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Evolution of “On-Barcode”Luminescence Oxygen Channeling
Immunoassay by Exploring the Barcode Structure and the Assay
System
Zuying Feng, Qingsheng Guo,*Yao Wang, Yunfei Ge, Zhiying Zhang, Yan Wu, Qilong Li,
Hajar Masoomi, Hongchen Gu, and Hong Xu*
Cite This: https://doi.org/10.1021/acsomega.1c06236
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sıSupporting Information
ABSTRACT: The multiplexed luminescence oxygen channeling
immunoassay (multi-LOCI) platform we developed recently that
combines conventional LOCI and suspension array technology is
capable of realizing facile “mix-and-measure”multiplexed assays
without tedious washing steps. However, previous work lacks
comprehensive studies of the structure−performance relationship
of the host−guest-structured barcode, which may obstruct the
evolution and further translation of this exciting new technology to
practical applications. Accordingly, this work revealed that
polyelectrolyte interlayers played a crucial role in tuning the
packing density of guest acceptor beads (ABs). More interestingly,
we noticed that “sparse”barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with “compact”
ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and
flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the
concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in
sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-
LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed
LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique “on-barcode”assay format.
1. INTRODUCTION
Multiplexed assays are of great interest to life sciences and
biomedical fields including gene analysis,
1,2
drug delivery and
screening,
3
and disease diagnostics
4−8
due to their capacity of
detecting multiple targets simultaneously within one reaction.
Compared with traditional planar-array-based biochips that are
widely used in the discovery of biomarkers and semi-
quantitative detection, encoded bead-based suspension arrays,
which are capable of addressing and capturing corresponding
multiple target molecules, high reaction efficiency, high
flexibility in target selection, and tremendous coding
capability,
9,10
are a promising technology for multiplex
quantitative assays. After Luminex Corporation successfully
launched xMAP technology,
11−13
more efforts have continu-
ously been made toward developing new coding strategies for
the sake of expanding the barcode libraries.
9,14−17
In addition to proposing new encoding strategies, remark-
ably fast-growing progress has also been made in developing
novel detection methodologies based on barcodes, aiming at
higher performance or ease of operation.
18−21
Recently,
through the combination of encoded beads with luminescence
oxygen channeling immunoassay (LOCI) technology, our
group developed a novel suspension array platform, called
“multi-LOCI”.
22
As shown in Scheme 1a, a dual-functional
multi-LOCI barcode consists of two isolated building blocks:
(a) encoded host beads (EHBs) that offer barcode signals for
the identification of multiple analytes and (b) LOCI acceptor
beads (ABs), which are immobilized onto the surface of EHBs
(EHB@ABs) and provide LOCI signals to quantify specific
analyte concentrations. For a typical multi-LOCI assay, the
analytes of interest are specifically captured by EHB@ABs,
further leading to the specific bounding of biotinylated
detection antibodies and streptavidin-coated donor beads
(DB-SA) via formation of immune complexes. Upon excitation
of DB-SA at 680 nm, singlet oxygen molecules transfer from
DB-SA to ABs and trigger strong chemiluminescence emission
at 615 nm,
23,24
and both barcode and LOCI signals are
acquired via image analysis processing. This strategy achieves a
Received: November 5, 2021
Accepted: December 23, 2021
Article
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unique multiplexed assay following a simple “mix-and-
measure”protocol without the need for tedious washing
steps, which is very promising for the development of
multiplexed point-of-care test technology.
The multi-LOCI technique exhibits the capacity of both
high multiplexing and ease of operation. However, the lack of
comprehensive studies of the structure−performance relation-
ship of the unique host−guest barcodes hinders the evolution
of this promising technology to a universal and practical
method. First, this study systematically investigated the
structure−performance relationship of multi-LOCI barcodes
from two aspects that are shown in Scheme 1b. On the one
hand, the barcode size was explored because it might be closely
related to the encoding capacity, reaction kinetics, and the
accuracy of imaging-based decoding. On the other hand, by
adjusting the number of polyelectrolyte interlayers, the packing
density of ABs was found to be precisely controlled, providing
a guideline to establish a robust and flexible multi-LOCI
barcode fabrication protocol. More importantly, this discovery
would pave a novel but universal pathway for construction of
host−guest-structured micro- and nanocomposites. A deeper
investigation on the relationship between packing density and
assay performance was explored since the number of ABs
loaded on a single barcode was supposed to directly influence
the LOCI signal. Second, there is still much room for detection
performance improvement of the multi-LOCI assay system,
which is not investigated in detail in our previous study. As
shown in Scheme 1c, the multi-LOCI assay system including
DB-SA concentration, the 680 nm laser power, and the
concentrations of biotinylated detection antibodies for specific
analytes was comprehensively optimized to clarify the potential
relationship between these parameters and the detection
performance, which in turn enabled improved sensitivity.
Through the elaborate investigations of both the barcode
structure and the assay system, the multi-LOCI was supposed
to be demonstrated as a more powerful platform with high
robustness and excellent performance.
2. EXPERIMENTAL SECTION
2.1. Materials. Multi-LOCI EHBs with a diameter of 3 and
6μm were prepared following the procedure described in our
previous studies.
14,25
By adjusting the fluorescence intensities
of fluorescein isothiocyanate (FITC) and rhodamine iso-
thiocyanate (RITC), 3 μm EHBs with 28 different barcodes
(combinations of seven levels of FITC and four levels of
RITC) and 6 μmEHBswith30different barcodes
(combinations of six levels of FITC and five levels of RITC)
were obtained, respectively. Poly(sodium 4-styrenesulfonate)
(PSS, Mw= 70k), poly(diallydimethylammonium chloride)
(PDDA, 100k ≤Mw≤200k), N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccini-
mide (NHS), 2-(N-morpholino)ethanesulfonic acid monohy-
drate (MES), 4-(dimethylamino)pyridine (DMAP), 2-imino-
thiolane hydrochloride (ITL), 5,5′-dithiobis(2-nitrobenzoic
acid) (DTNB), DL-dithiothreitol (DTT), and poly-
(ethylenimine) (PEI, Mw= 10k) were purchased from
Sigma-Aldrich. Traut’s Reagent was prepared by dissolving
ITL (40 mM) and DMAP (40 mM) in ethanol. Ellman’s
Reagent was a PBS (10 mM, pH 7.4) buffer containing 3.53
mM DTNB and 1 mM EDTA. Cysteine was bought from
Scheme 1. Schematic Illustration of (a) the Principle of Multi-LOCI for Multiplexed Assay and Key Issues That Were
Investigated in This Research Including the (b) Multi-LOCI Barcode Structure and (c) Assay System as Indicated
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B
Macklin. Carboxyl-modified ABs and DB-SA were obtained
from PerkinElmer. Anti-IFN-γcapture antibody, anti-IL-10
capture antibody, and biotinylated anti-IL-10 detection
antibody were obtained from Biolegend. Anti-IFN-γdetection
antibody, IL-6, IL-17A, IL-10, and the corresponding antibod-
ies of IL-6 and IL-17A were provided by R&D Systems. IFN-γ
was provided by Hytest.
2.2. Preparation of Multi-LOCI Barcodes. 2.2.1. Depo-
sition of Polyelectrolyte Multilayers on Encoded Host Beads.
Through the electrostatic interaction between the positively
charged PDDA and negatively charged PSS,
26
multilayered
polyelectrolytes were deposited on silica-encapsulated EHBs
(EHB@SiO2), and the preparation process was as follows.
EHB@SiO2was dispersed in PDDA solution (5 mg/mL, in 0.5
M NaCl), rotated for 20 min, and then washed with deionized
water. The obtained beads were then dispersed in PSS solution
(5 mg/mL, in 0.5 M NaCl) with rotation for 20 min and were
further washed with deionized water. By repeating this
procedure several times, PDDA and PSS were alternately
encapsulated onto the surface of EHBs. The obtained beads
were denoted as EHB@(PSS/PDDA)n, where n(from 0 to 4)
indicates the number of PSS/PDDA pairs. Finally,
EHB@(PSS/PDDA)n@PEI was obtained by suspending
beads in PEI solution (50 mg/mL, in 10 mM MEST) with
rotation for another 20 min, followed by washing six times.
2.2.2. Quantification of Surface Primary Amino Groups of
EHB@(PSS/PDDA)n@PEI. The ITL/DTNB method was used
to quantify the surface primary amino groups.
27−29
ITL and
DTNB are known as Traut’s Reagent and Ellman’s reagent,
respectively. Typically, 0.5 mg of EHB@(PSS/PDDA)n@PEI
(nfrom 0 to 4) were washed with ethanol twice and dispersed
in 400 μL of Traut’s Reagent. After the mixture reacted for 1 h,
beads were washed with ethanol, 1 mmol/L DTT, ethanol, and
PBS sequentially. Then, the beads were suspended in 203.5 μL
of PBS and reacted with 10 μL of Ellman’s reagent for 15 min.
By comparing the sample’s absorbance at 412 nm measured by
a SpectraMax i3 multimode microplate reader (Molecular
Devices, CA) with a standard curve of cysteine, the content of
sulfhydryl groups was calculated, and then the density of the
amino group on the surface of EHB@(PSS/PDDA)n@PEI
was obtained.
2.2.3. Fabrication of Host−Guest-Structured Multi-LOCI
Barcodes. Through a carbodiimide reaction, carboxylated ABs
were immobilized covalently on amino-functionalized
EHB@(PSS/PDDA)n@PEI host beads to achieve multi-
LOCI barcodes. The detailed assembly process of this host−
guest-structured multi-LOCI barcodes was as follows. First, 1.3
mg of EHB@(PSS/PDDA)n@PEI dispersed in 150 μL of 100
mM MES was dropwise added into a suspension containing
0.6 mg of ABs, followed by rotation for 30 min. Then, 100 μL
of 100 mg/mL EDC and NHS were, respectively, added into
the mixture followed by rotation for 3 h at room temperature.
Afterward, 2.5 mM sodium hydroxide (NaOH) and water were
used to sequentially wash the as-synthesized host−guest-
structured EHB@(PSS/PDDA)n@PEI@ABs (denoted as
EHB@ABs). To remove the residual amino group of the
multi-LOCI barcodes, 150 μL of PAA solution (1 mg/mL, in
10 mM MEST, pH 5.0) was added to the as-synthesized
EHB@ABs with rotation for 30 min at room temperature.
Then, 150 μL of EDC solution (10 mg/mL, in 10 mM MEST,
pH 5.0) was added into the mixture and reacted for 2 h, and
the beads were washed and stored in water finally.
2.3. Bioconjugation and Immunoassay. 2.3.1. Biocon-
jugation of EHB@ABs with Capture Antibody. Capture
antibodies were conjugated to EHB@ABs via the carbodiimide
reaction, and the protocol was as follows. First, 2.6 mg of
EHB@ABs was washed and dispersed in 400 μL of MEST
buffer (10 mM, pH 5.0) containing 10 mg of EDC and 10 mg
of NHS. The mixture was reacted for 20 min and then was
washed with MEST buffer twice. After the activation of
carboxyl groups on the beads, 0.1 mg of capture antibodies
were added and incubated for 2 h at 37 °C. The beads were
washed using PBS twice after the reaction. To block the
unreacted active NHS, beads were suspended in 400 μL of PBS
containing 0.5 wt % bovine serum albumin (BSA) and 0.3 wt
% glycine, followed by overnight incubation at 4 °C. After the
blocking process, the conjugated beads were washed and
stored in a PBS solution containing 0.1 wt % BSA. The amount
of immobilized capture antibodies of EHB@ABs was
quantified by the BCA protein quantification kit through the
depletion method. The amounts of conjugated capture
antibodies for IFN-γ, IL-17A, IL-10, and IL-6 were calculated
to be 1.51, 1.12, 1.43, and 1.43 pg/bead, respectively.
2.3.2. Multi-LOCI Assay. In general, the immunoassay
procedure of multi-LOCI was as follows. Four kinds of multi-
LOCI EHB@ABs (4000 for each type EHB@ABs) conjugated
with four specific capture antibodies (IFN-γ, IL-6, IL-10, IL-
17A) and the corresponding biotinylated detection antibodies
were added into 100 μL of PBS buffer containing a series
concentration of analytes. After incubation for 1 h at 37 °C,
DB-SA were added and incubated for another 1 h at 37 °C. A
fluorescence microscope was used to acquire barcode
information and the mean LOCI intensity of the correspond-
ing barcode, and then the obtained data was analyzed through
customized MATLAB (MathWorks) software. In addition, for
the optimized experiment of establishing the multiplexed
calibration curves, the incubation time of both steps was 30
min.
2.3.3. Conventional LOCI Assay. First, 15 μL of ABs
conjugated with anti-IFN-γcapture antibody (33 μg/mL),
IFN-γ, and biotinylated anti-IFN-γdetection antibody (2 μg/
mL) was mixed and incubated for 1 h at 37 °C. Then, 35 μLof
DB-SA (60 μg/mL) was added. After being incubated for
another 1 h at 37 °C, 35 μL of the sample was pipetted into a
384-well plate, followed by a signal readout on the microplate
reader.
2.3.4. Characterizations. The morphology and size of
EHBs and EHB@ABs were characterized using a Zeiss Ultra
Plus field emission scanning electron microscope (SEM, Carl
Zeiss AG, Germany) operated at an accelerating voltage of 5
kV. ζ-Potential was recorded using a Zetasizer Nano ZSP
(Malvern, U.K.). The number of beads was calculated via flow
cytometry analysis on a NovoCyte 2040R instrument (ACEA).
Acquisition of multi-LOCI signal was carried out via a
fluorescence microscope (IX83, Olympus) equipped with a
680 nm laser diode (Changchun New Industries, Changchun,
China) and an LED light (Lumencor) using a 10×objective.
In short, the LOCI signal was excited by a 680 nm laser and
collected by an sCMOS camera (Prime BSI, Photometrics) at
615/20 nm. The barcode signals and FITC and RITC
fluorescence were excited by the LED light at 466/40 and
554/23 nm with emission collected at 525/50 and 609/54 nm
by the camera, respectively. To evaluate the uniformity and
amount of assembled ABs, the fluorescence of ABs loaded on
EHBs was directly excited by the LED light at 378/52 nm with
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emission collected at 615/20 nm. Two images including
barcode signals from FITC and RITC channels were,
respectively, acquired, followed by image acquisition of
LOCI signal. Then, image analysis was realized by the
customized MATLAB software. First, all images were pre-
treated to reduce the background noises. Then, the fluorescent
intensities of all beads in FITC and RITC channels were
recognized by an appropriate threshold and then screened by
setting proper parameters of the recognized regions including
diameter, area, and circularity of the barcodes in images.
Afterward, gray values of the recognized beads in the FITC,
RITC, and LOCI channels were, respectively, extracted. The
extracted gray values in the FITC and RITC channels were
used to identify which code they were, and the gray values in
the LOCI channel were used to determine the concentration
level of the corresponding analytes.
3. RESULTS AND DISCUSSION
3.1. Effect of Barcode Size on the Detection Perform-
ance of the Multi-LOCI System. As the core element of
multi-LOCI, EHB@ABs, the host−guest-structured multi-
LOCI barcode acts as a dual-functional microcarrier that
enables high multiplexed capability and a facile detection
strategy. Thus, it is essential to investigate the structural
property of EHB@ABs, as well as its effect on the performance
of the platform. As mentioned in the scheme before, the size of
EHBs should be properly controlled in an appropriate range
considering the coding capacity, reaction kinetics, and accuracy
of imaging decoding. Large EHBs possess lower reaction
efficiency; thus, they require vigorous mixing to ensure
themselves in a uniform suspension state, which may damage
target molecules and even the EHBs.
30
However, if the EHBs
are too small, the following problems cannot be ignored: (a)
encoding capacity is limited as smaller beads provide less space
for embedding fluorescent dyes,
25
(b) a higher-magnification
objective lens is needed to guarantee decoding accuracy, which
increases the cost and complexity of the equipment, and (c)
the number of ABs loaded on a single barcode is limited due to
the smaller surface area, which may influence the detection
LOCI signal. Therefore, considering both the previous reports
on barcodes
31
and the specific requirements of multi-LOCI
barcodes described above, the appropriate size for multi-LOCI
EHBs is in the range of 3−10 μm. As a demonstration,
barcodes with diameters of 3 and 6 μm were employed to
explore how the barcode size influenced the detection
performance. As shown in SEM images (Figure 1a), both 3
and 6 μm multi-LOCI EHBs are well-assembled with high-
density ABs. It is worth noting that both 3 and 6 μm multi-
LOCI EHBs show high decoding accuracy, as 28 and 30
clusters from 3 and 6 μm barcodes can be clearly distinguished,
respectively (Figure 1b,c). Furthermore, multi-LOCI assays of
3 and 6 μm EHB@ABs were compared using 0 and 1 ng/mL
Figure 1. (a) Scheme and SEM images of 3 and 6 μm multi-LOCI barcodes. (b, c) Scatter plots of the library of the 3 and 6 μm barcodes obtained
by imaging-based decoding, respectively. (d) Mean LOCI intensities of 3 and 6 μm multi-LOCI barcodes detecting 1 and 0 ng/mL IFN-γat
different time points. (e) Bar plot of the corresponding signal-to-background ratios in (d).
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IFN-γas analytes under different incubation times. Images
were collected and analyzed to obtain barcode and LOCI
signal intensities of each EHB@ABs. However, because the
surface area of a 6 μm bead is four times larger than that of a 3
μm bead, the pixel numbers of 3 and 6 μm beads in images are
different (Figure S1). To make the data of different-sized beads
comparable, the mean gray value of all pixels of every EHB@
ABs was calculated as its LOCI intensity, and the mean LOCI
intensities from all EHB@ABs were plotted in Figure 1d. It is
observed that with extended incubation time, both 3 and 6 μm
EHB@ABs exhibit a consistent increase in LOCI intensity in
the positive detection group, while no increase is observed for
EHB@ABs in the control group. Especially, the signal-to-
background ratio of 3 μm EHB@ABs is slightly higher than
that of 6 μm EHB@ABs at each time point (Figure 1e),
possibly because smaller beads have higher reaction kinetics
that is derived from fast Brownian motion. Therefore, 3 μm
EHBs were employed as the host beads of multi-LOCI
barcodes for further investigation.
3.2. Effect of Polyelectrolyte Multilayers on Packing
Density of the Host−Guest-Structured Multi-LOCI
Barcode. According to the preparation process of host−
guest-structured EHBs that our group previously reported,
multilayer polyelectrolytes with opposite charges are alter-
nately deposited on the surface of EHBs, to provide binding
sites for further assembly of guest ABs.
9,15,22
However, the
relationship between the number of adsorbed polyelectrolyte
layers and the ABs loading density is yet to be uncovered.
Therefore, a number of PSS/PDDA pairs were coated onto the
surface of silica-coated EHBs to form EHB@(PSS/PDDA)n(n
Figure 2. (a) ζ-Potential of 3 μm EHBs continuously modified with four PDDA/PSS pairs and one layer of PEI. (b) Bar plot of densities of surface
amino groups on EHB@(PSS/PDDA)n@PEI (nfrom 0 to 4) measured using the ITL/DTNB method. (c) Bar plot of fluorescence intensities of
EHB@(PSS/PDDA)n@PEI@ABs (nfrom 0 to 3) with excitation at 378/52 nm. (d) SEM and fluorescence images of EHB@(PSS/PDDA)n@
PEI@ABs (nfrom 0 to 3) of barcode F0R1. Fluorescence images illustrate both the barcode channels and the LOCI channel excited at 554/23 and
378/52 nm, respectively.
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ranging from 0 to 4) before carrying out the ABs assembling
process. ζ-Potential analysis was performed to verify the
multilayered deposition procedure of polyelectrolyte. As
depicted in Figure 2a, PDDA with positive charge is deposited
on the negatively charged silica surface through electrostatic
interactions, resulting in ζ-potential changing from −37 to 38
mV. After the following adsorption of PSS, the ζ-potential
returned to a negative value of −29 mV. The subsequent zigzag
trend of the ζ-potential values proves that PDDA/PSS pairs are
alternatively assembled as expected. Meanwhile, with the
increasing number of PDDA/PSS pairs, the surface morphol-
ogy of EHB@(PSS/PDDA)nin SEM images seems to become
smoother and less grainy compared with bare EHBs (Figure
S2). These results indicate that a controllable number of
polyelectrolyte multilayers are successfully coated onto the
surface of EHBs.
Afterward, the prepared EHB@(PSS/PDDA)nwere func-
tionalized with PEI, which was confirmed by a change in the ζ-
potential value from −46 mv to positive 31 mV (Figure 2a).
To clarify whether the deposited PDDA/PSS pairs would
affect the abundance of covalent binding sites (primary amino
group) provided by PEI, the relationship between the number
of PDDA/PSS pairs and the surface primary amino density was
further explored. As described in Figure 2b, the amino density
of EHBs directly modified with PEI through electrostatic
interaction between the hydroxylated silica shell and PEI
(EHB@(PSS/PDDA)n@PEI, n= 0) is measured to be 36.2
μmol/g, demonstrating a high density of amino groups on the
branched PEI chains. Notably, if one pair of PDDA/PSS is
introduced as the interlayer between EHBs and PEI
(EHB@(PSS/PDDA)n@PEI, n= 1), the surface amino
groups exhibit a remarkable increase in density to 95.6
μmol/g. The possible reason for this is that the multilayered
polyelectrolytes provide more binding sites for subsequent PEI
adsorption compared with the bare silica surface. The layer-by-
layer assembly of polyelectrolytes may form a crisscross
branched polymer surface that offers more abundant binding
sites for subsequent adsorption of polyelectrolytes with reverse
charge, for example, PDDA, PSS, and PEI are alternately
adsorbed in turn. In contrast, the number of binding sites (only
hydroxyl groups) on the bare silica surface is relatively low.
With a further increasing number of PDDA/PSS pairs, the
Figure 3. Fluorescence images of EHB@ABs−DB-SA complexes with various concentrations of IFN-γusing (a) “compact”and (b) “sparse”
barcodes. Fluorescence images illustrate both the barcode channels and the LOCI channel excited at 554/23 and 680/20 nm, respectively. Scale
bar: 20 μm. (c) Calibration curves for IFN-γquantification using “compact”and “sparse”barcodes, respectively. (d) Bar plot of the corresponding
signal-to-background ratios with various concentrations of IFN-γin (c). (e) Schematic illustration of the two different labels of DB-SA on “sparse”
and “compact”barcodes.
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increase in density slows down and finally reaches a plateau
when n= 3. This result is attributed to the assumption that the
number of the outer PSS chains tends to be consistent and
uniform after consecutive modification of PDDA/PSS pairs,
offering a steady surface for PEI deposition.
To explore the effect of the surface amino density on
subsequent ABs loading density, EHB@(PSS/PDDA)n@PEI
with a different number of PSS/PDDA pairs (nvaried from 0
to 3, since the increase of surface amino density reached a
plateau when n= 3) was further coated with ABs following a
carbodiimide-based coupling approach, as described in the
previous study.
22
The SEM images of the fabricated
EHB@(PSS/PDDA)n@PEI@ABs are shown in Figure 2d.
An increase of the ABs packing density is observed with the
increased number of deposited PSS/PDDA pairs, which is also
in accord with the increase in surface amino density of
microbeads. In addition, the fluorescence images of
EHB@(PSS/PDDA)n@PEI@ABs were also measured, where
ABs were excited at 378/52 nm (Figure 2c). In agreement with
SEM results, the fluorescence signal of ABs exhibits a linear
elevation with an increased number of PSS/PDDA pairs,
owing to more ABs being assembled on microbeads.
Meanwhile, the coefficient of variation (CV) for the
fluorescence intensity decreases sharply from 25% to 9%
when nranges from 0 to 3, suggesting that a high ABs packing
density results in a more uniformed ABs assembly. These
results demonstrate that the ABs packing density of host−
guest-structured multi-LOCI barcodes is strongly relevant to
the deposition state of polyelectrolytes, owing to the
multilayered polyelectrolyte substrate providing rich surface
amino groups and a soft landing surface for ABs.
15
3.3. Detection Performance of Multi-LOCI Barcodes
with Different Packing Densities. To study the effect of
ABs packing density on detection performance, two types of
barcodes with low and high ABs loading densities (EHB@
PEI@ABs and EHB@(PSS/PDDA)2@PEI@ABs, referred to
as “sparse”and “compact”, respectively) were selected as they
represent two typical barcodes with a distinct packing density
and the simplicity of a practical fabrication process. The multi-
LOCI assay performance of the two barcodes was then
compared. Fluorescence images in barcode and LOCI channel
for IFN-γquantification were acquired (Figure 3a,b). It can be
observed that LOCI signals of either “sparse”or “compact”
barcode show a consistent growth with the increase of IFN-γ
Figure 4. (a) Bar plot of LOCI intensities and the corresponding signal-to-background ratios of 0 and 1 ng/mL IFN-γwith five different DB-SA
concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL). (b) Bar plot of LOCI intensities and the corresponding signal-to-background ratios of 0 and 1 ng/
mL IFN-γwith five different biotinylated detection antibody concentrations (0.25, 0.5, 1, 2, 4 μg/mL) (c) Calibration curves for IFN-γ
quantification using both LOCI and multi-LOCI methods, respectively. (d) Plots showing the RITC fluorescence intensities of EHB@ABs−DB-
SA. (e) Schematic of the “on-barcode”multi-LOCI assay and LOCI signals in the bulk solution.
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concentration, which is attributed to more DB-SA loaded onto
the barcodes. Furthermore, the two calibration curves are
plotted to compare the detection performance of “sparse”and
“compact”barcodes (Figure 3c). LOCI intensities of
“compact”barcodes are slightly higher than those of “sparse”
barcodes under a high concentration of IFN-γ(0.2, 4, 80 ng/
mL). This is possibly ascribed to more available ABs on the
surfaces of “compact”barcodes, resulting in higher LOCI
intensity when sufficient DB-SA is deposited on ABs via the
formation of immunocomplexes. In addition, “compact”
barcodes show a slightly higher signal-to-background ratio
compared with that of “sparse”barcodes. This is ascribed to
more ABs of “compact”barcodes being “turned on”in the
presence of analytes, while the background signals for both
“compact”and “sparse”are similar in the absence of analytes
(Figure 3d). With respect to the overall performance, however,
the two barcodes exhibit comparable sensitivity, dynamic
range, and precision. This unexpected result that the
performance of “sparse”barcodes is comparable with that of
“compact”barcodes may be due to the following possible
reason illustrated in Figure 3e. For “sparse”barcodes, there are
two surface states after immune reaction: (1) DB-SA is bound
directly on the surface of guest ABs of multi-LOCI barcode via
affinity interaction between SA and biotinylated detection
antibody and (2) DB-SA is caught on the exposed barcode
surface that is embedded in the middle of two ABs since the
barcode surface also has the immobilized capture antibodies.
For “compact”barcodes, however, only state 1 exists.
Undoubtedly, LOCI signals under state 1 for “compact”
barcodes are higher than those for “sparse”barcodes due to
more ABs being available. Nevertheless, DBs under state 2 for
“sparse”barcodes could also induce luminescence oxygen
channeling signal since their distances to ABs are still in close
proximity, which acts as an extra pathway to provoke LOCI
signals from ABs. Therefore, the “sparse”and “compact”
barcodes exhibit similar LOCI intensities. Thanks to the
unique tailor-designed structure of ABs being enriched on
individual multi-LOCI barcodes, the DB-SA labeling site
becomes more diverse and flexible. Hence, it is no longer only
dependent on the direct immune reaction between ABs and
DB-SA to induce LOCI signals. In contrast, for conventional
LOCI where ABs are uniformly dispersed in bulk solution,
LOCI signals are provoked only when ABs and DB-SA are
directly bonded. This interesting result inspires us to think that
the multi-LOCI barcodes are highly flexible and robust,
tremendously reducing complexity and elaboration during the
fabrication process.
3.4. Optimization of the Multi-LOCI Assay System
and Detection Performance Evaluation of Multiplexed
Assay. Apart from the barcode structure, the assay system
including the power of 680 nm laser diode and the
concentrations of DB-SA and detection antibody also plays a
decisive role in the final multi-LOCI detection performance.
For the multi-LOCI detection system, DB-SA, a key
component that generates singlet oxygen, is labeled onto the
surface of the barcode mediated by detection antibody via
biotin−streptavidin interaction. It is inevitable that the
concentration of DB-SA directly affects its labeling process,
further influencing the LOCI signal. Herein, the relationship
between the amount of DB-SA and the multi-LOCI detection
performance was investigated carefully by adjusting DB-SA
concentrations ranging from 0.1 to 0.5 mg/mL. As shown in
Figure 4a, the LOCI signal of the IFN-γpositive group
increases sharply when the concentration of DB-SA increases
from 0.1 to 0.2 mg/mL, indicating that more DB-SA loaded
onto EHB@ABs can cause more effective production of singlet
oxygen. The LOCI signal remains consistent as the
concentration of DB-SA varies from 0.2 to 0.5 mg/mL,
suggesting a saturated status of DB-SA labeling. However, the
background signal also shows consistent growth with an
increased amount of DB-SA. Thus, the highest signal-to-
background ratio is observed at 0.2 mg/mL of DB-SA, which is
chosen as the relatively optimal concentration.
To explore the relationship between the power of 680 nm
laser diode and the multi-LOCI performance, we measured the
LOCI signal intensity of different concentrations of IL-10 at
laser powers of 5, 4, 3.5, 3, 2.5, and 1.9 W, respectively (Figure
S3, Supporting Information). It is shown that LOCI intensities
increased with enhanced laser power within the whole IL-10
concentration range, demonstrating that more singlet oxygens
were generated by DB-SA with higher laser energy. Besides, the
background signal also increased with an increase of laser
power (Figure S3a, Supporting Information). It should be
noted that the signal-to-background ratios also increased with
enhanced laser power within the whole IL-10 concentration
range (Figure S3b, Supporting Information). Thus, we can
conclude that higher laser power results in better assay
performance. Since the highest power of the 680 nm laser
diode that we used here is 5 W, it is chosen as the optimized
laser power for all multi-LOCI experiments.
Moreover, the investigation of an optimized detection
antibody concentration is critical because the detection
antibody bridges analyte with DB-SA via specific antigen−
antibody binding and biotin−streptavidin interaction. A series
of experiments were carried out to explore the optimal
conditions of detection antibody by comparing the LOCI
signal of 1 ng/mL cytokine with that of the control, with the
candidate detection antibody concentration varying from 0.25
to 4 μg/mL. Taking IFN-γas an example, the LOCI signal
increases as the concentration of the detection antibody
increases from 0.25 μg/mL to 2 μg/mL (Figure 4b). This
phenomenon is ascribed to the formation of more immune
complexes, providing more biotin sites for the loading of DB-
SA. The LOCI signal decreases when the concentration of the
detection antibody further increases to 4 μg/mL, suggesting
that excess free biotinylated detection antibody in reaction
solution competes with the conjugated detection antibody
bound onto the surface of barcode, weakening the deposition
quantity of DB-SA and further reducing LOCI signals.
Meanwhile, the amount of detection antibodies shows no
influence on the background signals. Hence, the optimized
detection antibody concentration is 2 μg/mL according to its
highest signal-to-background ratio. Similar phenomena are
observed for detection antibodies corresponding to the other
three cytokines. The optimized concentrations of different
targets are in the range of 0.25−2μg/mL (Figure S4a−c,
Supporting Information).
After the optimization of the assay system, multi-LOCI
performance was demonstrated by quantifying four cytokines
with a series of concentrations. As can be seen from the
calibration curve for detecting IFN-γ(Figure 4c), the multi-
LOCI performance shows a remarkable improvement
compared with our previous work with the limit of detection
(LOD) decreasing from 55.6 to 1.3 pg/mL.
22
LODs of IL-6,
IL-10, and IL-17A are determined to be 3.8, 16.6, and 62.9 pg/
mL, respectively, flowing the 3σcriteria. IL-10 and IL-17A also
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demonstrated higher sensitivity than our previous work (LOD
of IL-10 is 43.1 pg/mL and LOD of IL-17A is 91.6 pg/mL)
(Figure S4d−f).
22
Moreover, the barcode signals remain stable
with the CVs of intensities as low as 3.2% regardless of the
varied analyte concentrations, which ensures the decoding
accuracy (Figure 4d). Besides, the assay performance of multi-
LOCI using IFN-γas a model is further compared with the
conventional LOCI approach, which is well-known as a
homogeneous assay with high sensitivity. As shown in Figure
4d, multi-LOCI exhibits even higher sensitivity than the
classical LOCI (LOD to be 3.8 pg/mL). This result surprised
us because theoretically, LOCI has higher reaction kinetics
since 200 nm sized ABs and DB-SA are homogeneously
dispersed in solution, compared with multi-LOCI whose
barcode size is in the micron scale. The reasons for this
unexpected high sensitivity are speculated as follows. First,
Figure 5. (a) Fluorescence image of four-plexed multi-LOCI EHBs. (b) Scatter plot of the library of the barcodes obtained by the imaging-based
decoding. (c) Proportion of each type of barcode obtained by both imaging-based decoding and flow cytometry decoding. (d) Histogram of the
mean LOCI intensities of mixed barcodes in the presence of 1 ng/mL IFN-γ, IL-17A, IL-10, and IL-6 and a mixture of the four cytokines as
indicated. (e) Scheme of the multi-LOCI “mix-and-measure”protocol for the multiplexed assay. (f) Calibration curves for multiplexed detection of
IFN-γ, IL-17A, IL-10, and IL-6 using the multi-LOCI approach, respectively.
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with respect to signal acquisition for conventional LOCI,
overall signals from the bulk solution that contains both cross-
linked ABs-DB-SA complexes that generate LOCI signals and
large amounts of free ABs and DB-SA particles that generate
background signals are simultaneously collected. Thus, the
LOCI signal is more susceptible to the background signal. In
contrast, for on-barcode multi-LOCI, in situ LOCI signals from
individual barcodes are collected via the imaging approach,
significantly reducing the interference of background noises
from the bulk solution. Second, LOCI signals localized on
individual barcodes are more concentrated for on-barcode
multi-LOCI assay, compared with conventional LOCI whose
signals are diluted in the bulk solution (Figure 4e). Thanks to
the unique on-barcode assay format where the LOCI process is
conducted in situ on individual barcodes, we suppose that
multi-LOCI assay could overcome the shortage of relatively
lower reaction kinetics and achieve even better sensitivity than
conventional LOCI.
More importantly, the multi-LOCI platform not only
provides a rapid and simple quantification method but also
realizes high throughput and multiplexed assays in one
reaction. As a proof of concept, four types of barcodes,
denoted as F0R1, F1R1, F2R1, and F3R1 (obtained by
adjusting the ratio of FITC and RITC), were assigned to
detect IFN-γ, IL-17A, IL-10, and IL-6, respectively. The
capacity of decoding based on imaging was verified for the
multi-LOCI platform. After automation and high throughput
image analysis, four clusters are clearly distinguished without
overlaps (Figure 5b). Moreover, the proportion of each type of
barcode gained by imaging was 30.6, 23.4, 22.2, and 23.8%,
which is in accordance with that acquired by flow cytometry
(Figure 5c, 29.4, 23.7, 22.4, 24.5%, respectively). These results
demonstrate that the imaging-based decoding strategy has high
accuracy and throughput in multiplexed assays.
To evaluate the specificity of the multi-LOCI platform for
multiplexed assay, six groups of experiments were carried out
(blank group: no cytokine was added; IFN-γ, IL-17A, IL-10,
and IL-6 groups: each of the four cytokines added separately;
mixture: all four cytokines were simultaneously added). It was
found out that the LOCI signal in a specificbarcode
significantly increased only when its corresponding cytokine
was added (Figure 5d), and for the blank group, no LOCI
signal was observed for each barcode, illustrating high
specificity for the multi-LOCI platform.
Moreover, calibration curves for multiplexed detection were
established using samples containing a mixture of the four
cytokines in different concentrations (Figure 5e). As shown in
Figure 5f, in accordance with the performance of the single-
plexed assay, higher sensitivity of the multiplexed assay is
observed than our previous work after comprehensive
optimization of the assay system developed in this work, and
the LODs of IFN-γ, IL-17A, IL-10, and IL-6 are calculated to
be 3.6, 354.3, 1.2, and 1.9 pg/mL, respectively. The
phenomenon of the sensitivity of IL-17A for multiplexed
detection being lower than that of the single-plexed assay may
due to the competition response between IL-17A with the
other three cytokines. Importantly, detection of these multiple
analytes was accomplished within one reaction without the
need for washing steps, which remarkably reduces require-
ments of sample volume and improves detection throughput,
demonstrating a facile biodetection platform combining both
high multiplexing and sensitivity.
4. CONCLUSIONS
In summary, through a deeper investigation on the structure−
performance relationship of multi-LOCI barcodes, this work
found out that the number of surface amino groups that
originated from PEI showed significant growth if polyelec-
trolyte pairs were employed, resulting in a uniform and high-
density assembly of ABs onto the EHBs. Interestingly, the
detection performance of multi-LOCI between the “sparse”
and “compact”barcodes was neck and neck. Besides, it was
worth noting that multi-LOCI exhibited comparable sensitivity
with the classical LOCI after comprehensive optimization of
the assay system. These unexpected results benefitted from the
ingenious structural design of the multi-LOCI barcode, making
this novel “on-barcode”multi-LOCI platform highly flexible
and robust. It is believed that with the advantages of high
multiplexing and a facile “mix-and-measure”protocol, together
with the aid of portable microfluidics or devices, the multi-
LOCI platform will open new pathways toward robust, highly
sensitive, and multiplexed point-of-care diagnostics.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.1c06236.
Three-dimensional (3D) mesh diagrams originating
from a single 3 and 6 μm barcode in fluorescence
images (Figure S1); SEM images of EHBs (Figure S2);
and optimization results of 680 nm laser power and
detection antibody concentration (Figures S3 and S4)
(PDF)
■AUTHOR INFORMATION
Corresponding Authors
Qingsheng Guo −School of Biomedical Engineering, Shanghai
Jiao Tong University, Shanghai 200030, P. R. China;
Email: shockwow@sjtu.edu.cn
Hong Xu −School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, P. R. China;
orcid.org/0000-0002-2787-5806; Email: xuhong@
sjtu.edu.cn
Authors
Zuying Feng −School of Biomedical Engineering, Shanghai
Jiao Tong University, Shanghai 200030, P. R. China;
orcid.org/0000-0002-9236-3030
Yao Wang −School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, P. R. China;
orcid.org/0000-0003-0470-9511
Yunfei Ge −School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, P. R. China
Zhiying Zhang −School of Biomedical Engineering, Shanghai
Jiao Tong University, Shanghai 200030, P. R. China
Yan Wu −School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, P. R. China
Qilong Li −School of Biomedical Engineering, Shanghai Jiao
Tong University, Shanghai 200030, P. R. China
Hajar Masoomi −School of Biomedical Engineering, Shanghai
Jiao Tong University, Shanghai 200030, P. R. China; School
of Integrated Technology, Gwangju Institute of Science and
Technology, Gwangju 61005, South Korea; orcid.org/
0000-0002-5776-6141
ACS Omega http://pubs.acs.org/journal/acsodf Article
https://doi.org/10.1021/acsomega.1c06236
ACS Omega XXXX, XXX, XXX−XXX
J
Hongchen Gu −School of Biomedical Engineering, Shanghai
Jiao Tong University, Shanghai 200030, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.1c06236
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
The project was supported by the China Postdoctoral Science
Foundation (BR0820016), the National Natural Science
Foundation of China (Grant Nos. 21874091, 31927803, and
32101158), and the Innovation Research Plan supported by
the Shanghai Municipal Education Commission (Grant No.
ZXWF082101). The authors acknowledge the Instrumental
Analysis Center of Shanghai Jiao Tong University for the
characterization of materials.
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