Development of Microfluidic / Nanofluidic Sensors Using Catalytic DNA for Heavy Metal Detection

Tulika S. Dalavoy1,2, Paul W. Bohn4, Charles S. Henry3, Yi Lu2, Jonathan V. Sweedler2, Bruce Flachsbart2, Mark Shannon2, Irene MacAllister1, Donald M. Cropek1*

1Construction Engineering Research Laboratory (CERL), U.S. Army Corps of Engineers, Champaign, IL, USA; 2Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA; 3Colorado State University, Fort Collins, CO, USA, 4University of Notre Dame, Notre Dame, IN, USA

*Email: Donald.M.Cropek@usace.army.milER-1459, Poster 163

Abstract

Heavy metal detection is an important issue for environmental assessments, drinking water monitoring, and soldier health protection. In order to develop a reliable and sensitive device for in situ measurement of heavy metals (in this case, lead in water), this work employs catalytic DNA as the sensing moiety and microfluidic chips for fluid control and transport. On-chip electrophoretic separations remove contaminants and interferents from the Pb(II) band. The DNA reacts selectively with Pb(II) to produce a fluorescent signal. This device also contains nanoscale fluidic molecular gates that further manipulate fluid flows and perform molecular separations on tiny volumes of material. This device is the first step toward a robust real-time unattended field sensor capable of multianalyte detection with a single injection by intrachannel immobilization of different DNAzymes.

 Sensing Molecule

AA

GCT

G| C

A| T

T| A

G| C

A| T

G| C

A| T

A| T

G•T

G| C

rA A| T

T| A

A| T

T| A

C|

G

C|

G

A| T

T| A

-5’

-3’

3’-

5’-

C|

G

AA

GCT

G| C

A| T

T| A

G| C

A| T

G| C

A| T

A| T

G•T

G| C

rA A| T

T| A

A| T

T| A

C|

G

C|

G

A| T

T| A

-5’

-3’

3’-

5’-

C|

G

G| C

A| T

T| A

G| C

A| T

G| C

A| T

A| T

G•T

G| C

rA A| T

T| A

A| T

T| A

C|

G

C|

G

A| T

T| A

-5’

-3’

3’-

5’-

C|

G

Enzyme

Pb2

+

Cleavage Site

Catalytic DNA Strand

Lead (Pb2+) Specific Catalytic DNA

0

50

100

150

200

250

300

350

400

Pb Co Zn Mn Ni Cd Cu Mg Ca

vfl

uo

(co

un

ts/s

)

0

10000

20000

30000

40000

50000

0 100 200 300 400

time (s)

inte

ns

ity a

t 580 n

m (

co

un

ts)

Specificity of Pb(II) DNAzyme. Pb(II) shows highest activity, little interference from other cations in solution (top) and immobilized in PMMA device (bottom).

Depleted Uranium (UO22+) Specific Catalytic

DNA

Detection Limit: 45 pM = 11 ppt, lower than ICP-MS.

Over 1 million fold selectivity.

Device Construction

Capacitor 1

Capacitor 2

0.00E+00

2.00E-08

4.00E-08

6.00E-08

8.00E-08

1.00E-07

80 130 180 230 280 330

Time (s)

Con

duct

ivity

(S)

Fe3+

Capacitance Detection

• A new adhesive was created – adhesive resolution improved, chemical stability improved, and a lower bond temperature achieved.

• New polymer layer material formulated – higher cross-linked polymer, improved chemical inertness, and lower fabrication temperature.

• Processing temperatures have been reduced from a maximum of 200 °C to a maximum of 90 °C.

• Reduction of pinholes in the polymer dielectric layer has been achieved for improved electrical integrity.

Successful immobilization of DNAzymes onto PMMA and demonstration of Pb detection activity and selectivity

We exploit the strong biotin-avidin affinitychemistry to bind the enzyme DNA strandto PMMA surface.

polycarbonate block

PMMA via reduction layer

separation channel layer

nanoporous polycarbonate track-etched membranedetection channel layer

Kemery, P. J.; Steehler, J. K.; Bohn, P. W. Langmuir 1998, 14, 2884

Li, J.; Lu, Y. J. Am. Chem. Soc.,2000, 122, 10466

Chang, I-H; Tulock, J. J.; Liu, J.; Kim, W-S; Cannon, D. M., Jr.; Lu, Y.; Bohn, P. W.; Sweedler, J. V.; Cropek, D. M. Environ. Sci. Technol. 2005, 39, 3756

Dalavoy, T. S.; Wernette, D. M.; Gong, M.; Flachsbart, B. R.; Bohn, P. W.; Shannon, M. A.; Sweedler, J. V.; Cropek, D. M. Lab on a Chip, 2008, 8(5), 786-793

Swearingen, C. B.; Wernette, D. P.; Cropek, D. M.; Lu, Y.; Sweedler, J. V.; Bohn, P. W. Anal. Chem., 2005, 77, 442-448

Wernette, D. P.; Swearingen, C. B.; Cropek, D. M.; Yi Lu, Sweedler, J. V.; Bohn, P. W. Analyst, 2006, 131, 41-47

Piruska, A.; Branagan, S.; Cropek, D. M.; Sweedler, J. V.; Bohn, P. W. Lab on a Chip, 2008, 8, 1625-1631

Detection of Pb2+ in a Nanocapillary Interconnected Microfluidic Channel - PMMA

References

Floating ON, 40s

ON

OFF OFF, 4s OFF, 30s

ON OFF

Conductivity detection

Separation of 4.68 mM Cu2+ and 5.32 mM Fe3+ in 5mM His/3mM HIBA at 800 V.

PMMA

NH

HN

S O

OO

NH

HN

S O

OO

NH

HN

S O

OO

NH

HN

S O

OO

Pb2+

streptavidin

Biotin-DNAzyme-FAM

BA C

D E F

(A) non-biotinylated DNA on streptavidin-coated PMMA; (B) biotinylated DNAzyme on PMMA without streptavidin; (C) FITC-labeled streptavidin on PMMA; (D) fluorescently-tagged, biotinylated enzyme strand immobilized on streptavidin-coated PMMA; (E) substrate of panel (D) after hybridization with quencher-labeled substrate DNA; and (F) substrate of panel (E) after exposure to 10 M Pb2+ for 1 h.

PCTE membrane

separation channel

detection channel

ground

V1

ground ground

V1

ground

V2V2

V2 > V1/2

Immobilization, hybridization, Pb2+ detection and regeneration.

0

0.5

1

1.5

2

2.5

Flu

ore

sc

en

ce

inte

ns

ity

ra

tio

Effect of applied potential

0

1

2

3

4

5

6

7

8

9

Bkg 0h 1h 2h 3h

Flu

ore

sc

en

ce

inte

ns

ity

ra

tio

(A) and (B); upper and lower half of the detection channel obtained after injection of buffer solution for 2 h in the absence of Pb2+, Fluorescence images of the channel after 0 h (C), 1 h (D), 2 h (E) and 3 h (F) of injection of 10 M Pb2+.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Bkg 0h 2hFlu

ore

sc

en

ce

inte

ns

ity

ra

tio

(A) and (B); upper and lower half of the detection channel after obtained after injection of buffer solution for 2 h in the absence of Pb2+; Fluorescence images of the channel after 0 h (C) and 2 h (D) of injection of 10 M Pb2+.

A B

C D E F

A B C D

V1= 150V, V2= 100V V1= 50V, V2= 30V

Detection of Pb2+ using quantum dot labeled DNAzyme – Elimination of photobleaching

Method 1

Bio-5T(7)-17Ea-AmEDC, PBS, 2h

Ultrafiltration to removeunreacted DNA

Method 2

Streptavidin coatedglass coverslip

Immobilization of QD functionalized DNAzyme

Bio-5T(7)-17Ea-Am

QD, EDC, PBS, 2h

Wash with buffer to remove unreacted QD

Method 1 Method 2

QD on SSG DNA +QD on SSG (no EDC) QD + EDC on SSG (no DNA)

Control experiments indicates no non-specificbinding of QD on SSG surface in the absence of EDC, but QD in the presence of EDC reactswith amine groups on both streptavidin and DNAzyme.

EDC- N-ethyl aminomethyl carbodiimide hydrochloride

Fluorescence Resonance Energy Transfer (FRET) with QD as donor and Texas Red as acceptorwas used for quantification of Pb2+

Hybridization with Tex-(7)17DSa 1 μM Pb2+ 10 μM Pb2+ 100 μM Pb2+

I525/I620 0.66 0.73 0.93 0.68

Fluorescence intensity ratio of 525 nm to 620 nm should be proportional to Pb2+ concentration.

While the fluorescence signal intensities at 525 nm and 620 nm increase for 1 and 10 M Pb2+ as expected, 100 M Pb2+ shows an unexpected decrease, likely indicating a loss of enzyme DNA from the channel. We are investigating stability issues to solve this problem.

Conclusions and Future work

1. We have a mild and effective method for the immobilization of DNAzyme on PMMA, involving the reaction between biotin-modified DNAzyme and streptavidin physisorbed on the PMMA surface.2. DNAzyme activity and selectivity for Pb2+ in a PMMA microfluidic-nanofluidic device has been demonstrated. 3. Regeneration and repeated use of the device for Pb2+ detection has been demonstrated.4. Work is in progress to obtain analytical figures of merit for the immobilized DNAzyme system and to incorporate separation of other metal ions using capillary electrophoresis in the separation channel prior to Pb2+ detection.5. Chip designs are being tested for multianalyte detection of Pb2+ and the uranyl ion, UO2

2+.

Substrate DNA

CdSe/ZnS/PEG

Cu2+