Biosensors and Bioelectronics 55 (2014) 32–38

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Improved activity of immobilized antibody by paratope orientation controller: Probing paratope orientation by electrochemical strategy and surface plasmon resonance spectroscopy Wei-Ching Liao a,b, Ja-an Annie Ho a,b,n a BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan b Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan

art ic l e i nf o

a b s t r a c t

Article history: Received 3 August 2013 Received in revised form 16 October 2013 Accepted 26 October 2013 Available online 1 December 2013

Electrochemical method and surface plasmon resonance (SPR) spectroscopic analysis are utilized herein to investigate antibody immobilization without and with orientation control for site-positioning paratopes (antigen binding site) of the antibody molecules. Biotin and its antibody were selected in current study as model. Such an approach employed thiophene-3-boronic acid (T3BA) as paratope orientation controller, (i) enabled site orientation of the antibody molecules reducing the hiding of paratopes, and (ii) maintained the activity of the captured antibodies, as confirmed by electrochemical and SPR anlysis. Anti-biotin antibody (a glycoprotein) was covalently bound to a self-assembled monolayer of T3BA modified on a nanogoldelectrodeposited screen-printed electrode through boronic acid–saccharide interactions, with the boronic acid units specifically binding to the glycosylation sites of the antibody molecules. The immunosensor functioned based on competition between the analyte biotin and biotin-tagged, potassium hexacyanoferrate (II)-encapsulated liposomes. The current signal produced by the released liposomal FeðCNÞ46  , measured using square wave voltammetry, yielded a sigmoidally shaped dose–response curve that was linear over eight orders of magnitude (from 10–11 to 10–3 M). Furthermore this biosensing system fabricated based on T3BA approach was found to possess significantly improved sensitivity, and the limit of detection toward biotin was calculated as 0.102 ng mL–1 (equivalent to 6 μL of 4.19  10–10 M biotin). & 2013 Elsevier B.V. All rights reserved.

Keywords: SPR analysis Electrochemistry Boronate affinity Site-oriented immobilization Immunosensor

1. Introduction Rapid and sensitive immunoassays are in demand because of the ever-increasing testing for biological analytes in clinical (Malhotra et al., 2010; Wu et al., 2007), environmental (Ahn et al., 2009; Medintz et al., 2003; Zhang et al., 2010), and bio-industrial fields (Luong et al., 2008; Rivas et al., 2008). One of the main issues in the development of an immunosensor is maintaining the immunorecognition capability of the antibody after it has been immobilized on the sensing surface. The available methods for attaching antibodies onto solid surfaces are mainly based on three interactions (Camarero, 2008; Danczyk et al., 2003; Rusmini et al., 2007; Willner and Katz 2000): (i) adsorption,

Abbreviations: CV, cyclic voltammetry; SWV, square wave voltammetry; SPE, screen-printed electrode; SPR, surface plasmon resonance; Ab, antibody; HRP, horseradish peroxidase; Fab, fragment antigen-binding n Corresponding author at: BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan. Tel.: þ 886 2 3366 4438. E-mail address: [email protected] (J.-a. Annie Ho). 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.054

(ii) covalent attachment, and (iii) affinity binding. When antibody molecules are adsorbed onto surfaces and stabilized through electrostatic, hydrophobic, and polar intermolecular interactions (Rusmini et al., 2007), the attachment layer is likely to be heterogeneous and weakly bound, resulting in a random orientation of the paratopes. Covalent methods for immobilizing proteins onto solid substrates usually involve the use of available functional groups (e.g., amino, carboxyl) of accessible amino acids or the formation of gold–thiol bonds to construct self-assembled monolayers (SAMs). Although the formation of a covalent bond between an immobilized protein and a solid substrate surface can improve the reproducibility of protein immobilization to some extent, the available functional groups are presented randomly on the antibody molecule and the attachment may occur simultaneously through many available residues, thereby producing a high degree of heterogeneity in the population of immobilized proteins (Rusmini et al., 2007). In addition, covalent approaches often lead to loss of the biological function of the antibody because of either denaturing under the relatively harsh chemical conditions used in the attachment process or decreased immunorecognition ability resulting from the inappropriate orientation (e.g., paratopes facing away from the solution).

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Development of antibody immobilization methods with orientation control (Camarero et al., 2004; Mouri et al., 2010; Tajima et al., 2011; Werner and Machleidt, 1978) and evaluation of antibody orientation on surface (Chen et al., 2003; Liu et al., 2010; Song et al., 2011; Zhou et al., 2004) has been subjects of relentless efforts and endless discussions among many groups. It was exploited previously that the formation of reversible cyclic esters from boronic acids and the cis-diol units of saccharides in nonaqueous and aqueous media at ambient temperature (Ho et al., 2010; Springsteen and Wang, 2002) and could be used as intermediate molecules for protein orientation control. Moreover boronate affinity has been used widely for the development of aqueous sugar sensors (Akay et al., 2007; Edwards et al., 2007; James et al., 1995), carbohydrate and glycoprotein separation systems (Bossi et al., 2001, 2004; Jackson et al., 2008), glycoprotein (Abad et al., 2002; Chen et al., 2008; Lin et al., 2009; Liu et al., 2005, 2006; Liu and Scouten ,1996) and cell (Polsky et al., 2008) immobilization systems, transporters of ribonucleotides and carbohydrates through lipid membranes (Paugam et al., 1996; Westmark et al., 1996; Westmark and Smith, 1996), and glucose-responsive controlled drug delivery systems (De Geest et al., 2006; Zhao et al., 2009). To minimize the loss of activity of the antibody resulting from structural deformation and to increase the analyte binding capacity of the immunosensor, we became interested in fabricating immunosensors with a site-specific orientation of the antibody units—based on boronic acid–saccharide interactions—aligned on the sensing surface, and evaluate the immobilization performance by measuring the conformation of the antibody on the surface. In this study, we implemented an easier and more convenient approach to better control paratope orientation and to facilitate the fabrication of immunosensing surface. Futhermore, we attempted to employ electrochemical method and SPR analysis to probe the antibody orientation/conformation on either a nanogold-electrodeposited screen-printed electrode or planar gold surface, that were pre-assembled a monolayer of thiophene-3boronic acid (T3BA) (Park et al., 2008), stabilized through gold– thiol linkages, as compared with the orientations of antibody units surface-immobilized using (i) direct adsorption, and (ii) gold–thiol linkage. As a result of boronate affinity, the boronic acid moiety of T3BA binds covalently with oligosaccharides, which are attached to the CH2 domains in most immunoglobulins (Suzuki et al., 2003; Yoo et al., 2002), leading to site-positioning of paratopes and preservation of the binding ability of paratopes (Lin et al., 2009). The new immobilization method described herein is confirmed to be less laborious, less time-consuming, and minimizes the probability of hiding the binding sites of antibody (i.e., it regulates the orientation of the paratopes in the antibody molecules).

2. Materials and methods 2.1. Reagents and materials All chemicals and organic solvents were of reagent grade or better. Potassium chloride, sodium chloride, acetic acid, potassium ferrocyanide, biotin, thionin acetate, hydrochloric acid, 2-mercaptoethylamine hydrochloride (2-MEA), and ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA) were purchased from SigmaAldrich (St. Louis, MO). Hydrogen tetrachloroaurate(III) was obtained from Alfa Aesar (Ward Hill, MA). Tris (base), HEPES, potassium phosphate monobasic (KH2PO4), and sodium bicarbonate (NaHCO3) were obtained from J. T. Baker (Phillipsburg, NJ). Potassium phosphate dibasic (K2HPO4) and sodium carbonate (Na2CO3) was purchased from Riedel-de Haën (Seelze, Germany). Thiophene3-boronic acid (T3BA) was obtained from Frontier Scientific (Logan, UT). The enzyme substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Neogen K-blue enhanced activity substrate, containing H2O2) was

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obtained from Neogen (Lexington, KY). Dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were acquired from Avanti Polar Lipids (Alabaster, AL). N-((6-(biotinoyl) amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Biotin-X-DHPE) was purchased from Molecular Probes (Eugene, OR). The IgG (immunoglobulin G) fraction of antibiotin (rabbit) was acquired from Rockland Immunochemicals (Gilbertsville, PA). Goat F(ab')2 polyclonal secondary antibody to rabbit IgG-(Fab')2 (HRP) (HRP-conjugated anti-Fab antibody) was obtained from Abcam (Cambridge, UK). Biotin-HRP was acquired from Invitrogen (Carlsbad, CA). Dialysis tubing (MWCO: 12,000–14,000) was purchased from Spectrum Laboratories (Rancho Dominguez, CA). All solutions were prepared with deionized water having a resistivity not less than 18 MΩ cm (Milli-Q, Bedford, MA). 2.2. Apparatus Cyclic voltammetry (CV), square wave voltammetry (SWV) and amperometric i–t curve measurements were performed using a CHI 633 electrochemical analyzer/workstation (CH Instruments, Austin, TX). Disposable electrochemical screen-printed electrodes (SPEs), comprising a carbon working electrode (3 mm diameter), carbon counter electrode, and Ag/AgCl pseudo-reference electrode, were purchased from Zensor R&D (Taichung, Taiwan). The effective diameter and zeta potential of the liposomes were measured using a Brookhaven 90Plus Nanoparticle Size Analyzer and Zeta Potential Analyzer, respectively (Brookhaven Instruments, Holtsville, NY). Surface plasmon resonance (SPR) experiments were performed using a Biacore T100 system (Biacore, Uppsala, Sweden).

2.3. Fabrication of Ab/T3BA/nanoAu/SPE Prior to modification, the working electrode of the SPE was preconditioned electrochemically by cycling the potential repeatedly between  0.6 and þ0.6 V at 0.5 V s–1 in PBS buffer (0.1 M potassium phosphate, 0.15 M NaCl, pH 7.2). The pre-conditioned SPE was then immersed in a solution of 10 mM HAuCl4 containing 0.1 M KCl, followed by electrodeposition (at  0.66 V for 10 s) to fabricate nanostructured gold on the electrode surface (Ho et al., 2009). Six microliters of 50 mM T3BA in distilled deionized (D.D.) water was placed onto the working electrode and left to react for 6 h at ambient temperature. After rinsing with D.D. water, the SPE was dried in air. Subsequently, a droplet (6 μL) of 0.2 mg mL–1 antibiotin antibody (Ab) in 0.1 M carbonate buffer (pH 8.5) was applied on the T3BA-modified surface and incubated at 4 1C overnight to complete the preparation of the Ab-modified SPEs. The blocking of non-specific binding sites on the immunosensing surface was initiated by rinsing with PBS buffer to remove the noncovalently bound antibody; subsequently, 1% casein (10 mg mL–1 in PBS, 6 μL) was placed on the working electrode and left to react for 10 min at room temperature. Finally, the electrode was rinsed thoroughly with PBS buffer and stored at 4 1C until required for use.

2.4. Evaluation of orientation of immobilized antibody using three different approaches Electrochemical strategy adopted herein employed the HRPconjugated anti-Fab antibody to evaluate the spatial position of the paratopes on the immobilized antibody. The boronate affinity immobilization procedure, approach with orientation control, was compared with two other antibody immobilization procedures— direct adsorption (one without orientation control) and gold–thiol assembly (one with orientation control)—in terms of the resulting binding capacity of the immobilized antibody, where the sensing

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surfaces prepared through (i) direct adsorption (denoted Ab/nanoAu/ SPE) involved directly adding the antibody solution (0.2 mg mL–1, 6 μL) onto the surface of the nanogold-fabricated SPE and (ii) thiol–gold linkages (denoted HalfAb/nanoAu/SPE) involved placing a drop of the half-antibody (0.2 mg mL–1, 6 μL) onto the surface of a nanogold electrode produced using 2-mercaptoethylamine (2-MEA) (Yoshitake et al., 1979), a mild reducing agent for selectively cleaving hinge-region disulfide bonds between the heavy chains of IgG molecules. Both modified electrodes were rinsed with PBS buffer to remove unbound antibody and subsequently blocked with 1% casein in PBS buffer. Finally, the HRP-conjugated anti-Fab antibody (0.2 mg mL–1 in 0.02 M phosphate buffer containing 0.15 M NaCl, pH 7.2) was placed on the three different antibody-modified electrodes (based on boronate affinity, direct adsorption, and gold–thiol linkages) and left to incubate (30 min) under refrigeration. After rinsing with 0.02 M PBS buffer, electrochemical measurements were performed in 0.1 M PBS buffer (0.15 M NaCl, pH 8.5) containing 20 μM thionine and 6 mM H2O2 using cyclic voltammetry (CV) (Liu et al., 2005; Zhang et al., 2008). SPR analyses were also conducted to further confirm the antibody orientation. The Au surface of a Biacore sensor chip (SIA Kit Au) was rinsed with 1 M NaCl containing 50 mM NaOH. The SPR measurement was initiated by injection of the running buffer (0.1 M carbonate buffer, pH 8.5) through the system at a rate of 30 μL min–1 until the baseline became stable. Two channels were used simultaneously: one channel, with no immobilized anti-biotin antibody, was used as a reference surface; the other, which served as a sensing channel, was modified with anti-biotin antibody as described below. To test the boronate affinity modification approach, 50 mM T3BA in D.D. water (50 μL) was injected into both channels at a rate of 10 μL min–1 and then unbound T3BA was washed off under running buffer. Anti-biotin antibody (50 μg mL–1, 5 μL) was dispensed onto the sensing channel, followed by the blocking of both channels with running buffer containing 0.001% (10 μg mL–1) casein. For comparison, antibody molecules were also immobilized on the sensor chips prepared using the other two immobilization approaches (direct adsorption and gold–thiol linkage). Briefly, anti-biotin antibody or halfantibody (50 μg mL–1, 5 μL) was introduced into the sensing channel and subsequently subjected to the blocking procedure with 0.001% casein. Finally, HRP-conjugated anti-Fab antibody (50 μg mL–1, 5 μL) was introduced into both the reference and sensing channels to investigate the effect of the spatial orientation of the Fab portions of the immobilized antibodies on their immunorecognition availability. The binding response was measured in resonance units (RU), proportional to the amount of injected antiFab antibody bound to the immobilized anti-biotin antibody on the sensing surface (1 RU¼1 pg of protein/mm2 of protein density on the surface). The difference between the binding responses in the sensing and reference channels was noted.

3. Results and discussion 3.1. Using electrochemical strategy to confirm antibody orientation To verify that the immobilization of the antibody on T3BA/nanoAu/ SPE through boronic acid–saccharide interactions did indeed optimize the site-orientation of the paratopes for better immunosensing, we compared the performance of this boronate affinity approach with those of direct adsorption and gold–thiol linkage approaches. Limited by currently available techniques, we were unable to visualize the real orientation and conformation of the immobilized antibody units on the electrodes, providing direct evidence. We were, however, able to discern whether the paratopes of the immobilized antibody units did undeniably face away from the matrix, by taking advantage of the specific immunorecognition ability of the anti-Fab antibody (Sabín et al., 2009). The antibody IgG has a Y-shaped structure; the upper branches of the Y contain the paratopes (antigen binding sites), termed Fab. For the immobilized antibody to be appropriately oriented and functioning well, its Fab fragments must face toward the solution, thereby favoring binding with the anti-Fab antibody. Herein, we chose HRP-conjugated anti-Fab antibody as the secondary antibody to produce electrocatalytic response. HRP catalyzes the oxidation of thionin by H2O2 and subsequently increases the electrocatalytic current. The electrocatalytic signal was generated through the following mechanism (Berglund et al., 2002; Zhang et al., 2008): HRP(Fe3 þ ) þH2O2-Compound 1 þH2O Compound 1 þThionin(Red)-Compound 2 þThionind(Ox) Compound 2 þThionind(Ox)-HRP(Fe3 þ )þ Thionin(Ox) Thionin(Ox) þ2e  þ2H þ -Thionin(Red) Therefore, in the presence of the same amount of thionin, a greater amount of HRP-conjugated anti-Fab Ab bound to the antibiotin Ab on the electrode would cause a larger catalytic current to be obtained. Fig. 1 displays the cyclic voltammograms of Ab/nanoAu/SPE, HalfAb/nanoAu/SPE, and Ab/T3BA/nanoAu/SPE before and after incubating with HRP-conjugated anti-Fab Ab. The current signal (μA) was acquired in 0.1 M PBS buffer (pH 8.5) containing 20 μM thionin and 6 mM H2O2. The change in the reduction current, 0.76 mA, obtained from Ab/T3BA/nanoAu/SPE, was larger than those of the other two electrodes (HalfAb/nanoAu/SPE, 0.31 mA; Ab/nanoAu/ SPE, 0.16 mA). Because the electrocatalytic current intensity is related to the amount of HRP-conjugated anti-Fab antibody bound on the electrode, a greater number of HRP-conjugated anti-Fab antibody units must have been presented on the Ab/T3BA/nanoAu/SPE system. Therefore, boronic acid–saccharide interactions appear to preserve the antigen binding capacity of the immobilized antibody by orienting the paratopes toward the solution. 3.2. Using SPR analysis to verify antibody orientation

2.5. Evaluation of assay performance for biotin detection A droplet (6 mL) of a mixture containing target biotin and an appropriately diluted liposome (preparation details shown in Supporting information) solution was placed onto the anti-biotin Ab-modified immunosensing surface and then the mixture was incubated for 30 min at room temperature with continuous shaking. The sensing surface was rinsed with PBS buffer and dried in air to break the liposomes prior to electrochemical measurement, conducted in 10 μL 0.1 M acetate buffer (0.15 M KCl, pH 4.0). The oxidation signal of K4Fe(CN)6 was measured using SWV by scanning from  0.1 to þ0.35 V with an amplitude of 25 mV and a step potential of 4 mV at 15 Hz. Moreover, the effect of antibody density (antibody immobilization efficiency) on current signal was assessed (experimental details shown in Supporting information).

SPR analysis is known to allow real-time, label-free monitoring of antigen and antibody interaction, enabling the quantitative observation for the antibody immobilization process and antigen–antibody binding process. We used SPR to confirm the orientations of the anti-biotin antibody units presented after performing the three immobilization approaches. Because of the low molecular weight of biotin (244 g mol–1), it was difficult to observe the specific binding of the anti-biotin antibody with this analyte based on changes in the SPR angle (units: RU). Therefore, instead of biotin, for this study we employed anti-Fab antibody to obtain a significant change in RU during SPR analysis. The value of ΔRU (difference in response) represents the amount of anti-Fab antibody bound to the sensing channel after subtracting for the nonspecific binding of the anti-Fab antibody on reference channel.

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Fig. 1. Cyclic voltammograms of (A) Ab/T3BA/nanoAu/SPE, (B) HalfAb/nanoAu/SPE, and (C) Ab/nanoAu/SPE in 0.1 M PBS (pH 8.5) containing 20 μM thionine and 6 mM H2O2 before (solid curve) and after (dashed curve) incubating with 0.2 mg mL–1 HRP-conjugated anti-Fab antibody. SPR sensorgrams of the interactions of anti-Fab antibody with anti-biotin antibodies immobilized on the surfaces of Au sensor chips (D) under the same experimental conditions and (E) with equivalent amounts of anti-biotin antibody modified on the sensor chips.

Fig. 1(D) displays the change in sensor response plotted with respect to time after introduction of the anti-Fab antibody; the signal increase of 1000 RU on the Ab/T3BA/AuChip system was greater than those on the HalfAb/AuChip (700 RU) and Ab/AuChip (400 RU) systems. Thus, under the same experimental conditions, in terms of immunorecognition capability, the surface prepared using T3BA modification provided the greatest performance, the gold–thiol linkage method was second, and the direct adsorption approach ranked third. In the experiment above, however, the amounts of antibody units modified on the three different sensing channels were not identical, even though we employed the same concentration and volume of the anti-biotin antibody and the same rate of flow through the sensing channels. Therefore, in a further experiment, we modified our chips by adjusting the injection time of the anti-biotin antibody and half-antibody to allow similar amounts of these antibody molecules to attach onto the chips so that we would obtain an increase in sensor response of approximately 2000 RU prior to introduction of the anti-Fab antibody. Fig. 1(E) reveals a greater value of ΔRU for the Ab/T3BA/AuChip (ΔRU¼590) than for the HalfAb/ AuChip (ΔRU¼ 483), again proving our hypothesis that the antibody units immobilized on the sensing surface using the T3BA approach tended to have more of their paratopes directed toward the assay solution. In addition, confocal fluorescence microscopy was exploited herein to analyze antibody orientation; similar results to those of electrochemical strategy and SPR analysis were acquired (details shown in Supporting information, Fig. S1). Therefore, we conclude that antibody immobilization through boronic acid–saccharide interactions has a greater chance of retaining the immunorecognition ability of the immobilized antibody as a result of superior orientation of the antibody binding sites. 3.3. Effect of immobilized antibody density on current signal The effect of immobilized antibody density on current signal cued by immunorecognization is illustrated in Fig. 2. Electrochemical

currents were acquired by two different detecting probes, biotinylated horseradish peroxidase (biotin-HRP) and K4Fe(CN)6-encapsulated biotin-derivatized liposome. HRP catalyzed the reduction of hydrogen peroxide and generated quantitative electrochemical current signals in the presence of the substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), reflecting to the amount of available binding sites on immobilized antibody. Liposome, on the other hand, holds the versatility of being surface-derivatized with specific ligands and is able to encapsulate diversified detectable markers. Amount of potassium ferrocyanide released from immobilized antibody-bound K4Fe(CN)6-encapsulated, biotin-derivatized liposomes again represented the accessible binding sites of antibody on the surface. Our results indicated that the antibody densities for the Ab/ T3BA/nanoAu/SPE and HalfAb/nanoAu/SPE (2.46 and 2.28 mg/cm2) are quite similar, but only represented half of that on Ab/nanoAu/ SPE (5.01 mg/cm2). However, the electrocatalytic signal acquired from the Ab/T3BA/nanoAu/SPE was higher than those from the HalfAb/nanoAu/SPE and Ab/nanoAu/SPE. The current signal attributed to the released liposomal K4Fe(CN)6 on Ab/T3BA/nanoAu/SPE is three times higher than that of Ab/nanoAu/SPE and 1.5-fold higher than that of HalfAb/nanoAu/SPE. It presumably could be considered that boronate affinity method reduces the randomly oriented antibodies on the surface, and the antibody molecules immobilized on the Ab/T3BA/nanoAu/SPE were less denatured, which in turn preserved their higher immunobinding affinity and, therefore, led to a greater number of binding sites being available for the immunorecognition of the biotinylated-HRP and biotintagged liposomes. 3.4. Fabrication and optimization of assay system Once the boronate affinity method was confirmed to be an efficient orientation controlling approach, we attempted to fabricate an immunosensor based on this protocol. The optimal amounts of anti-biotin antibody and liposome required to perform the immunoassay were investigated. We coated various concentrations of the

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Biotin-HRP detecting probe

Liposomal detecting probe

Protein density (µg/cm2)

SD

Current (µA)

SD

Current (µA)

SD

Ab/nanoAu/SPE

5.01

0.048

18.6

1.17

0.19

0.02

Ab/T3BA/nanoAu/SPE

2.46

0.049

23.7

0.52

0.57

0.04

HalfAb/nanoAu/SPE

2.28

0.023

21.3

0.35

0.37

0.02

Fig. 2. Comparison between antibody densities and signal intensities of three Ab-modified electrodes using three different immobilization approaches: Ab/nanoAu/SPE was prepared through direct adsorption; Ab/T3BA/nanoAu/SPE was prepared using the boronate affinity approach; and HalfAb/nanoAu/SPE was prepared via thiol–gold assembly.

it difficult to observe the competition (i.e., no significant difference in the signal ratio percentage). Table 1 summarizes the effect of the dilution factor of the added liposome on the performance of the electrochemical immunosensor toward the detection of the target biotin. After testing samples of the liposome solution that had been diluted two-, four-, six-, and eight-fold, we found that the optimal concentration of the added liposome preparation was obtained after eight-fold dilution; a 1 μL sample of such a solution contained approximately 1.1  108 liposomes.

0.6

Current (µA)

0.5 0.4 0.3 0.2

3.5. Competitive determination of biotin target

0.1

Under the optimized conditions, we performed competitive immunoassays (detection scheme is illustrated in Scheme S1) to quantify the target biotin. In these experiments, the target biotin and liposomal biotin competed for the available binding sites of the anti-biotin antibodies immobilized on electrodes. We varied the target biotin concentration from 10–13 to 10–3 M, performing triplicate analyses using independent electrodes; we used SWV to measure the current signals of the released liposomal FeðCNÞ46  . An increase in the concentration of the target biotin resulted in fewer liposome-labeled biotins binding with the immobilized anti-biotin antibody, leading to a decrease in the peak current (Fig. 4A). In this study, we found competition between sample biotin and biotin-tagged liposomal biolabels could still be observed at sample biotin concentration of 1  10–3 M, while biotin-tagged liposomal competitors was fixed at 1.8  10–10 M, six orders of magnitude lower than biotin, meaning that current signal released from the electrode-bound liposomes was sensible. We suspected that this is due to the multivalency effect (Badjić et al., 2005; Martinez-Veracoechea and Frenkel, 2011; Wang et al., 2010), the simultaneous binding of multiple ligands on one entity to multiple receptors on another. The multivalent biotin-tagged liposome has stronger binding affinity toward anti-biotin antibody than monovalent biotin. Furthermore, the variations in current signal provided a sigmoidally shaped dose–response curve, the linear range of which extended over eight orders of magnitude, ranging from 10–11 to 10–3 M (Fig. 4B). The proposed immunosensor offers excellent sensitivity, with a limit of detection (LOD) of

0.0 0.0

0.1

0.2

0.3

0.4

0.5

[Anti-biotin antibody] (mg/mL) Fig. 3. Effect of anti-biotin antibody concentration on assay performance. Current signals were obtained from various concentrations of anti-biotin antibody-modified T3BA/nanoAu/SPE using SWV. All prepared electrodes were incubated with an excess of biotin-tagged liposomes.

antibody onto the electrodes and let the system react with an excess of the biotin-tagged liposomes prior to signal acquisition. When the antibody concentration was greater than or equal to 0.2 mg mL–1, the current signal was optimized at its equilibrium value (Fig. 3). Therefore, we chose an antibody content of 0.2 mg mL–1 as the optimal concentration for antibody immobilization. Next, we examined the optimal dilution of the liposome preparation for use with the concentration-optimized Ab-modified electrodes. The biotin-tagged liposomes served not only as signal reporters but also as competitors of the sample biotin units for binding with the corresponding antibody; therefore, determining their concentration is critical in the design of liposomal immunoassays. If too few liposomes were present in the reaction solution, incomplete binding would occur, leaving unoccupied paratopes of the antibody and leading to incomplete signal amplification. In contrast, too many liposomes in the reaction solution would make

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Table 1 Relationship between the degree of dilution of the biotin-tagged liposomes and the corresponding electrochemical signal obtained from the experimental (in the presence of 1 mM biotin) and control (in the absence of target biotin) groups. The signal ratio percentage was calculated in terms of the current signal obtained from the experimental group divided by the current obtained from the control group. Biotin-tagged liposome diluted concentration

Control (μA)

Experiment (μA)

Signal ratio percentage

1/2 1/4 1/6 1/8

0.572 ( 7 0.070) 0.597 ( 7 0.058) 0.367 ( 7 0.038) 0.284 (7 0.022)

0.405 (7 0.065) 0.206 ( 70.020) 0.131 ( 7 0.028) 0.040 ( 70.006)

70.72 34.50 35.78 14.11

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the favorable site-orientation of the antibody, leading to the improved LOD for the proposed immunosensor.

4. Conclusions We employed electrochemistry and SPR strategies to probe the orientations of surface-immobilized antibody units using (i) direct adsorption, (ii) gold–thiol linkage, and (iii) boronate affinity approaches. The results obtained using both strategies confirmed that the immunosensors fabricated through the boronate affinity approach exhibited better orientation control than did those prepared through gold–thiol linkage and direct adsorption, validating the hypothesis that appropriate orientation of the antibody units leads to the presentation of more paratopes facing toward the solution. Furthermore an easier and more convenient approach using T3BA as paratope controller to direct paratope orientation and to facilitate the fabrication of immunosensing surface was demonstrated herein. Boronate affinity approach was adopted, taking advantage of boronic acid–saccharide interactions, for the development of an electrochemical immunosensor for biotin. Because no harsh chemical treatment was involved during antibody immobilization, the biological function of the antibody was well preserved. Immunosensors fabricated using various antibody immobilization methods for the detection of biotin are summarized in Table S1 (Supporting information). The boronate affinity approach resulted in the fabrication of an electrochemical immunosensor with excellent diagnostic ability for biotin, with the as-obtained current signal decreasing in a dose-dependent manner with respect to the concentration of the target within the range from 10–11 to 10–3 M —a wide dynamic range of at least eight orders of magnitude—and provided an LOD of 0.61 pg.

Acknowledgment We thank the National Science Council of Taiwan for financial support through Projects 98-2113-M-002–025-MY3, 101-2113-M002–003-MY3, and 100-2113-M-002–016-MY2.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.054. References

Fig. 4. (A) SWV traces acquired using the competitive immunosensor to detect different amounts of the target biotin. (B) Dose–response curve for the biotin target. Each data point represents an average7 1 standard deviation of three replicates. Inset: linear range of the logarithmic calibration graph of the current (μA) plotted with respect to the biotin concentration (M).

0.61 pg (equivalent to 6 μL of 4.19  10–10 M biotin), defined by subtracting three times the standard deviation of the control (free of target biotin) from its average value, which surpasses that of previously developed sensor (Hayakawa et al., 2008; Ho et al., 2009; Kuramitz et al., 2000). Therefore, we suspect that the immobilization of the antibody through boronate affinity assisted

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Improved activity of immobilized antibody by paratope orientation controller: probing paratope orientation by electrochemical strategy and surface plasmon resonance spectroscopy.

Electrochemical method and surface plasmon resonance (SPR) spectroscopic analysis are utilized herein to investigate antibody immobilization without a...
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