pubs.acs.

org/acssensors Article

On-Chip Analysis of Protein Secretion from Single Cells Using

Microbead Biosensors
Diana F. Cedillo-Alcantar, Roberto Rodriguez-Moncayo, Jose L. Maravillas-Montero,
and Jose L. Garcia-Cordero*
Cite This: ACS Sens. 2023, 8, 655−664 Read Online

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ABSTRACT: The profiling of the effector functions of single

Downloaded via ISTANBUL TEKNIK UNIV on December 12, 2023 at 11:22:36 (UTC).

immune cells�including cytokine secretion�can lead to a deeper

understanding of how the immune system operates and to
potential diagnostics and therapeutical applications. Here, we
report a microfluidic device that pairs single cells and antibody-
functionalized microbeads in hydrodynamic traps to quantitate
cytokine secretion. The device contains 1008 microchambers, each
with a volume of ∼500 pL, divided into six different sections
individually addressed to deliver an equal number of chemical
stimuli. Integrating microvalves allowed us to isolate cell/bead
pairs, preventing cross-contamination with factors secreted by
adjacent cells. We implemented a fluorescence sandwich immuno-
assay on the biosensing microbeads with a limit of detection of 9
pg/mL and were able to detect interleukin-8 (IL-8) secreted by single blood-derived human monocytes in response to different
concentrations of LPS. Finally, our platform allowed us to observe a significant decrease in the number of IL-8-secreting monocytes
when paracrine signaling becomes disrupted. Overall, our platform could have a variety of applications for which the analysis of
cellular function heterogeneity is necessary, such as cancer research, antibody discovery, or rare cell screening.
KEYWORDS: single cell, immune cells, secretion analysis, cytokines, immunoassay, microbeads, microfluidics, hydrodynamic traps

C ells secrete a myriad of products, including proteins,

peptides, metabolites, and microvesicles.1,2 These
secreted products can participate in cell signaling and cell−
cell communication, mediating a broad range of responses on
target cells, such as migration, proliferation, and differ-
entiation.3,4 Other secreted factors can alter the local
microenvironment by remodeling the extracellular matrix,
either by producing proteins that compose the extracellular
matrix (ECM) or by secreting enzymes that degrade it.5,6 The
ability to detect secreted factors can have potential clinical
applications by employing them as molecular biomarkers for
disease diagnosis, prognosis, and therapy evaluation.7,8 For
example, CTCs are known to secrete metalloproteases that
modify the ECM surrounding the tumor, thus increasing their
metastatic potential.9,10 In addition, in cell-based immuno-
therapy, such as chimeric antigen receptor (CAR)-engineered
T cells, measurement of cytokine secretion is necessary to
assess the cell’s activation state since immunosuppressive
cytokine-secreting cells reduce the treatment efficacy.11,12
Furthermore, from a biotechnology stance, secretion profiling
of B cells allows the identification of antibody-producing cells
specific against a particular antigen.13,14
Single-cell secretion analysis has allowed to measure the
heterogeneity in cell response, providing relevant insights as to
how cells regulate their behavior based on both environmental
cues and intrinsic factors.15,16 Moreover, it has allowed the
identification of cell subpopulations with dysregulated
behaviors that cause disease or prevent therapy effective-
ness.17,18 Two widely employed methods to measure single-cell
secretion are the enzyme-linked immunospot (ELISpot) and
immunocytochemistry coupled with flow cytometry.19,20
ELISpot is a semiquantitative approach that lacks throughput
because only a single condition can be analyzed at a time.21
Flow cytometry, on the other hand, can analyze hundreds of
cells per second, making it a high-throughput approach.
Nonetheless, it requires multiple sample-processing steps,
including treatment with secretion inhibitors, and cell fixation,
making it incompatible with live-cell analysis.19 Disappoint-
ingly, in these techniques, while cells are challenged, they are
maintained in volumes that are too large compared to their
volume, which causes the secreted soluble factors to be diluted,
Received: October 2, 2022
Accepted: January 17, 2023
Published: January 30, 2023

© 2023 American Chemical Society https://doi.org/10.1021/acssensors.2c02148

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Figure 1. Microfluidic device for analysis of protein secretion from single cells. (a) Photograph of the assembled device. The flow layer is shown in
blue while the control layer is shown in red. Scale bar is 5 mm. (b) A 3D schematic of a microchamber showing a single-cell secreting protein into a
biosensing microbead. Inset shows the trapping sieve. (c) Device schematic showing three functional units. A single cell and an antibody-
functionalized microbead are immobilized by hydrodynamic forces in their respective trap. Microfluidic valves isolate microchambers from each
other. The bottom row shows a micrograph of three microchambers with a closeup of a microbead and a cell trapped by the sieves. Scale bar is 20
μm for the inset.

impairing cell signaling.22 Overall, an ideal approach to assess
the secretory response of single cells should comprise at least
two elements. First, cells should be enclosed in a small volume
(nL) where they can secrete soluble factors that accumulate in
the milieu. Second, the approach should integrate a protein
detection method for local cellular monitoring that is both
quantitative and highly sensitive.
Microfluidics can facilitate the isolation of single cells by
providing enclosed chambers where secreted factors can
accumulate.19,20,23 For example, microwell arrays can capture
individual cells, which can then be sealed with a coverslip
functionalized with capture antibodies against the desired
cytokines.24−27 Alternatively, microchambers with microvalves
can be used to isolate individual cells with antibody-
functionalized microbeads or barcoded antibody micro-
arrays.28−31 Another common approach used is droplet-based
microfluidics, which can encapsulate cells along with nano- or
microbeads in micrometer-sized droplets.32−36 Although all of
these strategies are suitable to isolate single cells and measure
protein secretion, they are currently limited two-fold. First,
only a single stimulus can be evaluated at a time in these
platforms. Second, the solutions cannot be exchanged on
demand, either in a droplet or an isolated microchamber,
which prevents (i) supplying fresh nutrients to the cells, (ii)
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providing stimulus dynamically, or (iii) introducing washing
steps during immunoassays, to name a few.
Here, we present an integrated microfluidic platform to
quantify cytokine secretion from single cells. The device makes
use of hydrodynamic traps and microvalves to capture and
isolate single cells, along with antibody-coated microbeads. We
characterized cells and beads capture efficiency at different
conditions. We also characterized the process to replace the
microchamber solution once the beads had been isolated.
Finally, we demonstrate the utility of our device by measuring
the secretion of cytokines from individual primary monocytes
and evaluated differences in cellular responses under paracrine
and autocrine signaling by stimulating cells cultured in bulk or
under isolation, respectively.
■ RESULTS AND DISCUSSION
Microfluidic Device with Hydrodynamics Traps to
Capture Microbeads and Single Cells. Microfluidic
hydrodynamic traps have been widely employed to capture
single cells since they offer the ability to confine single cells
from a large population in a specific location without
modifying the material’s surface properties or using external
forces.37−40 On the other hand, integration of microvalves in a
device allows automation, parallel processing, and multi-
plexing.41,42 Hence, we exploited these key advantages to
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Figure 2. Assay workflow. (1) Microvalves on the bead-side channel are activated, and cells are immobilized in the cell traps. Next, (2) valves on
the bead-side channel are deactivated, and beads are immobilized on bead traps. (3) Channels are flushed with fresh media. (4) Microvalves on the
central channels and cell-side channels are actuated, and solutions with stimulant and detection antibodies are then delivered to the cells. (5) All
the microvalves are actuated, followed by 24 h incubation to allow the cells to release cytokines. Finally, (6) the formation of immunocomplexes is
evaluated through fluorescence microscopy.

develop a microfluidic device integrating hydrodynamic traps
and microvalves using multilayer soft lithography. The device
allows the precise immobilization and confinement of a single
cell in tandem with an antibody-functionalized microbead to
quantitate cytokine secretion.
Our multilayer device consists of a control layer, for fluidic
control, and a flow layer that contains hydrodynamic traps,
Figures 1a and S1a−c. The device possesses six inlets, two of
which have an integrated array of posts that traps aggregates
when introducing the cells and microbeads. The microfluidic
device is divided into six sections (Figures 1a and S2), each
with an independent inlet, to introduce an equal number of
chemical stimuli, and three control lines with microvalves to
isolate and prevent cross-contamination from adjacent micro-
chambers. In addition, each of the six sections includes six
central channels with 28 cell-microbead trap pairs (for a total
of 1008 pair traps in the device) connected to the side
channels through sieves (3 μm × 3 μm) that help capture cells
and microbeads on opposite sides of the main channel, Figure
1b,c. Activation/deactivation of microvalves on the side
channels allows to control the fluid flow and directs cells or
beads to their specific capture sites.
To trap cells and beads, the cells are flowed first through the
central channels of the device, followed by the beads. The
reason for this order is because the cells are deformable and
viscoelastic43 and thus can completely occlude the sieves
preventing the beads from being captured in the cell traps
while they are injected. Conversely, microbeads are rigid and
thus do not completely block off the sieves, allowing some flow
through the sieves, which could drag cells into the traps.
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More specifically, capturing cells and microbeads in the chip
to detect secreted proteins consists of six steps that also involve
actuating the valves at a specific sequence, Figure 2. Initially,
(i) microvalves on the bead-side channel are activated to
prevent flow through the bead-trapping sieves. Then, cells are
injected through the central channel and trapped in the cell
traps. Next, fresh media is injected through the central
channels to remove all the cells remaining in the central
channel. After that, (ii) valves on the bead-side channels are
deactivated, allowing flow through the bead sieves, while
microbeads are introduced through the central channel for
their capture in the bead’s traps. (iii) Central channels are then
flushed with fresh media to eliminate any cells or microbeads
outside of the traps. (iv) Microvalves on the central channels
and cell-side channel are actuated afterward to partially isolate
the microchambers and to prevent cells and beads from
moving to contiguous chambers. Solutions containing the
stimuli and the detection antibodies are delivered using the
bead-side channels that lead to the microbead traps. The
immunocomplex formation initiates on the beads’ surface at
this point. (v) All the microvalves are actuated to completely
isolate the cell/bead pairs in the microchambers, followed by
incubation under standard culture conditions (37 °C and 5%
CO2) for the duration of the assay. (vi) Finally, brightfield and
fluorescence micrographs of the microchambers are acquired
for further analysis.
Several strategies are used to detect protein secretion from
single cells in microfluidic devices, including droplets with
particles,32,33,35,36 microchambers with beads28,29,31 or barc-
odes,30,44 and surface-modified microwells.24,26,27 However,
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Figure 3. Characterization of trap occupancy and cellular viability. (a) Cell-trapping characterization. Plots show the increase in the percentage of
cell-occupied traps through time for three different flow rates. (b) Cellular viability assay. Representative micrographs of trapped cells stained with
Hoechst and EthD. (c) Plot shows the percentage of viable primary monocytes trapped at flow rates of 1 and 2 μL/min at 0 and 24 h. (d) Bead
trapping characterization. Plots show the increase in the percentage of bead-occupied traps through time for three different flow rates. Number of
microbeads per trap over time at flow rates of 0.5 (e), 1 (f), and 2 (g) μL/min. (h) Percentage of chambers occupied with a single cell and one,
two, or three beads using different flow rates: 2 μL/min (red), 1 μL/min (blue), or 0.5 μL/min (green). (i) Micrographs of representative
microchambers pairing a cell with single or multiple beads.

some of these methods lack the ability to evaluate different
stimuli in parallel,24,26−28,30−33,35,36 are difficult to automate
and to replace the media once cells have been iso-
lated,24,26,27,30−33,35,36,44 or can only analyze a few cells
(∼40) per chip.29 Compared to these approaches, our highly
integrated device allows to pair hundreds of cells and sensing
beads to interrogate cell secretion function in an automated
fashion. Furthermore, it allows the simultaneous assessment of
up to six different chemical stimuli, which are delivered into
the microchambers through the hydrodynamic traps’ sieves.
Characterization of Single-Cell Occupation. It is well
known that the cell capture efficiency of hydrodynamic traps is
affected by the flow rate.45,46 Because our device is pressure-
driven, we first determined the flow rates in the device
generated at different pressures, Figure S3. Next, we
characterized the trap occupancy�defined as the percentage
of traps occupied by either cells or microbeads�at flow rates
ranging from 0 to 2 μL/min through the central channels. We
used NALM-6 cells, which in suspension are small round cells
with an average diameter of ∼10 μm. As shown in Figure 3a,
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after introducing the cells in the device, the trap occupancy
increases rapidly and reaches a plateau after 8−9 min. Under a
flow rate of 0.5 μL/min, only 50% of the traps were occupied
after 1 min and reached a maximum occupancy of 72% after 8
min. On the other hand, under flow rates of 1 and 2 μL/min,
the trap occupancies reached 73 and 83% after 1 min,
respectively. The maximum occupancies were obtained
employing flow rates of 1 μL/min (83%) and 2 μL/min
(94%) after 9 min. The volumes used to obtain 72, 83, and
94% cell occupancy correspond to 5, 10, and 20 μL,
respectively. Given the cell density employed (1.5 × 106
cells/mL), the total number of cells delivered to the device
were approximately 7500, 15,000, and 30,000 cells, respec-
tively. Similar occupancies were obtained for blood-derived
monocytes and Jurkat cells, Figure S4a,b. In addition, we
measured the size of cells captured in one section of the device
and determined that the smallest cell diameter that can
efficiently block the sieves and become trapped is 6.5 μm,
Figure S5.
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Figure 4. Characterization of solution exchange and immunoassay performance. (a) Solution exchange protocol: (1) microbeads are immobilized
and a replacement solution can be delivered through the side channel. (2) This generates a diffusive flow from the side channel, through the sieves,
and into the chamber. (3) Additional replacement solution can be injected. (4−6) Cycles are repeated until the complete exchange is achieved. (b)
Mass transfer characterization. Plots show the equalization of solution for multiple injection cycles when different number of beads are immobilized
per trap (n = 56). (c) Micrographs of representative microchambers with single or multiple beads for interleukin-8 (IL-8) immunoassay. (d) IL-8
calibration curve for different numbers of beads per microchamber. Bar graphs show the differences in fluorescent intensity between beads per
chamber (n = 24).

Simulation of Shear Stress on Captured Cells and
Evaluation of Cell Viability. High shear stress might activate
cells, causing undesired behaviors47 or even inducing
apoptosis.48 Normal shear stress values in the veins range
between 1 and 6 dynes/cm.2,49 To understand the shear stress
that cells are subjected to in the traps, we performed finite
element simulations (Figure S6 and Table S1). The simulated
shear stress experienced by the cells at flow rates of 0.5 and 1
μL/min are 1.3 and 3 dynes/cm,2 respectively, values which
are comparable with other methods such as microwells (0.1−5
dynes/cm2).50,51 Flow rates equal or greater than 2 μL/min
cause higher shear stresses (>6 dynes/cm2) on the cells, which
could interfere with cellular function. Overall, our results agree
with other studies where authors have employed similar flow
rates to capture single cells in hydrodynamic traps.45,52
Next, we assessed the effects of flow rate on cell viability.
Monocytes isolated from peripheral blood were stained with
Hoechst and introduced into the device. Cells were captured
and incubated with a medium containing EthD-1, and
micrographs were acquired at 0 and 24 h. The percentage of
dead cells at the beginning of the assay was 94 and 96% for 1
and 2 μL/min, respectively, and decreased to 87 and 78% after
24 h for the same flow rates, Figure 3b,c. Cellular viability
obtained using 1 μL/min was comparable to previous reports
using peripheral blood mononuclear cells and monocytes (8953
and 85%54 at 24 h), suggesting that most of the cells trapped in
our device remain functional after 1 day.
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Characterization of Microbeads Occupation. Next, we
evaluated the occupancy of microbeads when the cells had
already been captured in their respective traps. We observed
that the percentage of occupied traps increased at higher flow
rates, with 80, 88, and 93% traps occupied at 0.5, 1, and 2 μL/
min, respectively, Figure 3d. Furthermore, bead capture was
achieved rapidly, reaching a plateau after only 25 s at the
highest flow rate.
As shown in Figure 3e, when microbeads were injected at a
flow rate of 0.5 μL/min for 20 s, 53% of the traps had either
one, two, or three beads captured, while 26% of the traps had
more than three beads. Similarly, when beads were injected at
1 μL/min, a maximum occupancy of 53% was achieved with
two to three beads; however, the capture time decreased to 10
s, Figure 3f. The highest percentage (58%) of traps occupied
with one, two, or three beads was achieved when beads were
injected at 2 μL/min for 3 s, Figure 3g. Since polystyrene
microbeads are rigid, they do not completely block the sieves;
therefore, fluid can still flow through the gaps, leading to the
attraction of more than one microbead in a trap. In summary,
an increase in both trapping time and flow rate leads to a
higher number of beads captured per trap.
From the above experiments, we measured and plotted the
pairing efficiency of a single cell with at least one or up to three
beads for various flow rates and trapping times, Figure 3h,i.
The single-cell bead pairing was also performed rapidly, and
the efficiency varied with the flow rate. We achieved a
maximum pairing of 42% after 15 s at 0.5 μL/min, 45% after
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10 s at 1 μL/min; however, the maximum efficiency was
achieved at 2 μL/min after 3 s with 56% of microchambers
containing a single cell and bead. Most notably, our cell/bead
pairing system (Figure S7) is superior to other approaches,
such as droplet encapsulation or microcompartment isolation,
which show pairing efficiencies of 19−2732,33 and 37.3%,31
respectively. Other droplet-based microfluidics strategies have
been employed for single-cell transcriptomics applications. For
example, using embryonic stem cells and deformable hydrogel
microspheres carrying barcoded primers achieved co-encapsu-
lation efficiencies of over 90%.55 Using hydrogel microspheres
could be an interesting approach to apply in our microfluidic
system for single-cell proteomics applications. For example,
encapsulating antibody-coated polystyrene microspheres in
hydrogel microspheres could increase the occupancy of the
traps since their characteristic deformability will allow them to
block the sieve efficiently.
Solution Exchange in Microchambers. Fluorescent
immunoassays implemented either in microwells,24 reconfig-
urable microchambers,31 or microdroplets32,33 are the most
commonly employed microfluidic-based methods to quantify
protein secretion from single cells. Although they have
provided insights regarding immune response at the single-
cell level,20 a main drawback of these methods is the inability
to perform solution exchange once cells have been isolated,
and thus, in these approaches, cells can only be kept alive for a
few hours due to the depletion of nutrients. Therefore, the
frequent delivery of fresh media is paramount to maintain
healthy and viable cells.56 Furthermore, solution exchange can
also enable dynamic stimulation, which can provide insights
into the underlying mechanisms related to information
processing within cells.29 Last, the ability to exchange solutions
in the microchambers facilitates, for example, washing steps
during immunoassays (removing detection antibodies), to
decrease background signal.
In comparison to other microfluidic platforms, one major
advantage of our device is that it can replace the micro-
chambers’ solutions once the cells and beads have been
trapped. This is done through the implementation of a solution
exchange protocol, which consists of a sequence of cycles. Each
cycle consists of loading a new solution via the bead-side
channels for 1 min, followed by the actuation of valves for 30 s
to allow the solutes to diffuse from the bead-side channel
through the sieves and into the microchamber. In this fashion,
at the end of each cycle, the solution in the bead-side channel
is replenished with fresh solution that then diffuses into the
microchamber, Figure 4a. We characterized this diffusion
process when different numbers of beads (1−3) are
immobilized per trap. As shown in Figure 4b, independently
of the number of beads present in the chamber, a plateau is
reached after 10 cycles, indicating that the solution in the
chamber has been completely exchanged and, given that each
cycle lasts 1.5 min, the total solution replacement time is 15
min. This capability of replacing the microchambers’ solutions
after cell/bead isolation provides a significant advantage over
other previously reported microfluidics systems,32,35,36 since it
could allow dynamic stimulation as well as maintain the cells
viable for extended periods of culture time.
On-Chip Immunoassays. Next, to assess the performance
of our microfluidic bead-based immunoassay method and
determine its limit of detection, we carried out fluorescent
sandwich immunoassays on microbeads isolated in micro-
chambers. For this purpose, we performed calibration curves
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against Interleukin-8 (IL-8). IL-8 is a pro-inflammatory
cytokine mainly produced by monocytes and macrophages57
and a potent chemoattractant of neutrophils.58 Anti-IL-8
modified microbeads were captured in the traps, and known
concentrations of recombinant IL-8, ranging from 0 to 12.5
ng/mL, were then delivered through the central channels at 1
μL/min and incubated for 1.5 h. Next, fluorescently labeled
detection antibodies were injected through the side channels
using the solution exchange protocol (10 cycles of 1:30 min at
1 μL/min). As shown in Figure 4c,d, the fluorescence intensity
values correlate with the protein concentration yielding a
coefficient of determination (R2) of 0.92, 0.95, and 0.95 when
one, two, or three beads are isolated per chamber, respectively.
Furthermore, the limit of detection (LOD, calculated as the
mean value of the blank value plus three standard deviations)
was 11 pg/mL, independent of the number of beads present.
Conflicting reports have documented the effects that have
the number of microbeads in cytokine detection, with some
implying that the presence of up to three beads does not
impact the sensitivity of the assay,31 while others suggest that it
creates a significant variability due to possible competition
between the bead’s binding site for free cytokines.59 Therefore,
we assessed this variability in our assay by partitioning the
calibration curves for one, two, and three beads per chamber.
As evidenced in Figure 4d, for some IL-8 concentrations (0.2,
1.6, 3.1, and 12.5 ng/mL), the signal intensity is higher when
only one bead is present and significantly decreases when three
beads are immobilized per chamber (15% less signal for 12.5
ng/mL and 18% less for 3.1 ng/mL). Despite these differences
in signal intensities and regardless of the number of sensing
beads per chamber, our bead-based immunoassay performs
comparable to other methods employed in microfluidic
devices, which have achieved LODs between 7.5 and 75 ng/
mL for IL-8.60,61
Detection of IL-8 Secreted from Single Blood-
Derived Human Monocytes. Microfluidics technology
provides unique means to perform in vitro single-cell analysis,
including cytokine secretion from immune cells.24,29−31
Nonetheless, in vivo conditions rely on a complex interplay
of signaling events that coordinates the immune response and
dictates the response a cell will exhibit, such as paracrine and
autocrine signaling. Indeed, a disadvantage of traditional
microfluidic approaches for the analysis of single-cell secretion
is that by isolating the cells in small compartments, paracrine
signaling becomes disrupted, influencing the outcome of the
experiments.62
To test whether our device could help understand the effect
autocrine and paracrine signaling has on cytokine secretion at
the single-cell level, we devise an experiment to assess the
secretion of IL-8 from blood-derived human monocytes
challenged with LPS, while subjected to only autocrine
signaling or to both autocrine and paracrine signaling. We
chose IL-8 because it is a potent chemoattractant factor for
neutrophils, playing a major role in acute inflammation.58 In
response to an inflammatory stimulus, such as bacterial LPS,
activated cells�mainly monocytes and macrophages�pro-
duce IL-8 to initiate the inflammatory response, by recruiting
and activating neutrophils, homing them to the site of
inflammation.63 Although IL-8 is not a major chemotactic
factor for monocytes or macrophages, these cells abundantly
express IL-8 receptors.64 The high expression of IL-8 receptors
suggests that both monocytes and macrophage functional
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Figure 5. Detection of proteins secreted from single cells. (a) Human monocytes purified from peripheral blood were stimulated to evaluate IL-8
secretion while subjected to only autocrine signaling or to both paracrine and autocrine signaling. The secretory response of single monocytes was
assessed under different concentrations of LPS (b) without paracrine communication by stimulating the isolated cells and (c) with paracrine
communication by stimulating first in bulk. (d) Autocrine and paracrine signaling assessment in monocyte secretory response using the same
device. p > 0.01 (ns), p < 0.01 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

activity is modulated via paracrine or autocrine IL-8 signal-
ing.64,65
To assess the effect of paracrine signaling, blood-derived
human monocytes were maintained under bulk culture
conditions to allow cell-to-cell communication via soluble-
secreted factors. Cells were then challenged with LPS for 4 h in
a petri dish (1.5 mL), collected, and seeded to the
microchambers. For the autocrine signaling experiment, the
monocytes were first isolated and then seeded in the
microchambers. Next, the cells were stimulated with LPS
employing the reagent exchange protocol previously described.
In both cases, cells were challenged with different concen-
trations of LPS (0, 12.5, 50, and 100 μg/mL) and incubated
for a total period of 24 h to assess the secretory response of
individual cells, Figure 5a.
When subjected to only autocrine signaling, 61% of
monocytes did not secrete IL-8 while 39% displayed a
secretory phenotype for all LPS concentrations, Figure 5b.
This was further confirmed by running a Mann−Whitney
statistical test, which did not show significant differences
between the negative control group and the LPS-treated
groups. In contrast, while subjected to both paracrine and
autocrine signaling, LPS-challenged cells exhibited a dose-
dependent secretory response showing significant differences
between all treated groups and the negative control, Figure 5c.
These results are consistent with previous reports where
individual monocyte-derived macrophages, also stimulated
with LPS, not only exhibited heterogeneous levels of secretion
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for different cytokines (including tumor necrosis factor-α
(TNF-α) and IL-8) but also showed a considerable increase in
the secretion of these factors when subjected to paracrine
signaling.62 The considerable reduction in IL-8 secreting cells
under autocrine signaling could be due to the disruption of
paracrine signaling mediated by other cytokines or soluble
factors.
To verify that there was no experimental bias due to
different samples, we carried out an experiment in which the
monocytes isolated from the same sample were subdivided into
two groups, cells with only autocrine signaling and another
group where cells were allowed to maintain both paracrine and
autocrine signaling. Then, the cells were analyzed in the same
device where the secretory response was evaluated, Figure 5d.
We observed a significant difference in the secretory response
between the two groups. The group that allowed paracrine
signaling showed more cells with a high secretion profile, on
average 2.5-fold higher than the group with only autocrine
signaling. Overall, our results suggest that while performing
single-cell secretory analysis, regulatory signaling conveyed by
either autocrine or paracrine signaling should be carefully
considered to prevent bias arising from the experimental design
from interfering with the final results.
■ CONCLUSIONS
We presented a microfluidic device to analyze the cytokine
secretion of single immune cells via an immunoassay
conducted on microbeads functionalized with capture anti-
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bodies. By integrating microvalves and hydrodynamic traps
into our device, we were able to control the fluid flow to direct
cells and microbeads into their respective capture site, as well
as to isolate the chambers to avoid cross talk of secreted
soluble factors. Additionally, our device allows us to challenge
cells from the same sample with different chemical stimuli in
parallel. One key advantage of our platform is the possibility to
exchange the chambers’ solutions once the cells have been
isolated, which could enable dynamic cell stimulation or long-
term experimentation by replenishing the cells with fresh
medium. Furthermore, we showed that our biosensing method
(antibody-coated microbeads) provided the sensitivity re-
quired to detect IL-8 secreted from single blood-derived
human monocytes. Last, we proved the versatility of our
platform by assessing the response of single human monocytes
under different concentrations of LPS and evaluated differ-
ences in the distribution of secreting cells subjected to either
paracrine/autocrine signaling or solely autocrine signaling. Our
device could have a wide range of applications in the analysis
of cellular function heterogeneity, such as antibody discovery
or investigation of rare cells.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acssensors.2c02148.
Complete methodology for master mold fabrication;
device fabrication; assembly and setup; cell line culture;
microbead functionalization; calibration curve gener-
ation; primary human monocyte isolation, culture,
viability analysis, and on-chip stimulation; protein
detection, imaging, and result analysis; shear stress
experienced by cells; images of representative traps with
actuated or unactuated traps; chip designs; flow rate
characterization under various driving pressures; trap
occupancy using different cell lines; and shear stress
simulations (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Jose L. Garcia-Cordero − Laboratory of Microtechnologies for
Biomedicine, Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional (Cinvestav),
Monterrey 66628 Nuevo León, Mexico; Roche Institute for
Translational Bioengineering (ITB), Roche Pharma Research
and Early Development, Roche Innovation Center Basel,
Basel 4058, Switzerland; orcid.org/0000-0002-3868-
7405; Email: jose_luis.garcia_cordero@roche.com
Authors
Diana F. Cedillo-Alcantar − Laboratory of Microtechnologies
for Biomedicine, Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional (Cinvestav),
Monterrey 66628 Nuevo León, Mexico
Roberto Rodriguez-Moncayo − Laboratory of
Microtechnologies for Biomedicine, Centro de Investigación y
de Estudios Avanzados del Instituto Politécnico Nacional
(Cinvestav), Monterrey 66628 Nuevo León, Mexico; Present
Address: Research Laboratory of Electronics,
Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States; orcid.org/0000-
0001-8775-5921
662
Article
Jose L. Maravillas-Montero − Red de Apoyo a la
Investigación, Universidad Nacional Autónoma de México e
Instituto Nacional de Ciencias Médicas y Nutrición Salvador
Zubirán, Mexico City 14080, Mexico
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssensors.2c02148
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was funded by Mexico’s National Science and
Technology Council (CONACyT Grants No. 102963, and
CB-286368) and by Cinvestav (Grant No. SEP-Cinvestav
104).
Notes
The authors declare the following competing financial
interest(s): J.L.G.C is now an employee of the Roche Institute
for Translational Bioengineering.
■ ACKNOWLEDGMENTS
The authors would like to thank Rocio J. Jiménez-Valdés and
Alan M. Gonzalez-Suarez for helpful discussions.
■ ABBREVIATIONS
EthD-1, ethidium homodimer-1; IL-8, interleukin-8; LOD,
limit of detection; LPS, lipopolysaccharide; PBMC, peripheral
blood mononuclear cells; PDMS, poly(dimethylsiloxane); PE,
phycoerythrin; TNF-α, tumor necrosis factor-α
■ REFERENCES
(1) Takemura, R.; Werb, Z. Secretory Products of Macrophages and
Their Physiological Functions. Am. J. Physiol.: Cell Physiol. 1984, 246,
C1−C9.
(2) Turola, E.; Furlan, R.; Bianco, F.; Matteoli, M.; Verderio, C.
Microglial Microvesicle Secretion and Intercellular Signaling. Front.
Physiol. 2012, 3, No. 149.
(3) Ngangan, A. V.; Waring, J. C.; Cooke, M. T.; Mandrycky, C. J.;
McDevitt, T. C. Soluble Factors Secreted by Differentiating
Embryonic Stem Cells Stimulate Exogenous Cell Proliferation and
Migration. Stem Cell Res. Ther. 2014, 5, No. 26.
(4) Ojima, K.; Oe, M.; Nakajima, I.; Muroya, S.; Nishimura, T.
Dynamics of Protein Secretion during Adipocyte Differentiation.
FEBS Open Bio 2016, 6, 816−826.
(5) Kusindarta, D. L.; Wihadmadyatami, H. The Role of
Extracellular Matrix in Tissue Regeneration. In Tissue Regeneration;
IntechOpen Ltd., 2018; Vol. 65.
(6) Lu, P.; Takai, K.; Weaver, V. M.; Werb, Z. Extracellular Matrix
Degradation and Remodeling in Development and Disease. Cold
Spring Harbor Perspect. Biol. 2011, 3, No. a005058.
(7) Stastna, M.; Van Eyk, J. E. Secreted Proteins as a Fundamental
Source for Biomarker Discovery. Proteomics 2012, 12, 722−735.
(8) Welsh, J. B.; Sapinoso, L. M.; Kern, S. G.; Brown, D. A.; Liu, T.;
Bauskin, A. R.; Ward, R. L.; Hawkins, N. J.; Quinn, D. I.; Russell, P. J.;
Sutherland, R. L.; Breit, S. N.; Moskaluk, C. A.; Frierson, H. F.;
Hampton, G. M. Large-Scale Delineation of Secreted Protein
Biomarkers Overexpressed in Cancer Tissue and Serum. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 3410−3415.
(9) Dhar, M.; Lam, J. N.; Walser, T.; Dubinett, S. M.; Rettig, M. B.;
Di Carlo, D. Functional Profiling of Circulating Tumor Cells with an
Integrated Vortex Capture and Single-Cell Protease Activity Assay.
Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 9986−9991.
https://doi.org/10.1021/acssensors.2c02148
ACS Sens. 2023, 8, 655−664
ACS Sensors pubs.acs.org/acssensors
(10) Winkler, J.; Abisoye-Ogunniyan, A.; Metcalf, K. J.; Werb, Z.
Concepts of Extracellular Matrix Remodelling in Tumour Progression
and Metastasis. Nat. Commun. 2020, 11, No. 5120.
(11) Xhangolli, I.; Dura, B.; Lee, G. H.; Kim, D.; Xiao, Y.; Fan, R.
Single-Cell Analysis of CAR-T Cell Activation Reveals A Mixed TH1/
TH2 Response Independent of Differentiation. Genomics, Proteomics
Bioinf. 2019, 17, 129−139.
(12) Xue, Q.; Bettini, E.; Paczkowski, P.; Ng, C.; Kaiser, A.;
McConnell, T.; Kodrasi, O.; Quigley, M. F.; Heath, J.; Fan, R.;
Mackay, S.; Dudley, M. E.; Kassim, S. H.; Zhou, J. Single-Cell
Multiplexed Cytokine Profiling of CD19 CAR-T Cells Reveals a
Diverse Landscape of Polyfunctional Antigen-Specific Response. J.
Immunother. Cancer 2017, 5, No. 85.
(13) Gérard, A.; Woolfe, A.; Mottet, G.; Reichen, M.; Castrillon, C.;
Menrath, V.; Ellouze, S.; Poitou, A.; Doineau, R.; Briseno-Roa, L.;
Canales-Herrerias, P.; Mary, P.; Rose, G.; Ortega, C.; Delincé, M.;
Essono, S.; Jia, B.; Iannascoli, B.; Richard-Le Goff, O.; Kumar, R.;
Stewart, S. N.; Pousse, Y.; Shen, B.; Grosselin, K.; Saudemont, B.;
Sautel-Caillé, A.; Godina, A.; McNamara, S.; Eyer, K.; Millot, G. A.;
Baudry, J.; England, P.; Nizak, C.; Jensen, A.; Griffiths, A. D.; Bruhns,
P.; Brenan, C. High-Throughput Single-Cell Activity-Based Screening
and Sequencing of Antibodies Using Droplet Microfluidics. Nat.
Biotechnol. 2020, 38, 715−721.
(14) Jin, A.; Ozawa, T.; Tajiri, K.; Obata, T.; Kondo, S.; Kinoshita,
K.; Kadowaki, S.; Takahashi, K.; Sugiyama, T.; Kishi, H.; Muraguchi,
A. A Rapid and Efficient Single-Cell Manipulation Method for
Screening Antigen-Specific Antibody-Secreting Cells from Human
Peripheral Blood. Nat. Med. 2009, 15, 1088−1092.
(15) Altschuler, S. J.; Wu, L. F. Cellular Heterogeneity: Do
Differences Make a Difference? Cell 2010, 141, 559−563.
(16) Zenobi, R. Single-Cell Metabolomics: Analytical and Biological
Perspectives. Science 2013, 342, No. 1243259.
(17) Jindal, A.; Gupta, P.; Jayadeva; Sengupta, D. Discovery of Rare
Cells from Voluminous Single Cell Expression Data. Nat. Commun.
2018, 9, No. 4719.
(18) Perkins, T. J.; Swain, P. S. Strategies for Cellular Decision-
Making. Mol. Syst. Biol. 2009, 5, No. 326.
(19) Bucheli, O. T. M.; Sigvaldadóttir, I.; Eyer, K. Measuring Single-
Cell Protein Secretion in Immunology: Technologies, Advances, and
Applications. Eur. J. Immunol. 2021, 51, 1334−1347.
(20) Chen, Z.; Chen, J. J.; Fan, R. Single-Cell Protein Secretion
Detection and Profiling. Ann. Rev. Anal. Chem. 2019, 12, 431−449.
(21) Kouwenhoven, M.; Ö zenci, V.; Teleshova, N.; Hussein, Y.;
Huang, Y. M.; Eusebio, A.; Link, H. Enzyme-Linked Immunospot
Assays Provide a Sensitive Tool for Detection of Cytokine Secretion
by Monocytes. Clin. Diagn. Lab. Immunol. 2001, 8, 1248−1257.
(22) Chowdhury, M. M.; Fujii, T.; Sakai, Y. Importance of a
Diffusion-Dominant Small Volume to Activate Cell-Secreted Soluble
Factor Signaling in Embryonic Stem Cell Culture in Micro-
bioreactors: A Mathematical Model Based Study. J. Biosci. Bioeng.
2013, 116, 118−125.
(23) Nikoloff, J. M.; Saucedo-Espinosa, M. A.; Kling, A.; Dittrich, P.
S. Identifying Extracellular Vesicle Populations from Single Cells. Proc.
Natl. Acad. Sci. U.S.A. 2021, 118, No. e2106630118.
(24) Han, Q.; Bradshaw, E. M.; Nilsson, B.; Hafler, D. A.; Love, J. C.
Multidimensional Analysis of the Frequencies and Rates of Cytokine
Secretion from Single Cells by Quantitative Microengraving. Lab Chip
2010, 10, 1391−1400.
(25) Mcwhorter, F. Y.; Smith, T. D.; Luu, T. U.; Rahim, M. K.;
Haun, J. B.; Liu, W. F. Integrative Biology Macrophage Secretion
Heterogeneity in Engineered Microenvironments Revealed Using a
Microwell Platform. Integr. Biol. 2016, 8, 751−760.
(26) Shirasaki, Y.; Yamagishi, M.; Suzuki, N.; Izawa, K.; Nakahara,
A.; Mizuno, J.; Shoji, S.; Heike, T.; Harada, Y.; Nishikomori, R.;
Ohara, O. Real-Time Single-Cell Imaging of Protein Secretion. Sci.
Rep. 2014, 4, No. 4736.
(27) Varadarajan, N.; Kwon, D. S.; Law, K. M.; Ogunniyi, A. O.;
Anahtar, M. N.; Richter, J. M.; Walker, B. D.; Love, J. C. Rapid,
Efficient Functional Characterization and Recovery of HIV-Specific
663
Article
Human CD8 + T Cells Using Microengraving. Proc. Natl. Acad. Sci.
U.S.A. 2012, 109, 3885−3890.
(28) Armbrecht, L.; Müller, R. S.; Nikoloff, J.; Dittrich, P. S. Single-
Cell Protein Profiling in Microchambers with Barcoded Beads.
Microsyst. Nanoeng. 2019, 5, No. 55.
(29) Junkin, M.; Kaestli, A. J.; Cheng, Z.; Jordi, C.; Albayrak, C.;
Hoffmann, A.; Tay, S. High-Content Quantification of Single-Cell
Immune Dynamics. Cell Rep. 2016, 15, 411−422.
(30) Ma, C.; Fan, R.; Ahmad, H.; Shi, Q.; Comin-Anduix, B.;
Chodon, T.; Koya, R. C.; Liu, C. C.; Kwong, G. A.; Radu, C. G.;
Ribas, A.; Heath, J. R. A Clinical Microchip for Evaluation of Single
Immune Cells Reveals High Functional Heterogeneity in Phenotypi-
cally Similar T Cells. Nat. Med. 2011, 17, 738−743.
(31) Son, K. J.; Rahimian, A.; Shin, D. S.; Siltanen, C.; Patel, T.;
Revzin, A. Microfluidic Compartments with Sensing Microbeads for
Dynamic Monitoring of Cytokine and Exosome Release from Single
Cells. Analyst 2016, 141, 679−688.
(32) Bounab, Y.; Eyer, K.; Dixneuf, S.; Rybczynska, M.; Chauvel, C.;
Mistretta, M.; Tran, T.; Aymerich, N.; Chenon, G.; Llitjos, J. F.;
Venet, F.; Monneret, G.; Gillespie, I. A.; Cortez, P.; Moucadel, V.;
Pachot, A.; Troesch, A.; Leissner, P.; Textoris, J.; Bibette, J.; Guyard,
C.; Baudry, J.; Griffiths, A. D.; Védrine, C. Dynamic Single-Cell
Phenotyping of Immune Cells Using the Microfluidic Platform
DropMap. Nat. Protoc. 2020, 15, 2920−2955.
(33) Chokkalingam, V.; Tel, J.; Wimmers, F.; Liu, X.; Semenov, S.;
Thiele, J.; Figdor, C. G.; Huck, W. T. S. Probing Cellular
Heterogeneity in Cytokine-Secreting Immune Cells Using Droplet-
Based Microfluidics. Lab Chip 2013, 13, 4740−4744.
(34) Eyer, K.; Doineau, R. C. L.; Castrillon, C. E.; Briseño-roa, L.;
Menrath, V.; Mottet, G.; England, P.; Godina, A.; Brient-litzler, E.;
Nizak, C.; Jensen, A.; Griffiths, A. D.; Bibette, J.; Bruhns, P.; Baudry, J.
Single-Cell Deep Phenotyping of IgG-Secreting Cells for High-
Resolution Immune Monitoring. Nat. Biotechnol. 2017, 35, 977−982.
(35) Konry, T.; Golberg, A.; Yarmush, M. Live Single Cell
Functional Phenotyping in Droplet Nano-Liter Reactors. Sci. Rep.
2013, 3, No. 3179.
(36) Mazutis, L.; Gilbert, J.; Ung, W. L.; Weitz, D. A.; Griffiths, A.
D.; Heyman, J. A. Single-Cell Analysis and Sorting Using Droplet-
Based Microfluidics. Nat. Protoc. 2013, 8, 870−891.
(37) Chung, K.; Rivet, C. A.; Kemp, M. L.; Lu, H. Imaging Single-
Cell Signaling Dynamics with a Deterministic High-Density Single-
Cell Trap Array. Anal. Chem. 2011, 83, 7044−7052.
(38) Kimmerling, R. J.; Szeto, G. L.; Li, J. W.; Genshaft, A. S.; Kazer,
S. W.; Payer, K. R.; de Riba Borrajo, J.; Blainey, P. C.; Irvine, D. J.;
Shalek, A. K.; Manalis, S. R. A Microfluidic Platform Enabling Single-
Cell RNA-Seq of Multigenerational Lineages. Nat. Commun. 2016, 7,
No. 10220.
(39) Banaeiyan, A. A.; Ahmadpour, D.; Adiels, C. B.; Goksör, M.
Hydrodynamic Cell Trapping for High Throughput Single-Cell
Applications. Micromachines 2013, 4, 414−430.
(40) Skelley, A. M.; Kirak, O.; Suh, H.; Jaenisch, R.; Voldman, J.
Microfluidic Control of Cell Pairing and Fusion. Nat. Methods 2009,
6, 147−152.
(41) Cedillo-Alcantar, D. F.; Han, Y. D.; Choi, J.; Garcia-Cordero, J.
L.; Revzin, A. Automated Droplet-Based Microfluidic Platform for
Multiplexed Analysis of Biochemical Markers in Small Volumes. Anal.
Chem. 2019, 91, 5133−5141.
(42) Dettinger, P.; Frank, T.; Etzrodt, M.; Ahmed, N.; Reimann, A.;
Trenzinger, C.; Loeffler, D.; Kokkaliaris, K. D.; Schroeder, T.; Tay, S.
Automated Microfluidic System for Dynamic Stimulation and
Tracking of Single Cells. Anal. Chem. 2018, 90, 10695−10700.
(43) Zak, A.; Merino-Cortés, S. V.; Sadoun, A.; Mustapha, F.;
Babataheri, A.; Dogniaux, S.; Dupré-Crochet, S.; Hudik, E.; He, H. T.;
Barakat, A. I.; Carrasco, Y. R.; Hamon, Y.; Puech, P. H.; Hivroz, C.;
Nüsse, O.; Husson, J. Rapid Viscoelastic Changes Are a Hallmark of
Early Leukocyte Activation. Biophys. J. 2021, 120, 1692−1704.
(44) Lu, Y.; Chen, J. J.; Mu, L.; Xue, Q.; Wu, Y.; Wu, P. H.; Li, J.;
Vortmeyer, A. O.; Miller-Jensen, K.; Wirtz, D.; Fan, R. High-
https://doi.org/10.1021/acssensors.2c02148
ACS Sens. 2023, 8, 655−664
ACS Sensors pubs.acs.org/acssensors Article

Throughput Secretomic Analysis of Single Cells to Assess Functional (63) Agarwal, S.; Piesco, N. P.; Johns, L. P.; Riccelli, A. E.
Cellular Heterogeneity. Anal. Chem. 2013, 85, 2548−2556. Differential Expression of IL-1β, TNF-α, IL-6, and IL-8 in Human
(45) Kobel, S.; Valero, A.; Latt, J.; Renaud, P.; Lutolf, M. Monocytes in Response to Lipopolysaccharides from Different
Optimization of Microfluidic Single Cell Trapping for Long-Term Microbes. J. Dent. Res. 1995, 74, 1057−1065.
on-Chip Culture. Lab Chip 2010, 10, 857−863. (64) Browning, D. D.; Diehl, W. C.; Hsu, M. H.; Schraufstatter, I.
(46) Deng, B.; Li, X. F.; Chen, D. Y.; You, L. D.; Wang, J. B.; Chen, U.; Ye, R. D. Autocrine Regulation of Interleukin-8 Production in
J. Parameter Screening in Microfluidics Based Hydrodynamic Single- Human Monocytes. Am. J. Physiol.: Lung Cell. Mol. Physiol. 2000, 279,
Cell Trapping. Sci. World J. 2014, 2014, No. 929163. L1129−L1136.
(47) Nomura, S.; Tandon, N. N.; Nakamura, T.; Cone, J.; Fukuhara, (65) Corre, D. Interleukin-8 (IL-8): An Autocrine Paracrine Growth
S.; Kambayashi, J. High-Shear-Stress-Induced Activation of Platelets Factor for Human Hematopoietic Progenitors Acting in Synergy with
and Microparticles Enhances Expression of Cell Adhesion Molecules CSF-1 to Promote Monocyte-Macrophage Growth and Differ-
in THP-1 and Endothelial Cells. Atherosclerosis 2001, 158, 277−287. entiation. Exp. Hematol. 1998, 26, No. 802.
(48) Leytin, V.; Allen, D. J.; Mykhaylov, S.; Mis, L.; Lyubimov, E. V.;
Garvey, B.; Freedman, J. Pathologic High Shear Stress Induces
Apoptosis Events in Human Platelets. Biochem. Biophys. Res. Commun.
2004, 320, 303−310.
(49) Papaioannou, T. G.; Stefanadis, C. Vascular Wall Shear Stress:
Basic Principles and Methods. Hell. J. Cardiol. 2005, 46, 9−15.
(50) Jimenez-Valdes, R. J.; Rodriguez-Moncayo, R.; Cedillo-
Alcantar, D. F.; Garcia-Cordero, J. L. Massive Parallel Analysis of
Single Cells in an Integrated Microfluidic Platform. Anal. Chem. 2017,
89, 5210−5220.
(51) Gonzalez-Suarez, A. M.; Peña-del Castillo, J. G.; Hernández-
Cruz, A.; Garcia-Cordero, J. L. Dynamic Generation of Concen-
tration- and Temporal-Dependent Chemical Signals in an Integrated
Microfluidic Device for Single-Cell Analysis. Anal. Chem. 2018, 90,
8331−8336.
(52) Aljaghtham, M. S.; Liu, Z. L.; Guo, J. J.; He, J.; Celik, E.
Numerical Simulations of Cell Flow and Trapping within Microfluidic
Channels for Stiffness Based Cell Isolation. J. Biomech. 2019, 85, 43−
49.
(53) Cui, X.; Liu, Y.; Hu, D.; Qian, W.; Tin, C.; Sun, D.; Chen, W.;
Lam, R. H. W. A Fluorescent Microbead-Based Microfluidic
Immunoassay Chip for Immune Cell Cytokine Secretion Quantifica-
tion. Lab Chip 2018, 18, 522−531.
(54) Rodriguez-Moncayo, R.; Jimenez-Valdes, R. J.; Gonzalez-
Suarez, A. M.; Garcia-Cordero, J. L. Integrated Microfluidic Device Recommended by ACS
for Functional Secretory Immunophenotyping of Immune Cells. ACS
Sens 2020, 5, 353−361. Cryopreservable Through-Hole Arrays for the High-
(55) Klein, A.; Mazutis, L.; Akartuna, I.; Cell, N. T. Droplet Throughput Three-Dimensional Smartphone-Based Cell
Barcoding for Single-Cell Transcriptomics Applied to Embryonic Colorimetric Assay
Stem Cells. Cell 2015, 161, 1187−1201.
(56) Halldorsson, S.; Lucumi, E.; Gómez-Sjöberg, R.; Fleming, R. M. Chunqi Chang, Zhen Xu, et al.
T. Advantages and Challenges of Microfluidic Cell Culture in JANUARY 27, 2023

Polydimethylsiloxane Devices. Biosens. Bioelectron. 2015, 15, 218− ACS SENSORS READ
231.
(57) Duque, G. A.; Descoteaux, A. Macrophage Cytokines: Alleviating Cell Lysate-Induced Inhibition to Enable RT-
Involvement in Immunity and Infectious Diseases. Front. Immunol. PCR from Single Cells in Picoliter-Volume Double Emulsion
2014, 5, No. 491. Droplets
(58) Turner, M. D.; Nedjai, B.; Hurst, T.; Pennington, D. J.
Margarita Khariton, Bo Wang, et al.
Cytokines and Chemokines: At the Crossroads of Cell Signalling and
JANUARY 04, 2023
Inflammatory Disease. Biochim. Biophys. Acta, Mol. Cell Res. 2014,
ANALYTICAL CHEMISTRY READ
1843, 2563−2582.
(59) Hsu, W.; Shih, Y. T.; Lee, M. S.; Huang, H. Y.; Wu, W. N. Bead
Number Effect in a Magnetic-Beads-Based Digital Microfluidic Measurement of Protein–Protein Interaction Dynamics Using
Immunoassay. Biosensors 2022, 12, No. 340. Microfluidics and Particle Diffusometry
(60) Kamińska, A.; Winkler, K.; Kowalska, A.; Witkowska, E.; Hui Ma, Tamara L. Kinzer-Ursem, et al.
Szymborski, T.; Janeczek, A.; Waluk, J. SERS-Based Immunoassay in a OCTOBER 31, 2022
Microfluidic System for the Multiplexed Recognition of Interleukins ANALYTICAL CHEMISTRY READ
from Blood Plasma: Towards Picogram Detection. Sci. Rep. 2017, 7,
No. 10656. Ultraportable Flow Cytometer Based on an All-Glass
(61) Zhou, L.; Chen, P.; Simonian, A. Advanced Biosensing Microfluidic Chip
Towards Real-Time Imaging of Protein Secretion from Single Cells.
In Biosensors-Current and Novel Strategies for Biosensing; IntechOpen, Jiayu Li, Qin Li, et al.
2020. JANUARY 18, 2023

(62) Xue, Q.; Lu, Y.; Eisele, M. R.; Sulistijo, E. S.; Khan, N.; Fan, R.; ANALYTICAL CHEMISTRY READ
Miller-Jensen, K. Analysis of Single-Cell Cytokine Secretion Reveals a
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