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Article
A Numerical Investigation of Enhancing Microfluidic
Heterogeneous Immunoassay on Bipolar Electrodes
Driven by Induced-Charge Electroosmosis in
Rotating Electric Fields
Zhenyou Ge 1 , Hui Yan 1, *, Weiyu Liu 2, * , Chunlei Song 1 , Rui Xue 1 and Yukun Ren 1,3
1 School of Mechatronics Engineering, Harbin Institute of Technology, West Da-zhi Street 92, Harbin 150001,
China; gezhenyou1997@163.com (Z.G.); sclei@hit.edu.cn (C.S.); 15145011764@163.com (R.X.);
rykhit@hit.edu.cn (Y.R.)
2 School of Electronics and Control Engineering, Chang’an University, Middle-Section of Nan’er Huan Road,
Xi’an 710064, China
3 State Key Laboratory of Robotics and System, Harbin Institute of Technology, West Da-Zhi Street 92,
Harbin 150001, China
* Correspondence: yanhui@hit.edu.cn (H.Y.); liuweiyu@chd.edu.cn (W.L.)

Received: 11 July 2020; Accepted: 27 July 2020; Published: 30 July 2020
Abstract: A unique approach is proposed to boost on-chip immuno-sensors, for instance,

immunoassays, wherein an antibody immobilized on the walls of a microfluidic channel binds
specifically to an antigen suspended freely within a working fluid. The performance of these sensors
can be limited in both susceptibility and response speed by the slow diffusive mass transfer of
the analyte to the binding surface. Under appropriate conditions, the binding reaction of these
heterogeneous immuno-assays may be enhanced by electroconvective stirring driven by external AC
electric fields to accelerate the translating motion of antigens towards immobilized antibodies. To be
specific, the phenomenon of induced-charge electroosmosis in a rotating electric field (ROT-ICEO)
is fully utilized to stir analyte in the vicinity of the functionalized surface of an ideally polarizable
floating electrode in all directions inside a tri-dimensional space. ROT-ICEO appears as a consequence
of the action of a circularly-polarized traveling wave signal on its own induced rotary Debye screening
charge within a bipolar induced double layer formed on the central floating electrode, and thereby
the pertinent electrokinetic streamlines exhibit a radially converging pattern that greatly facilitates the
convective transport of receptor towards the ligand. Numerical simulations indicate that ROT-ICEO
can enhance the antigen–antibody binding reaction more effectively than convectional nonlinear
electroosmosis driven by standing wave AC signals. The effectiveness of ROT-ICEO micro-stirring is
strongly dependent on the Damkohler number as well as the Peclet number if the antigens are carried
by a continuous base flow. Our results provide a promising way for achieving a highly efficient
heterogeneous immunoassay in modern micro-total-analytical systems.
Keywords: on-chip immunoassay; induced-charge electroosmosis; rotating electric field; bipolar

floating electrode; microfluidics
1. Introduction
Immunoassays, which is known as the specific binding reaction between the free antigens
suspended in a fluid flow and the immobilized antibodies at a functionalized surface, have been
broadly applied in medical diagnostics, quality control, biomarker identification, and environment
monitoring for their high selectivity [1,2]. Traditional immunoassays, including microarrays and
Micromachines 2020, 11, 739; doi:10.3390/mi11080739 www.mdpi.com/journal/micromachines

Micromachines 2020, 11, 739 2 of 20
enzyme-linked immunosorbent assay (ELISA), usually require a vast sample volume and long
cultivation time due to the complicated fluid handling steps involved at different stages of the
assay [3–6]. Some representative label-free biomolecule detection approaches have been applied to
probe specific proteins and bacteria [7,8]. Nevertheless, the dependence of the test performance on the
interfacial binding reaction, which is routinely limited by slow diffusion rate and long detection time,
limits their broad application in various situations [9,10]. In addition, the low throughput constrains
the application of immunoassay in time-pivotal circumstances [11]. With the rapid development of
microfabrication techniques, embedding the functional biosensors into micro-total-analytical systems
(µTAS) has captured great attention from the microfluidic society [12]. In stark contrast with the
conventional immunoassay approaches, microfluidic biosensors provide enormous advantages to
transport the antigen sample onto the binding surface in a continuous-flow mode, in terms of imposing
a quite low demand on the sample volume and generating high-throughput while with a much shorter
analysis time and an enhanced sensitivity [13]. The mass transfer process where the specific analyte
is conveyed from the bulk of the liquid suspension to the reaction surface is negatively influenced
by the molecular diffusion effect across a concentration gradient on account of a small Reynolds
number in microsystems [13]. Consequently, on-chip immunoassays still suffer from restrictions in both
response speed and detection sensitivity, due to the slow diffusive transport of target antigens to the
functionalized surface with immobilized antibodies, which determines a non-ideal device performance
in diffusion-limited binding reactions with fast surface reactions [14].
A lot of physical mechanisms have been applied to result in stirring flow patterns in microfluidic
channels for on-chip biomedical diagnosis, such as hydrodynamic pressure [15], electrokinetics [16–19],
magnetic effect [20], and optical forces [21]. For instance, Selmi et al. calculated the binding
kinetics using a microchannel-based flow confinement strategy with an orthogonal complementary
stream to guide the translating motion of analyte molecules towards the binding surface [22].
Alternatively, AC electrokinetic (ACEK) phenomenon has become a popular way to drive fluid
motion and manipulate colloid samples within to expected locations in the presence of a low
voltage supply [23–26]. ACEK mainly includes dielectrophoresis (both particles and stratified liquid
contents) [27], AC electrothermal induced flow (ACET) [28–30], AC electroosmosis (ACEO) [31–33],
and induced-charge electroosmosis (ICEO) [34–36]. Both ACET and ACEO have been theoretically
and experimentally exploited for accelerating the convective transport of antigens towards specific
antibodies for enhancing the binding rate on functionalized electrode surfaces [37,38]. On the other
hand, ICEO has emerged as a brand new tool for manipulation of both fluid and particle motion in
microfluidic channels [39]. Like ACEO, ICEO is originated by the action of the applied electric field
on its own induced charge within a thin induced double layer (IDL) on a polarizable solid surface
immersed in an electrolyte solution and has been broadly applied for pumping [40], mixing [41–43],
and particle handing [44,45]. Though ICEO has a similar mechanism with ACEO, the introduction
of floating conductors (namely, floating electrodes) endows ICEO the traits of flexible configuration,
locally addressable, free from external wiring, and easy for device integration as compared to ACEO.
Our group have recently employed ICEO vortex flow field to trap and enrich microscale particle
samples in both static and dynamic conditions with a low voltage supply [46–49]. Pascall et al. designed
a standard electrode structure for actuating ICEO slipping fluid motion on the ideally polarizable
surface of a floating metal strip in the center of the gap between a pair of conducting probes inserted
into the reservoirs on both sides, and demonstrated by strict experimental measurement and physical
argumentation that the presence of a dielectric coating layer and an ion adsorption effect on the central
floating electrode (FE) was responsible for the larger ICEO slipping velocity predicted by the standard
RC circuit theory than that from experiments [50].
As a result, when the reaction rate is essentially much quicker than the diffusive transport of
target analytes to the sensor surface in microchannels, it is necessary to explore a label-free and
highly integrated detection strategy to boost the binding rate between antigen and antibody in
diffusion-limited cases. To address this issue, in this study, we propose an approach to transport the
Micromachines 2020, 11, x 3 of 20

target antigen sample to the binding surface in a dilute electrolyte (typically with electric conductivity
thanthan
less 0.050.05
S/m,S/m,
so as
so to
as evade double-layer
to evade double-layer shrinkage
shrinkage and steric
and stericeffect
effectatathigher
higherionion concentrations)
concentrations)
and improve the device performance of microfluidic heterogeneous immunoassays
and improve the device performance of microfluidic heterogeneous immunoassays by using by using ICEO
ICEO
electroconvective streaming on ideally polarizable surfaces of FE. In particular,
electroconvective streaming on ideally polarizable surfaces of FE. In particular, a traveling-wave a traveling-wave
voltage signal
voltage signalwith
witha 90
a 90° phase
◦ phase shift
shift is imposed
is imposed sequentially
sequentially to a circular
to a circular electrodeelectrode
array ofarray of four
four discrete
discrete phases. ICEO fluid motion with a radially converging flow pattern is induced
phases. ICEO fluid motion with a radially converging flow pattern is induced on the central FE by on the central
such
aFErotating
by suchelectric
a rotating
field,electric field,
namely, thenamely, the phenomenon
phenomenon of ROT-ICEO. of ROT-ICEO.
By numerical Bymodeling,
numericalROT-ICEO
modeling,
ROT-ICEO
is shown to is
beshown to be more
more effective effective the
in delivering in delivering the antigen
antigen samples samples in
in all directions and allsuppressing
directions and
the
suppressing the length of the diffusion boundary layer than ordinary nonlinear electroosmosis
length of the diffusion boundary layer than ordinary nonlinear electroosmosis driven by AC standing driven
by ACsignals,
wave standing wave
due to thesignals,
actiondue totri-dimensional
of its the action of itschaotic
tri-dimensional
streamlines.chaotic streamlines.
2. Theory and Methods
2.1. Device Geometry

2.1. Device Geometry
To investigate the the vortex
vortexflow
flowpattern
patternofofROT-ICEO
ROT-ICEO andandits its important
important rolerole in accelerating
in accelerating the
the binding rate in an immuno-transducer, a microfluidic chip
binding rate in an immuno-transducer, a microfluidic chip with a coplanar array of with a coplanar array of thin-film
microelectrode is designed
designed and and displayed
displayedin inFigure
Figure1.1.AAsquare
squareconducting
conductingFE FEwithwitha awidth
width WW
ofof F is
F
is deposited
deposited onon thethe bottom
bottom surface
surface of aofmicrofluidic
a microfluidic chamber
chamber with with a height
a height of H ofC H C and
and a length
a length of Lof
C,
L C , respectively.
respectively. A circular
A circular array
array of of 4 rectangulardriving
4 rectangular drivingelectrodes
electrodes(DE) (DE)isisdisposed
disposed on on the channel
bottom surface, and surrounds the central FE. By imposing a 90°-phase-shifted ◦
90 -phase-shifted travelling wave (TW)
voltage signal
voltage signal to the 4 DE in sequence, a rotating
sequence, rotating (ROT) (ROT) electric field is produced
produced and runs runs throughout
throughout
the chamber. Since the TW signal rotates circularly, instead of propagating in
chamber. Since the TW signal rotates circularly, instead of propagating in a linear route, a linear route, the resulted
the
electric
resultedfield is infield
electric effect
is aincircularly polarizedpolarized
effect a circularly rotating (ROT)
rotating electric
(ROT)field. It isfield.
electric noteworthy that the
It is noteworthy
rotating field rotates
that the rotating field counterclockwise in the direction
rotates counterclockwise of the of
in the direction decreasing
the decreasingvoltage phase,
voltage so we
phase, socall
we
it a counterclockwise
call it a counterclockwise rotating electric
rotating field.
electric Figure
field. Figure2 is
2 isananadvanced
advanceddevice devicedesign
designunder
under dynamic
incoming laminar streams, which will be be introduced
introduced in in detail
detail in
in Section
Section 2.3.
3.6.
Figure
Figure 1. A systematic
1. A schematic of
systematic schematic of improving
improving the
the specific
specific binding
binding reaction
reaction between
between immobilized
immobilized
antibodies
antibodies and freely suspended antigens in the dilute electrolyte by induced-charge electroosmosis
and freely suspended antigens in the dilute electrolyte by induced-charge electroosmosis
driven by an externally-imposed rotating electric field (ROT-ICEO) on the ideally polarizable surface
driven by an externally-imposed rotating electric field (ROT-ICEO) on the ideally polarizable surface
of a central floating electrode (FE). (a) A 3D sketch for enhancing inhomogeneous immunoassay in a
of a central floating electrode (FE). (a) A 3D sketch for enhancing inhomogeneous immunoassay in a
static microchamber; (b) fluidic samples before electrical powering; (c) electroconvective transport of
static microchamber; (b) fluidic samples before electrical powering; (c) electroconvective transport of
antigens towards the functionalized FE surface accelerates the interfacial binding reaction.
antigens towards the functionalized FE surface accelerates the interfacial binding reaction.
Figure
Figure 2.2. AA3D3Dschematic
schematic illustration
illustration of ROT-ICEO-mediated
of ROT-ICEO-mediated antigen-antibody
antigen-antibody specificspecific
binding binding
reaction
reaction on the conducting
on the conducting surface ofsurface of the
the central FE central FEby
encircled encircled by a four-phase
a four-phase DE under
DE strip array, strip array, under
the dynamic
the dynamic condition that an external pressure-driven laminar flow injects
condition that an external pressure-driven laminar flow injects the target antigen samples from the the target antigen
samples from
inlet to the the inlet tooutlet.
downstream the downstream outlet.
Once they pass onOnce
top ofthey
the pass on FE,
central topthe
of the centralROT-ICEO
localized FE, the localized
vortex
ROT-ICEO vortex flow
flow field disturbs field disturbs
the laminar the laminar
streamlines streamlines
and transports the and transports
antigens to the the
FE’santigens to the FE’s
ideally polarizable
ideally
surface,polarizable
giving rise surface,
to more giving
chance rise to more
for the chance
binding for the
reaction binding
between reaction
antigens between
and antigens
antibodies. (a) Aand3D
antibodies.
schematic of(a)theA dynamic
3D schematic of the dynamic
immuno-sensor in theimmuno-sensor in the presence
presence of continuous of continuous
base flow. (b) A magnifiedbase
flow.
view of(b)the
A magnified
upstream flowviewpassage
of the upstream
before theflow passageantigens
suspended before the suspended
pass antigens
by the reaction pass by
region. (c) the
An
reaction
amplifiedregion.
view of(c)
theAn amplified
critical view
reaction of the
region on critical reaction
top of the region
electrode arrayonaffected
top of by
theelectroconvection
electrode array
affected by electroconvection of ROT-ICEO.
of ROT-ICEO.
The external
The external ROT
ROT field
field injects
injects bipolar
bipolar counterionic
counterionic charges
charges into
into aa thin
thin Debye
Debye layer
layer at at the
FE/electrolyte interface by Ohmic conduction, in the presence of a
FE/electrolyte interface by Ohmic conduction, in the presence of a normal normal field component on the
component on
blocking electrode
blocking electrode at
at the
the early
early stage.
stage. After
After aa characteristic
characteristic RC
RC time scaleτRC
time scale τ RC==R
RC σ ff ((11++δ δ) ) for
CDD /σ for
double-layer capacitive charging, the bulk electric field lines are fully repelled, and a stable bipolar
double-layer capacitive charging, the bulk electric field lines are fully repelled, and a stable bipolar
induced double layer (IDL) is developed on the ideally polarizable surface of the FE in the field center
induced double layer (IDL) is developed on the ideally polarizable surface of the FE in the field center
due to complete field-induced Debye screening as shown in Figure 3a. From the perspective of an
due to complete field-induced Debye screening as shown in Figure 3a. From the perspective of an
observer, this renders the conducting surface manifest as a perfect insulator beyond a characteristic
observer, this renders the conducting surface manifest as a perfect insulator beyond a characteristic
distance scale of the Debye screening length, giving rise to a pair of ICEO micro-vortices in opposite
distance scale of the Debye screening length, giving rise to a pair of ICEO micro-vortices in opposite
rotating directions on top of the central FE due to the interaction of the tangential field components
rotating directions on top of the central FE due to the interaction of the tangential field components
with the dipolar induced ionic charges inside the bipolar IDL (Figure 3a). The above physical picture is
with the dipolar induced ionic charges inside the bipolar IDL (Figure 3a). The above physical picture
valid only under a steady DC bias. For a low-frequency rotating electric field, however, the situation
is valid only under a steady DC bias. For a low-frequency rotating electric field, however, the
becomes more subtle. As the field vector rotates counterclockwise within each complete AC voltage
situation becomes more subtle. As the field vector rotates counterclockwise within each complete AC
cycle, with the axis of rotation constantly fixed at the field center, the bipolar charge accumulated
voltage cycle, with the axis of rotation constantly fixed at the field center, the bipolar charge
within the IDL rotates synchronously in the direction of the electric field as well. In this way, the ICEO
accumulated within the IDL rotates synchronously in the direction of the electric field as well. In this
slipping fluid motion switches alternatively between two complementary convection modes, in which
way, the ICEO slipping fluid motion switches alternatively between two complementary convection
the flow direction is along the x- and y-axis, respectively (Figure 3). After time average operation,
modes, in which the flow direction is along the x- and y-axis, respectively (Figure 3). After time
the ROT-ICEO fluidic eddies ought to exhibit a convergent profile, with the liquid molecules sucked
average operation, the ROT-ICEO fluidic eddies ought to exhibit a convergent profile, with the liquid
from surrounding medium in all outer directions to the center of the conducting surface of the FE and
molecules sucked from surrounding medium in all outer directions to the center of the conducting
then ejected to the bulk fluid along the channel depth direction, as indicated by the 3D toroidal arrows
surface of the FE and then ejected to the bulk fluid along the channel depth direction, as indicated by
in Figure 1. This kind of electroconvective stirring flows on top of the bipolar FE may be in favor of
the 3D toroidal arrows in Figure 1. This kind of electroconvective stirring flows on top of the bipolar
enhancing antigen-antibody specific binding reactions on the functionalized interface of the FE.
FE may be in favor of enhancing antigen-antibody specific binding reactions on the functionalized
interface of the FE.
Figure 3. Theoretically-predicted bi-dimensional phase diagram of the applied rotating electric field
Figure 3. Theoretically-predicted bi-dimensional phase diagram of the applied rotating electric field
lines, induced surface charge, and the resulted transient ROT-ICEO surface slipping flow at different
lines, induced surface charge, and the resulted transient ROT-ICEO surface slipping flow at different
time instants within a complete AC cycle for case (i): (a) t = nT, (b) t = nT + T/4, (c) t = nT + T/2, and (d)
time instants within a complete AC cycle for case (i): (a) t = nT, (b) t = nT + T/4, (c) t = nT + T/2, and (d)
t = nT + 3T/4.
t = nT + 3T/4.
2.2. Basic Theory of ICEO Electroconvection at the Metal/Electrolyte Interface
2.2. Basic Theory of ICEO Electroconvection at the Metal/Electrolyte Interface
To account for the occurrence of transient ICEO streaming on metal electrodes with analytical
To account for the notation
occurrence of transient ICEO streaming on metal electrodes with analytical
convenience, complex is invoked for various electric field variables. For instance, φ(t) =
convenience, complex notation is invoked for various electric field variables.
of theFor instance,

A cos(ωt + θ) = Re Ae e jωt jθ = Re φee jωt , where φ e is the complex amplitude transient AC
( ) φ(t(), and Re()
φvoltage
t = A cos ω t + θ = Re (Ae e) = Re ( 
φ e )
) the real part operator. A,, where
jωt jθ jω t 
φ
ω and θ denote
is the the
complex amplitude
amplitude, offield
angular the frequency
transient
and phase angle of the imposed AC voltage signal, respectively. Under sinusoidal steady-state,
AC voltage φ ( t ) , and Re() the real part operator. A, ω and θ denote the amplitude, angular field
the charge conservation equation within the liquid domain is reduced to the Laplace equation:
frequency and phase angle of the imposed AC voltage signal, respectively. Under sinusoidal steady-
state, the charge conservation equation within ∇ the2 eliquid domain is reduced to the Laplace equation:
φ=0 (1)
∇ 2 φ = 0 (1)
Under the Debye–Huckel limit, the IDL behaves like a thin capacitor skin being charged by the
Under the
conduction Debye–Huckel
current limit, the of
from the resistance IDL behaves
liquid bulk.like a thin capacitor
Consequently, the skin
Ohmicbeing charged
current bybulk
in the the
conduction current from the resistance of liquid bulk. Consequently, the Ohmic current
should be continuous with the displacement current running across the thin boundary layer at the in the bulk
should be continuousinterface:
electrode/electrolyte with the displacement current running across the thin boundary layer at the

electrode/electrolyte interface: σn·∇φ e = jωC0 φ e− φ
e0 (2)
(
σ n ⋅∇φ = jωC0 φ − φ 0 ) (2)
where n denotes the unit outward vector normal to the thin-film electrode, pointing from the reaction
surface to the bulk of the liquid suspension, and σ the electrolyte conductivity. C0 = CD /(1 + δ) is the
total double-layer capacity, which is essentially a series connection of the diffuse layer capacitance
CD = ε/λD and Stern layer capacitance Cs = 0.8 F/m2 , in terms of the surface capacitance ratio
δ = CD /CS . φe and φe0 are the electrostatic potential in the bulk fluid right outside the IDL and the
equal potential of the metal electrode, respectively.
The peripheral DE array located adjacent to the channel sidewalls is powered by a TW voltage
signal, with the specific voltage sequence φ10 = Acos(ωt), φ20 = Acos(ωt + 90◦ ), φ30 = Acos(ωt + 180◦ ),
φ40 = Acos(ωt + 270◦ ) imposed to the four DE strips along the clockwise direction, while the central
square electrode is floating in potential, as shown in Figure 1.
Only the voltage drop across the diffuse layer eζ=φ e0 − φ/1
e +δ serves as the effective induced zeta
potential (IZP) that contributes to the induced electrokinetic flows. The time-averaged ICEO slipping
under AC forcing is derivable from the generalized Helmholtz–Smoluchowski formula, which is
subsequently inserted into the Navier–Stokes equation as a leaking wall boundary condition on the
ideally polarizable surfaces of all the metal electrodes:
D E ε 1 ee∗ ε 1 ∗
uslip = − Re ζEt = Re φ e− φ
e0 e E −e
E·n·n (3)
η2 2η 1 + δ
where η is dynamic viscosity of water, < . . . > the time-average operator for calculating the averaged
value within one sinusoidal voltage cycle, and the asterisk * the complex conjugate.
Fluid flows within the microchannel obeys the simplified Navier–Stokes equation for water-based
incompressible Newtonian fluids:
− ∇p + η∇2 u = 0 (4)
∇·u = 0 (5)
where p denotes the hydrostatic pressure, and u the vector field of flow velocity originated by
combined ICEO and external pressure gradient. It is well known that ICEO fluid motion vanishes
in high conductivity buffer solutions due to both double layer shrinkage and ion overcrowding
phenomenon inside the IDL. So, we prefer to study herein the effect of ROT-ICEO on improving
microfluidic immunoassays in dilute electrolyte with electric conductivity usually no more than
0.02 S/m. The aqueous electrolyte is a typical Newtonian fluid, and has a constant dynamic viscosity,
serving as the most appropriate liquid medium for suspending the free antigens in the present analysis.
On the other hand, recently, the necessity of manipulation of biofluids in small confinements has
triggered a renewed interest in the dynamics of non-Newtonian fluid with a shear-rate-dependent
viscosity. However, this is beyond the scope of current work with the purpose to provide a utilitarian
reference for the selection of the parametric space in experiments with water-based Newtonian fluid.
As a result, a uniform viscosity value η = 0.001 Pa·s is used in the simulation to reconstruct the actual
mechanical behavior of target antigens monodispersed in water solution. Please refer to Ref. [51–53]
for a systematic knowledge of both Newtonian fluid and non-Newtonian fluid.
2.3. Mass Transfer of Antigen and Binding Reaction Enhancement

Mass conservation of the target antigens freely suspended in the liquid flow can be mathematically
described by the standard convection-diffusion equation:
∂c
+ ∇·J = 0 (6)
∂t
J = uc − D∇c (7)
where c stands for the concentration of antigens in the bulk fluid, and D their mass diffusivity. An initial
background concentration of antigens is introduced to the microchamber in the static case (Figure 1) or
to the channel inlet in the dynamic condition (Figure 2).
The specific binding reaction between the antigen in liquid suspension and the antibodies adhered
to the surface of the FE can be presumed to abide by the first-order Langmuir adsorption model:
∂B
= kon Cw (RT − B) − koff B (8)
∂t
where B represents the surface concentration of the antigen bound on the reaction surface in mol/m2 ,
kon and koff denote the association rate constant and the dissociation counterpart, respectively. Cw is the
antigen volumetric concentration just on top of the reaction surface, and RT the surface concentration
of the fixed antibody in mol/m2 .
To enable the quantification of the feasibility of ROT-ICEO micro-stirring in elevation of the
antigen-antibody binding rate, we define the transient binding enhancement factor, Be(t) = BV (t)/B0 (t),
in which BV (t) and B0 (t) are the bound antigen concentration after introducing ROT-ICEO slipping
flow and without voltage supply at time node t, respectively. It has been reported that the binding rate
is also highly dependent on the nondimensional Damkohler number:
Da = kon RT HC /D (9)
which is the ratio of the reaction speed to the diffusion speed. The Da number is usually employed
to judge whether the biosensor is restricted by diffusion or by reaction. If the reaction rate is quicker
than the diffusion transport of target analyte to the sensor surface, the whole binding process is
diffusion-limited. On the contrary, the binding rate will be reaction-limited in the situation whereby
the analyte diffusion is fast but the reaction speed cannot match with the rate of diffusion.
3. Results and Discussion
3.1. Binding Reaction Enhancement by ROT-ICEO Micro-Stirring

Three distinct convection modes of ICEO in this four-phase rotating electrode array are compared
in detail in the Supplementary Information (SI, Figures S1–S6), with the corresponding powering
schemes explicitly presented in Table 1. As the optimum ICEO slipping flow profile is created by case
(i), we then focus on the effect of ROT-ICEO micro-stirring on the antigen-antibody binding reaction on
the functionalized surface of the central FE. Before the DE array is energized, in the absence of ICEO
micro-stirring, the concentration of antigen freely suspended in the fluid bulk is primarily depleted by
the molecular diffusion effect. The mass transfer limitation constrains severely the binding reaction
between the free antigen and the fixed antibody, and results in the rapid growth of the antigen-depleted
diffusion boundary layer (Figure 4a), the thickness of which determines the detection performance of
the proposed immunosensor. When the four metal strips in the peripheral DE array are excited by a
TW voltage sequence with neighboring electrodes of a 90◦ phase shift at voltage amplitude V 0 = 8 V
and field frequency f = 200 Hz, the transversal ROT-ICEO recirculating vortex flow field stirs effectively
the fluidic sample and thereby redistributes the depleted antigen concentration on top of the central
square FE. As displayed in Figure 4b, the rotating electrokinetic whirlpools enhance the convection
effect on the functionalized surface of the FE, facilitating greatly the efficient transport of free antigens
in the bulk to the reaction surface. Consequently, the depletion boundary layer shrinks in thickness,
and depletion occurs only in the central region of the FE surface (Figure 4b), which provides a sufficient
chance for a binding interaction between the free antigen and immobilized antibody, leading to an
enhancement of both the association and disassociation rates.
Table 1. AC voltage sequence for the three distinct power supply modes.
Power Supply Table 1. AC voltage sequence for the three distinct power supply modes.
1st Terminal 2nd Terminal 3rd Terminal 4th Terminal
Modes
Power Supply Modes 1st Terminal 2nd Terminal 3rd Terminal 4th Terminal
(i) (i) V1 = V0cos(ωt)
V 1 = V 0 cos(ωt)V2 = V0cos(ωt + 90°)
V 2 = V 0 cos(ωt + 90◦ ) V3 =VV0cos(ωt
3 = V 0
+
cos(ωt 180°)
+ 180 ◦) V4V =
4 =V0cos(ωt
V 0 cos(ωt ++270
270°)
◦)
(ii) (ii) V 1 = V 0 cos(ωt)

V1 = V0cos(ωt) V 2 = V 0 cos(ωt)
V2 = V0cos(ωt) 3 = V 0 cos(ωt
V3 =VV0cos(ωt ++180°)
180◦ ) V4V4==V0cos(ωt
V 0 cos(ωt ++180◦)
180°)
V 1 = V 0 cos(ωt) V 2 = V 0 cos(ωt + 180◦ ) = V 0 cos(ωt)
(iii)(iii) V1 = V0cos(ωt) V2 = V0cos(ωt + 180°) V3 =V3V0cos(ωt) V4V4==V0cos(ωt
V 0 cos(ωt + 180◦ )
+ 180°)
.
Figure4.4.Simulation
Figure Simulationresult
result of the
of the volumetric
volumetric concentration
concentration of suspended
of suspended antigens
antigens on top ofonthe
top of the
binding
surface t = 100 at
bindingatsurface t =the
s for 100following
s for the two
following two circumstances:
circumstances: (a) Concentration
(a) Concentration distributiondistribution
of suspended of
suspended
antigens in antigens
the absencein the
of absence
any AC of any AC
voltage voltage
supply supply
with onlywith onlydiffusive
analyte analyte diffusive
transporttransport
on the FE,on
the FE, toleading
leading a thick to a thick boundary
depletion depletionlayer.
boundary
(b) Thelayer. (b) Therecirculating
ROT-ICEO ROT-ICEO flowrecirculating flow stirs
stirs effectively the
depleted concentration
effectively the depletedafter the peripheral
concentration afterDE
thearray is powered
peripheral by a four-phase
DE array is poweredAC by voltage signalAC
a four-phase at
0 =
Vvoltage
8 V and f = 200 Hz.
signal at V0 = 8 V and f = 200 Hz.
To
Toexplore
explore thethe
dependence
dependence of theofbound antigenantigen
the bound concentration on the applied
concentration on the voltage magnitude,
applied voltage
we calculatedwe
magnitude, bycalculated
numericalby simulation
numerical thesimulation
binding rate theinbinding
the presence
rate inofthe
ROT-ICEO
presencemicro-stirring
of ROT-ICEO
under sinusoidal
micro-stirring steady-state,
under sinusoidal with respect to with
steady-state, the non-improved
respect to thesituation with no
non-improved voltage with
situation supply,
no
when Da number equals 660. As identified in Figure 5a, when the four DE
voltage supply, when Da number equals 660. As identified in Figure 5a, when the four DE strips strips are activated with
are
varying
activatedvoltage amplitude
with varying of 0 (the
voltage passive of
amplitude case), 2, 4,
0 (the 8, 16, and
passive 32 2,
case), V 4,
with a prescribed
8, 16, signal
and 32 V with frequency
a prescribed
of 200 Hz (the active case), the resulted binding rate becomes monotonously
signal frequency of 200 Hz (the active case), the resulted binding rate becomes monotonously higher with larger imposed
higher
voltages.
with larger In imposed
the low voltage
voltages.range
In the(0–8
lowV), the binding
voltage rate is
range (0–8 V),almost linearly
the binding proportional
rate to the
is almost linearly
proportional to the voltage amplitude, in that the enhanced ROT-ICEO whirlpool promotes the
voltage amplitude,
convective transportin ofthat theantigen
free enhanced to ROT-ICEO whirlpoolsurface
the functionalized promotesfor the convective transport
diffusion-limited bindingof
free antigen to the functionalized surface for diffusion-limited binding reactions.
reactions. In accordance with the classical RC-circuit theory of ICEO, our numerical modeling In accordance with
the classical
indicates thatRC-circuit theory of
the ROT-ICEO ICEO, our
slipping numerical
velocity has amodeling
quadratic indicates
dependencethat the
onROT-ICEO slipping
the imposed AC
velocity has a quadratic dependence on the imposed AC voltage amplitude,
voltage amplitude, and increases by 4-fold as the voltage is doubled (Figure 5b). As the voltageand increases by 4-fold as
the voltage is doubled (Figure 5b). As the voltage further increases beyond
further increases beyond 8 V, the growth trend of the binding rate slows down, since the reaction8 V, the growth trend of
the binding
speed on therate slows down,surface
functionalized since the reaction
cannot matchspeed
the on
fastthe functionalized
ROT-ICEO surface cannot
electroconvection, match the
namely the
fast ROT-ICEO electroconvection, namely the binding reaction transits from being
binding reaction transits from being diffusion-limited to being reaction limited as the time-averaged diffusion-limited to
being reaction
ROT-ICEO limited
slipping as theon
velocity time-averaged ROT-ICEO
the ideally polarizable slipping
surface velocity
under harmonic on the
ACideally
forcingpolarizable
exceeds a
surface under harmonic AC forcing exceeds a certain threshold value. In this sense,
certain threshold value. In this sense, there always exists an optimal AC voltage for the improvement there always exists
an optimal AC voltage for
of the specific binding reaction. the improvement of the specific binding reaction.
5. (a)
Figure 5.
Figure (a)Normalized
Normalized concentration evolution
concentration of theof
evolution bound
the antigen
bound with respect
antigen to the
with immobilized
respect to the
antibody on antibody
immobilized the functionalized surface of FE
on the functionalized affected
surface by affected
of FE ROT-ICEO under varying
by ROT-ICEO under AC voltage
varying ACat
f = 200 Hz.
voltage at f =(b) ROT-ICEO
200 slipping velocity
Hz. (b) ROT-ICEO slippingasvelocity
a function
as aoffunction
the applied voltage
of the applied = 200 Hz.
at f voltage at f = 200
Hz.
3.2. Effect of the Damkohler Number
3.2. Effect of the Damkohler
To investigate Number of ROT-ICEO rotating whirlpool in microfluidic immunoassays, we
the importance
calculate the binding enhancement factor Be of the binding reaction on the application of an AC signal
To investigate the importance of ROT-ICEO rotating whirlpool in microfluidic immunoassays,
with V 0 = 4 V and f = 200 Hz for a time period of t = 100 s. As shown in Figure 6a, the antigen-antibody
we calculate the binding enhancement factor Be of the binding reaction on the application of an AC
binding rate enhances globally with an increase of the Damkohler number, implying a higher binding
signal with V0 = 4 V and f = 200 Hz for a time period of t = 100 s. As shown in Figure 6a, the antigen-
efficacy yielding a factor of 21.5 higher binding rate for an applied TW voltage of 4 V at 200 Hz
antibody binding rate enhances globally with an increase of the Damkohler number, implying a
with a Da number of 1000. For a small Da number, ICEO vortex flow on top of the FE is not able to
higher binding efficacy yielding a factor of 21.5 higher binding rate for an applied TW voltage of 4 V
enhance the binding efficiency by transporting electro-convectively the free antigens to the binding
at 200 Hz with a Da number of 1000. For a small Da number, ICEO vortex flow on top of the FE is not
surface due to the slow reaction confinement. On the other hand, when Da is sufficiently large and
able to enhance the binding efficiency by transporting electro-convectively the free antigens to the
surpasses a certain threshold, the reaction rate becomes too fast, and therefore not enough time is left
binding surface due to the slow reaction confinement. On the other hand, when Da is sufficiently
for association. For instance, once the DE array is excited by 4 V at 200 Hz, the ROT-ICEO micro-stirring
large and surpasses a certain threshold, the reaction rate becomes too fast, and therefore not enough
is not potent enough, so that the binding performance reaches a plateau when Da number is beyond
time is left for association. For instance, once the DE array is excited by 4 V at 200 Hz, the ROT-ICEO
10,000 (Figure 6a). Under this situation, the voltage amplitude has to be further elevated, so as to get a
micro-stirring is not potent enough, so that the binding performance reaches a plateau when Da
larger ICEO flow velocity for convective delivery of free antigens towards the functionalized surface
number is beyond 10,000 (Figure 6a). Under this situation, the voltage amplitude has to be further
and lead to an obvious improvement in the binding rate. On the basis of the above analysis, a higher
elevated, so as to get a larger ICEO flow velocity for convective delivery of free antigens towards the
Da number (Da = 1000) is chosen for subsequent simulations.
functionalized surface and lead to an obvious improvement in the binding rate. On the basis of the
Considering the ROT-ICEO circulating fluid motion has a second-order dependence on the voltage
above analysis, a higher Da number (Da = 1000) is chosen for subsequent simulations.
magnitude imposed on the electrode array, we then study the relationship between the binding rate
enhancement and the AC voltage amplitude when the Da number is fixed at Da = 1000. As indicated in
Figure 6b, when an AC signal at an intermediate field frequency f = 200 Hz is applied to the peripheral
DE array, the interfacial binding rate rises quickly by adjusting the source voltage magnitude and is
almost linearly proportional to the background electric field intensity with the increment of the voltage
amplitude below 16 V (Figure 6b). The reason behind is that, since the reaction rate on the conducting
surface of the central FE is fast, the diffusive transport nature of the free antigen determines there
is no possibility for efficient binding reaction to occur. In the presence of ROT-ICEO micro-vortices,
however, electroconvection accelerates the mass transfer of antigens to the functionalized surface and
enhances the
Micromachines binding
2020, 11, x performance. 10 of 20
Figure
Figure 6.
6. Effect
EffectofofDa
Danumber
numberononthe
thebinding
bindingefficiency
efficiencyimprovement
improvementdue duetotothe
theaction
actionof
ofROT-ICEO
ROT-ICEO
slipping
slipping flow.
flow. (a)
(a) A
Adata
datapoint
pointplot
plotofofthe
thebinding
bindingenhancement
enhancementfactor
factorBe
Bewith
withrespect
respectto
toan
anincreasing
increasing
Da
Da number
number under AC voltage V = 4Vand
V00=4V andfield frequencyf f==200
fieldfrequency 200Hz.
Hz.(b)
(b)Voltage-dependent
Voltage-dependentBe Be for
for
different voltage
different voltage amplitudes at t = 100 s when Da = 1000.
= 100 s when Da = 1000.
With further the

Considering increase of the applied
ROT-ICEO circulatingvoltage,
fluidthe binding
motion hasrate enhances to adependence
a second-order slight degreeon as the
the
voltage magnitude
voltage exceeds 16 V, which can
imposed on thebe explained
electrode by the excessively
array, we then study largetheROT-ICEO
relationship slipping
between velocity
the
in the lateral
binding direction on the
rate enhancement andideally
the ACpolarizable surface of
voltage amplitude FE inthe
when contrast to the is
Da number limited
fixed at reaction rate.
Da = 1000.
In fact,
As the larger
indicated applied
in Figure voltage
6b, when anisACcertainly
signal atimportant for the field
an intermediate acceleration
frequency of the
f = 200binding
Hz is reaction,
applied
while
to the actual binding
the peripheral DE array, ratethe
may be negatively
interfacial bindinginfluenced
rate risesby a sufficiently
quickly large voltage
by adjusting the source because
voltagethe
reaction rateand
magnitude is not able to linearly
is almost match with the moving
proportional tovelocity of the freeelectric
the background antigens to the
field binding
intensity surface
with the
any longer.ofThat
increment the is, the antigen-antibody
voltage amplitude below binding
16 V reaction
(Figure turns fromreason
6b). The being behind
diffusion-limited
is that, sinceto being
the
reaction rate
reaction limited at large
on the voltages.
conducting So, it will
surface becentral
of the meaningless to simply
FE is fast, increasetransport
the diffusive the voltage amplitude
nature of the
for boosting
free the bindingthere
antigen determines response
is no of the microfluidic
possibility immunosensors.
for efficient binding reaction It isto
then
occur.necessary for us to
In the presence
seek
of other possible
ROT-ICEO ways to achieve
micro-vortices, however, the electroconvection
same goal. accelerates the mass transfer of antigens to
the functionalized surface and enhances the binding performance.
3.3. Frequency-Dependent Binding Reaction
With further increase of the applied voltage, the binding rate enhances to a slight degree as the
voltage Theexceeds 16 V, which
ideal operating can beof
condition explained by the excessively
the immunosensor is supposed largetoROT-ICEO
depend on the slipping
signalvelocity
frequency, in
the lateral direction on the ideally polarizable surface of FE in contrast to the
in that the ROT-ICEO slipping velocity itself is very susceptible to the field frequency of the applied limited reaction rate. In
fact, the larger
TW voltage applied
(Figure voltage
S1 and Figureis certainly important
S2b). As shown for the7b,
in Figure acceleration
the ROT-ICEO of thefluidbinding
motion reaction,
attains
while the actual
a localized binding
relaxation rateat
peak may
an be negatively frequency
intermediate influencedbetween
by a sufficiently largeRC
the inverse voltage because the
time constant for
reaction rate is not able to match with the moving velocity of the free antigens
electrochemical polarization of the peripheral DE array and that of the central FE. Namely, fRC ≤ to the binding DE
surface
any FE , which
fideallonger.
≤ fRC That is,agrees
the antigen-antibody
well with the precedingbinding reaction
analysisturns from 3.1.
in Section being diffusion-limited
According to being
to the simulation
reaction
result in limited
Figure 7b,at large voltages.
the ideal working So, it will be meaningless
frequency of ICEO is fideal = 200 Hz,
to simply increase the voltage
any further amplitude
deviation of the
for
fieldboosting
frequency the from
bindingthisresponse of thewould
critical value microfluidic
make the immunosensors.
ICEO flow velocity It is then
decay necessary
to a greatforextent
us to
seek
whateverother thepossible
applied ways to achieve
voltage is 4 V,the8 V,same
or 16goal.
V. For instance, the slipping fluid motion decreases by
almost 50% as the signal frequency increases from the ideal frequency of 200 Hz to a higher value of
3.3.
1000Frequency-Dependent
Hz (Figure 7b). Although Binding the Reaction
binding enhancement factor Be is also maximized at the same ideal
frequency
The ideal operating conditionmore
200 Hz, it decreases no of thethan 50% with the increment
immunosensor is supposed in frequency
to dependfrom on the200signal
Hz to
1000 Hz (Figure
frequency, in that7a),
thewhich can be ascribed
ROT-ICEO slipping to the factitself
velocity that not onlysusceptible
is very the ROT-ICEO to theslipping fluid motion
field frequency of
but also the molecular diffusion effect contribute to the immuno-reaction.
the applied TW voltage (Figure S1 and Figure S2b). As shown in Figure 7b, the ROT-ICEO fluid In practical experiments, it is
quite suitable
motion attainsfor us to raiserelaxation
a localized the field frequency
peak at antointermediate
1000 Hz or even 2000–10,000
frequency between Hz the(Figure 7a), RC
inverse since the
time
binding rate
constant decays much more
for electrochemical slowly than
polarization of the
the ROT-ICEO
peripheral slipping
DE arrayfluidandmotion
that ofitself as the field
the central FE.
DE FE
Namely, f RC ≤ fideal ≤ f RC , which agrees well with the preceding analysis in Section 3.1. According
to the simulation result in Figure 7b, the ideal working frequency of ICEO is fideal = 200 Hz, any further
deviation of the field frequency from this critical value would make the ICEO flow velocity decay to
a great extent whatever the applied voltage is 4 V, 8 V, or 16 V. For instance, the slipping fluid motion
at the same ideal frequency 200 Hz, it decreases no more than 50% with the increment in frequency
from 200 Hz to 1000 Hz (Figure 7a), which can be ascribed to the fact that not only the ROT-ICEO
slipping fluid motion but also the molecular diffusion effect contribute to the immuno-reaction. In
practical experiments, it is quite suitable for us to raise the field frequency to 1000 Hz or even 2000–
10,000 Hz (Figure 7a), since the binding rate decays much more slowly than the ROT-ICEO slipping
fluid motion
frequency itself as
increases the field
(Figure frequency
7b). By increases
doing this, (Figure
we can insist on 7b). By doing
improving this, weperformance
the binding can insist on of
improving the binding
the microfluidic performance
immuno-sensors of the microfluidic
without immuno-sensors
having to know without
the accurate ideal havingfrequency
operating to know the in
accurate
advance ideal operating
and evade frequency
the bipolar in advance
electrode and
reaction evade
and theproduction
bubble bipolar electrode reaction
on top of and bubble
the central FE that
production
tend to occur onbelow
top of1000
the central FE same
Hz at the that tend
time.to occur below 1000 Hz at the same time.
Figure 7.
Figure Effect of
7. Effect of the
the AC
AC signal
signal frequency
frequency on binding reaction with Da =
on the binding 1000. (a)
= 1000. (a) A
A data
data point
point
plot of
plot of the
the binding
binding enhancement
enhancement factor Be versus
factor Be versus AC AC signals
signals of
of varying
varying oscillation
oscillation frequencies
frequencies when
when
the voltage
the voltage amplitude
amplitude takes
takes the
the value
value of V00==4 4V,V,88V,V,and
of V and16
16V,
V,respectively.
respectively. (b)
(b)ROT-ICEO
ROT-ICEO slipping
slipping
velocity as
velocity as aa function
function of
of the
the field
field frequency for distinct voltage amplitude.
3.4. Effect of Geometric Arrangement of the Floating Electrodes

3.4. Effect of Geometric Arrangement of the Floating Electrodes
In this section, we study how the geometric configuration of the floating electrodes in the field
In this section, we study how the geometric configuration of the floating electrodes in the field
center exerts an influence on the binding efficiency enhancement at a given TW voltage amplitude
center exerts an influence on the binding efficiency enhancement at a given TW voltage amplitude of
of V = 4 V. In preceding discussions, the square FE in the center of the rotating electric field has a
V0 = 04 V. In preceding discussions, the square FE in the center of the rotating electric field has a given
given edge length of W = 100 µm, being comparable in size with the channel depth HC = 200 µm.
edge length of WF = 100 Fμm, being comparable in size with the channel depth HC = 200 μm. So, the
So, the first choice for changing the geometry is to adjust the edge length of the individual FE while
first choice for changing the geometry is to adjust the edge length of the individual FE while
maintaining its quantity.
maintaining its quantity.
As shown in Figure 8b, the ideal working frequency shifts to lower values as the FE’s
As shown in Figure 8b, the ideal working frequency shifts to lower values as the FE’s edge length
edge length
rises from 100 rises μm
from to100300µm to 300keeping
μm, µm, keeping consistent
consistent withwith
the the theoreticalprediction
theoretical prediction of of
fideal = σλD (1 + δ)/2πεR, in which an increment of the characteristic macroscopic length scale R
fideal = σλD (1 + δ ) 2π ε R , in which an increment of the characteristic macroscopic length scale R for
for electrochemical polarization of the FE results in a smaller RC charge relaxation frequency at the
electrochemical polarization
FE/electrolyte interface. of the
It is well FE results
known in a flow
that ICEO smaller RC charge
velocity becomes relaxation
faster as frequency at the
the size of the FE
FE/electrolyte
increases, as evidenced by the simulation result of ROT-ICEO slipping fluid motion for different FE
interface. It is well known that ICEO flow velocity becomes faster as the size of the FE
increases,
edge lengths as evidenced
(Figure 8b)by asthe simulation result of ROT-ICEO slipping fluid motion for different FE
well.
edge As lengths (Figure
for the 8b) as well.
corresponding enhancement of binding performance, however, a marked difference
has emerged (Figure 8a). Although the ideal operating frequency for the microfluidic immunosensor
shifts to lower frequency values just like the ideal ICEO flow rate, the maximum binding enhancement
factor Be decreases as the FE becomes larger in size (Figure 8a). A plausible reason for this particular
difference is that the ROT-ICEO slipping velocity is indeed fastest on the surface of the largest FE
(the blue line in Figure 8b), but the ICEO vortex flow field in the bulk fluid tends to be suppressed
to a great degree by a vertical confinement effect as the size of FE approaches and even exceeds the
channel depth (Figure 8c). Consequently, the recirculating fluid motion in the liquid bulk due to the
action of ROT-ICEO cannot be fully developed in the presence of a finite channel height (Figure 8c),
which may exert an adverse effect on the binding reaction enhancement. This conclusion suggested
that it is not necessary to deliberately enlarge the size of the square FE for accelerating the analyte
mass transfer towards the functionalized surface, and there exists an intermediate edge size on the
order of the channel depth serving as the best choice for such purpose.
Figure
Figure8.8.Effect
Effectofofthe
theFE’s size
FE’s onon
size thethe
binding
bindingreaction improvement
reaction improvement at aatgiven voltage
a given amplitude
voltage V0
amplitude
=V40 V.
= (a)
4 V.Frequency-dependent
(a) Frequency-dependent Be forBedifferent FE’s edge
for different FE’s length; (b) Frequency-dependent
edge length; (b) Frequency-dependent ROT-
ICEO slipping
ROT-ICEO velocity
slipping when when
velocity the edge
thelength of the of
edge length central FE takes
the central FE the value
takes of 100of
the value μm,
100200 μm,
µm, 200and
µm,
300 μm, respectively. (c) A cross-sectional plot of the suspended antigen concentration
and 300 µm, respectively. (c) A cross-sectional plot of the suspended antigen concentration on top on top of a 300
of a
μm
300×µm
300×μm 300FE
µm with an AC
FE with anvoltage supply
AC voltage of V0 of
supply = 4 Vf =and
= 4VV0 and = 150
150f Hz at tHz at ts.= 100 s.
= 100
The
As forsecond way to reconfigure
the corresponding the geometry
enhancement of the
of binding central FE ishowever,
performance, to alter the number difference
a marked of discrete
floating
has emergedelectrodes
(Figurelocated in the center
8a). Although of the
the ideal microchamber.
operating frequencyAs forshown in Figure 9c,
the microfluidic we introduce
immunosensor
a 3 × to
shifts 3 coplanar FE array to
lower frequency take the
values justplace
like of
thethe original
ideal ICEO individual
flow rate, FE the
surrounded
maximum by binding
the four
peripheral DE
enhancement metal
factor Bestrips. In the
decreases as absence of AC power,
the FE becomes larger inthesize
target analytes
(Figure 8a). Asuspended in the bulk
plausible reason for
are consumed by the diffusion-limited interfacial binding reaction, resulting in
this particular difference is that the ROT-ICEO slipping velocity is indeed fastest on the surface of the the formation of one
thick depleted
largest boundary
FE (the blue line inlayer
Figureon the
8b),ideally
but thepolarizable
ICEO vortex surfaces of each
flow field in of
thethe 9 floating
bulk conductors
fluid tends to be
respectively,
suppressed toas shown
a great in Figure
degree by a9c. On switching
vertical confinementthe multiphase
effect as thefunction
size of FE generator
approacheson, ROT-ICEO
and even
whirlpools
exceeds appear on
the channel all the
depth FEs, 8c).
(Figure which are constantly
Consequently, thesucked from the
recirculating surrounding
fluid motion in the medium
liquid to the
bulk
electrode
due to the center,
action then ejected upward
of ROT-ICEO cannottobethe topdeveloped
fully surface of the fluidic
in the chamber,
presence of a and
finitefinally
channelspread out
height
in all directions
(Figure 8c), whichtomay form closed
exert fluid loops.
an adverse effectAsona the
consequence, all the enhancement.
binding reaction depletion boundary layers are
This conclusion
reshaped by
suggested thatROT-ICEO vortex flow
it is not necessary field, becoming
to deliberately enlargemuchthethinner on the
size of the electrode
square FE forsurface with most
accelerating the
of the depletion
analyte mass transferregion shifted the
towards above the center ofsurface,
functionalized the floating
and conductors
there exists(Figure 9d), whichedge
an intermediate indicates
size
anthe
on enhancement
order of thein electroconvective
channel depth serving transport of the
as the best freefor
choice antigens to the functionalized surfaces,
such purpose.
The second way to reconfigure the geometry of the central FE is to alter the number of discrete
floating electrodes located in the center of the microchamber. As shown in Figure 9c, we introduce a
3 × 3 coplanar FE array to take the place of the original individual FE surrounded by the four
bringing about more opportunities for the binding reaction between the immobilized antibody and
target analyte.
Figure
Figure 9. The
9. The effectofofthe
effect thenumber
numberofof FE
FE in
in the
thediscrete
discreteelectrode
electrodearrangement
arrangement onon
thethe
degree of binding
degree of binding
reaction improvement when the voltage amplitude of the applied rotating
reaction improvement when the voltage amplitude of the applied rotating electric fields electric fields is V0 = 4V. (a)
is V 0 = 4V.
The binding
(a) The bindingenhancement
enhancementfactorfactorBeBeononeach
eachelectrode
electrodein in
thethe
FEFE
array versus
array ACAC
versus signals of different
signals of different
field frequencies at t = 100 s. (b) Frequency-dependent ROT-ICEO slipping velocity on each electrode
field frequencies at t = 100 s. (b) Frequency-dependent ROT-ICEO slipping velocity on each electrode in
in the FE array. (c) Multiple cross-sectional plots of the suspended antigen concentration with no
the FE array. (c) Multiple cross-sectional plots of the suspended antigen concentration with no external
external voltage supply at t = 100 s. (d) Multiple cross-sectional plots of the suspended antigen
voltage supply at t = 100 s. (d) Multiple cross-sectional plots of the suspended antigen concentration at
concentration at t = 100 s with an imposed rotating electric field at V0 = 4 V and f = 200 Hz.
t = 100 s with an imposed rotating electric field at V 0 = 4 V and f = 200 Hz.
3.5. Binding Reaction Enhancement in a Pressure-Driven Flow
Even so, the binding rate is different from one another on the 9 FE. The binding enhancement
factor Be is the least for the middlemost FE (Be = 16 for the 5th electrode at f = 200 Hz), and a bit
larger on the outer ones (Be ranges from 18 to 25 for the 1st–4th and 6th–9th electrodes at f = 200 Hz).
This unexpected growth phenomenon of the bound antigen concentration on the peripheral eight
electrodes can be accounted for by the difference in ICEO slipping velocity within the microfluidic
system (Figure 9b). As shown in Figure 9b, the ICEO slipping fluid motion is weakest on the middlemost
FE as well, corresponding to the least improvement of the binding efficiency (Figure 9a). The unequal
electrokinetic flow velocity on top of the 9 FE can be ascribed to the following two reasons: (1) The
discrete arrangement of the coplanar FE array makes the electric field unevenly distributed in the
fluidic chamber, which is stronger on the outer eight FEs and weakest on the middlemost FE. Since
the electroosmotic flow velocity is linearly proportional to the intensity of the tangential electric field
component that reaches a local minimum in the field center, the nonlinear ROT-ICEO slipping velocity
is suppressed to a certain degree on the 5th FE. (2) The ACEO fluid motion has a net flow component
that propagates in the direction of the rotating electric field (Supplementary Figure S3a,b), which can be
effectively superimposed with the ICEO vortex flow field on the outer eight electrodes, but diminishes
gradually along the field gradient due to the action of finite penetration depth, resulting in a higher
electroosmotic flow rate on the surface of the 8 FE close to the DE array than that on the middlemost
one that is most far away from chamber sidewalls. The above two factors coact to make the ideal Be
number Be = 16 (the purple line in Figure 9a) on the functionalized surface of the central FE lower than
Be = 22 (the black line in Figure 8a) with merely a single FE. However, the largest Be = 27 on the 1st FE in
the 3 × 3 discrete FE arrangement is still much higher than Be = 22 in the 1 × 1 configuration. The large
variance of bound antigen concentration on different FEs in the advanced device design (Figure 9a,d)
may be useful under certain circumstances that desire a range of binding rates simultaneously at
distinct positions of the inhomogeneous immunoassay.
3.5. Binding Reaction Enhancement in a Pressure-Driven Flow

To deal with a more realistic situation in a microfluidic device with continuous sample injection,
we have to check the effectiveness of the radially converging ROT-ICEO slipping fluid motion on
top of the FE in a straight microchannel under the influence of an axial pressure gradient on the
antigen-antibody binding reaction. For such, we prescribed the boundary condition at the upstream
channel entrance (Figure 10) with a paraboloidal profile of a mean inlet flow rate u0 = 25–200 µm/s in
the full scale tri-dimensional computational domain. In this integrated device design, two straight
branch channels on both sides are bridged by a microchamber in the center, as shown in Figure 10.
In contrast to the static condition, there is a uniform concentration of the analyte in the solution at the
initial time, and it is assumed that all the analyte sample is brought in by the incoming laminar flow,
so that a fixed analyte concentration c = c0 is prescribed at the channel inlet, while no analyte is present
in the bulk for the initial condition.
As shown in Figure 10a, without AC power, the axial laminar flow driven by the externally-imposed
pressure difference constantly injects the free antigens into the microchannel, and the base flow is
paraboloidal in essence (Figure 11a). So, the supplement of reacted analyte adjacent to the binding
surface in the center of the chamber bottom surface mainly depends on the axial convective transport
of the laminar streaming and transverse mass diffusion. On the application of a phase-shifted TW
signal to the four peripheral DE metal strips, however, the rotating ROT-ICEO whirlpool appears
(Figure 11b) and severely distorts the free antigen concentration on top of the central FE, providing
more chance for binding interaction between the ligand and receptor in terms of a much thinner
depletion boundary layer on the FE surface (Figure 10b). Consequently, the transient motion of target
analytes is a consequence of the combined action of laminar inflow, molecular diffusion effect, and ICEO
micro-stirring under the dynamic condition. In particular, the vortex flow field of ROT-ICEO is of the
greatest importance in delivering the suspended antigens transversely to the functionalized reaction
of the FE in a straight microchannel under the influence of an axial pressure gradient on the antigen-
antibody binding reaction. For such, we prescribed the boundary condition at the upstream channel
entrance (Figure 10) with a paraboloidal profile of a mean inlet flow rate u0 = 25–200 μm/s in the full
scale tri-dimensional computational domain. In this integrated device design, two straight branch
channels on2020,
Micromachines both11,sides
739 are bridged by a microchamber in the center, as shown in Figure 10. In contrast15 of 20
to the static condition, there is a uniform concentration of the analyte in the solution at the initial time,
and it is assumed that all the analyte sample is brought in by the incoming laminar flow, so that a
surface and refreshing
fixed analyte concentrationthe depleted sample as signified
c = c0 is prescribed by theinlet,
at the channel non-negligible analyte
while no analyte is concentration
present in the
gradient above the FE surface
bulk for the initial condition. (Figure 10b), giving rise to enhanced binding reaction kinetics.
Figure 10.
Figure 10. Concentration
Concentration distribution
distribution of
of the
the incoming
incoming antigen
antigen injected
injected from
from the inlet of
the inlet of aa straight
straight
fluidic channel at t = 80 s, which is modified by the ROT-ICEO vortex flow field on the
fluidic channel at t = 80 s, which is modified by the ROT-ICEO vortex flow field on the top of the square top of the
square FE of 300 μm in edge length inside the central microchamber, when the peripheral
FE of 300 µm in edge length inside the central microchamber, when the peripheral DE array was excited DE array
was excited by TW voltage signals of (a) V 0 = 0 V, and (b) V0 = 8 V at f = 200 Hz with an average inlet
by TW voltage signals of (a) V 0 = 0 V, and (b) V 0 = 8 V at f = 200 Hz with an average inlet flow rate of
flow
50 rate(unit:
um/s. of 50 um/s.
mol/m(unit:
3 ). mol/m3).
As shown
Since therein Figure
is no 10a, without
background ACconcentration
analyte power, the axial laminar
in the flow driven
bulk before by the externally-
the incoming electrolyte
imposed pressure difference constantly injects the free antigens into the microchannel,
carries the sample into the channel, it takes a finite time for the biosensor to make a definite response and the base
to
flowanalyte
the is paraboloidal in essence
flow. The specific time(Figure 11a).
duration So, the
equals the supplement of reacted
ratio of the distance analyte
(from adjacent
the channel to the
entrance
binding
to surface
the flat FE) toin the
the center
inlet flowofrate.
the chamber
So, a lowerbottom
pump surface
speedmainly
impliesdepends
a largeron the axialtime
response convective
for the
binding reaction (Figure 12a). For instance, with a low inlet flow rate of u0 = 25 µm/s, the surface
transport of the laminar streaming and transverse mass diffusion. On the application of a phase-
shifted TW signal
concentration to the antigen
of bound four peripheral
begins toDEincrease
metal strips, however,
observably onlytheafter t = 100
rotating ROT-ICEO
s. On thewhirlpool
contrary,
appears
as (Figure
the pump 11b) isand
velocity raised to u0 =
severely distorts the the
200 µm/s, freerelaxation
antigen concentration
time needed for onthe
topbinding
of the central
reactionFE,
to
make a response to the applied voltage decreases sharply from t = 100 s to t = 10 s.
providing more chance for binding interaction between the ligand and receptor in terms of a much
thinner depletion
Under boundary layer
the circumstances with on theinlet
a net FE surface (Figure
flow rate, 10b).
the ratio of Consequently, the transient
convection to diffusion motion
rate, namely,
the Peclet number Pe = uH/D, has to be introduced to depict the correlation between the binding
kinetics and inlet flow rate. We calculate the binding enhancement factor Be as a function of Da number
for distinct Peclet number, namely, 400, 1000, 2000, and 4000, which correspond to a pump speed of
20 µm/s, 50 µm/s, 100 µm/s, and 200 µm/s, respectively (Figure 12b). As shown in Figure 12b, the value
of Be enhances as the Da number increases under a given inlet flow rate, which agrees well with the
previous discussions on the Da-dependence of the sensor performance in static condition (Figure 6a).
The influence of Pe number on the binding reaction is much more pronounced than that of Da number
(Figure 12b). With an increasing Pe number, the value of Be first rises when Pe is no more than 1000, and
then declines as Pe further increases from 1000 to 4000. This non-monotonous varying trend of Be as a
function of Pe number is attributed to the following two reasons: (1) Since Be is calculated at a specific
time node of t = 300 s, the binding reaction cannot be well activated by the imposed AC voltage as the
samples are not sufficiently transported to the functionalized surface for a small Pe (Pe ≤ 1000). (2) In
the presence of a large enough pump speed (Pe > 1000), the free antigens can amply arrive at the top of
the binding surface. Although a higher pump speed is able to boost the analyte transportation along
the channel length direction, a finite ICEO vortex flow field in the lateral direction is less efficient for
of target analytes is a consequence of the combined action of laminar inflow, molecular diffusion
higher inlet flow rates by causing a lower probability in the binding reaction between the immobilized
effect, and ICEO micro-stirring under the dynamic condition. In particular, the vortex flow field of
antibody and the dynamic antigens that pass too quickly over the functionalized surface. So, in a
ROT-ICEO is of the greatest importance in delivering the suspended antigens transversely to the
practical experiment, the microfluidic immunoassay should operate at a moderate Peclet number or
functionalized reaction surface and refreshing the depleted sample as signified by the non-negligible
inlet flow velocity to achieve an observable binding rate enhancement, and either a lower or higher
analyte concentration gradient above the FE surface (Figure 10b), giving rise to enhanced binding
pump rate may deteriorate the device functionality in biosensing.
reaction kinetics.
Figure
Figure 11.11. Influence
Influence of
of ROT-ICEO
ROT-ICEO electroconvection
electroconvection on
on top
top of
of the
the central
central FE
FE on
on the
the incoming
incoming laminar
laminar
stream
stream under the dynamic
under the dynamic condition,
condition, when
whenthe
thedriving
drivingvoltage
voltageamplitude (a)VV0 0== 00 V,
amplitudeisis(a) V,and
and(b)
(b)VV00 =
=
88 V
V at
at ff ==200
200HzHzwith
withananaverage
averageinlet
inletflow
flowrate
rateof
of50
50um/s
um/s (unit:
(unit: m/s).
m/s).
Since there is no background analyte concentration in the bulk before the incoming electrolyte
carries the sample into the channel, it takes a finite time for the biosensor to make a definite response
to the analyte flow. The specific time duration equals the ratio of the distance (from the channel
entrance to the flat FE) to the inlet flow rate. So, a lower pump speed implies a larger response time
for the binding reaction (Figure 12a). For instance, with a low inlet flow rate of u0 = 25 μm/s, the
surface concentration of bound antigen begins to increase observably only after t = 100 s. On the
contrary, as the pump velocity is raised to u0 = 200 μm/s, the relaxation time needed for the binding
reaction to make a response to the applied voltage decreases sharply from t = 100 s to t = 10 s.
Figure 12.
Figure Thequantitative
12. The quantitativestudy studyofofthe theeffect
effectofof ROT-ICEO
ROT-ICEO on on the
the degree
degree of
of binding
binding reaction
reaction
improvement in the dynamic flow condition. (a) The normalized surface
improvement in the dynamic flow condition. (a) The normalized surface concentration of bound concentration of bound
antigen with respect to the immobilized antibody under a voltage contrast between 0 V and 8 V 8forV
antigen with respect to the immobilized antibody under a voltage contrast between 0 V and
for different
different inletinlet
flowflow rate.
rate. (b) (b) Influence
Influence of of
DaDa number
number onon thebinding
the bindingenhancement
enhancementfactor Beafter
factorBe after
applyingaarotating
applying rotatingelectric fieldofofVV
electricfield = 8 V and f = 200 Hz at t = 300 s for distinct Peclet number.
0 =0 8 V and f = 200 Hz at t = 300 s for distinct Peclet number. The
The simulation file for an inlet flow
simulation file for an inlet flow rate of 100 rate of 100 µm/s
μm/s using
using Comsol
Comsol Multiphysics5.5
Multiphysics 5.5isisincluded
includedininthe
the
Supplementary Information.
Supplementary Information.
4. Conclusions
Under the circumstances with a net inlet flow rate, the ratio of convection to diffusion rate,
namely,In summary,
the Pecletanumber
unique ROT-ICEO
Pe = uH/D,method has to isbeproposed
introducedherein for thethe
to depict enhancement
correlation of the specific
between the
binding reaction between free antigens and immobilized antibody on a
binding kinetics and inlet flow rate. We calculate the binding enhancement factor Be as a function functionalized surface ofof
conducting
Da number floating electrodes
for distinct depositednamely,
Peclet number, in the center of the2000,
400, 1000, channel
andbottom
4000, whichsurfacecorrespond
for performingto a
microfluidic heterogeneous immunoassays. The lateral ROT-ICEO whirlpools
pump speed of 20 μm/s, 50 μm/s, 100 μm/s, and 200 μm/s, respectively (Figure 12b). As shown in can create more chances
for the12b,
Figure binding reaction
the value of Beby convectively
enhances as the transporting the free under
Da number increases antigens to the
a given transducer
inlet flow rate,surface,
which
resulting
agrees in with
well an acceleration
the previousof both the association
discussions and dissociation of
on the Da-dependence processes.
the sensor ROT-ICEO
performance in a circularly
in static
polarized (Figure
condition electric field favors
6a). The a slipping
influence of Peflow pattern
number onwhereby the reaction
the binding electroosmotic
is much streamlines converge
more pronounced
radially
than thatfrom
of Daallnumber
outer directions to the
(Figure 12b). electrode
With center, and
an increasing is therebythe
Pe number, more
value advantageous to achieve
of Be first rises when
full-scaled micro-stirring in comparison with the conventional ICEO slipping
Pe is no more than 1000, and then declines as Pe further increases from 1000 to 4000. This non- modes. The dependence
of binding efficiency
monotonous varyingenhancement
trend of Be ason AC voltage
a function amplitude,
of Pe number is signal frequency,
attributed to theand discretetwo
following arrangement
reasons:
of the
(1) floating
Since electrodes with
Be is calculated or without
at a specific timeannode
externalof t pump
= 300 s,fluid
the motion
bindingisreaction
analyzed in detail.
cannot It is
be well
believed
activated bythat the detection susceptibility and limit of detection are both enhanced
imposed AC voltage as the samples are not sufficiently transported to the by implementing
the ROT-ICEOsurface
functionalized vortex flow
for a field
smallonPethe (Peideally polarizable
≤ 1000). (2) In thesurface
presence of of
thea blocking
large enoughFE. The pumpnumerical
speed
computations
(Pe > 1000), thereported here can
free antigens demonstrate
amply arrive thatatROT-ICEO
the top of micro-stirring
the binding surface.can serve as a utilitarian
Although a higher
technique
pump speedforisboosting the binding
able to boost rate in
the analyte microfluidic along
transportation heterogeneous
the channel assays,
lengthespecially
direction, when the
a finite
reaction
ICEO process
vortex flowisfield
dominated by a slow
in the lateral mass is
direction diffusion.
less efficient for higher inlet flow rates by causing a
lower probability in the binding reaction between the immobilized antibody and the dynamic
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/11/8/739/s1,
antigens
Figure S1:that
(a,b)pass
Time too quickly
sequence over
of the thediagram
phase functionalized surface.
for case (ii) So, two
with every in aDEphase
practical experiment,
shifted the
by 180◦ , (a) at
t = nT, and (b)immunoassay
microfluidic at t = nT + T/2.should operate at aofmoderate
(c,d) Time-sequence Peclet number
the phasediagram or inlet
for case (iii) with flow velocity
adjacent DE being to
oppositely
achieve anpolarized,
observable t = nT, and
(a) atbinding rate at t = nT + T/2. Figure
(b)enhancement, S2: A comparison
and either a lower orofhigher
the frequency-dependence
pump rate may
of electrokinetic flow velocity between different convection modes ((i), (ii) and (iii)) at a given voltage amplitude
deteriorate the device functionality in biosensing.
of V = 4 V. Figure S3: Simulation result of ROT-ICEO flow field inside the static fluidic chamber for case (i) under
0
a given voltage amplitude V 0 = 4 V. Figure S4: A surface and arrow plot of ROT-ICEO slipping fluid motion on
the
4. central FE for case (i) at distinct signal frequencies: (a) f = 10 Hz; (b) f = 200 Hz; (c) f = 500 Hz; (d) f = 5000 Hz.
Conclusions
(unit: m/s). Figure S5: Simulation result of nonlinear electroosmotic flow field within the microchamber for case
f =summary,
(ii) atIn 200 Hz anda V 0 = 4 V.ROT-ICEO
unique method
(a) A top view is proposed
of tri-dimensional herein for
streamline the
plot ofenhancement of theand
the combined ACEO specific
ICEO
binding reaction between free antigens and immobilized antibody on a functionalized surface the
vortex flow field within the fluidic chamber. (b) A surface and arrow plot of ACEO slipping fluid motion on of
electrode array. (c) A surface and arrow plot of ICEO slipping fluid motion on the central FE. (unit: m/s). Figure S6:
conducting floating electrodes deposited in the center of the channel bottom surface for performing
Simulation result of nonlinear electroosmotic flow field within the microchamber for case (iii) at f = 300 Hz and
V 0 = 4 V. (a) Aheterogeneous
microfluidic immunoassays.
top view of tri-dimensional Theplot
streamline lateral
of theROT-ICEO
combined ACEO whirlpools
and ICEOcanvortex
create more
flow field
within the fluidic chamber. (b) A surface and arrow plot of ACEO slipping fluid motion
chances for the binding reaction by convectively transporting the free antigens to the transducer on the electrode array.
(c) A surface and arrow plot of ICEO slipping fluid motion on the central FE. (unit: m/s).
surface, resulting in an acceleration of both the association and dissociation processes. ROT-ICEO in
a circularly polarized electric field favors a slipping flow pattern whereby the electroosmotic
Author Contributions: Writing—original draft preparation, Z.G.; supervision and project administration, H.Y.;
methodology, W.L.; conceptualization, C.S.; software, R.X.; Literature retrieval, Y.R. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (No. 11702035,
No. 11672284, No. 11702075), Natural Science Foundation of Shaanxi Province (No. 2019JQ-073), Henan provincial
department of transportation science and technology project (No. 2019G-2-5), National or Provincial College
Students’ Innovation and Entrepreneurship Training Program (No. 201910710069), and Self-Planned Task
(SKLRS201803B) of State Key Laboratory of Robotics and System (HIT).
Acknowledgments: This project is financially supported by the National Natural Science Foundation of China
(No. 11672284, No. 11702035, No. 11702075), Natural Science Foundation of Shaanxi Province (No. 2019JQ-073),
Henan provincial department of transportation science and technology project (No. 2019G-2-5), National or
Provincial College Students’ Innovation and Entrepreneurship Training Program (No. 201910710069), and
Self-Planned Task (SKLRS201803B) of State Key Laboratory of Robotics and System (HIT).
Conflicts of Interest: The authors declare no conflict of interest.
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Quality Management System and ISO 90012008 CertificationBarriers and Challenges To SMEsEspacios

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micromachines

Abstract: A unique approach is proposed to boost on-chip immuno-sensors, for instance,

Keywords: on-chip immunoassay; induced-charge electroosmosis; rotating electric field; bipolar

Micromachines 2020, 11, 739; doi:10.3390/mi11080739 www.mdpi.com/journal/micromachines

Micromachines 2020, 11, x 3 of 20

2. Theory and Methods

2.1. Device Geometry

2.3. Mass Transfer of Antigen and Binding Reaction Enhancement

3. Results and Discussion

3.1. Binding Reaction Enhancement by ROT-ICEO Micro-Stirring

(ii) (ii) V 1 = V 0 cos(ωt)

With further the

3.4. Effect of Geometric Arrangement of the Floating Electrodes

3.5. Binding Reaction Enhancement in a Pressure-Driven Flow

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