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Enhanced immunoassay using a rotating paper platform for
quantitative determination of low abundance protein
biomarkers
Abootaleb Sedighi and Ulrich. J. Krull*
Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359
Mississauga Road, Mississauga, Ontario, Canada, L5L 1C6
Abstract:
The changing concentrations of circulating protein biomarkers have been correlated with a
variety of diseases. Quantitative bioassays capable of sensitive and specific determination of
protein biomarkers at low levels can be essential for therapeutic treatments that can improve
outcomes for patients. Herein, we describe the investigation of a rotating paper device (RPD) for
quantitative determination of targeted proteins at the fM concentration level. The RPD consists
of two circular papers each separately supported with a plastic disc. Protein detection is
conducted via enhanced immunoassay using amplification in a sequential workflow, which
includes a sandwich immunoassay in the upper paper and a signal amplification reaction in the
lower paper. The sandwich immunoassay is conducted using bio-barcode nanoparticles (BNPs)
and results in the release of reporter oligonucleotides from BNPs. These oligonucleotides are
transferred to the bottom paper, where they engage in a target recycling methodology that leads
to the production of a colorimetric signal. The assay was evaluated for quantitation of
Interleukin-6 (IL-6), a cytokine biomarker in serum. A limit of detection of 63 fM and a dynamic
range of 63 200 fM - 8 pM was observed for the assay. The specificity of the assay was
successfully verified against several common protein biomarkers.
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1. Introduction:
Protein biomarkers play an important role in modern medicine.1 A large number of proteins have
been identified where abundance in bodily fluids (e.g. blood, urine, saliva, tears) is used as an
indication of the state of health of an individual.2,3 Sensitive and selective quantitative
determination of certain biomarkers allows for disease diagnostics, selection of therapeutic
treatments, and accurate monitoring of responses to such therapies.4,5 A challenge when
considering the use of such biomarkers in the clinic is the low levels of protein biomarkers in
bodily fluids, 6 which are to be measured in the presence of high levels of interfering proteins
(e.g. plasma proteins). The background matrix renders the assays susceptible to false positive
and false negative results, hence demanding highly sensitive and specific protein bioassays.7
The gold standard method for quantitative determination of protein biomarkers is the enzyme-
linked immunosorbent assay (ELISA), which provides for the high sensitivity and selectivity
required for protein biomarker determinations. ELISA relies on a multi-step workflow consisting
of multiple blocking and washing steps that enhance assay sensitivity and specificity by
minimizing nonspecific adsorption. This multi-step workflow causes ELISA to be a labor-
intensive and slow as a bioassay platform.8,9 Other common platforms with simpler workflows
that are used for protein detection are lateral flow immunoassays (LFIA),10,11 and paper-based
analytical devices (PAD). 12–15 These platforms take advantage of properties of paper substrates
such as facile fabrication, surface modification and flow transport using capillary action.16
However, paper-based devices are commonly used to achieve qualitative and semi-quantitative
bioassays rather than quantitative determinations. Also, the limit of detection (LOD) achieved in
PADs tend to be in the nM range while the biologically relevant levels of many protein
biomarkers are at the pM-fM range.17
Herein, we report an investigation of a solid-phase enzyme amplification scheme by means of a
rotating paper device (RPD) for quantitative determination of protein biomarkers at the pM-fM
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range. RPD consists of two rotating circular papers each supported by a plastic disk (Figure 1).
Sandwich immunoassays using bio-barcode nanoparticles (BNPs) were conducted in the
immunoassay (IA) zones of the upper paper, while colorimetric detection was achieved using a
Foerster resonance energy transfer (FRET)-based signal amplification method in the bottom
paper. Several strategies were used to enhance the protein bioassay and enable reliable
quantification at low levels: (1) A BNP approach was coupled with a FRET-based signal
amplification strategy; (2) the surfaces of paper substrates and BNPs were passivated using
polyethylene glycol (PEG) layers to reduce nonspecific adsorption; (3) an internal calibration
method was used to improve accuracy and precision for quantitative assays. Inteleukin-6, an
important cytokine biomarker with meaningful diagnostic levels at pM-fM range for a variety of
diseases,18 was chosen as a model protein to evaluate the performance of the enzyme
amplification using the RPD system.
2. Experimental Section
2.1. Materials. An ELISA kit containing recombinant human IL-6 standard, anti IL-6
capture antibody, anti IL-6 detection antibody and blocking buffer (10% fetal bovine
serum in PBS) was from Thermo Fischer Scientific (San Diego, CA, USA). Exonuclease
III (EXO) and 10X CutSmart buffer were from New England Biolabs (Ipswich, MA, USA)
and used without further purification. Green-emitting CdSe/ZnS core/shell quantum dots
(PL at 518 nm) were from Cytodiagnostics (Burlington, ON, Canada). Diethylaminoethyl
(DEAE)-functionalized magnetic beads (MB, 1 μm) were from Bioclone Inc. (San Diego,
CA, USA). Hexahistidine-maleimide peptide sequences were from Canpeptide Inc.
(Montreal, QC, Canada). Prostate cancer antigen (PSA), EpCAM recombinant human
protein was from Thermo Fischer Scientific (Burlington, Canada). llustra NAP-5 size
exclusion chromatography columns were from GE Life Sciences (Quebec, Canada).
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Recombinant Protein G was from Abcam (Ontario, Canada). Amicon Ultra-0.5
centrifugal filters were from Fisher Scientific (Ontario, Canada). Polyethylene glycols of
different sizes (800, 2k, and 5k Da), Whatman® cellulose chromatography papers
(Grade 1, CHR-1, 200 × 200 mm), sodium tetraborate, L-glutathione (GSH, reduced,
≥98%), avidin, DTT, tetramethylammonium hydroxide solution (TMAH, 25% w/w in
methanol), sodium (meta)periodate (NaIO4, ≥ 99%), 1-(3-aminopropyl)imidazole (API,
98%), gold nanoparticles of 15 and 40 nm in diameter, 4-(2-hydroxyethyl)piperazine-1-
ethanesulfonic acid (HEPES, ≥ 99.5%), sodium cyanoborohydride (NaCNBH3, 95%),
and albumin from bovine serum (BSA, ≥ 98%) were from Sigma Aldrich (Oakville, ON,
Canada). All buffer solutions were prepared using a water purification system (Milli-Q,
18 MΩ cm−1), and were autoclaved prior to use. The buffer solutions included 100 mM
tris-borate buffer (TB, pH 7.4), 50 mM borate buffer (BB, pH 7.4), and phosphate buffer
(PB, pH 7.4), borate buffer saline (BBS, borate buffer 5 mM, pH 9.2, 100 mM NaCl), and
phosphate buffer saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4).
All oligonucleotides were from Integrated DNA Technologies (Coralville, IA, USA), and
are identified in Table S1.
Table 1. The oligonucleotide sequences
Name Sequence
MB 5'- /SH/-CTGAGCACAGTCCTCAGCGAAA -/Cy3/-3' R-oligo 5'- TTTCGCTGAGGACTGTTTTT -3'C-oligo 5'- /SH/ -AAA AAC AGT CCT CAG CGA AA -3'
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2.2. Preparation of barcode nanoparticles (BNPs). Conjugation of oligonucleotides to the
surface of AuNPs of 15 and 40 nm diameter were done using the magnetic bead-loading (MBL)
method that wasa previously reported methodpreviously.19,20 In this method, the negatively-
charged nanoparticles and DNA oligonucleotides are electrostatically loaded on the surfaces of
the positively-charged magnetic beads. The accumulation of oligonucleotides in the vicinity of
nanoparticles at the magnetic bead surface lead to high density nanoparticle-DNA conjugates
within seconds.19,20 Briefly, 0.3 mg magnetic beads (MBs) were dispersed in 200 µL TBS buffer
(tris-borate 100 mM, 1 M NaCl, pH 7.4) in a 2-ml Eppendorf tube, vortexed and isolated using a
magnet. The procedure was repeated once again in TBS buffer and then twice in phosphate
buffer (PB, 10 mM, pH 7.4). 500 fmol of 15 nm AuNPs or 100 fmol of 40 nm AuNPs dispersed in
PB were added to the washed MBs in 100 µL PB and the tube was vortexed for 30 s. C-oligo
(600 pmol) was added and the tube was vortexed for 1 min. The MBs were isolated using a
magnet, then re-dispersed in 100 µL PBS. 600 pmol reporter oligonucleotide (R-oligo) was
added and the solution was agitated for 20 min. The MBs were isolated and washed in BBS
(borate buffered saline, 50 mM, 200 mM NaCl, pH 9.2) twice. To release NPs, MBs were
dispersed in elution buffer (borate buffer, 50 mM, 1M NaCl, pH 10), vortexed for 30 s and
isolated using a magnet. The supernatant containing oligonucleotide coated AuNPs (BNP-2)
was diluted 10 times in PBST (PBS plus 0.02% tween 20) and centrifuged. The centrifugation
was done at 7000 rpm for 5 min for AuNP-40 and at 13000 rpm for 15 min for AuNP-15. To
produce BNP-3, 500 ng protein G was added to BNP-2 in PBST. After 1 h incubation, BNP-3
was centrifuged and re-dispersed in PBST. BNP-4 was prepared by incubation of BNP-3 in 100
µL of IL-6 detection antibody solution for 60 min. Then, NPs were centrifuged and re-dispersed
in 100 µL PBST. The concentration of NPs was obtained using absorption spectroscopy.21 To
PEGylate NP surfaces, the BNP-4 was incubated in PBST solutions of PEG-thiol with molecular
weight of 800, 2k and 6k for 1 h. Finally, the NPs were purified twice by centrifugation, re-
dispersed in PBST and stored at 4 ˚C for later use.
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24.3. Preparation of molecular beacon probes (MB). A 22-mer oligonucleotide that was
modified with Cy3 dye at the 3’-end and a thiol group at the 5’-end was the molecular beacon
(MB) probe. The thiol group was first reduced via 500× DTT in 1x PBS for 2 h. The unreacted
DTT was then removed by ethyl acetate extraction (4 times). The molecular beacon-
quantum dot conjugates (MB-QDs) were prepared using the magnetic bead loading (MBL)
method as described in the previous section. To prepare MB-QD probesBriefly, the MB
oligonucleotide was first functionalized with hexahistidine tags (H6) by incubation with 5 molar
equivalents of a maleimide functionalized peptide (Maleimide-G(Aib)GHHHHHH), for 24 h.
Unreacted peptide was removed by running the sample through two consecutive NAP-5
desalting columns.
Water-soluble glutathione-coated QDs (GSH-QD) were prepared using a previously reported
method.22 The immobilization MB probes on QD surfaces was done using the magnetic loading
method.19 Briefly, 5 pmol GSH-QD was added to 0.1 mg MB in 100 µL TBS buffer (Tris-borate
100 mM, pH 7.4) and the tube was agitated for 30 s. Then 50 pmol of H6-MB was added to the
solution and the tube was agitated for another 30 s. The MBs were isolated using a magnet and
re-dispersed in BBS. The MBs were again isolated using a magnet and re-dispersed in 50 uL
release buffer (borate buffer 50 mM, pH 10, with 1 M NaCl). The MBs were isolated again and
the concentration of the MB-QDs was determined using absorption spectroscopy.
2.4. Preparation of paper substrate. The upper and lower circular papers were prepared using
a method previously described by our group.23,24 Briefly, chromatography paper grade 1
substrates were patterned with wax using a Xerox ColorQube 8570DN solid ink printer. The
patterned circular paper sheets of 120 mm diameter were cut using a compass cutter. The
upper paper contained two alternating radial arrays of 8 by 3 circular zones of 5 mm diameter,
which included one array of immunoassay (IA) reaction zones and another array of holes that
allowed for addition of amplification mix to the lower paper. The lower paper contained two
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radial arrays including one 8 by 3 array of 5 mm detection zones and another 8 by 3 array of 10
mm circular washing zones. The wax printed papers were subsequently incubated in an oven at
120 C for 2.5 min to melt and affix the wax. The upper and lower support discs were treated
with Repel Silane (Sigma Aldrich, Oakville, Canada), and the upper paper was then loaded on
the RPD. In order to activate the immunoassay zones for immobilization of capture antibody, the
cellulose surface was functionalized with aldehyde groups by two consecutive additions of 10 μL
of aqueous solutions of NaIO4 (50 mM) and LiCl (700 mM) followed by incubation of the paper at
50 C for 30 min.25 The paper was then washed with DI water and left to dry for 30 min. Capture
antibodies were immobilized on the IA zones by adding 10 μL of 4 μg/ml solutions with reaction
for 1 hour. To wash the IA zones, they were aligned on top of the washing zones (in the lower
paper) and the central spring was pushed down to place the two papers at a close distance (2
mm). 200 μL of wash solution (PBST) was gradually pipetted on the IA zones and allowed for
flow and absorption into the cotton packing underneath the washing zone paper. The spring was
released and the paper was allowed to dry in desiccator for 20 min. This washing procedure
was used in all subsequent steps of IA reactions. In order to passivate IA zones, 10 μL of
amine-functionalized PEG (MW 750 Da, 1 μg/mL) was added to the IA zones and the reaction
was allowed to proceed for 30 min. The IA zones were washed using the procedure described
above. This washing procedure was adopted throughout the enzyme amplification after each
step of the reactions.
In order to immobilize MB-QDs onto the amplification zones in the bottom paper, the paper
zones were modified with imidazole groups in two subsequent steps. First, the cellulose paper
was modified with aldehyde groups by 2 cycles of additions of aqueous solutions of NaIO4 (50
mM) and LiCl (700 mM) and incubation of the paper at 50 C for 30 min. Next, the papers were
functionalized with imidazole groups by spotting 10 μL of a solution containing API at 200 mM
and NaCNBH3 at 300 mM, in HEPES buffer pH 8. The reactions were allowed to proceed at
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room temperature for 30 min. MB-QDs (300 nM,10 μL) were added to the signal amplification
zones and the reactions were allowed to proceed for 30 min. The papers were then rinsed with
BB and loaded on the RPD.
2.5. Enhanced immunoassay procedure. Different concentrations of IL-6 standards in PBS
were added to IA zones (10 µL/zone), and the reactions were allowed to proceed for 10 min.
The zones assigned to internal calibration standard including NC, LS, MS and HS were spotted
with solutions containing 0, 0.2, 2 and 8 pM of IL-6 standards in PBS, respectively. The IA
zones were washed once using the washing procedure described in the previous section. Next,
10 µL of BNP solutions (1 nM in PBS) was added to each zone and the reactions were allowed
to proceed for 20 min. Then, the IA zones were washed once using the optimized washing
procedure. The upper disc was rotated once to align the IA zones on top of the amplification
zones. To dehybridize release R-oligos (bound through DNA hybridization with C-oligos) and
release them from BNPs, 15 µL MilliQ water was added to each IA zone. After 10 min of
reaction time, the RPD was rotated to align IA zones on top of the amplification zones in the
lower paper and the central spring was pushed down to transfer the liquid to the detection zone.
Then, the upper disc was rotated for the second time to align the holes on top of the
amplification zones. Next, the immunoassay signal was amplified using a target recycling
strategy, called the EXO method, in which the released R-DNAs serve as the templates (See
section 3.5).26 3 µL of The amplification mix (3 µL) containing 15 unit/µL EXO and 5x CutSmart
buffer (250 mM potassium acetate, 100 mM tris-acetate, 50 mM magnesium acetate, 500 μg/ml
BSA, pH 7.9) was added to each amplification zone and the amplification reactions were
allowed to proceed for 30 min.
Digital color images from the bottom paper were acquired using an iPhone 7 (Apple, Cupertino,
CA, U.S.A.). Papers were illuminated at a distance of 20 cm with an ultraviolet (UV) lamp
(UVGL-58, LW/ SW, 6W The Science Company, Denver, CO, U.S.A.) operated at the long
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wavelength (365 nm) setting. The digital images were split into corresponding R-G-B color
channels using ImageJ software and the amplification signal was quantified by ratiometric
analysis of each zone using equation 1:
Amplification (%)=( IGI R )S−( IGIR )NC
( IGI R )NC×100(1)
where IG and IR are the mean color intensity of green channel (G) and red channel (R) for a
given zone, respectively. The subscript S denotes a measurement made in the presence of the
analyte, while NC denotes the negative control.
3. Results and Discussion:
Figure 1: Schematic representation and photograph of the rotating paper device (RPD)
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3.1. RPD design. The RPD consisted of two circular papers that were wax printed to create
zones of 5 mm diameter to constrain liquids. One circular paper was placed over the other, each
being supported by a plastic disc. The upper paper contained two series of 24 reaction zones of
5 mm diameter. One series of zones were used for immunoassay reactions (IA zones), and the
other series were punched to allow for pipetting the amplification mix into the lower paper. The
upper paper was supported by a 2 mm thick plastic disc containing 48 circular holes, each
aligned on one paper zone. The lower paper also contained two series of circular paper zones.
One series included 24 paper zones of 10 mm diameter that were used as the drain zones for
washing. The second series included 24 zones of 5 mm diameter intended as the signal
amplification zones. The lower paper was supported from the bottom by a 10 mm thick plastic
disc with an identical design to the lower paper. The 10 mm holes of the lower disc were filled
with cotton wadding to hold the wash solutions. The upper paper was positioned 10 mm above
the lower paper using a central spring. To transfer solutions from the upper to the lower paper
for washing and transfer of reporter oligonucleotides, the upper disc was pushed down to let the
solution drain into the lower zones by wicking action. To proceed from the immunoassay step to
the signal amplification step, the upper disk was rotated 22˚ to align the immunoassay zones
with the amplification zones.
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Figure 2: Schematic representations of different steps of the assay including (A) preparation of
barcode nanoparticles (BNPs), (B) sandwich Immunoassay reaction, and (C) signal
amplification using the EXO method.
3.2. Barcode nanoparticle (BNP) design. Figure 2A shows different steps for the preparation
of BNPs that involve immobilization of different ligands on the surface of gold nanoparticles
(AuNPs). The two functional reagents are the detection antibodies (D-Abs) used in the sandwich
immunoassay reactions and the reporter oligonucleotides (R-Oligos) that serve as the template
in the exonuclease III DNA amplification (EXO) method. Both the antibodies and
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oligonucleotides are immobilized onto AuNPs through auxiliary ligands that are directly
immobilized on the AuNP surfaces. R-Oligo is attached by hybridization with its complementary
immobilized strand (capture oligonucleotide, C-Oligo), and D-Ab is immobilized by interaction
with Protein G. Given the critical role of BNPs in the sensitivity and specificity of the enzyme
amplification method, surface immobilization strategies have been used that allow for adequate
control of the packing density and orientation of these reagents. An interfacial NP decoration
method using magnetic beads, called the magnetic bead loading (MBL) method,19 that we have
recently reported was used to immobilize capture oligonucleotides (C-oligos, BNP-1) and to
subsequently hybridize R-oligo to the surface of AuNPs (BNP-2). In addition to the rapid
immobilization kinetics, tThe MBL is method allowed for maximization of the packing density of
C-oligos, and hence maximizing the R-Oligo loading capacity of BNPs. The average loading of
R-Oligos on the AuNP surfaces as well as the fraction of those R-Oligos released upon
dispersion of NPs in deionized (DI) water were determined using previously reported methods.19
We have determined that the average loading was 94 ± 12 and 326 ± 29 R-oligos on the
surfaces of AuNPs of 15 nm and 40 nm diameter, respectively. It was determined that on
average, 34 ± 6 R-oligos from 15 nm AuNPs and 103 ± 13 R-Oligo from 40 nm AuNPs were
released upon dispersion of NPs in deionized (DI) water. In the MBL method, a portion of the
NP surface area is unavailable due to contact with the magnetic bead surfaces during the
process of oligonucleotide immobilization. This unreacted area becomes available for
conjugation after release of the nanoparticles from the surface of magnetic beads. According to
our previous results the fraction of the surface available for further conjugation is 18-30% of the
total surface area of the NPs.19 This available surface area was then used for immobilization of
D-Ab, which was conjugated via thiol-functionalized Protein G. This immobilization strategy
allowed for optimum surface orientation of D-Ab which was necessary to enhance its antigen
binding efficiency.27
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A sandwich immunoassay reaction was developed for detection of IL-6, which was localized in
the immunoassay reaction zones (Figure 2B). First, the capture antibody (C-Ab) was
immobilized on aldehyde-modified paper substrates of the IA zones. Then, the solid-phase
immunoassay was conducted by sequential addition of IL-6 sample solutions and BNP solutions
to the IA zones. Finally, the R-oligos were released from BNPs by the addition of deionized (DI)
water to the IA zones. A particular feature of the RPD is the washing procedure, which offers
simplicity, speed and maintains the selectivity of the assay. In the assembled device, each IA
zone associated with the upper paper was aligned with a circular 10 mm diameter drain zone in
the lower paper. To wash an immunoassay zone the upper plastic disc was pressed against the
lower disc, forcing contact between the upper and lower papers, allowing for the continuous
drainage of the wash buffer into the drain zone. Wash buffer (20 times the reaction volume) was
then added to the IA zone. The drain zones of the lower disc were packed with cotton to
facilitate the movement of the wash buffer via wicking action. In a single wash step, this
dynamic wash procedure adequately eliminated interference that would arise from nonspecific
adsorption (See Figure S1), presenting an advantage over conventional ELISA methods as the
latter typically requires multiple washing steps.
3.3. PEGylation. The prevention of nonspecific adsorption of BNPs onto the paper matrix of the
IA zone is a crucial factor for assay specificity. Therefore, surface modification of both the paper
substrates of the IA zones and the BNPs was implemented to suppress nonspecific interactions.
Polyethylene glycol and bovine serum albumin (BSA) are the most common passivation agents
used to reduce nonspecific adsorption onto the surfaces of biosensors and nanoparticles.28–30
The surfaces of BNPs were passivated using BSA, or by polyethylene glycol methyl ether
(mPEG) using polymer of 0.8, 2 or 6 kDa size. The IA zones were passivated either by physical
adsorption of BSA or by covalent immobilization of methoxy-PEG-amine (mPEG 750, MW 750
Da) following the immobilization of capture antibody. In order to assess the nonspecific
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adsorption of BNPs onto the paper, BNPs were added to the IA zones in the absence of IL-6
(i.e. negative control solution). After washing, the R-oligos were released from residual BNPs
and were analysed using gel electrophoresis. The gel electrophoresis results in Figure 3A
indicate significant adsorption of non-passivated BNPs (BNP-4) regardless of the modification
on the paper surface. The intensity of the R-oligo bands seen in the gel images were reduced
when BNPs were modified with BSA and 0.8 kDa PEG (PEG-800), and complete suppression of
the band was achieved when BNPs were coated with PEG of 2 or 6 kDa size (PEG-2k, PEG-6k)
and the IA zones were coated with mPEG 750. In another approach, colorimetric quantification
of BNPs was used to determine nonspecific adsorption and to verify the findings derived from
gel electrophoresis. Figure 4A and 4B show the colorimetric signals obtained from IA reactions
on mPEG 750-modified IA zones where sample solutions were the negative control or contained
5 nM IL-6. When BNP-4 or BNP-5 PEG-800 were used, significant signals were observed on
the negative control (NC) zones. Use of BNP-5 PEG-2k and BNP-5 PEG-6k resulted in
complete suppression of the colorimetric signal. These results are consistent with the gel
electrophoresis analysis of the released R-Oligos indicating that a complete suppression of
nonspecific adsorption is only achieved when BNPs were passivated with the larger PEGs
(Figure 3A). The compromise was that the BNP-5 PEG-2k and BNP-5 PEG-6k resulted in a
23% and 52% reduction in signal, respectively, in comparison to unmodified BNP-5, indicating
that the immunoassay sensitivity was reduced by the presence and the size of the PEG
polymers. The differences in specificities and sensitivities induced when BNPs were coated with
PEGs of different sizes may be attributed to thickness of PEG layer relative to the size of other
ligands on the NP surfaces. The thickness of PEG coating increased with the molecular size of
the PEG molecules as well as with the NP surface density (i.e. cumulative density of all the
ligands on the NP surface). For example, Abou-Saleh et al. reported that the thickness of a
PEG-2k layer on NP surfaces ranged from 2.7 nm in a mushroom conformation to 6.9 nm in a
dense brush conformation.31 In comparison, the thickness of PEG-5k was reported to be in the
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range of 5.5-20 nm at different surface densities.32 Dynamic light scatter (DLS) data showed that
the hydrodynamic diameter (dh) of BNPs significantly increased upon passivation by PEG-2k
and PEG-6k but not when PEG-800 was coated on the surface. This suggested that only the
larger PEGs created a sufficiently thick layer to block the non-specific binding sites on BNP
surfaces located at the IA zones. It is clear that a sufficiently thick PEG layer will also block
some of the surface-immobilized antibody preventing the binding between the antibody and IL-
6. These results suggest that there will be an optimal PEG polymer size to maximize sensitivity
and specificity, and for the BNPs in this study the optimal selection was 2 kDa PEG.
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Figure 3: Investigation of the nonspecific adsorption of BNPs on paper substrates. A Gel
electrophoresis data indicates the released R-oligos from C-Ab functionalized immunoassay
zones after addition of different BNPs and subsequent washing. B and C show the gel
electrophoresis and hydrodynamic diameter of BNPs after addition of different ligands,
respectively. D Schematic shows the relative sizes of PEG compared to other ligands on NP
surfaces (only approximate scales). The abbreviations represent AuNPs after sequential
conjugation of C-oligo (BNP-1), R-oligo (BNP-2), protein G (BNP-3), D-Ab (BNP-4) and
subsequent modification with different blocking agents including BSA (BNP-5 BSA) and thiol-
PEGs with molecular weights of 800 Da (BNP-5 PEG-800), 2 kDa (BNP-5 PEG-2k) and 6 kDa
(BNP-5 PEG-6k).
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Figure 4. The effect of PEGylation on the nonspecific adsorption of BNPs in the IA zones. A and
B show the optical image and the corresponding histogram bar graph of the immunoassay
reactions with BNPs passivated with different PEG layers. Reactions in rows 1-4 and rows 5-8
were done using 0 and 5 nM of IL-6 solutions. Different BNPs used were BNP-4 (1, 5), BNP-5
PEG-800 (2, 6), BNP-5 PEG-2k (3, 7), and BNP-5 PEG-6k (4, 8).
3.4 .Influence of AuNP size. The effect of AuNP size was investigated by studying the kinetics
and sensitivity of IA reactions when BNPs were prepared using AuNPs of 15 and 40 nm
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diameter. Figure 5A shows the time-based colorimetric IA signals obtained for the IL-6 binding
step (Step 4 in Fig. 2B) and the subsequent BNP addition step (Step 6 in Fig. 2B) as the
reactions were allowed to proceed for 2-60 min. The results in Figure 5A show that the IA signal
reached a plateau when IL-6 was allowed to react with a zone coated with C-Ab for ~10 min.
This relatively rapid reaction33 is attributed to the fast mass transport in the paper matrix
facilitated by capillary action. A comparison of time-based signal evolution from BNPs prepared
using AuNPs of 15 nm and 40 nm diameters (Figure 5B) shows that the reaction kinetics
decrease with AuNP size, which was expected considering the slower mass transport of the
larger nanoparticles. In addition to faster kinetics, the smaller AuNPs also provided for a higher
sensitivity. Figure 5C shows the signals obtained from the gel electrophoresis analysis of R-
DNAs released from the reaction zones. The results show more than a 2-fold increased signal
when 15 nm AuNPs were used as compared to 40 nm AuNPs for the IA analysis. It was
reported already that a smaller number of R-oligos are released from each 15 nm BNP as
compared to the larger 40 nm BNPs (Section 3.2). Therefore, this observation of higher total
released R-DNA obtained from the 15 nm BNPs indicates the higher efficacy of binding between
15 nm BNP and the captured IL-6, resulting in a higher overall signal intensity. Thus, 15 nm
AuNPs were selected for preparation of BNPs and used in the optimized enhanced
immunoassay reaction.
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0
Figure 5. (A) and (B) Time-based colorimetric immunoassay signals obtained from IA zones
where the IL-6 (A) and BNP solutions (B) were allowed to react for 2-60 min. (C) The
normalised signal from gel electrophoresis analysis of the R-oligos released from BNP prepared
from AuNPs of 15 and 40 nm in diameter. A sample solution containing 5 nM IL-6 was used in
all experiments.
3.5. Colorimetric detection using exonuclease-assisted amplification (EXO). Signal
amplification and colorimetric detection were done using an exonuclease III target recycling
approach (EXO method) that we have recently reported for paper substrates.26 Here, the R-
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oligos released from the BNPs were transferred to the detection zones in the lower paper where
they hybridized to a molecular beacon probe (MB, Table 1). The MB operated by FRET and
possessed a green-emitting quantum dot donor (gQD) at the 5’-end and a Cy3 acceptor dye at
the 3’-end (Figure 1C). Upon hybridization of the probe to R-oligo, the EXO enzyme initiated to
remove nucleotides from the 3’-end of the probe strand, resulting in the release of Cy3-dye and
a reduction in the FRET signal. Colorimetric detection was conducted by irradiating the lower
paper with UV light and capturing an image using a cellphone camera. The changes in red-to-
green intensity ratio in RGB images (see experimental section), which indicated the changes in
the FRET signal, were correlated with R-oligo concentration. The curve shown in Figure S2 was
obtained from the amplification of R-oligos of 1-5000 pM using the EXO method. The curve
shows that the amplification signal linearly increased with the R-DNA concentration across the
range of 20 - 1000 pM.
3.6. IL-6 quantification using an internal calibration method. In order to conduct a complete
assay, R-oligos released from immunoreaction zones were transferred into the detection zones
where they were detected and quantified by the EXO method. An external calibration method
was based on a calibration curve prepared using standards added to one paper with the linear
response equation being used to quantify samples tested on different papers. For internal
calibration, both the standards and samples were determined using a single paper.
An external calibration method showed that the EXO response linearly increased with IL-6
concentration in the range of 0.2 - 8 pM (Figure S46). However, the quantification was limited by
the experimental variability as the range of RSD% for the replicates within one paper were
<15% while the RSD% from multiple papers were 33-79%. To address this challenge, an
internal calibration system for the assays was designed which allowed for quantification using 4
sets of standards that were concurrently analysed with samples. These standards included a
negative control (NC), and three calibration solutions containing IL-6 concentrations of 0.2 (LS,
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low sensitivity), 2 (MS, medium), and 8 (HS, high) pM representing the dynamic range for IL-6.
Figure 6A 7A and 6B 7B present green and red channel images, respectively, of the lower
paper that includes the results from the four sets of calibration standards as well as the four IL-6
samples. A 3-point calibration curve was created and used to determine the concentrations of
IL-6 in the samples (Figure 6C7C). The recovery using spiked samples and the RSD% were
evaluated to represent accuracy and precision of the assay, respectively. The results in Figure
6D 7D show recoveries of 85-116% and RSD of 7-14% for the method. The limit of detection
(LOD) of the method was calculated to be 63 fM using 3 standard deviations of the background
as the statistical criterion. The robustness was verified by conducting the assay in goat serum
(95% v/v), which resulted in an assay response that was within the experimental error of the one
conducted in PBS buffer (Figure 6E7E). To evaluate the specificity of the assay, IL-6 was
replaced with various proteins including BSA, prostate specific antigen (PSA), avidin, and
epithelial cell adhesion molecule (EpCAM). The minimal signals obtained for all proteins other
than IL-6 support the specificity of the assay.
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Figure 6. The external calibration curve prepared for determination of IL-6 using enhanced immunoassay in the RPD. (A) shows the entire range of IL-6 concentration (1 fM – 100 pM) that was investigates, and (B) shows the linear range at 0.2 – 8 pM. (C) shows the assay recoveries (Recovery%) and RSD% for four different quality control samples. The recovery% was
calculated using as the following equation: Recovery%= determined conc .actual concentration
×100.The
samples were tested in triplicate using three different papers.
Figure 67. Quantitative determination of IL-6. A & B show the green (QD) and red (Cy3)
channels of the optical images from the lower paper of the RPD. The signals were obtained
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from 4 sets of calibration standards including negative control (NC), and standard solutions
containing 0.2, 2, and 8 pM of IL-6 as well as 4 IL-6 sample solutions. (C) shows a 3-point
internal calibration curve prepared using signal intensities of the standard solutions. (D)
Recovery % and RSD % values for four IL-6 samples at different point of dynamic range. The
recovery% was calculated using as the following equation: ❑❑(E) assay signals when 1 pM IL-6
was in PBS and goat serum solutions or when 10 pM of other proteins were dissolved in PBS in
absence of IL-6.
4. Conclusion
A rotating paper device has been investigated for quantification of protein biomarkers at the fM
level using an immunoassay method with subsequent amplification. Several strategies were
combined to enable accurate and precise quantification of protein biomarkers at low
concentration levels. A bio-barcode method was coupled to a signal amplification approach to
enhance sensitivity. Paper substrates and barcode nanoparticle surfaces were passivated with
PEG layers to suppress nonspecific adsorption. An internal calibration approach was used to
enhance reproducibility and accuracy.
A number of novel immunoassay-based technologies have recently been developed that aim to
provide an improvement in simplicity and speed over ELISA while maintaining the same level of
LOD and multiplexing capabilities. Table 1 provides a summary of some of these technologies.
A comparison of different qualities show that tThe RPD offers simplicity and speed fits well
within the list of these successful attempts. While the method provides a significant reduction in
the analysis time and the number of steps, a moderate simplicity remains the primary limitation
of the device. of the paper-based analytical devices, while the analytical figures of merit and the
multiplexing capabilities are competitive with ELISA methods. The performance of the device for
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real clinical samples needs to be verified, however, the device presents qualities that suggest
promise for quantitative determination of low abundance protein biomarkers.
Table 1. Examples of recently developed methods for detection of IL-6
Protein LOD (fM)
Analysis Time1
Simplicity2
Multiplexing capability
ELISA34 IL-6 42 3h – 2 days
Complex High
Bio-barcode assay35 Multiple cytokines
5 8-10 h Complex High
~D4 Assay36 IL-6 265 ~2 h Simple HighPhotoelectrochemical
immunoassay37
IL-6 0.0013 >1h moderate low
Digital microfluidic assay38 IL-6 50,000 ~90 min moderate highMicrofluidic biochip
platform39
IL-6 5,200 ~5 min moderate low
RPD IL-6 63 ~2 h moderate high1 The assay times were estimated based on the reported protocols.2 The simplicity was evaluated based on the number / complexity of steps in analysis, detection instruments, and complexity of data analysis.
5. Acknowledgements
We are grateful to the Natural Sciences and Engineering Research Council of Canada for
financial support of this work (Grants STPGP 479222-15; RGPIN-2014–04121).
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