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Controllable formation of aromatic nanoparticles in a

three-dimensional hydrodynamic flow focusing
Cite this: RSC Advances, 2013, 3,
17762 microfluidic device3
Liguo Jiang,a Weiping Wang,{b Ying Chauab and Shuhuai Yao*ac

Herein we investigate the formation of aromatic organic nanoparticles in a three-dimensional (3D)

Received 26th April 2013,
Accepted 24th July 2013
DOI: 10.1039/c3ra42071j
www.rsc.org/advances
hydrodynamic flow focusing microfluidic device. We demonstrate a microfluidic based solvent/non-
solvent exchange technique that enables controllable formation of aromatic nanoparticles with tunable
size and size distribution. The 3D focusing is achieved by hydrodynamically focusing the sample stream
with sheathed streams in both horizontal and vertical directions, and solvent/non-solvent exchange
happens between the solvent contained sample stream and non-solvent contained horizontally sheathed
streams. The 3D focusing effect was visualized using florescence confocal microscopy and the dynamics of
solvent (DMF) depletion in the focused stream was calculated using a finite element computation software
package COMSOL Multiphysics. By analyzing the results of self-assembled aromatic nanoparticles in the
microfluidic device, we find that the speed of DMF depletion strongly influences the size and size
distribution of self-assembled aromatic nanoparticles, and a rapid depletion of DMF is critical for achieving
small aromatic nanoparticles with narrow size distribution. This work suggests that our 3D hydrodynamic
flow focusing microfluidic device with the ability to precisely control the convective-diffusion process, and
continuously vary the fluid flow conditions is promising for studying the formation of nanomaterials.

1 Introduction Recently we designed a tripodal molecule (FTAEA) by

Aromatic interactions, consisting of van der Waals, hydro-
phobic and electrostatic forces1,2 are ubiquitous in chemical
and biological systems and play an important role in
stabilizing the structures of DNA, RNA and proteins.3–5 They
also play a significant role in the self-assembly of amyloid
fibrils.6 And the interactions with aromatic rings are key
processes in chemical and biological recognition.7 Although
aromatic interactions are theoretically not fully understood,8,9
the utilization of aromatic residues to mediate molecular self-
assembly to form well-ordered novel nano-structure materials
has received notable achievements.10–15 It is assumed that the
p stacking interactions between aromatic moieties provide
energetic contribution as well as order and directionality for
the formation of well-ordered nanostructures.10,11
a
Division of Biomedical Engineering, Bioengineering Graduate Program, Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
b
Department of Chemical and Biomolecular Engineering, Hong Kong University of
Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
c
Department of Mechanical Engineering, Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: meshyao@ust.hk
3 Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ra42071j
{ Current address: The David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
coupling 9-fluorenylmethoxycarbonyl (Fmoc) groups with
three legs of tris(2-aminoethyl)amine (TAEA) to fabricate
nanoparticles (NPs) by self-assembly.16,17 This idea was
inspired by clathrin molecules which can assemble into a
polyhedral lattice surrounding the membrane vesicle for
trafficking in cells,18 and fully employs the concept of p–p
interactions mediated molecular self-assembly. Such aromatic
NPs self-assembling from small molecules can be functiona-
lized in a versatile manner for targeted drug delivery.17
For nanoparticle drug delivery systems, rapid clearance of
circulating NPs is a critical issue. The size of NPs has been
demonstrated to be one of the key factors affecting the
biodistribution and clearance of the circulating system.19 This
is due to the fact that particle size not only dictates their
transport and adhesion in blood vessels but also mediates the
cellular response.20,21 From this point of view, controllable
formation of stable NPs with tunable size is critical for the
application of NPs as drug delivery carriers. Based on the
classical nucleation and growth theory,22,23 the formation of
NPs in solution is engaged in two processes: nucleation and
growth. And the kinetics of two processes is governed by the
saturation conditions of the solution, which is determined by
the monomer concentration and the solvent/non-solvent
(dimethylformamide (DMF) is the solvent and water is the
non-solvent fluid in our system) ratio in solution. When the

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solution is undersaturated, in which the monomer concentra-

tion is lower than the critical concentration or the solubility of
the monomer at the presented solvent/non-solvent ratio,
aromatic NP nuclei may be formed, but it rapidly dissipates
due to the high nucleation barrier.23 In such situations, either
increasing the monomer concentration or decreasing the
solvent/non-solvent ratio will create a supersaturation condi-
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tion in solution, which leads to aromatic NP nucleation and

growth. And decreasing the solvent/non-solvent ratio can be
readily accomplished by exchanging or replacing solvent
molecules with non-solvent molecules. Thus, we expect that
controlling the solvent/non-solvent ratio is a straightforward
way to control the formation of aromatic NPs. Conventionally,
organic NPs are self-assembled by bulk nanoprecipitation
method,24 which involves dropwise addition of stock solution
in solvent into a larger volume of non-solvent and mixing
solutions by vortex. Although the nanoprecipitation method is
simple, this approach is by no means to control the
microscopic solvent/non-solvent ratio due to the uncontrol-
lable nature of chaotic and turbulent mixing, resulting in
uncontrollable size and size distribution. The other issue of
the current NP formation method comes from the conflict
between the slowly bulk mixing by vortex and the rapid self-
assembly of aromatic NPs. This conflict may hurdle the ability
to investigate the dynamic process of the NP formation and
thus limit us in understanding the fundamental of the self-
assembly mechanism.
Hydrodynamic flow focusing (HFF)25–27 micromixers, in
which the central stream is hydrodynamically focused to a
narrow jet by two side streams at a cross junction under
Fig. 1 (a) A schematic shows the concept of hydrodynamic flow focusing.
laminar flow condition (Fig. 1a), provide an alternative
Solvent/non-solvent exchange is achieved by solvent molecules diffusing into
platform for nanomaterial formation.28–33 Hydrodynamic the focused stream and non-solvent molecules diffusing out of the focused
focusing reduces the stream width (wf) to a few micrometers stream. (b) A schematic of the microfluidic device showing the concept of
even submicrometers, and consequently reduces the diffusion 3DHFF. Cross section A shows the sample stream focused in vertical direction,
path (wf), where solvent molecules diffuse out and non-solvent and cross section B shows the sample stream focused in both vertical and
horizontal direction.
molecules diffuse in. Therefore, the time for solvent extraction
from its initial solvent/non-solvent ratio to a critical ratio,
when nuclei starts to be formed and growth, can be greatly
two-level layers35 or a single layer with sequential inlets.34 In a
shortened by the diffusion based extraction in HFF.
3DHFF system, the reagents and precipitating NPs are isolated
Furthermore, the diffusion path can be controlled by the
from the channel walls, therefore aggregation and/or clogging
applied pressure or flow rate and thus the speed of the solvent
can be minimized. In addition, by constraining the sample
depletion can be varied to offer different stable reaction
stream in the center of the microchannel where flow velocity
environments for organic nanoparticle nucleation and growth.
reaches the maximum with less variation, the 3D focused
This technique has already been successfully applied to
sample stream is expected to have a uniform width and thus
control the liposome and block copolymer vesicle forma-
improved the uniformity of the solvent/non-solvent ratio.
tion,29,30 polymeric nanoparticle self-assembly,31 noisome and
In this work, we used a simple 3DHFF microfluidic device36
liposome-hydrogel hybrid nanoparticle self-assembly,32,33 and
made of two layers of PDMS for nanoparticle formation.
rubrene nanocrystal formation.28 However, most HFF systems
FTAEA NPs with tunable size and narrow size distribution were
using two-dimensional (2D) focusing, in which the reagent in
obtained by controlling the microfluidic flow conditions. The
the central stream is only focused in the horizontal plane, may
encounter several problems such as sticking of molecules on size distribution of the NPs collected at different flow
the channel walls34 (see ESI3 Fig. S1) and non-uniform solvent/ conditions was measured using dynamic light scattering
non-solvent exchange in the vertical direction. To address (DLS). Based on the classic nucleation and growth theory, we
these problems, a three-dimensional hydrodynamic flow explored how the size of self-assembled FTAEA NPs is related
focusing (3DHFF) configuration in both horizontal and vertical to the dynamics of the solvent (DMF) depletion in the focused
planes has been implemented in microfluidic systems using stream.

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2 Experimental section
2.1 Reagents and materials
FTAEA dissolved in dimethylformamide (DMF) stock solution
was prepared as described in the previous work,16 and stored
at 220 uC prior to usage. Depending on the experimental
requirements, the stock solution was dissolved in DMF–water
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(75 : 25, volume ratio) mixture to make FTAEA solutions at
different concentrations. Poly(dimethylsiloxane) (PDMS)
(Sylgard 184 silicone elastomer) was purchased from Dow
Corning. Photoresist SU-8 2025 was purchased from
MicroChem Corp. FITC-dextran-10K and fluorescein were
purchased from Invitrogen.
2.2 Microfluidic device fabrication and implementation
Fig. 1b schematically shows the concept of the fabricated
3DHFF microfluidic device which consists of two layers of
microchannels.36 Within the microchannel network, the
sample stream (FTAEA in DMF–water solution, red color) is
first vertically focused by two streams of DMF–water (green
color) to form a parallel central stream (see cross section A) in
a 10 mm width central microchannel. And the laminar central
stream is horizontally focused by two water streams (blue
color) from both sides, resulting in an isolated sample stream
(see cross section B) in a 20 mm width microchannel. The
microfluidic device was fabricated with two layers of PDMS
using a standard soft lithography process. For PDMS replica
molding, a negative photoresist SU-8 (SU-8 2025) was spin-
coated on a silicon wafer in a thickness of 40 mm, and then it
was photolithographically patterned and developed, yielding a
mold master of 40 mm deep microchannels. A PDMS mixture
(in a 10 : 1 weight ratio of monomer and curing agent) was
poured over the molds, degassed in a vacuum chamber, and
cured in an oven at 85 uC for 2 h. The resulted PDMS layers are
4 mm and 0.8 mm in thickness, respectively. After the PDMS
replicas were removed from molds, individual device was cut,
and inlet, outlet holes were punched by a pan head needle on
the thick PDMS replica. The two PDMS replicas were treated
with oxygen plasma and aligning bonded to seal microchan-
nels. Before an experiment, the device was fixed on the chip-
holder and reagents were preloaded in glass (Hamilton, Reno,
NV) and plastic (BD) syringes with polyetheretherketone
(PEEK, Upchurch Scientific) capillary tubing connecting the
syringes to microdevice access holes. Reagents were pumped
into the microdevice using syringe pumps (KD Scientific) and
the self-assembled products were collected from the outlet.
2.3 Confocal laser scanning microscopy and epifluorescence
microscopy
Cross sections of 3D focused sample streams were imaged by
the Zeiss laser scanning confocal microscope (LSM7 DUO)
with 0.5 mm resolution in both horizontal and vertical
directions. An Argon ion laser at 488 nm wavelength was
employed as the excitation source. The excitation beam was
focused into the chip by an air immersion objective lens (406/
0.6 NA) to excite FITC-dextran-10K labeled sample stream and
the emission light was collected by the same objective and
detected by a photomultiplier tube (PMT) detector. ZEN 2009
analysis software (Zeiss) and ImageJ (National Institutes of
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Health) were used to analyze and extract the cross-sectional
images. An inverted epifluorescence microscope (Eclipse Ti,
Nikon) with an objective lens (206/0.45 NA) was also used to
observe microfluidic mixing in the device. A mercury lamp
with a filter cube (B-2A, Nikon) was used for fluorescence
excitation/emission. A highly sensitive CCD camera (EXi Blue,
Q-IMAGING) was mounted on the microscope to capture the
images.
2.4 DLS measurement
The nanoparticle number average diameter and distribution
measurements were performed on Zeta Plus (Brookhaven
Instruments Corp.) with the use of the BIC Particle Sizing
Software. The collected product solution (y80 mL) was put into
a cuvette (Eppendorf UVette) and tested at 25 uC with a
detection angle of 90u and a wavelength of 659 nm. Each
sample was run for 5 times with 60 s per run. The data of each
run were collected only when its baseline index was larger than
or equal to 5.
3 Numerical simulation section
The microfluidic flow and DMF depletion dynamics in the
central stream sheathed by two adjacent water streams in a
hydrodynamic flow focusing device were simulated using a
commercial finite element code, COMSOL MULTIPHYSICS 4.1
(Comsol, Inc.). The fluid flow in microfluidic channels is
governed by the incompressible steady-state Navier–Stokes
equations, and the diffusion between DMF and water is
governed by the steady-state convective-diffusion equation:37–41
A
+?V = 0 (1)
A A A
r(V ?+)V = 2+P + m+2V (2)
A
V ?+C = D+2C (3)
A
where V is the flow velocity, r is the fluid density, P is the
pressure, m is the fluid dynamic viscosity, C is the concentra-
tion of DMF, and D is the mutual diffusion coefficient of DMF
in water. In the simulation, the fluid density, dynamic
viscosity, and diffusion coefficient are a function of the DMF
concentration and are expressed by a fourth order polynomial
fitting density data, viscosity data, and diffusion coefficient
data at 20 uC, as reported by Volpe et al.42
To quantify the DMF depletion dynamics in the central
stream, we traced 20 streamlines across the focused central
stream in the midplane of the microchannel. We follow the
suggestion from Hertzog et al.39 and Park et al.27 to define the
time ti of DMF depletion at each streamline in our system, of
which the clock starts when the concentration of DMF along
the streamline has decrease to the 99% of the initial
concentration in the central stream, and the average time of
DMF depletion ,t> at x location is calculated by summing and
averaging the contribution of the depletion times from all the
streamlines:
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ð
x dx
ti ðxÞ~
x0 ,c~0:99c0 ui ðxÞ
P
i ti ðxÞwi ðxÞ
P
StðxÞT~
i wi ðxÞ
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And the standard deviation of the depletion time, which
represents the uniformity of DMF concentration across the x
location, is defined as:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
i ðti ðxÞ{St ðxÞTÞ2 wi ðxÞ
sðxÞ~ P ðti w0Þ (6)
i wi ðxÞ
where ui is the x component of the flow velocity in the ith
streamline, wi is the width of the ith streamline, and C0 is the
initial concentration of DMF. All the simulated data are
analyzed using customer-built programs in MATLAB.
4 Results and discussion
4.1 Cross-sectional images of 3D focused stream
Due to the fact that the 2D focused stream may spread out of
the midplane (over the depth of the channel) and its speed
varies from zero near the channel walls to a maximum at the
midplane, 3D focusing is preferred to achieve a nearly uniform
velocity in the focused stream and therefore nearly uniform
solvent/non-solvent ratio for the sample molecules carried in
the focused stream. To visualize the 3D focusing effect of the
sample stream, we labeled the sample stream with FITC-
dextran-10K, and obtained the cross sectional images of the
sample stream at different flow conditions in the 3DHFF
microdevice using confocal laser scanning microscopy. The
flow rates of the sample stream and the two vertical focusing
streams were set at 0.5, 1, and 1 mL min21, respectively, while
the total flow rate of water streams varied from 20, 40 to 60 mL
min21, and the corresponding Reynolds number is 0.83, 1.66,
and 2.49, respectively. The confocal scans were taken at 40 mm
downstream of the outlet channel at the location of cross
section B, as indicated in Fig. 1b, and the results are shown in
Fig. 2a–c. They clearly reveal several important facts. Firstly, all
three focused streams are isolated from the channel walls.
Secondly, the focused stream profile has relatively uniform
width which ensures the nearly uniform molecular diffusion
across it. Thirdly, the focused stream becomes narrower at
higher water flow rate conditions, thus the time for water
molecules to diffuse across the focused stream is shorter,
according to the Fick’s law of diffusion.43 We also took the
cross section images at a fixed total flow rate and a fixed flow
rate of Qwt while changing the flow rate of Qbf1, Qbf2, and QSa,
as shown in ESI3 Fig. S2. We have found that the aspect ratios
of the focused streams vary but the widths of focused streams
are almost identical, which imply that the processes of the
diffusion based solvent depletion in these conditions are
similar.
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(4)
(5)
Fig. 2 Cross section images of 3D focused streams at different flow conditions,
taken at 40 mm downstream at cross section B in Fig. 1b. Flow rates in
experiments: Qbf1–Qbf2–QSa–QWt ml min21 (a) 1–1–0.5–20; (b) 1–1–0.5–40; (c) 1–
1–0.5–60.
4.2 Focused stream width determined by FRR
As the time for a molecule to diffuse across the focused stream
is primarily determined by the width of the focused stream, it
is necessary to investigate the governing parameters for the
width of the focused stream. As shown in the confocal scans,
the artifact of the non-uniform profile is negligible in the
3DHFF focused stream and the stream is mostly focused in the
horizontal direction. Therefore, we use a 2D theoretical model
(Fig. 1a) for predicting the width of the focused stream in a
rectangular channel.26,29 We combine the sample stream and
the two vertical streams as the central stream. According to the
mass conservation, the width of the focused central stream
can be derived as
w0
Wf ~ (7)
cð1zFRRÞ
where w0 is the width of the outlet channel, c is the ratio of the
average velocity in the focused central stream to the average
velocity in the outlet channel, and the flow rate ratio (FRR) is
defined as the ratio of the total flow rate of the two side
streams to the flow rate of the central stream. Hence, wf is
governed by FRR and the velocity ratio (c). The theoretical
analysis results26 show that the velocity ratio c approximately
equals to 1.5 when the aspect ratio (e, height to width) of the
outlet channel is larger than 1.78 (e = 2 in our device).
Therefore, the diffusion path wf is mainly governed by the FRR
in a given fluidic network. To verify the relationship between
wf and FRR, we experimentally measured the midplane width
of the focused stream in the confocal scans at different FRR
conditions. The FITC-dextran-10K labeled sample solution
remains as a focused stream at the center of the channel and
width of the focused stream is determined when the intensity
drops to half of maximum value in the lateral direction. As
shown in Fig. 3, the experimental data match well with
theoretical calculation by eqn (7). This indicates that FRR,
apparently, is the dominated parameter that governs the width
of the focused stream, and hence the solvent depletion
dynamics in HFF system. Thus for all experiments and
simulations presented in this article, we set the flow rate of
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Fig. 3 3D focused stream width versus FRR. Symbols represent the experimen-
tally measured data and the solid line is the theoretical prediction by eqn (7) (c =
1.5).
the two vertical focusing streams and the sample stream at 1, 1
and 0.5 mL min21, respectively, which resulted in a 2.5 mL
min21 of the central stream. We varied the flow rate in the two
side channels to vary the value of FRR.
4.3 Numerical analysis of mixing performance
Under laminar flow condition and along with flow boundary
and concentration boundary conditions in microfluidic HFF
system, the flow and concentration fields are governed by the
steady-state Navier–Stokes equations and convective-diffusion
equation. Use of 2D flow simulation in our analysis, that
greatly reduces computational time, is justified for the
uniform width of the 3D focused stream. Since the aspect
ratio of the focused stream is more than 10, the DMF depletion
dynamics at the midplane can be approximated using a 2D
model (Fig. 4a–d). Meanwhile, the 2D simulation results can
be qualitatively validated by the epifluorescence microscopic
Fig. 4 Comparison of numerically simulated DMF depletion (a–d) and experimentally observed DMF depletion (e–h) in the focused stream at different FRR conditions.
The width of the focused central stream decreases as the FRR increases, which results in faster depletion of DMF in the focused stream.
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images (Fig. 4e–h). In the experiments, the FITC labeled water
streams were pumped into the two side inlets, and DMF/water
solvent streams were pumped into the device as the central
stream. Both simulated and experimentally captured images at
the midplane show a reasonable coherence at the same FRR
conditions, indicating the accuracy of our simulation results.
When changing the FRR condition, we observed that the
focused central stream reached the steady-state flow within
one minute and the focused central stream was very stable.
Fig. 4 clearly illustrates the speed of DMF depletion at
different FRR conditions along the channel. At low focusing
conditions, the intensity of the focused stream slightly
changes along the channel, indicating the slowly exchanging
of DMF and water molecules across the relatively wide jet. In
contrast, the intensity of the focused stream rapidly changes at
high focusing conditions due to the fast extraction of DMF
molecules out of the narrow focused jet. At high FRR
conditions, another important phenomenon was observed in
experiments that two slightly brighter belts (Fig. 4g and h)
appear around the central stream probably due to the
changing of the fluorescence yield when microfluidic environ-
mental polarity and viscosity change upon the curved
interfaces.44 We believe that the curved interface is caused
by the larger flow velocity gradients near the channel top and
bottom walls.
As the formation of NPs is determined by the microenviron-
ment of reagent molecules and this microenvironment is
mediated by the diffusion based extraction of DMF in the
stream, to carry out controllable formation of NPs, we
investigated the dynamics of DMF depletion in the hydro-
dynamic focused stream as described in the simulation
section. Fig. 5a shows the depletion of DMF in the central
stream with the time defined in eqn (5) at different FRR
conditions. The results clearly reveal that, at low FRR
conditions, the volume fraction of DMF in the focused stream
depletes gently. In contrast, at high FRR conditions, the
volume fraction of DMF deplete rapidly within sub-millise-
cond timescale. In addition, at different FRR conditions, the
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Fig. 5 (a) Simulation results show the depletion of the DMF along the focused
central stream at different FRR conditions, and the depletion timescale is on the
order of submillisecond to millisecond. (b) Simulation results show the standard
deviations of the depletion time along the outlet channel. Small values in the
standard deviation in time ensure uniform DMF concentrations across the
focused stream.
values of standard deviations of the depletion time defined in
eqn (6) are shown in Fig. 5b varying from 24 to 42 ms, which
means that the DMF concentration within the focused stream
Fig. 6 DLS measurements of self-assembled FTAEA NPs at different FRR conditions with the sample stream containing an FTAEA initial concentration of 20 mM. (a)
For FRR , 20, the mean size and size distribution of NPs are highly dependent on FRR. (b–d) For FRR ¢ 20, the mean size and size distribution of NPs are similar. (e) &
(f) Aromatic NPs formed by conventional bulk nanoprecipitation show a wide size distribution.
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is very uniform to provide homogeneous reaction environ-
ments.
4.4 Dependence of FTAEA NP size and size distribution on FRR
We investigated the formation of FTAEA NPs in terms of the
size and size distribution by controlling the depletion of DMF
mediated by different FRR conditions in the 3D HFF
microfluidic device. Fig. 6 shows the results of self-assembled
FTAEA NPs at different FRR conditions with the sample stream
containing an FTAEA initial concentration of 20 mM in DMF–
water (75 : 25, volume ratio). For FRR , 20, the size and size
distribution of self-assembled FTAEA NPs are highly sensitive
to the FRR condition. As shown in Fig. 6a, at FRR = 8, relatively
larger FTAEA NPs with a mean size of y140 nm and a wide
size distribution are obtained, while increasing FRR results in
FTAEA NPs with smaller mean size and an improved size
distribution. For FRR ¢ 20, the size and size distribution of
self-assembled FTAEA NPs remain nearly unchanged for
different FRR conditions (Fig. 6b–d), all showing a mean size
of y40 nm and a similar narrow size distribution. For
comparison, aromatic NPs were formed by conventional bulk
nanoprecipitation, and the results are presented in Fig. 6e and
f. The testing samples have the same finial FTAEA concentra-
tion and the DMF–water ratios correspond to FRR = 12 (Fig. 6e)
and FRR = 32 (Fig. 6f). Slow and uncontrollable exchange of
solvent/non-solvent in bulk nanoprecipitation results in
aromatic NPs with a relatively large size and a broad size
distribution. A similar trend of NP size and size distribution
changing with different FRR conditions were obtained in the
3DHFF microfluidic device when the sample stream contains
an FTAEA initial concentration of 10 mM (see ESI3 Fig. S3).
Combining the theory of nucleation and growth in solutions
and the numerical analysis of DMF depletion dynamics
presented in Fig. 4 and 5, we try to explain the results of
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Fig. 7 The mean size of self-assembled NPs versus FRR for two different initial
FTAEA concentrations. The trends of FRR conditions controlling the NP
formation in the nucleation and growth progress are clearly illustrated.
self-assembled FTAEA NPs in the 3D HFF microfluidic device.
Fig. 7 plots the mean size of self-assembled NPs versus the FRR
conditions. The dashed lines and dotted lines illustrate the
trends of the FRR conditions controlling the NP formation at
two different FTAEA initial concentrations. Apparently, a
higher initial concentration of FTAEA results larger aromatic
NPs at the same FRR condition. One possibility is that in a
micrometer or submicrometer confined space the number of
nuclei are likely the same at a certain FRR condition.
Therefore, for higher concentration with more monomer
molecules in the focused stream, larger NPs were formed.
The assumption of the same number of nuclei is quantitatively
verified by the fact that the volume (pR3/6, R is the mean size of
NPs) of NPs formed with FTAEA initial concentration of 20 mM
is approximately two times larger than that formed with
FTAEA initial concentration of 10 mM when the FRR condition
is the same. Furthermore, the dependence of self-assembled
NPs on FRR is also obvious. At low FRR conditions, the side
sheath flow forms a relatively wide focused sample stream,
across which the exchange of solvent molecules slowly takes
place. In the solutions of the focused sample stream, the DMF
fraction is still high, where less FTAEA nuclei are formed and
abundant FTAEA molecules remain solubilized. Thereby, the
nuclei of FTAEA NPs are growing in size due to the aromatic
stacking interaction of free FTAEA molecules on the nuclei
during gentle depletion of DMF. Therefore, the size of FTAEA
NPs highly depends on the FRR condition. In contrast, at high
FRR conditions, rapid extraction of DMF molecules takes place
by diffusion of water molecules across a tightly focused
stream. As a result, a steep depletion of DMF occurs in the
focused central stream along the downstream which provides
nearly homogeneous microenvironment for FTAEA NP forma-
tion. The steep depletion of DMF induces a larger number of
FTAEA nuclei formed, leading to less dissolved FTAEA free
molecules in the focused stream for NP growth. As a result,
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when the value of FRR is larger than 20, smaller FTAEA NPs
with similar mean size and size distribution are obtained.
5 Conclusions
In this paper, we demonstrate controllable formation of FTAEA
NPs by nanoprecipitation enabled by solvent/non-solvent
exchange in a 3D HFF microfluidic device. From the cross-
sectional view, the focused sample stream is isolated from the
channel walls by the sheath flow in both vertical and
horizontal directions, thereby preventing surface sticking
and channel clogging problems. The stable, uniformly focused
stream ensures a homogenous reaction environment.
Numerical simulation results show that the dynamics of
DMF depletion can be mediated by the flow rate in the
microchannels. Based on the self-assembled NP size distribu-
tion and the classical nucleation and growth theory, we
speculate that rapid depletion of DMF is critical to achieve
small and uniform aromatic NPs. At low water-to-sample flow
rate ratio, gentle depletion of DMF in the focused central
stream leads to a lot of solubilized FTAEA molecules for nuclei
growing, resulting in larger FTAEA NPs. In contrast, at high
flow rate ratio, rapid depletion of DMF induces the formation
of a larger number of FTAEA nuclei. Thus, few free FTAEA
molecules are left for nuclei growth, resulting in smaller
FTAEA NPs with narrower size distribution. The formation of
aromatic NPs with a desired size can be achieved using the
demonstrated 3DHFF microfluidic system, which benefits
biomedical applications with the NPs used as drug delivery
carriers. The developed 3DHFF microfluidic system is com-
pletely applicable for fast self-assembly of nanomaterials with
or without sticking problem on the surface wall, for the solvent
depletes rapidly in the narrowly focused stream, 3D focused
stream is isolated from surface walls, and the uniform width of
the 3D focused stream ensures a relatively homogenous
reaction environment for the formation of nanomaterials.
Acknowledgements
We thank the Nanoelectronics Fabrication Facility, Biosciences
Central Research Facility, Materials Characterization and
Preparations Facility, Advanced Engineering Material Facility
and Professor Baoling Huang’s group at HKUST for their
technical support. This work was supported by HKUST Research
Project Competition Grant (RPC10EG30).
References
1 C. A. Hunter and J. K. M. Sanders, J. Am. Chem. Soc., 1990,
112, 5525–5534.
2 M. L. Waters, Curr. Opin. Chem. Biol., 2002, 6, 736–741.
3 M. E. W. Saenger, Principles of Nucleic Acid Structure,
Springer Advanced Texts in Chemistry, 1984.
4 S. Burley and G. Petsko, Science, 1985, 229, 23–28.
This journal is ß The Royal Society of Chemistry 2013

RSC Advances
5 C. A. Hunter, J. Singh and J. M. Thornton, J. Mol. Biol.,
1991, 218, 837–846.
6 E. GAZIT, FASEB J., 2002, 16, 77–83.
7 E. A. Meyer, R. K. Castellano and F. Diederich, Angew.
Chem., Int. Ed., 2003, 42, 1210–1250.
8 M. O. Sinnokrot and C. D. Sherrill, J. Phys. Chem. A, 2006,
110, 10656–10668.
9 S. Grimme, Angew. Chem., Int. Ed., 2008, 47, 3430–3434.
Published on 25 July 2013. Downloaded by Institute of Macromolecular Chemistry on 06/11/2017 15:58:36.
10 M. Reches and E. Gazit, Science, 2003, 300, 625–627.
11 M. Reches and E. Gazit, Nano Lett., 2004, 4, 581–585.
12 Y. Zhang, H. Gu, Z. Yang and B. Xu, J. Am. Chem. Soc., 2003,
125, 13680–13681.
13 V. Jayawarna, M. Ali, T. A. Jowitt, A. F. Miller, A. Saiani, J.
E. Gough and R. V. Ulijn, Adv. Mater., 2006, 18, 611–614.
14 R. Orbach, L. Adler-Abramovich, S. Zigerson, I. Mironi-
Harpaz, D. Seliktar and E. Gazit, Biomacromolecules, 2009,
10, 2646–2651.
15 A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R.
F. Collins, M. L. Turner, A. Saiani and R. V. Ulijn, Adv.
Mater., 2008, 20, 37–41.
16 W. Wang and Y. Chau, Chem. Commun., 2011, 47,
10224–10226.
17 W. Wang and Y. Chau, Chem. Mater., 2012, 24, 946–953.
18 T. Kirchhausen, Annu. Rev. Biochem., 2000, 69, 699–727.
19 F. Alexis, E. Pridgen, L. K. Molnar and O. C. Farokhzad,
Mol. Pharmaceutics, 2008, 5, 505–515.
20 S. Mitragotri and J. Lahann, Nat. Mater., 2009, 8, 15–23.
21 W. Jiang, Y. S. KimBetty, J. T. Rutka and C.
W. ChanWarren, Nat. Nanotechnol., 2008, 3, 145–150.
22 M. Perez, M. Dumont and D. Acevedo-Reyes, Acta Mater.,
2008, 56, 2119–2132.
23 D. Kashchiev and G. M. van Rosmalen, Cryst. Res. Technol.,
2003, 38, 555–574.
24 H. Fessi, F. Puisieux, J. P. Devissaguet, N. Ammoury and
S. Benita, Int. J. Pharm., 1989, 55, R1–R4.
25 J. B. Knight, A. Vishwanath, J. P. Brody and R. H. Austin,
Phys. Rev. Lett., 1998, 80, 3863–3866.
26 L. Gwo-Bin, C. Chih-Chang, H. Sung-Bin and Y. Ruey-Jen, J.
Micromech. Microeng., 2006, 16, 1024.
This journal is ß The Royal Society of Chemistry 2013
View Article Online
Paper
27 H. Y. Park, X. Qiu, E. Rhoades, J. Korlach, L. W. Kwok, W.
R. Zipfel, W. W. Webb and L. Pollack, Anal. Chem., 2006,
78, 4465–4473.
28 V. Génot, S. Desportes, C. Croushore, J.-P. Lefèvre, R.
B. Pansu, J. A. Delaire and P. R. von Rohr, Chem. Eng. J.,
2010, 161, 234–239.
29 A. Jahn, W. N. Vreeland, M. Gaitan and L. E. Locascio, J.
Am. Chem. Soc., 2004, 126, 2674–2675.
30 J. Thiele, D. Steinhauser, T. Pfohl and S. Förster, Langmuir,
2010, 26, 6860–6863.
31 R. Karnik, F. Gu, P. Basto, C. Cannizzaro, L. Dean, W. Kyei-
Manu, R. Langer and O. C. Farokhzad, Nano Lett., 2008, 8,
2906–2912.
32 C. T. Lo, A. Jahn, L. E. Locascio and W. N. Vreeland,
Langmuir, 2010, 26, 8559–8566.
33 J. S. Hong, S. M. Stavis, S. H. DePaoli Lacerda, L.
E. Locascio, S. R. Raghavan and M. Gaitan, Langmuir,
2010, 26, 11581–11588.
34 M. Rhee, P. M. Valencia, M. I. Rodriguez, R. Langer, O.
C. Farokhzad and R. Karnik, Adv. Mater., 2011, 23,
H79–H83.
35 C. Simonnet and A. Groisman, Appl. Phys. Lett., 2005, 87,
114104.
36 C. Chih-Chang, H. Zhi-Xiong and Y. Ruey-Jen, J. Micromech.
Microeng., 2007, 17, 1479.
37 A. Jahn, S. M. Stavis, J. S. Hong, W. N. Vreeland, D.
L. DeVoe and M. Gaitan, ACS Nano, 2010, 4, 2077–2087.
38 Z. Wu and N.-T. Nguyen, Sens. Actuators, B, 2005, 107,
965–974.
39 D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago,
S. Weiss and O. Bakajin, Anal. Chem., 2004, 76, 7169–7178.
40 D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin and J.
G. Santiago, Anal. Chem., 2006, 78, 4299–4306.
41 S. Yao and O. Bakajin, Anal. Chem., 2007, 79, 5753–5759.
42 C. D. Volpe, G. Guarino, R. Sartorio and V. Vitagliano, J.
Chem. Eng. Data, 1986, 31, 37–40.
43 A. Fick, Ann. Phys. Chem., 1855, 170, 59–86.
44 D. Magde, G. E. Rojas and P. G. Seybold, Photochem.
Photobiol., 1999, 70, 737–744.
RSC Adv., 2013, 3, 17762–17769 | 17769