Enhanced Immunoassay Using a Rotating Paper Platform for

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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 Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Analytical Chemistry

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. Email: [email protected] 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 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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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 enzymelinked 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 laborintensive 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 2 ACS Paragon Plus Environment

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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). Recombinant 3 ACS Paragon Plus Environment


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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 trisborate 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.

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 was reported previously.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


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Briefly, 0.3 mg magnetic beads 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. 2.3. 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 5 ACS Paragon Plus Environment


previous section. Briefly, the MB oligonucleotide was first functionalized with hexahistidine tags (H6) by incubation with 5 molar equivalents of a maleimide functionalized peptide (MaleimideG(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 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 6 ACS Paragon Plus Environment

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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 aminefunctionalized 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 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 7 ACS Paragon Plus Environment


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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 Roligos (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 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 (UVGL58, LW/ SW, 6W The Science Company, Denver, CO, U.S.A.) operated at the long 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:

() () () IG

IR

Amplification (%) =

―

S

IG

IR

IG

IR

NC

× 100

NC


(1)

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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) 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 11 ACS Paragon Plus Environment


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 the MBL method allowed for maximization of the packing density of Coligos, and hence maximizing the R-Oligo loading capacity of BNPs. The average loading of ROligos 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



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 14 ACS Paragon Plus Environment

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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 PEG2k 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 IL6. 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.



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 thiolPEGs 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 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 17 ACS Paragon Plus Environment


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 RDNAs 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-



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-togreen 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 6). However, the quantification was limited by the experimental variability as the range of RSD% for the replicates within one paper were 1h

Simple moderate

High low

IL-6 IL-6 IL-6

50,000 5,200 63

~90 min ~5 min ~2 h

moderate moderate moderate

high low high

3ELISA

Bio-barcode

assay34

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IL-6

D4 Assay35 Photoelectrochemical immunoassay36 Digital microfluidic assay37 Microfluidic biochip platform38 RPD 1The

assay times were estimated based on the reported protocols. 2The simplicity was evaluated based on the number / complexity of steps in analysis, detection instruments, and complexity of data analysis. 3Data shown in Figure S3.

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).

6. Supporting Information. The oligonucleotide sequences, the immunoassay wash cycle optimization data, calibration curves of the EXO method, and the calibration curve of ELISA are supplied as Supporting Information.

7. References:

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