH%

2 5 2 6 2 7 2 8 2 9 3 0 3 11 3 6 0 0 0

1 3 6 1 0 0

1 3 6 2 0 0

1 3 6 3 0 0

1 3 6 4 0 0

1 3 6 5 0 0

R e l a t i v e H u m i d i t y ( % )

b y d e s i c c a n t f l o w i n g H

2O / N

2 m i x t u r e

3rd peak

Slope=70 Hz/RH%

∆m/ ∆f ~10-12 g/Hz

Piezoelectric Microcantilever FabricationAntibodypad on Au

Au/Ti driving electrode Au/Ti sensing

electrode

SiO2PMN-PT/PZT

Al2O3

Si3N4

100 µm

PZT/Mo microcantilevers

1µm

-800 -600 -400 -200 0 200 400 600 800

-80

-60

-40

-20

0

20

40

60

80

Pr=27.5µC/cm2

Pmax=69µC/cm2

PZT (50 Layers)

P

olar

izat

ion

(µC

/cm

2 )

Drive Field (kV/cm)

Dielectric constant >1500

∆m/∆f = 10-15 g/Hz for 50 µm long cantilever

ConclusionsHigh piezoelectric PMN-PT films have been successfully made with

better than specially cut single-crystal piezoelectric properties

Array PMN-PT/Cu microcantilevers 500 µm long were made showing better than 10-12 g/Hz

we demonstrated in-situ real time detection of Salmonella in standingwater with PZT/glass and PZT/Ti cantilevers with various immobilization schemes

0.4 mm long PZT/glass cantilevers of 3x10-11 g/Hz sensitivity 3-4 times more sensitive than 10MHz QCM in protein/molecular detection, 6-7 times more sensitive than 10MHz QCM’s planar detection in cell detection

The better than 104 cfu/ml detection sensitivity comparable to a 10 MHz QCM, better than commercial ELISA, and tapered optical fiber sensors

Further sensitivity improvement includes using PMN-PT/Cu microcantilevers, using a flow cells

Ceramic Processing and Sensor Group

Students:

Q. Zhu, J.-P. McGovern, H. Luo, C. Martorano, Z. Shen, H. Li, H.D. Li, H. Yegingil, J. Bermudez, Y. Chiu,

Collaborators:

R. Mutharasan, I. Chaiken, N. Robertson, R. Rest, P. Loll, A. Saunders, A. Brooks, F. Garcia, E. Papazoglou, B. Onaral, R. Lec, K. Pourrezai, Compex Inc., ExxonMobil

R829604Other FundingNSF, NIH, NASA, NTI, DOT, PA