development of microfluidic / nanofluidic sensors using catalytic dna for heavy metal detection
DESCRIPTION
A. B. C. Capacitor 1. D. E. F. Capacitor 2. Fe 3+. V 1. V 1. PCTE membrane. ground. ground. V 2. ground. separation channel. ground. detection channel. polycarbonate block. Enzyme. Pb 2+. PMMA via reduction layer. separation channel layer. nanoporous polycarbonate - PowerPoint PPT PresentationTRANSCRIPT
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: [email protected], 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
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300
350
400
Pb Co Zn Mn Ni Cd Cu Mg Ca
vfl
uo
(co
un
ts/s
)
0
10000
20000
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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
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4
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7
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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+