Immunosensors for C-Reactive Protein Based on Ultrathin Films of

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Immunosensors for C-reactive protein based on ultrathin films of carboxylated cellulose nanofibrils Yanxia Zhang, and Orlando J. Rojas Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01681 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Immunosensors for C-reactive protein based on ultrathin films of carboxylated cellulose nanofibrils Yanxia Zhang,*,§, Orlando J. Rojas*,†,‡ §

Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University, Suzhou, Jiangsu, 215007, P. R. China. †

Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, FI-00076 Aalto, Finland.

‡

Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States.

ABSTRACT C-reactive protein (CRP) is an acute phase protein that has been widely used as a predictor of cardiovascular diseases. We report herein the synthesis of immunosensors based on carboxylated cellulose nanofibrils (CNF) for CRP detection, as demonstrated by quartz crystal microgravimetry (QCM). QCM sensors carrying ultrathin films of carboxylated CNF were prepared by using two protocols: (i) spin coating of CNF on QCM resonators followed by carboxylation via in situ oxidation with 2,2,6,6-tetramethyl-piperidine 1-oxyl (TEMPO) and, (ii) carboxymethylation of CNF in aqueous dispersion followed by spin coating deposition on the QCM resonators. Protein A was conjugated to the carboxylated CNF via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide 1

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hydrochloride/N-hydroxysuccinimide (EDS/NHS) and used as ligand for oriented immobilization of anti C-reactive protein (anti-CRP). The different carboxyl group density of the two oxidized CNF surfaces influenced Protein A binding and, subsequently, the available immobilized anti-CRP molecules. The detection efficiency for CRP, specificity and concentration range displayed by the carboxylated CNF-based immunosensors coupled with oriented and unoriented anti-CRP were determined and compared.

Keywords: Immunosensors; C-reactive protein; Anti-CRP; Protein A; Bioactive CNF; Carboxylated cellulose nanofibrils; EDC/NHS coupling; Human blood serum.

INTRODUCTION C-reactive protein (CRP) is well known as a human blood serum biomarker for infections and inflammatory processes, whose levels can increase as high as 1000-fold under acute inflammatory stress.1-5 CRP has also emerged as one of the most powerful indicators of cardiovascular disease.6-10 Clinically, the blood serum concentration of CRP in the ranges 3 µg/mL is generally adopted to indicate low, moderate or high risk of cardiovascular disease, respectively.11-13 Individuals with CRP >10 µg/mL should be examined for sources of infection or inflammation.12,13 Therefore, it is of great importance to detect CRP concentration in patients’ serum as an early diagnosis of cardiovascular disease. Several techniques have been developed, including enzyme linked

immunosorbent

assay

(ELISA),2

fluoro-immunoassay,14

chemiluminescent-based

immunoassay,15 and label-free methods based on quartz crystal microgravimetry (QCM),16,17 surface plasmon resonance (SPR)18-20 and electrochemical impedance spectroscopy21.

2


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Compared with other types of biosensors, paper-based biosensors (or bioactive papers) have attracted considerable attention due to their advantages such as low-cost, simplicity, portability and disposability.22 Particularly, low-cost biosensors consisting of cellulose are expected to be most useful for medical, environmental, and food analyses and for detection in home health-care or remote settings.22-24 However, related systems have a high porosity and limited mechanical performance, which have prevented applications in diagnostic.25 Cellulose nanofibril (CNF) (also referred to as nanofibrillar cellulose, NFC) is typically produced from wood fibers via mechanical disintegration after optional chemical or enzymatic pretreatment.26,27 Besides the inherent advantages of cellulose, such as good biocompatibility, biodegradability and indicated non-toxicity, CNF present unique nanostructures in films that also display high mechanical strength.28,29 In addition, CNF films display small porosity and high density.25 CNF in films and other forms is entering different fields, such as pharmaceutics, cell culture and tissue engineering, antimicrobial agents, and biosensors and diagnostics.30 CNF has both amorphous and crystalline cellulose I regions as presented in native cellulose.31,32 Moreover, CNF-based ultrathin films or “nanopapers” can be used as a model system to study molecular interactions.25,33,34 A critical component of a biosensor is the bio-recognition agent, which is able to specifically bind to or react with a target analytes (CRP in our case). Agents for recognition of CRP include antibodies,9,16,35 phosphocholines36,37 or aptamers.38,39 Among these, antibodies are most widely used in biosensors. An antibody can be immobilized on a biosensor matrix through chemical covalent binding or affinity interaction in unoriented or oriented configurations. Immobilization of antibody via affinity binding provides more reproducible results and makes available the active site of the 3


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antibody for interaction with the analyte.16,40,41 Proteins such as Protein A, can bind strongly with the Fc portion of antibodies and thus has been widely used as ligand for oriented immobilization to achieve maximum activity and detection.41,42 In this work, we developed a carboxylated CNF ultrathin film with immobilized anti-C reactive protein antibody (anti-CRP) as immunosensor for CRP detection. Carboxylated CNF films were prepared by two different methods, followed by conjugation of Protein A, which served as a ligand for oriented immobilization of anti-CRP. The modification process was mainly monitored using QCM. Our results suggest that the developed bioactive CNF films can specifically detect CRP, offering an option for designing cellulose-based immunosensors and facilitating the way for simple, portable, disposable, and low-cost sensing devices for biomarker detection in the field of medical analyses.

MATERIALS AND METHODS Materials. CNF dispersion with 1.6% (wt/wt) solids was prepared as previously reported.25 Briefly, bleached hardwood birch fibers were mechanically treated with a Masuko grinder (5 passes) and then further disintegrated by micro-ﬂuidization (20 passes, M110P fluidizer, Microfluidics Corp., Newton, MA, USA); the CNF charge density was 65 µmol/g. Carboxymethylated CNF dispersion was also prepared at a solid content of 1.27%. Briefly, bleached birch fibers were first carboxymethylated43 and converted to the sodium form according to Swerin et al.44 Thereafter, they were further disintegrated by microﬂuidization (Microfluidics M110P, Microfluidics Corporation, MA) using 5 passes. The charge density of the dispersion was measured to be 315 µmol/g. QCM gold chips were supplied from Q-Sense (Göteborg, Sweden). Sodium bromide (NaBr, >99%) was obtained from Acros Organics. Fibrinogen (plasminogen-depleted, human plasma) was obtained 4


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from Calbiochem (La Jolla, CA) as a lyophilized powder. Goat anti-human C-reactive protein (anti-CRP) was purchased from Bethyl Laboratories (Montgomery, TX). C-reactive protein from human plasma (CRP), Protein A from Staphylococcus aureus (≥95%), albumin from human serum, N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide N-hydroxysuccinimide

(NHS,

98%),

sodium

hydrochloride hypochlorite

(EDC,

≥98.0%),

solution

(NaClO),

2,2,6,6-tetramethyl-piperidine 1-oxyl (TEMPO), and poly(ethyleneimine) (PEI, 600 -1000 kDa molecular weight 50% in water) were all purchased from Sigma-Aldrich (Milwaukee, WI) and used as received. Phosphate-buffered saline (PBS 10 mM, pH 7.4) and acetic acid buffer (HAc 10 mM, pH 4.5) were prepared freshly. For clarity, Table 1 includes a summary of the acronyms used in this work. Table 1. Abbreviations used in this paper Abbreviation PEI TEMPO EDC NHS CRP Anti-CRP HSA Fg HAc

Description Poly(ethyleneimine) 2,2,6,6-tetramethyl-piperidine 1-oxyl N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride N-hydroxysuccinimide C-reactive protein from human plasma Anti C-reactive protein Human serum albumin Fibrinogen Acetic acid buffer

Preparation of CNF-based immunosensor. PEI pre-adsorbed QCM surface. QCM gold chips were cleaned with “piranha” solution (H2SO4/H2O2 = 7:3 (v/v)) (Caution: Piranha solution reacts violently with organic materials and should be handled carefully) at room temperature for 5 min to remove the organic residues on the surfaces. The surfaces were then rinsed with copious amounts of Milli-Q water and dried under a 5


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flow of nitrogen. Finally, the surfaces were exposed to UV irradiation for 30 min just before use. The cleaned QCM chips were immersed into 1 g/L PEI aqueous solutions for 15 min to obtain Au-PEI surfaces. They were rinsed carefully with Milli-Q water and dried gently with nitrogen gas and stored in desiccators prior to use. Ultrathin films of carboxylated CNF. Carboxylated CNF films were prepared by two different methods as indicated in Scheme 1: Method I: a dispersion of unmodified CNF was spin-coated onto Au-PEI surfaces, which offered a branched and highly cationic structure for CNF attachment.25,31,45 Briefly, CNF aqueous dispersion (1.67 g/L) were homogenized with microtip-sonicator (10 min, 25% amplitude, Branson s-450 sonicator), and then centrifuged (10,400 rpm, 45 min, 25 oC) to collect supernatant CNF suspension.

The

CNF

suspension

was

spin-coated

(Laurell

Technologies

Corporation

WS-400-6NPP\LITE) at 3,000 rpm for 1 min onto the Au-PEI surface. The spin-coating process was repeated twice in order to obtain chips fully covered with CNF. The obtained CNF surfaces were rinsed carefully with Milli-Q water and dried gently with nitrogen gas and stored in desiccators prior to use. The hydroxyl groups on CNF surfaces were then carboxylated by using TEMPO-mediated oxidation in situ.25,34,46 Briefly, TEMPO (0.13 mmol) and NaBr (4.7 mmol) were dissolved in 20 mL water, followed by pipetting of NaClO (5.65 mmol) to achieve TEMPO-oxidation solution (the final pH was adjusted to 10 using 1 M HCl). The CNF surfaces were incubated in this solution at 25°Cfor 2 min and the oxidation was quenched using ethanol.34 The resultant surfaces were rinsed with abundant Milli-Q water to remove excess oxidation reagents and dried under a nitrogen flow to achieve the carboxylated (TEMPO-oxidized) CNF surface, which is referred thereafter as tCNF. 6


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Method II. Carboxymethylated CNF dispersions were directly spin-coated onto Au-PEI surfaces. Briefly, the diluted suspensions (1.25 g/L in water) were homogenized by frequent hand-shaking for 5 min, and then the suspensions were sonicated (FS30 Ultrasonic sonicator, Fisher Scientific, Pittsburgh, PA) for 3 min to remove gas bubbles. The spin coating of the suspension onto QCM chips followed the same procedure as indicated in Method I to obtain the carboxymethylated CNF surface, which is referred thereafter as cCNF.

Scheme 1. (Top panel) Schematic illustration of the preparation of tCNF and cCNF surfaces by Methods I and II, respectively. In method I, CNF was deposited on the solid support by spin coating followed by in-situ TEMPO oxidation (tCNF). In method II, previously obtained cCNF solution was 7


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spin coated on the solid support. The inset includes the main differences in the chemical structure of CNF, tCNF and cCNF. (Bottom panel) Nomenclature used for the developed sensor systems.

Conjugation of anti-CRP on carboxylated CNF surfaces. Oriented immobilization of anti-CRP on carboxylated CNF surfaces was achieved using Protein A as ligand as shown in Scheme 2. Binding of this protein onto the surface was monitored by QCM using a Q-sense E4 system (Göthenburg, Sweden). In this study, all QCM measurements were conducted at the third overtone number and the changes in frequency was reported (∆f = ∆f3/3).

Scheme 2. Immobilization of anti-CRP on carboxylated CNF surfaces (tCNF or cCNF) via EDC/NHS coupling for CRP detection. Depending on the presence of Protein A ligands, the anti-CRP immobilization was either oriented (top) or unoriented (random, bottom).

QCM chips coated with ultrathin films of carboxylated CNF, either tCNF or cCNF, were mounted in the QCM module, and then stabilized in HAc for 1 h in order to acquire a stable baseline. The

carboxyl

groups

on CNF were activated

by EDC/NHS chemistry to

generate

N-hydroxysuccinimide ester (NHS-ester) groups by injection of EDC (0.1 M) and NHS (0.4 M) in HAc solution at 50 µL/min for 20 min, thus obtaining NHS-CNF surfaces.25,47 HAc was then introduced to the system for 15 min to establish a second baseline. 8


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Protein A in HAc (1 mg/mL) was flowed through the NHS-CNF surface for 25 min forming amide bonds between amine groups of Protein A and NHS-ester groups on tCNF and cCNF, thus achieving Protein A conjugated tCNF (tCNF/A) and Protein A modified cCNF (cCNF/A) surfaces. To minimize the usage of Protein A, the injection speed was adjusted to 10 µL/min. HAc was finally passed through Protein A modified surface to remove the unbound or loosely attached Protein A and a third baseline was established. The frequency shift registered for the second and the third baselines reflected the binding of Protein A on the CNF surfaces. In order to prevent non-specific binding of unreacted NHS-ester with other proteins, the tCNF/A or cCNF/A surface was washed with ethanolamine aqueous solution (1 M, pH 8.5) for 10 min to block the unreacted NHS-ester groups. The surface was cleaned with HAc for 20 min, and then PBS was injected in the system until a constant QCM signal was obtained. Finally, anti-CRP in PBS (100 µg/mL) was injected onto the tCNF/A or cCNF/A surface for 25 min at 10 µL/min, obtaining

anti-CRP

binding

surfaces,

namely,

tCNF/A/anti-CRP

or

cCNF/A/anti-CRP

immunosensors. As a control, anti-CRP in HAc (100 µg/mL) was injected instead of Protein A, directly to sensor chamber containing NHS-CNF surfaces for 25 min at 10 µL/min (Scheme 2). Thus, unoriented, immobilized anti-CRP was achieved on the cCNF (cCNF/anti-CRP); the surface was then rinsed with HAc until a constant QCM signal was established. Surface Characterization Ellipsometry. The thickness of PEI and CNF layers deposited on the QCM sensor was measured using a spectroscopic ellipsometer (model M-2000 V, J. A. Woollam Co., Inc.) at angle of 70° and wavelengths from 400 to 800 nm. The data was fitted with Cauchy model with fixed (An, Bn) values 9


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of (1.46, 0.01). The thickness for each sample was reported as the average value from three replicates. Contact angle goniometry. Surface wettability was evaluated by the static water contact angles (WCA), which were measured using a contact angle goniometer (SEO Phoenix 300, Korea) operated at room temperature. Each contact angle average value reported was obtained from six replicates. Attenuated total reflection Fourier transform infrared spectroscopy. The changes in surface chemistry after the modification steps were determined via attenuated total reflection Fourier transform infrared (ATR-FTIR). The measurements were performed in a Thermo Nicolet 670 FTIR ESP spectrometer equipped with a Smart OMNI sampler (Nicolet Instrument Corp.). The data were collected 256 continuous scans with a resolution of 4 cm−1. Atomic Force Microscopy. Tapping-mode topographical images of CNF, tCNF and cCNF surfaces were acquired in air using a Digital Instruments D3000 atomic force microscope (AFM). The root-mean-square (RMS)surfaces roughness values were calculated using Nanoscope Analysis software. Quartz Crystal Microgravimetry. Using QCM (Q-sense E4 system, Göthenburg, Sweden), Zhu et al. found that the swelling of surface-tethered polyelectrolytes containing carboxyl groups leads to a decrease in frequency, and that the extent of such reduction depends on the amount of charged carboxylic groups.48 According to this observation, both tCNF and cCNF sensors were mounted in the QCM chamber and were exposed to water until a stable frequency signal (baseline) was obtained. Following, PBS (pH 7.4) was injected in the system, which facilitated deprotonation of the carboxyl groups and, consequently, produced a reduction in frequency due to swelling of the films. The extent

10


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frequency reduction is taken here as indirect evidence of the different carboxyl group density in the films. Nonspecific Protein Adsorption and CRP Detection. The nonspecific protein adsorption on CNF-based immunosensor was investigated using the quartz microgravimetry technique using a QCM (Q-Sense E4 system, Göthenburg, Sweden). Human serum albumin (HSA, MW= 66.5 kDa) and fibrinogen (Fg, MW= 341 kDa) were used as model proteins for the nonspecific protein resistance of the developed CNF-based immunosensors. First, PBS was exposed to the anti-CRP modified surface to establish a baseline. Then, HSA or Fg (100 µg/mL) were introduced to the system for 30 min at a flow rate of 10 µL/min. Finally, the solution was switched back to PBS. The frequency shift determined for the frequency signals acquired in the two PBS media was taken as a measured of the nonspecific protein adsorption on anti-CRP conjugated surfaces. The detection ranges of immunosensor against its antigen, CRP, was investigated. CRP was dissolved in PBS at different concentrations, from 1 to 100 µg/mL and circulated on the sensor surface. Similarly, this procedure was applied for HSA and Fg adsorption. The frequency changes between the signals acquired for the protein-free systems (in the PBS background electrolytes) was assumed to correspond to the extent of the specific CRP adsorption on sensor surfaces.

RESULTS AND DISCUSSION Previous studies have demonstrated that CNF exhibits good biocompatibility and excellent non-specific protein resistance, suggesting the potential for CNF in bioactive films or nanopapers for diagnostics.25,34,45 In our work, CNF-based immunosensors were prepared to detect CRP, as indicator

11


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of cardiovascular disease. Firstly, carboxylated CNF surfaces were prepared using two different protocols using tCNF and cCNF, as described earlier (Scheme 1). And then the two carboxylated CNF surfaces (tCNF and cCNF) were activated for conjugation of Protein A to obtain Protein A modified surface (tCNF/A and cCNF/A). Following, anti-CRP was immobilized to Protein A modified surface to achieve tCNF/A/anti-CRP and cCNF/A/anti-CRP (oriented systems), respectively. As a control, anti-CRP was immobilized to carboxylated CNF surface directly, in the absence of Protein A ligand, obtaining unoriented immobilized antibodies on the surface (Scheme 2). The surface properties and the binding ability of Protein A and anti-CRP on the two different carboxylated CNF surfaces were characterized and compared. Characterization of CNF-based immunosensors. The surface morphology of unmodified CNF and carboxylated CNF (tCNF and cCNF) on QCM gold chips was observed using AFM (Figure 1). The samples were scanned at several different locations and the images exhibited similar morphology, indicating typical network nanostructures and a homogenous deposition on the solid support.

Figure 1. Surface morphology of (a) CNF, (b) tCNF and (c) cCNF observed by AFM. The vertical Z contrast range (dark to light) is -15 nm to 15 nm (for (a) and (b)), and -10 nm to 10 nm (for (c)), respectively. 12


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The surface roughness (expressed as the root mean squared roughness, RMS) of the surface covered with CNF is 3.5 ± 0.2 nm. The nanofibrils, with an average width of 28 ± 3 nm, were randomly oriented (Note: in this size evaluation several tens of single nanofibrils from AFM images were used for section analyses and the average value and standard deviation are reported). The RMS for tCNF surface increased to 4.0 ± 0.1 nm while the fibrils diameter was similar to that of CNF (26 ± 3 nm). For cCNF surfaces, the RMS and average width were 2.4 ± 0.2 nm and 18 ± 2 nm, respectively. The smaller fiber diameter for cCNF is ascribed to the procedure used in its preparation. The changes in layer thickness, wetting and surface chemistry after each reaction step were determined by ellipsometry, contact angle goniometer and ATR-FTIR, respectively (Figure 2). The pre-adsorbed PEI produced a dry layer with thickness of 0.8 ± 0.2 nm and water contact angles (WCA) of 48±2o. Spin coating of CNF suspension on gold surfaces yielded a layer with thickness of 6.5 ± 0.2 nm and reduced WCA of 25±3o, in agreement with our previous reports.45Compared with Au-PEI support layers, CNF surfaces used in Method I displayed new characteristic bands at 3394 cm-1 (νO-H), 2904 cm-1 (νC-H), 1062 cm-1 (νC-O), 897 cm-1 (νC-O-C) and 1105 cm-1 (δC-O-C), indicating successful coating on the QCM sensor. After TEMPO-oxidation, the strong signal peak at 1641 cm-1 (νCOO-) confirmed the presence of carboxyl groups; the WCA decrease below the detection limit of 10o and the film thickness decreased to 4.9±0.2 nm, due to the possible degradation of CNF during TEMPO treatment.34 The ATR-FTIR spectrum of cCNF surfaces prepared by Method II showed similar FTIR peak features as those of the tCNF surfaces. The thickness and WCA of cCNF film was 8.2 ± 0.6 nm and21 ± 4o, respectively. The WCA of tCNF surface was significantly smaller than that

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of cCNF surface. The enhanced hydrophilicity might be attributed to the higher surface density of carboxyl groups of tCNF (Figure S1, Supporting Information).

Figure 2. Surface chemical characterization, wetting and thickness of CNF films following the different modification steps: (a) ATR-FTIR of Au-PEI; CNF, tCNF and cCNF surfaces. Figures (b) and (c) include, respectively, the contact angle and thickness of the surfaces: (I) Au-PEI, (II) CNF, (III) carboxylated CNF, (IV) carboxylated CNF after conjugation with Protein A, and (V) carboxylated CNF after conjugation with Protein A and binding of anti-CRP.

In summary, the results of AFM, ellipsometry, WCA and ATR-FTIR indicated that tCNF and cCNF films were coated homogenously on the QCM solid supports and were hydrophilic. Compared with cCNF surfaces, tCNF displayed a higher surface density of carboxyl groups, which can be used to couple biomolecules containing amine groups and therefore to build the bioactive cellulose.25 Binding of Anti-CRP on CNF Surfaces conjugated with Protein A. If antibodies are immobilized on a solid surface with no proper orientation, their antigen binding activity is expected to decrease due to steric hindrance, yielding poor reproducibility and sensitivity.49-51 Therefore, oriented immobilization of antibodies is necessary for sensitive and specific immunosensors. A promising and widely used approach to oriented immobilization of antibody is through capture ligand Protein A.41,42,52,53 When Protein A is immobilized on a surface, antibodies couple in a 14


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controlled and directional fashion through the Fc region, thereby allowing the Fab arms of the antibody to remain free for antigen binding.41,54 We compared the binding capability of anti-CRP and further detection ability of CRP of oriented and unoriented immobilization of anti-CRP onto CNF surface, depending if Protein A ligand was present or not. The results confirmed that oriented immobilization of anti-CRP on the surface of CNF produces nearly 3 times higher binding capability for CRP compared with the unoriented surface (Figure S2, Supporting Information). Protein A was coupled to carboxylated CNF surfaces via EDC/NHS chemistry. The whole processes of activation of carboxyl group, binding of Protein A, and binding anti-CRP to tCNF or cCNF surfaces were monitored by QCM (Figure 3). We note that the principle of QCM sensing relies on the piezoelectric effect, in which mass changes (from protein adsorption/desorption and water coupling) causes a shift in frequency, registered as ∆f in Figure 3.55,56

Figure 3. Time-resolved QCM isotherms for activation, adsorption and binding of Protein A and anti-CRP on tCNF and cCNF surfaces. (a) The QCM detection of Protein A immobilization with QCM sensor events that includes: 1, baseline in HAc; 2, activation of carboxyl groups from carboxylated CNF with EDC/NHS in HAc; 3, baseline in HAc; 4, coupling of Protein A; 5, injection of HAc; 6, deactivation of unreacted NHS-esters with ethanolamine; 7, injection of HAc; 8, injection of PBS buffer. (b) The difference of Protein A binding to tCNF and cCNF surfaces. (c) The binding of anti-CRP to tCNF/A and cCNF/A surfaces. Note that the range of ∆f values (vertical axis) in the sensograms is different in each case: (a) -160 Hz, (b) -80 Hz and (c) -27.5 Hz, respectively.

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As shown in Figure 3a, prior to coupling of Protein A, the carboxylated CNF surfaces were exposed to HAc buffer, until a stable frequency was achieved (step 1). Then terminal carboxyl groups were activated by injection of EDC/NHS solution to generate an active intermediate of NHS-esters (step 2, the detailed chemical changes are shown in Scheme 2). After activation, the surfaces were rinsed with HAc until a second baseline was obtained (step 3). The activation of EDC/NHS induced a ∆f of -18 ± 1 Hz and 1 ± 2 Hz for tCNF and cCNF surfaces, respectively, further indicating that the tCNF surface exhibited a higher density of available carboxyl groups, which were then activated. Protein A in HAc (1 mg/mL) buffer was coupled to carboxylated CNF upon injection. The frequency showed a gradual decrease with time of QCM signal, suggesting the binding of Protein A on the activated NHS-CNF surface. A plateau frequency values was reached within 25 min (step 4). Loosely bound Protein A was then rinsed out from the surface by injection of HAc solution over the sensor surface, producing another baseline (step 5). The difference between the baselines in step 3 and 5 indicates the extent of effective binding of Protein A on the carboxylated CNF surfaces. Here the frequency in step 3 was set to zero, as a reference point, and the changes in frequency after injection of Protein A are plotted in Figure 3b: the binding of Protein A induced a ∆f of -71± 1 Hz and -11± 2 Hz for tCNF and cCNF surfaces, respectively, suggesting higher binding of Protein A on tCNF compared to that for cCNF. The binding of Protein A resulted in a less hydrophilic surface, as suggested by the increase in WCA from 3 µg/mL, can be considered a risk sign of cardiovascular diseases. In the following experiments, the concentration of CRP ([CRP]) was varied from 1 to 100 µg/mL to study the detection range of the developed immunosensor (Figure 5). Figure 5a includes the reduction of frequency as a function of time in immunosensors exposed to different CRP concentrations. The adsorption process is complex, as clearly shown in the QCM sensograms. At [CRP] = 3 µg/mL, the adsorption region corresponds to -1 Hz frequency reduction. It should be noted that the QCM used in this work has a nominal mass sensitivity in liquid of ~1.8 ng/cm2 (corresponding to ~0.1 Hz in frequency change), which indicate that the measured ∆f= -1 Hz was produced by the specific binding of CRP. This conclusion was also supported by the results of non-specific protein adsorption from Figure 4. As [CRP] is increased to 30 µg/mL and 100 µg/mL different adsorption dynamics were observed. Two consecutive adsorption steps were observed before CRP adsorption reached a plateau; similar protein adsorption behavior have been observed by other groups using different sensors chemistries.59 We hypothesized that the first step of initial rapid adsorption involves specific binding of CRP on the anti-CRP immobilized surface. We speculate that the second step with a relatively slower adsorption rate is related to physical adsorption of a second layer of CRP on the surface, which are loosely bound (these physically adsorbed CRP molecules are removed upon injection of PBS). The CRP adsorption induces net frequency changes of -1.5 Hz and -3.9 Hz for [CRP] at 30 and 100 µg/mL, respectively. Indicating that higher CRP binding to cCNF/A/anti-CRP surfaces as [CRP] 19


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is increased. Based on these results, a calibration curve for cCNF/A/anti-CRP immunosensors was determined for the tested concentration range (Figure 5b): –∆f = 0.034 [CRP]+0.45 (R2 = 0.995) While ELISA detection limit in blood serum is 0.1 µg/mL,60 our results indicate that the CNF-based sensor can give a wide CRP detection range and thus, it is suitable to determine the [CRP] at slightly higher values.

Figure 5. Detection of CRP using cCNF/A/anti-CRP immunosensors. (a) Time-resolved QCM isotherms for CRP adsorption at different concentrations. (b) The frequency changes for CRP adsorption at concentrations range of 1 to 100 µg/mL. The dotted line is a linear fit to the data: –∆f = 0.034 [CRP]+0.45 (R2 = 0.995).

CONCLUSIONS We developed a carboxylated CNF-based immunosensor for CRP detection. Two methods were used to prepare carboxylated CNF surfaces (tCNF or cCNF) for conjugation of protein A, which served as a ligand for oriented immobilization of anti-CRP. Compared with unoriented anti-CRP attached to CNF, oriented immobilization of anti-CRP via Protein A ligand facilitates detection with three times higher specificity. Compared with cCNF, tCNF surfaces have a higher surface density of carboxyl groups and binds more Protein A and anti-CRP. However, CRP binding yielded similar 20


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results. Besides the specific binding ability with CRP, as low as 3 µg/mL in PBS, the cCNF/A/anti-CRP immunosensor resists non-specific protein adsorption (e.g., Fg and HSA). The significance of this study resides in specific detection of CRP with CNF-based biointerfaces, which can guide the design of paper-based immunosensors toward low-cost sensing devices. In sum, we demonstrated the feasibility of simple, portable, and disposable tools for biomarker detection in the field of medical analyses.

ASSOCIATED CONTENT Supporting Information Comparison of QCM sensograms of carboxylated CNF (cCNF and tCNF) in water and PBS (pH 7.4); descriptions of the difference in frequency changes of anti-CRP and CRP binding to cCNF surfaces with or without Protein A used as ligand. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (OJR), Tel.: +358-50-5124227; [email protected] (YZ).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

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This research was carried out with partial support from the National Natural Science Foundation of China (21604059), the Natural Science Foundation of Jiangsu Province (BK20160321) and Jiangsu Higher Education Institutions of China (16KJB430029) as well as the Academy of Finland Centers of Excellence Programme (2014–2019), project 264677.

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FOR TABLE OF CONTENTS USE ONLY Immunosensors for C-reactive protein based on ultrathin films of carboxylated cellulose nanofibrils Yanxia Zhang,*,§, Orlando J. Rojas*,†,‡ §

Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University, Suzhou, Jiangsu, 215007, P. R. China. †

Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, FI00076 Aalto, Finland.

‡

Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States.

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Scheme1 150x141mm (300 x 300 DPI)


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scheme 2 150x47mm (300 x 300 DPI)


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Fig.2 150x40mm (300 x 300 DPI)


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Fig.4 119x91mm (300 x 300 DPI)


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TOC 150x78mm (300 x 300 DPI)


Immunosensors for C-Reactive Protein Based on Ultrathin Films of

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