SIGNAL-GUIDED SEQUENTIAL ASSEMBLY OF
NANO-BIO-COMPONENTS IN THE COMPLETELY
PACKAGED MICROFLUIDIC ENVIRONMENT
Xiaolong Luo1,6, Angela T. Lewandowski2,3, Hyunmin Yi5, Gregory F. Payne3, Reza Ghodssi4,6, William E. Bentley1,3
and Gary W. Rubloff5,6
1Fischell Department of Bioengineering, 2Department of Chemical and Biomolecular Engineering, 3University of Maryland Biotechnology Institute
(UMBI), 4Department of Electrical and Computer Engineering, 5Department of Materials Science and Engineering, 6Institute for Systems Research,
University of Maryland, College Park, MD 20742, USA
Abstract
This paper reports the signal-guided sequential assembly of labile nanobio-components onto specific assembly sites inside a completely packaged
microfluidic environment. We demonstrate (a) programmable assembly
of biologically active green fluorescence protein (GFP) and (b) the
sequential DNA hybridization on a chitosan scaffold at specific assembly
sites in microchannels. The biomolecule assemblies in microfluidics are
spatially and temporally controlled, which provide a flexible scaffold for
biomolecular sensing, synthesis, and metabolic engineering applications.
Sequential DNA Hybridization
Microfluidic channel
a
Flo
w
chitosan film
electric
signal
chitosan
molecule
Microfluidic channel
b
Flow
Method
Chitosan electrodeposition
Glutaraldehyde
Chitosan film
H+
Microfluidic channel
c
Flow
Low pH
(soluble)
Probe DNA
+ +
+
+
+
Microfluidic channel
d
+
+ +
(Negatively biased)
1/2H 2
- - - -
Nano-bio-components assembly
¾Biofunctionalization in prefabricated
microfluidic devices
¾Spatially and temporally controlled
chitosan electrodeposition
¾Sequential assembly of nano-biocomponents onto chitosan scaffold
Flow
pH gradient
Mismatch DNA
pH=pKa
Prefabricated microfluidic device
e
F
F
F
Electric I/O
Fluidic I/O
Microfluidic channel
F
Flow
Match DNA
a
500 μm After chitosan deposition
d
e
activation
Static state
500 μm
60 min
255 Surface Plot
0
14
e ls
pix
ixels
234 p
(b) Control: GFP without tyrosinase
500 μm
0 min
20 min
40 min
255
Surface plot
Rinsed in PBS
Surface Plot
0
5
14
pix
500 μm
Experimental process
¾Electric signal guides
chitosan assembly
(spatial and temporal).
¾Tyrosinase activates
the pro-tag on GFP for
covalent binding onto
chitosan.
6
500 μm
f
GFP
tyrosinase
pro-tag
scaffold
electric signal
4A/m2, 4 min
30 min
After glutaraldehyde
Flow
(a) Experiment: GFP with tyrosinase
Surface plot
c
b
Programmable Protein Assembly
0 min
Results:
¾Matched ssDNA complementarily
hybridized to probe ssDNA on
chitosan scaffold
¾The hybridization saturated within
15 minutes
Bright field and fluorescent micrographs of sequential DNA hybridization
Electrodes
Microfluidic
channel
chitosan electrode
molecule
Experimental process:
¾First, chitosan solution was
introduced into a microchannel and
electrodeposited on a selected
electrode.
¾Second, glutaraldehyde was
introduced to activate the
electrodeposited chitosan.
¾Next, probe single string DNA
(ssDNA) was introduced to be
assembled onto the scaffold.
¾Then a mismatched ssDNA is
introduced into the microchannel
¾Finally, a matched target ssDNA is
introduced to hybridize onto the
probe ssDNA.
els
ixels
228 p
Results:
¾Experiment: GFP
covalently bonds to
spatially selective
chitosan scaffold, thus
withstands PBS rinsing.
¾Control: GFP is nonspecifically associates
with chitosan, thus is
easily rinsed away.
After probe DNA
After mismatch DNA
After match DNA
During introduction of match ssDNA over probe ssDNA on chitosan
0 min
1 min
3 min
5 min
10 min
Conclusions:
• Simple, robust and versatile biofunctionalization strategy
• Sequential assembly of labile proteins and DNA in microfluidic devices
• Spatial and temporal programmability of biomolecule assembly
Applications:
• The signal-guided programmability is attractive for multi-site/multi-step
bioreactions in metabolic engineering and other bioMEMS applications.
Acknowledgements
This work was supported in part by the NSF MRSEC DMR080008, NSF
IMI DMR0231291, NIST and the Laboratory for Physical Sciences.