Journal of Medical and Biological Engineering, 31(2): 121-127

121

Microfluidic Trapping of Antibody-secreting Cells

Sean F. Romanuik1,* Samantha M. Grist1 Moeed Haq1 Bonnie L. Gray1

Naveed Gulzar2 Jamie K. Scott2,3 Donna Hohertz4 Karen L. Kavanagh4

Rajinder Nirwan5 Christy Hui5 Alexandre G. Brolo5 Reuven Gordon6

1Microinstrumentation Laboratory, School of Engineering Science, Simon Fraser University, Burnaby, B.C. V5A 1S6, Canada 2Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C. V5A 1S6, Canada

3Faculty of Health Sciences, Simon Fraser University, Burnaby, B.C. V5A 1S6, Canada 4Department of Physics, Simon Fraser University, Burnaby, B.C. V5A 1S6, Canada 5Department of Chemistry, University of Victoria, Victoria, B.C. V8W 2Y2, Canada

6Department of Electrical and Computer Engineering, University of Victoria, Victoria, B.C. V8W 2Y2, Canada

Received 4 Sep 2010; Accepted 14 Dec 2010; doi: 10.5405/jmbe.841

Abstract

The development of antibody-based drugs typically begins with the isolation and screening of thousands of

antibody-secreting cells, often requiring 4-6 weeks to identify only 1-10 cell lines of interest. We are developing a

novel immunobiosensing device to afford direct and rapid assessment of the antibody production of each of thousands

of living cells captured on a single slide. Each cell shall be trapped near a designated surface plasmon-resonant

nanohole array immunobiosensor to detect the binding of the cell’s secreted antibodies to an immobilized target antigen.

This paper presents arrays of single-cell traps consisting of concave hydrodynamic structures and inset microwells.

Each design is fabricated using either SU-8 photoresist or poly(dimethyl siloxane) (PDMS) and then tested using

polystyrene microspheres and 17/9 mouse B cell hybridoma cells. Although we find that PDMS adheres more strongly

to our substrates than SU-8, we also find that channels fabricated in PDMS are wider than expected and thus fail to

limit cellular passage. The hydrodynamic tests agree with the predictions of finite element modeling using COMSOL®

Multiphysics. We find that the hydrodynamic designs featuring concave traps inset into spirals and serpentine bends

capture cells more efficiently than our other hydrodynamic designs. Testing also verifies the functionality of our

microwell traps, with 5-20 cells/well trapped following a 10 min settling time. Moreover, untrapped cells beyond the

microwells are removed via peristaltic pumping, with minimal trapped cell displacement. Furthermore, 4-24 hr

trapped-cell incubations verify the biocompatibility of each of our fabricated designs, provided that a Cr-free substrate

is used.

Keywords: Single-cell trap, Hydrodynamic cell trap, Microwell, Immunobiosensor, Therapeutic antibody

1. Introduction

There is a growing interest in the therapeutic use of human

and humanized monoclonal (M) antibodies (Abs) that bind to

antigens (Ags) related to human diseases. MAb development

typically begins with the labour-intensive isolation and

screening of thousands of antibody-secreting cells (ASCs),

often requiring 4-6 weeks to identify only 1-10 cell lines

producing the desired antibodies. There are currently no

automated large-scale methods for the identification, isolation

and cloning of such ASCs. We are developing a novel

immunobiosensing device affording rapid and simultaneous

* Corresponding author: Sean F. Romanuik

Tel: +1-778-7826966; Fax: +1-778-7824951

E-mail: sromanui@sfu.ca

direct assessment of the Ab production, dynamics and

characteristics of each of thousands of living ASCs. Each ASC

shall be microfluidically trapped near a designated

immunobiosensor, to detect the binding of the ASC’s secreted

Abs to an immobilized target Ag. This paper expands upon

previous conference papers [1,2] that presented our single-cell

trap array designs, utilizing both hydrodynamic concave

structures and inset microwells. Our hydrodynamic designs are

first evaluated using COMSOL® [3] simulations. Our

hydrodynamic and microwell trap arrays are then fabricated

using SU-8 photoresist (PR) [4] and poly(dimethyl siloxane)

(PDMS) [5]. Our fabricated traps are tested using

fluid-suspended polystyrene microspheres (PSS) [6] and 17/9

mouse B cell hybridoma cells. Our immunobiosensing system

should shorten the time required to identify the ASCs secreting

J. Med. Biol. Eng., Vol. 31 No. 2 2011

122

the Abs of interest and provide a platform affording the

isolation and cloning of Ab-genes from the individual ASCs.

2. Motivation

In response to pathogenic infection or immunization, a

subset of the human body’s lymphocytes, B cells, differentiate

into ASCs that produce and secrete Abs. Abs bind to a protein

or a site on the invading pathogen, referred to as an Ag,

neutralizing and/or flagging the pathogen for destruction. Over

time, the in vivo secretion of Abs in the absence of persistent

Ag develops an individual’s immunological memory,

providing protective immunity against pathogens bearing this

Ag. Immunological memory also serves as a biological

indicator of the individual’s past pathogenic exposure [7,8].

There is growing interest in the therapeutic use of human

and humanized MAbs that bind to human disease-related Ags.

Such MAbs have shown promise during the treatment of

cancer, autoimmune diseases and infectious diseases [9-12].

The production of therapeutic MAbs commonly begins with

the isolation and screening of a large ASC population isolated

from humans or transgenic mice. Hybridoma cell lines are

usually produced via the fusion of human or murine B cells

with myeloma cells [13,14]. Supernatants from these cultured

cell lines are then collected and tested for the presence of the

desired Ab(s). Using cloned expressed Ab-encoding genes,

cultured cells are then transformed into clonal ASC lines that

produce the desired MAb(s) in large quantities [12,15]. As

only a fraction of the B cells used to create the hybridoma cell

lines produce the desired Ab(s), thousands of ASCs must be

screened in a labour-intensive limiting step typically requiring

4-6 weeks to yield only 1-10 cell lines of interest.

Other groups have developed devices to isolate and

identify individual ASCs, secreting the desired Ab(s), within a

population of thousands [16,17]. However, these systems

neither detect this Ab-Ag binding in real time nor quantify its

affinity. We are developing a device ultimately intended to

monitor the Ab production by individually trapped ASCs at

this scale. This device shall measure, and quantify the affinity

of, the binding of secreted Abs to an immobilized target Ag in

real time. We envision a two-component system, consisting of:

an array of immunobiosensors, coated with immobilized target

Ag; and a polymeric microfluidic system that traps individual

ASCs near designated immunobiosensors [18,19].

Each immunobiosensor consists of an array of nanoholes

milled, via a focused Ga+ beam, through a gold film adhered to

a glass substrate [18,19]. Such metal-dielectric interfaces can

sustain coherent collective electronic oscillations called

surface plasmons (SPs) [20]. Optical excitation of SPs

generates quasi-particles called SP polaritons (SPPs). Many of

these SPPs decay into photons, resulting in an optical

transmission through the array of subwavelength nanoholes

exceeding classical aperture theory, a phenomenon termed

extraordinary optical transmission (EOT) [21]. EOT spectra

peak at resonant wavelengths, λSPR, dependent upon the

permittivities of the metallic and dielectric layers [22]. This

dependence has been widely exploited to monitor various

surface binding events [20,23]. The binding of Abs to Ag

immobilized onto the milled gold surface alters the

permittivity of the dielectric media contacting a nanohole array,

shifting λSPR by ∆λSPR. The affinity of this Ab-Ag binding can

be deduced by monitoring the rates of Ab binding and release

from the immobilized Ag via the time response of the

corresponding ∆λSPR shifts.

Before conducting ∆λSPR measurements on the desired

scale, we must first develop a system that individually captures

large ASC populations within arrays of single-cell traps.

Single-cell manipulation has been a major focus in

microfluidics research [24]. Di Carlo et al. [25] report u-shaped

concave structures facing an oncoming fluid flow,

hydrodynamically trapping suspended cells via perfusion.

Ogunniyi et al. [17] and Tokimitsu et al. [16] demonstrated

trapping fluid-suspended ASCs, settling under gravity, into

microwells inset into a planar surface. This paper presents our

initial work developing similar single-cell trap arrays.

3. Design and simulation of single-cell trap arrays

3.1 Design of hydrodynamic cell trap arrays

Figure 1 presents our first hydrodynamic cell trap design.

Fluid flow carries suspended cells into the concave openings

of heart-, c-, or v-shaped structures. The 10-µm-wide channel

bisecting each structure is too narrow to afford further cellular

passage, hydrodynamically trapping the captured cell against

the fluid flow via perfusion.

Figure 1. Fluid flow carries a cell into the concave opening of a heart-,

c-, or v-shaped cell trap bisected by a narrow channel,

hydrodynamically trapping the said cell via perfusion.

Figure 2 depicts our second hydrodynamic cell trap

design, which adds two 50-µm-by-50-µm chambers, referred

to as nanohole array shelters, connected to each bisecting

channel via 10-µm-wide channels. Each nanohole array shall

be positioned within a designated shelter, to be exposed to Abs

secreted by the nearby trapped ASC and partially shielded

from the Abs secreted by all other ASCs.

Figures 3-5 portray hydrodynamic cell trap designs with

concave cell traps and nanohole array shelters inset into curved

microfluidic channel walls. Centrifugal force Fc pushes cells

denser than the fluid out to the walls, where the fluid flow then

carries them into the concave cell traps.

Microfluidic Trapping of Antibody-secreting Cells

123

Figure 2. Hydrodynamic cell traps with two 50-µm-by-50-µm nanohole

array shelters connected via additional 10-µm-wide channels.

Figure 3. Hydrodynamic cell traps and nanohole array shelters inset into

a serpentine channel’s bends.

Figure 4. Hydrodynamic cell traps and nanohole array shelters inset

between a ramped channel’s bends.

Figure 5. Hydrodynamic cell traps and nanohole array shelters inset into

a spiraled channel.

Figure 3 presents concave cell traps and nanohole array

shelters inset into the walls of serpentine bends. This design is

inspired by the serpentine channels commonly utilized as

microfluidic mixers [26], capitalizing on the back and forth

motion of the normally laminar fluid flow as induced by Fc.

Notably, the wall to which Fc pushes a given cell changes after

a bend. Regardless, the commonness of the serpentine channel

lends itself as a logical starting point in the development of

curved channel designs.

Figure 4 depicts concave cell traps and nanohole array

shelters inset into ramps. As with the serpentine channel, the

wall to which Fc pushes cells changes following a bend. The

placement of the cell traps between the bends, rather than on

the bends themselves, makes this design a logical perturbation

of the serpentine channel.

Figure 5 portrays concave cell traps and nanohole array

shelters inset into a spiral. Unlike the designs of Figs. 3 and 4,

Fc always pushes cells towards the same wall. The spiraled

design is thus expected to trap cells more efficiently than the

serpentine and ramp designs.

Fluid shall be pumped through these designs during the

cell trapping stage only. After a sufficient number of cells have

been trapped, this fluid flow shall cease. As such, the dispersal

of the trapped ASCs’ supernatant shall be entirely diffusive.

Moreover, the distance separating the nanohole arrays is such

that any cross-talk induced by the supernatant of remote ASCs

is expected to be negligible compared to the signal induced by

the supernatant of the nearby trapped ASC.

3.2 Simulation of hydrodynamic cell trap arrays

The designs of Section 3.1 were modeled using

COMSOL® [3] Multiphysics 2D Incompressible Navier-Stokes

Module [27], to compute the respective fluid velocities ννννs by

solving the incompressible Navier-Stokes equation,

ρ (∂ννννs / ∂t) - ∇•{-P � + µ [∇ννννs + (∇ννννs)T]} + ρ ννννs •∇ννννs =Fv (1)

where ρ ≈ 1000 kg m-3 is fluid density, P is fluid pressure,

µ ≈ 0.001 kg s-1 m-1 is dynamic viscosity, Fv are the volumetric

body forces acting on the fluid and � is the identity matrix [27].

Simple 2D models erroneously estimate ννννs in channels

with a depth d much less than their length L, as they exclude

the effect of the un-modeled boundaries upon the flow [27].

The shallow channel approximation compensates via an

additional Fv term, Fv = -12 µ ννννs d-2. Our simulations use a

d = 100 µm shallow channel approximation. No additional Fv

terms beyond the shallow channel approximation were used.

Eq. (1) is constrained by: (i) the mass flow continuity

equation, ∇•ννννs = 0 [27]; (ii) no-slip channel wall boundary

conditions (BCs), ννννs = 0; and (iii) normal stress BCs along the

fluid ports’ edges,

{-P � + µ [ ∇ννννs + (∇ννννs)T ] } n = -F0 n (2)

where n is the outward unit vector normal to the edges and F0

is the stress vector magnitude along n [27]. In our simulations,

Eq. (2) implicitly constrains P such that P ≈ F0 [27]. F0 was

specified as: (i) zero along the outlets; and (ii) non-zero and

constant along the inlets.

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COMSOL® solved Eq. (1) using the Generalized Minimal

Residual Method (GMRES) solver, an incomplete LU

preconditioner, 0.0001 drop tolerance and automatic matrix

symmetry. The solver was stepped in time from t = 0-30 s in

0.1 s steps, with 0.01 and 0.001 relative and absolute

tolerances, respectively. Sufficient mesh density was assured

in each case.

To conserve memory, the designs of Section 3.1 were not

modeled in their entirety. For example, neither the nanohole

array shelters nor their channels were included within the

models. However, since ννννs is expected to be negligible in these

regions, the errors in the computed ννννs due to their absence are

also expected to be insignificant.

To verify the accuracy of the computed ννννs, we compared

the mean |ννννs| within the main 100-µm-wide channels to their

analytical values, |ννννa|, as predicted using well-known fluid

resistance theory [28]. To obtain the |ννννa| values, we first

compute each channel’s effective hydraulic diameter Deff,

Deff = 256 A / ( k P ) (3)

where A is the channel’s cross-sectional area, P is the

channel’s wetted perimeter and k ≈ 56.91 is a geometric factor

corresponding to the channel’s square cross-section. For our

channels, Deff ≈ 112 µm. We then compute the fluidic

resistance R of our simulated channels as

R = 128 µ L / ( π Deff4 ) (4)

We then compute the mean |ννννa| within the simulated

100 µm wide channels as

|ννννa| = ∆P / ( A R ) (5)

Table 1 presents the ∆P normal stress BCs used to

initialize each simulation (inlet P ≈ F0 relative to outlet

P ≈ F0 = 0), each channel’s L and R and the mean |ννννs| and |ννννa|

within the 100-µm-wide channels at t = 30 s. Table 1 verifies

the accuracy of the computed ννννs within each simulation.

Table 1. Predicted and computed mean |ννννs| and |ννννa| within the

100-µm-wide channels of each simulation at t = 30 s.

Design ∆P [Pa]

L

[mm]

R

[kg s-1 m-4] |ννννa|

[mm s-1] |ννννs|

[mm s-1]

♥-trap 20 9 2.29 × 1012 0.9 1.0

C-trap 20 9 2.29 × 1012 0.9 0.9

V-trap 20 9 2.29 × 1012 0.9 1.0

Traps with shelters

20 9 2.29 × 1012 0.9 0.9

Serpentine 15 10 2.54 × 1012 0.6 0.7

Ramped 20 11 2.80 × 1012 0.7 0.8

Spiralled 20 9.5 2.42 × 1012 0.8 0.9

The cellular trajectories within these ννννs profiles were

estimated via ννννs streamlines. The accuracy of these estimations

was later confirmed via particle tracing simulations.

Table 2 presents, for each entry in Table 1, the total

number of: cell traps, streamlines and cell traps bisected by a

streamline. The bisecting of a cell trap by a streamline

suggests that cells would likely be trapped within that

structure.

Table 2. Number of cell traps bisected by a ννννs streamline, for each

simulation, at t = 30 s.

Design ∆P [Pa]

No. ννννs streamlines

No. cups No. cups

bisected

♥-traps 20 20 12 5

C-traps 20 20 9 5

V-traps 20 20 10 7

Traps with shelters 20 30 6 0

Serpentine 15 12 12 12

Ramped 20 8 16 3

Spiralled 20 20 28 14

Table 2 suggests that, for a given ∆P, the designs of

Figs. 1, 3 and 5 should trap cells more efficiently than those of

Figs. 2 and 4. Moreover, |ννννs| is low within each simulated cell

trap, implying that the trapped cells would experience minimal

fluid drag and supernatant dispersal in the presence of the fluid

flow.

Figure 6 presents |ννννs| within the simulated spiral. The top

inset shows a streamline bisecting a cell trap, whilst the bottom

inset portrays a cell trap that is not bisected by a streamline.

|ννννs| as computed within the other designs of Section 3.1 are

presented elsewhere [2].

Figure 6. The |ννννs| field (color) and ννννs streamlines (white) within the

design of Figure 5 (at t = 30 s), simulated using COMSOL®

Multiphysics with ∆P = 20 Pa and 20 streamlines.

3.3 Design of microwell cell trap arrays

We have developed 7 × 7 arrays of microwells

(30-200 µm diameters, 60-80 µm depths and 3.77 mm

periodicity) inset into polymer films (400-700 µm thick).

Following the trapping of cells into these microwells and the

subsequent removal of untrapped cells, an immunobiosensing

nanohole array is to be aligned atop each microwell. The

distance separating the microwells is such that any cross-talk

induced by the supernatant of remote ASCs is expected to be

negligible compared to the signal induced by the supernatant

of the nearby trapped ASC.

Microfluidic Trapping of Antibody-secreting Cells

125

4. Polymeric fabrication of single-cell trap arrays

4.1 Fabrication of hydrodynamic cell trap arrays

Our hydrodynamic cell traps were fabricated on

RCA-cleaned, soda-lime glass slides (1” × 1.5” × 1 mm,

1” × 3” × 1 mm and 3” × 3” × 1 mm) with and without

sputtered Ti or Cr adhesion layers (5 nm thick) or Pyrex wafers

(4” diameter and 500 µm thick). SU-8 2035 PR [4] films

(30-120 µm thick) were spun onto these substrates and

soft-baked on a 65°C hot-plate. Each SU-8 film was then

photolithographically patterned using a Mylar contact mask

featuring all of the designs of Figs. 1-5. The SU-8 films were

then post-exposure baked on a 95°C hot-plate before being

developed using MicroChem’s SU-8 developer. Utilized spin

speeds and exposure and baking times were set by the desired

film thicknesses as based upon the SU-8 2025-2075 datasheet

[4], as subsequently optimized for our equipment.

Some SU-8 films were instead patterned with the said

mask’s negative, creating soft-lithographic molds to fabricate

the designs of Figs. 1-5 using Dow Corning’s Sylgard®

184 PDMS [5] (as per [29]). In addition to the aforementioned

substrates, RCA-cleaned Si wafers (4” diameter and 525 µm

thick) were also used during the fabrication of these molds.

10-15 µm wide SU-8 structures suffered from poor

adhesion (Fig. 7(b)), whereas similar PDMS structures did not

suffer from this poor adhesion. However, PDMS shrinkage

and/or poor SU-8 mold definition resulted in widened channels

that failed to prevent cellular passage (Fig. 8(b)). We were thus

unable to fabricate the hydrodynamic cell trap arrays presented

in Fig. 1.

Figure 7. (a) Hydrodynamic cell traps and nanohole array shelters inset

into a SU-8 based spiral. (b) Partial cell trap delamination,

due to poor SU-8 adhesion.

4.2 Fabrication of microwell cell trap arrays

Our 7 × 7 arrays of microwells (30-200 µm diameters,

60-80 µm depths and 3.77 mm periodicities) were fabricated in

SU-8 [4] and PDMS [5] (400-700 µm thick), as per

Section 4.1.

Figure 8. (a) PSS trapped in a cell trap inset into a PDMS-based spiral.

(b) 17/9 hybridoma cells densely populated along a PDMS

serpentine bend. Cells were able to enter the nanohole array

shelters.

5. Experimental testing of fabricated single-cell trap

arrays: results and discussion

5.1 Testing of fabricated hydrodynamic cell trap arrays

Our fabricated hydrodynamic cell trap arrays were

sonicated in phosphate-buffered saline (PBS, pH 7.4) and then

tested using: PSS (20 µm diameter) [6], suspended in

deionized water (DI H2O); and 17/9 mouse B cell hybridoma

cells (10-20 µm diameter) [7], re-suspended in cell culture

media (Dulbecco’s modified Eagle’s medium; DMEM).

Concentrations and flow rates on the order of 105/mL and tens

of µL/min, respectively, were used throughout these tests.

Optical microscopy showed that most cells flowed around

the SU-8 traps, with few becoming trapped. This is likely due

to the reduced flow through these cell traps as a consequence

of poor SU-8 adhesion.

Optical microscopy also demonstrated that the serpentine

and spiraled designs trapped cells most efficiently, as predicted

in Section 3.2. Moreover, cell trapping occurred along the

complete length of the serpentine and spiraled channels.

Figure 8 presents: (a) a PSS trapped in a cell trap inset

into a PDMS spiral; and (b) cells densely populated along a

PDMS serpentine bend, alongside cells that passed through

widened shelter channels. This cellular buildup suggests an

affinity between the cells and the PDMS, which may be

reduced by treating the PDMS with O2 plasma or bovine serum

albumin (BSA). Reducing the number of traps containing

multiple cells could be achieved via reducing the concentration

of the suspended cells, at the cost of a greater number of traps

containing no cells. To prevent cells from entering the

nanohole array shelters, the connecting channel width defined

within the photolithographic masks could be reduced to

compensate for the widening observed during fabrication, so

as to achieve the desired channel width that is too narrow to

afford cellular passage.

The removal of untrapped cells beyond the traps is

difficult using our current hydrodynamic cell trap designs.

Surface treatments and/or pressure bursting methods may

afford their removal. In addition, we intend to explore various

hydrodynamic flow steering and/or cell sorting methods for the

removal of specific trapped ASCs.

J. Med. Biol. Eng., Vol. 31 No. 2 2011

126

Optical microscopy confirms the viability of trapped cells

following a 4 hr incubation at 37°C in a 5% CO2 environment.

Cells in traps featuring Cr adhesion layers did not survive, but

remained viable in all other traps. Fortunately, we have also

shown that SU-8 adheres more strongly to Ti than Cr [30].

5.2 Testing of fabricated microwell cell trap arrays

The fabricated microwell arrays were tested with

17/9 mouse B cell hybridoma cells (10-20 µm diameter) [7]

re-suspended in DMEM, using a procedure adapted from

Ogunniyi et al. [17]. A PDMS film (670 µm thick) with a

7 × 7 array of inset microwells (200 µm diameter and

70-80 µm deep) was incubated in PBS with fetal bovine serum

(FBS, 25% v/v) for 24 hr at 4°C, to block nonspecific binding

sites, and then sonicated in PBS. Figure 9 presents optical

microscopy images of one microwell prior to-, and 10 min

after-, the deposition of 17/9 hybridoma cells (100 µL,

4x105 cells/mL). During this period, each microwell filled with

5-20 cells. Peristaltic pumping then removed untrapped cells

with minimal trapped cell displacement. Cells remained viable

in the microwells during 24 hrs of incubation at 37°C in a 5%

CO2 environment, as assessed via optical microscopy.

(a) After PBS sonication (b) After 10 min of settling

(c) After peristaltic pumping

(d) After 24 hr incubation

Figure 9. A microwell (200 µm diameter and 70-80 µm deep) inset

into PDMS (670 µm thick): (a) after sonication in PBS; (b)

10 min after depositing DMEM-suspended 17/9 hybridoma

cells (100 µL, 4 × 105 cells/mL); (c) after using a peristaltic

pump to remove untrapped cells; and (d) after 24 hrs of

incubation.

In contrast to our success trapping cells into PDMS-based

microwells, we had difficulty trapping cells into SU-8 based

microwells, likely due to SU-8’s surface charge or

hydrophobicity.

Although we are encouraged by our preliminary

microwell tests, further refinements are necessary to yield

arrays capable of individually trapping large numbers of ASCs

on a single slide. These refinements most likely include

reducing the size of the microwell traps and/or reducing the

concentration of the deposited cellular suspension. However,

even this experimentally observed trapping density is a

significant achievement as it affords examining the Ab

production of individual 5-20 cell subpopulations, narrowing

identification of the ASCs of interest to their associated

subpopulations. The subsequent removal of trapped ASCs

shall most likely be achieved via a micropipetting procedure.

6. Conclusions

We have designed various single-cell trap arrays for

individually capturing large ASC populations. Hydrodynamic

cell trap designs, fabricated using SU-8 [4] and PDMS [5], have

been verified via COMSOL® [3] simulations and experimental

testing using fluid-suspended PSS [6] and 17/9 hybridoma cells.

Although we find that PDMS adheres more strongly to our

substrates than SU-8, we also find that channels fabricated in

PDMS are wider than expected and thus fail to limit cellular

passage. The simulations and tests demonstrate that our

hydrodynamic designs featuring traps inset into serpentine

bends and spirals trap more efficiently than our other

hydrodynamic designs. Our PDMS based microwell traps were

also tested using fluid-suspended 17/9 hybridoma cells,

verifying their functionality. Furthermore, 4-24 hr trapped cell

incubations verify the biocompatibility of each of our fabricated

designs, provided that a Cr-free substrate is used.

Although encouraged by our preliminary results, our cell

trap designs and/or experimental methods must be further

refined to yield single-cell trap arrays capable of trapping large

ASC populations on a single slide. It shall also be necessary to

remove untrapped cells, to isolate each immunobiosensor from

the Abs secreted by ASCs other than the nearby trapped ASC.

In the case of microwell traps, untrapped cells have been

removed via peristaltic pumping, with minimal trapped cell

displacement. It shall also be necessary to remove specific

trapped ASCs.

We consider the results reported in this paper to be good

progress towards a polymeric microfluidic system capable of

individually trapping thousands of ASCs, each in the vicinity of

a designated nanohole array based immunobiosensor. The

successful development of our immunobiosensing device

should streamline the process of isolating and identifying ASCs

secreting desired Ab(s), which is a limiting step in the current

methods of therapeutic Ab-based drug development.

Acknowledgments

We thank Jody D. Berry (Cangene Inc.), Jasbir N. Patel

(Simon Fraser University; SFU) and Ajit Khosla (SFU) for

their assistance. We also acknowledge the finance and

resources provided by the Natural Sciences and Engineering

Research Council of Canada (NSERC), the Canada Foundation

for Innovation (CFI), the British Columbia Knowledge

Development Fund (BCKDF), CMC Microsystems, Cangene

Inc. and the Canada Research Chairs (CRC).

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