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pubs.acs.org/Langmuir
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669, Singapore
National Institute for Research and Development in Microtechnologies, IMT-Bucharest, Bucharest 077190, Romania
Laboratoire de Physique des Solides, Universite Paris-Sud, CNRS, 91400 Orsay, France
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077, Singapore
Singapore-MIT Alliance for Research and Technology, 65 Nanyang Dr, Singapore 637460, Singapore
#
Mechanobiology Institute, National University of Singapore, Singapore 119077, Singapore
INTRODUCTION
Recent developments in molecular biology and genomic
research have given a detailed characterization of genetic
diseases such as Alzheimers, Parkinsons, and Huntingtons.
Gene therapy, the transfection of therapeutic genes into cells,
oers the opportunity to treat, prevent, and control such
diseases. Despite the ethical debate regarding the use of gene
therapy in medical practice,1 the main challenges in gene
therapy are related to cell targeting, transfection eciency, gene
expression regulation, and safety issues related to gene carriers.2
Current research on gene delivery vector development focuses
on two major directions: viral and synthetic vectors. The use of
viral vectors have recorded some successful clinical trials,3,4 but
side eects such as immunogenic response5 or vector-induced
cancer6 have limited so far the development of this method.
Nonviral vectors such as lipid-based containers,7 inorganic
nanoparticles,8 and polymer-based carriers9,10 confer advantages in terms of simplicity of use, structural and chemical
exibility in exerting control over physical and chemical
properties, feasibility of large-scale production, lack of specic
immune response, and a larger gene-loading capacity.1,9 The
synthetic solutions must protect the DNA from intracellular
and extracellular degradation,10,11 neutralize the negative
charges of DNA, avoid the interactions with cell surface,12,13
and have an appropriate size for transfection. Large vector sizes
favor in vitro tests due to their sedimentation to the bottom of
the cell culture dish and at the same time exhibit a high
2014 American Chemical Society
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EXPERIMENTAL SECTION
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Figure 1. Rapid collapse of surfactantDNA complexes by hydrodynamic ow focusing in microuidic channel. A mixture of DNA, cationic
surfactant (DTAB) and 35% ethanol is focused between two streams of pure water. In 35% ethanol, DTAB and DNA are weakly bound (A) and as
the water content increases by transverse diusion, the binding becomes stronger (B). At high water content, the DTAB molecules in poor solvent
induce the collapse of DNA chains into nearly monomolecular nanoparticles (C).
Fabrication of the Microuidic Device. The microuidic
channel (40-m-depth) was fabricated in silicon using classical Deep
RIE Bosch process through a 5-m-thick photoresist, while the etchthrough holes were performed using same method through a
photoresist/SiO2 mask. A 150 nm-thick dry-oxide layer was grown
in a furnace on the silicon surface in order to achieve a permanent
hydrophilic surface of the microuidic channel. The silicon wafer was
anodically bonded to a glass wafer for the sealing of the microuidic
channels. Finally the bonded wafers were diced in individual chips and
Nanoport microuidic connectors (Upchurch Scientic) were
mounted for the inlets and outlet. Detailed considerations regarding
fabrication process are presented in our previous work.41
Microuidic Experimental Setup. A two-channel pumping
system MFCS-FLEX (Fluigent, France) was used for controlling the
ow of reactants through the microuidic chip. The system was
equipped with three pumping units and three ow-meters (0-50 L/
min), only two were used for the current experiment, and a software
that allowed a ne-tuning of the ow rates (by varying the applied
pressure) in the microuidic device. The chip was mounted on a chip
holder, and an optical microscope (Keydance) allowed the observation
of the ow in the microuidic device.
Computer Simulations. The numerical three-dimensional (3D)
geometry and the structured orthogonal mesh were generated using
GAMBIT preprocessor (ANSYS), consisting of 1 188 000 hexahedral
nite volumes. The dimensions of the reconstructed microuidic
channel were carefully selected to capture the diusion process with an
acceptable computational cost. Therefore, the length of the inlet
channels was 300 m, the length of the outlet channel was 3000 m,
and the cross sections were 60 40 m in all cases. The 3D pressuredriven ow in the microchannel conguration was simulated using the
+ (u)u = b p + 2(( ) E)
t
(1)
where is the uid density, b the specic mass force, E the strain rate
tensor, t the time, u the velocity vector, p the pressure, and the
viscosity function dependent on the shear rate . All the quantities
entering eq 1 varied with the position r and the time t. Along with the
momentum equation, the diusion equation was also solved in order
to obtain an accurate solution
(r, t )
= [D()(r, t )]
t
(2)
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Figure 2. (a) Exploded view of the microuidic device. (b) Optical image of the device. (c) Testing setup: two-channels MFCS-FLEX pumping unit
was used for owing the reagents and the water in a controlled manner through the microuidic channels.
by dening solution zones, the ux function, the unsteady function and
other parameters so as to obtain an accurate simulation of the diusion
process. The value for the diusion coecient of ethanol in water was
0.625 109 m2/s.44 No chemical reaction was taken into account and
the ethanol was therefore assumed to be a passive tracer in water.
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Figure 3. (a) Simulation results presenting the mixing process along the microuidic channel: near the conuence point, and at 1, 2, and 3 mm from
the focusing point. Attached to the simulated cross sections, optical images of the channel illustrate the mixing process (the width of the microuidic
channel is 60 m). (b) Calculated variations of the ethanol concentration in cross sections (maximal value, minimal value and the value at the center
of the channel) taken along the channel length. The values for channel lengths greater than 3 mm were obtained by extrapolating the simulation
results with a set of exponential functions.
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and
Q A + 2Q B = wovo h
(4)
where h denotes the height of the channel and vf and vo are the
average ow velocities in the focused and outlet streams. These
two equalities can be rearranged to yield an expression of the
ow rate ratio R 2QB/QA:
1 wo
1
R=
wf
(5)
where vf /vo is a velocity ratio to be found. Equation 5 is
obtained from the incompressibility of the uids in a regime of
laminar ow. As a result of the strong reduction of wf via R, the
time required for the pure water to diuse transversely across
the focused stream is considerably shortened. Since viscous
eects dominate, the ow in the outlet channel can be assumed
to be fully developed. In the approximation of large aspect ratio,
i.e., wo h, and by imposing a no-slip condition on the
channel wall, the longitudinal uid velocity v(x) obeys to the
Poisson equation:
x2
v(x) = vmax 1 4 2
wo
(6)
1
wf
wf /2
w /2 v(x) dx vmax
f
(7)
(3)
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wo 2
wf 2
4Dwater
9(1 + R )2 Dwater
(8)
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Figure 5. On-chip synthesis of DTAB/calf thymus DNA nanoparticles. The ow rate of the DTAB/DNA stream was 5 L/min and that of the water
stream 45 L/min. (a) Hydrodynamic diameter and (b) PDI as a function of DNA concentration for DTAB concentrations of 5 and 7 mM. (c)
Hydrodynamic diameter and (d) PDI as a function of DTAB concentration for 30 and 50 mg/L of DNA.
(9)
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size [nm]
STDEVb [nm]
PDIc
onchip
bulk
onchip
bulk
onchip
bulk
1/45 (45)
2/45 (22.5)
3/45 (15)
4/45 (11.25)
5/45 (9)
6/45 (7.5)
9/45 (5)
10/40 (4)
107
102
103
103
97
100
200
210
528
364
147
283
433
178
199
288
3.50
1.08
0.25
2.89
1.53
2.80
33.64
6.8
101
137
39
71
20
5.2
21
81
0.18
0.18
0.21
0.21
0.16
0.17
0.38
0.38
0.45
0.42
0.43
0.43
0.44
0.45
0.32
0.35
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Figure 6. On-chip synthesis of CTAB/calf thymus DNA nanoparticles. The ow rate of the CTAB/DNA stream was 5 L/min and that of the water
stream 45 L/min. (a) Hydrodynamic diameter and (b) PDI as a function of DNA concentration for a CTAB concentration of 6 mM. (c)
Hydrodynamic diameter and (d) PDI as a function of CTAB concentration for 3 mg/L of DNA.
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Figure 7. On-chip synthesis of DTAB/-DNA nanoparticles. The ow rate of the DTAB/-DNA stream was 5 L/min and that of the water stream
45 L/min. (a) Hydrodynamic diameter and (b) PDI as a function of -DNA concentration for a DTAB concentration of 6 mM. (c) Hydrodynamic
diameter and (d) PDI as a function of DTAB concentration for 1 mg/L of -DNA.
AUTHOR INFORMATION
Corresponding Authors
ACKNOWLEDGMENTS
The authors thank Mehdi Zeghal for performing measurements
of diusion coecients by pulsed-eld gradient NMR as well as
Anniina Salonen and Ming Ni for stimulating discussions. This
research was supported by the Institute of Bioengineering and
Nanotechnology (Biomedical Research Council, Agency for
Science, Technology, and Research, Singapore) (C.I., S.V., and
H.Y.), and partially by the France-Singapore Merlion Program
(Project Reference 8.02.10), and by CNRS through the
Interface Physique, Chimie, Biologie program (G.T.). C.M.
acknowledges support from the contract O2.1.2, no.209, ID
665.
CONCLUSIONS
In this report we successfully demonstrated the use of a
microuidic hydrodynamic ow focusing device in combination
with an on-chip rapid solvent quality change strategy, to yield
well-dened surfactantDNA nanoparticles with excellent
control over their hydrodynamic diameter (70 nm) and
distribution (PDI < 0.2). Straightforward and accurate control
on the morphological properties of surfactantDNA nanoparticles may have broad implications in gene delivery: it may
enhance the bioavailability of the delivery vectors and oer a
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REFERENCES
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(42) Johnson, B. K.; Prudhomme, R. K. Mechanism for rapid selfassembly of block copolymer nanoparticles. Phys. Rev. Lett. 2003, 91,
118302.
(43) Rudorf, S.; Radler, J. O. Self-Assembly of stable monomolecular
nucleic acid lipid particles with a size of 30 nm. J. Am. Chem. Soc. 2012,
134, 1165211658.
(44) Tyn, M. T.; Calus, W. F. Temperature and concentration
dependence of mutual diffusion coefficients of some binary liquid
systems. J. Chem. Eng. Data 1975, 20, 310316.
(45) Trabelsi, S.; Raspaud, E.; Langevin, D. Aggregate formation in
aqueous solutions of carboxymethylcellulose and cationic surfactants.
Langmuir 2007, 23, 1005310062.
(46) Weigl, B. H.; Yager, P. Microfluidic diffusion-based separation
and detection. Science 1999, 283, 346347.
(47) Castillo-Leon, J.; Rodriguez-Trujillo, R.; Gauthier, S.; Jensen, A.
C.; Svendsen, W. E. Micro-factory for self-assembled peptide
nanostructures. Microelectron. Eng. 2011, 88, 16851688.
(48) Iliescu, C.; Taylor, H.; Avram, M.; Miao, J.; Franssila, S. A
practical guide for the fabrication of microfluidic devices using glass
and silicon. Biomicrofluidics 2012, 6, 016505.
(49) Iliescu, C.; Tresset, G.; Xu, G. Continuous field-flow separation
of particle populations in a dielectrophoretic chip with three
dimensional electrodes. Appl. Phys. Lett. 2007, 90, 234104.
(50) Iliescu, C.; Tresset, G.; Xu, G. Dielectrophoretic field-flow
method for separating particle populations in a chip with asymmetric
electrodes. Biomicrofluidics 2009, 3, 044104.
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