sensors Article

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element Özlem Ertekin 1,2, *, Selma Öztürk 1 and Zafer Ziya Öztürk 3 1

2 3

*

˙ TÜBITAK, The Scientific and Technological Research Council of Turkey, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Gebze, 41470 Kocaeli, Turkey; [email protected] Department of Molecular Biology and Genetics, Gebze Technical University, 41400 Kocaeli, Turkey Department of Physics, Gebze Technical University, 41400 Kocaeli, Turkey; [email protected] Correspondence: [email protected]; Tel.: +90-26267-73314

Academic Editor: Alexander Star Received: 15 May 2016; Accepted: 29 July 2016; Published: 11 August 2016

Abstract: This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. Keywords: QCM immunosensor; IgA monoclonal antibody; aflatoxin immobilization; chemical blocking

1. Introduction Aflatoxins (AF) are secondary metabolites of fungi from Aspergillus spp. and can cause acute and chronic toxicity in both humans and animals when ingested [1,2]. They are amongst the most abundant food and feed contaminants, and directives are employed in order to prevent the associated health risks [3]. Internationally accepted precise AF quantification is conducted with laboratory based analytical methods such as HPLC, LC-MS/MS or ELISA, which require expensive, sophisticated equipment and trained staff [1,2]. The availability of rapid and on-site systems for the analysis of AF will both provide better control of AF contamination in food and feedstuff, and also decrease the related analytical costs. Biosensors, which have gained popularity during the past decade, are the most potent solutions towards this goal. A biosensor is defined as a bioanalytical device incorporating a molecular recognition element associated or integrated with a physicochemical transducer [4]. Various studies have been conducted in order to develop AF biosensors. Among these studies, immunosensors are widely preferred since they utilize the specificity, selectivity and affinity of the

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antibodies as sensing elements [5–7]. These properties of the antibodies are particularly noteworthy in order to detect analytes that have high toxicity at low concentrations like AF in complex media such as food matrices [8,9]. As a recognition element, the antibody is in close contact with a transducing element that converts the antigen–antibody binding into quantitative electrical or optical signals in biosensors. We used AF as a sensing layer on Quartz Crystal Microbalance (QCM) transducers, and antibodies as recognition elements in an inhibitory immunoassay format. A high quality antibody is critical in order to achieve a sufficiently low limit of detection in immunoassays. Antibodies delivered to the sensor surface are expected to interact with very small concentrations of the analyte, particularly in studies conducted with toxins such as AFB1, which has a 5 ng/mL maximum allowable legislative limit for most of the foodstuff in Europe [10]. An AF specific high affinity antibody is crucial for the development of an efficient biosensor. In addition, the detection of small molecule analytes such as AF usually requires various labels like enzymes, nanoparticles, or fluorescent molecules in order to increase sensitivity. QCM is a commonly used piezoelectric transducer for immunosensing. It is an extremely sensitive weighing device, which is based on measurement of the change in mechanic resonance of the quartz crystal with changing mass. Quartz crystal (QC) has piezoelectric properties, which, under mechanical stress, produce electrical voltage. On the contrary, when an electric voltage is applied to the crystal, it produces a resonance at a certain frequency, which is affected by the mass on the crystal. According to Sauerbrey’s equation [11] (∆f = ´2f0 2 ∆m/A(ρq µq )1/2 , where ∆f is the counted frequency change (Hz); f0 is the fundamental resonance frequency of the quartz oscillator; ∆m is the mass change; A is the area of the electrode; ρq is quartz density; and µq is the shear stress of quartz), the change in resonant frequency of a QCM is principally based on the mass of adsorbed material on the QCM surface. The shift in resonance frequency (∆f) is proportional to the surface mass (M) of the deposit [8,12]. This mechanism allows label-free detection of analytes. Other label free transducers such as surface plasmon resonance (SPR) sensors rely on optical detection of the molecules on the gold coated sensor chip. Although these systems were proven to be efficient for biosensing, they require relatively more expensive equipment, and the fabrication of sensor strips are more complex and expensive compared to QCM transducers [13]. While working with QCM sensors, structural character of the antibody, which is determined by its isotype, is also an overseen important criterion for signal enrichment. Immunoglobulin G (IgG) antibodies are high affinity antibodies, which are the most abundant isotype in mammals, and accordingly, they are the most easily developed monoclonal antibodies. IgA antibodies are also high affinity antibodies; however, their relatively low abundance make it harder, although possible, to develop as monoclonals. Thus, IgG and IgA antibodies are two notable high affinity choices to be utilized in detection systems. These antibodies differ in their molecular size and the number of antigen binding sites. IgG antibodies have two antigen binding sites with ~150 kDa MW, and IgA has four antigen binding sites with ~340 kDa MW [14]. Higher molecular weight of IgA antibodies can pose a significant advantage while working with QCM sensors, which are actually highly sensitive microbalances. With their higher MW compared to IgG, IgA antibodies are expected to generate a higher frequency shift in QCM transducer systems, which rely on the mass change on the sensor surface, and hence an increase in sensitivity can be expected. QCM based AF sensors utilized signal enhancers such as nanoparticles [10] or magnetic beads [11] and the use of IgA antibodies may compensate this requirement. Despite IgA antibodies being good candidates, there are few reports regarding their production and use in detection systems [15,16], and there are no records of their use in mycotoxin biosensor development yet. Previously known anti-AF antibodies are mostly IgG isotypes [17–19], where a limited number of reports presented monoclonal IgA antibodies [20,21]. However, AF biosensor development literature is based on either polyclonal antibodies [22,23] or monoclonal IgG antibodies [24,25]. Another aspect of antibody choice comprises the affinity of the antibody. Although high affinity antibodies have a major role to improve the limit of detection, they require harsh regeneration

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conditions; such as pH extremes, application of detergents or both. Thus, a sensor surface should be tolerant to different regeneration solutions [26]. Therefore, a properly functionalized surface allowing proper antibody-antigen interaction as well as effective regeneration is crucial. In previous studies, AF was immobilized to the sensor surface via its protein conjugates either by direct adsorption [25,27,28] or using self assembled monolayers (SAM) [29,30] for the development of AF biosensors employing inhibitory immunoassay. However, protein bearing surfaces are labile to harsh regeneration conditions, which may lead to protein denaturation or delocalization preventing successive regeneration cycles. Chemical immobilization of AFB1 to the sensor surface is a means of overcoming challenges related with reproducibility, stability and regeneration. Direct immobilization of AFB1 to a Biacore CM5 sensor chip (Buckinghamshire, UK) was previously described where carboxylated AFB1 derivative AFB1—o carboxymethyloxime (AFB1-oxime) was coupled to amine groups on the CM5 chip [5,31]. Surface preparation not only involves antigen immobilization, but also proper blocking of the surface in order to prevent non-specific adsorption of molecules. As a label free transducer, QCM detects not only specific binding, but also nonspecific binding. Therefore, selective binding and efficient blocking of the prepared sensor surface are crucial to avoid interferences with the original signal. This factor is particularly important for analytes with ng/mL level maximum allowable limits, such as AF. Sensor surfaces prepared by the immobilization of analytes via SAM are usually blocked with a nonspecific protein in order to minimize off-target readings [30,32,33]; however, protein blocking is sensitive to harsh regeneration conditions, and chemical blocking is more stable. In the above-mentioned Surface Plasmon Resonance (SPR) sensors, blocking of reactive carboxyl groups was achieved by ethanolamine [5]; however, blocking of unbound reactive amine groups on the AF immobilized surface was not evaluated. Optimization of the coupling reaction, as well as proper blocking of the sensor surface to prevent nonspecific protein binding is also essential for a good performance. This study is hitherto the first demonstration of the use of IgA isotype monoclonal antibodies in biosensors. We used a high affinity, AF specific IgA isotype monoclonal antibody as a recognition element to develop a QCM immunosensor. We prepared a protein free sensor surface compatible with the harsh conditions required for the regeneration of the surface from this high affinity antibody. As a result, AF was detected in one step, without any need for enrichment, using an IgA monoclonal antibody based inhibitory immunoassay. 2. Materials and Methods 2.1. Materials We used all chemical reagents from Sigma Aldrich, Taufkirchen, Germany with the exception of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Thermo Scientific, Waltham, MA, USA) and Aflatoxin B1 (AFB1) (Fermentek, Jerusalem, Israel). In addition, 5 MHz AT cut quartz crystals with gold-plated electrodes on both sides were purchased from KVG Quartz Crystal Technology GmbH, Neckarbischofsheim, Germany. For the immunoassay, we used in-house developed purified AF-specific murine monoclonal antibodies MAM-D12E2 of IgA isotype and MAM-8G8 of IgG isotype. 2.2. Preparation of the Gold Surface for AF Binding Gold coated quartz crystals were argon plasma cleaned with Diener Femto plasma cleaner (Ebhausen, Germany) for 3 min at 40 mV prior to surface modification. The coating of quartz was carried out under a sterile hood to avoid contamination. Crystals were soaked in the ethanol solution of 2 mM 11-mercaptoundecanoic acid (MUA) overnight at room temperature. Then, 400 mM EDC was mixed with equal volume of 100 mM N-hydroxysuccinimide (NHS) to obtain an EDC/NHS solution. MUA coated surface was activated with EDC/NHS for either 10 or 15 min. Carboxylic acid moieties of MUA were transformed to amine by incubation of activated surface with 1 M ethylenediamine (EDA), pH:8.5 for 7 min followed by blocking with 1 M ethanolamine (EOA) pH:8.5 for 2 min.

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Chemical BlockingofofActivated ActivatedSurfaces Surfaces 2.3.2.3. Chemical Blocking Unboundamine aminegroups groupson onthe the crystal crystal surface surface were in in thethe Unbound were blocked blockedwith withthe thecarboxylic carboxylicacid acid acetate buffer. Blockingefficiencies efficienciesofof60 60mM mM acetate acetate buffer, pH:4.8 were acetate buffer. Blocking buffer, pH:4 pH:4and and11MMacetate acetatebuffer, buffer, pH:4.8 were evaluated. Additionally, 60 mM acetate at pH:4 or pH:7 was activated with EDC/NHS solution for evaluated. Additionally, 60 mM acetate at pH:4 or pH:7 was activated with EDC/NHS solution for min with acetate:EDCratio, ratio,and andused used for for blocking blocking to blocking 10 10 min with 1:51:5 acetate:EDC to test testthe theeffect effectofofactivation activationonon blocking efficiency. The crystals prepared for comparison of 10 or 15 min EDC/NHS activation of MUA prior to efficiency. The crystals prepared for comparison of 10 or 15 min EDC/NHS activation of MUA prior to amine conversion were blocked with 1 M acetate buffer, pH:4.8. All experiments were carried out amine conversion were blocked with 1 M acetate buffer, pH:4.8. All experiments were carried out with with at least three independent replicas. at least three independent replicas. QCM200 Quartz Crystal Microbalance of Stanford Research Systems, Sunnyvale, CA, USA was QCM200 Quartz Crystal Microbalance of Stanford Research Systems, Sunnyvale, CA, USA was used for the measurement of frequency changes. Binding of nonspecific proteins to the prepared the used for the measurement of frequency changes. Binding of nonspecific proteins to the prepared the crystal surface was evaluated by sensor measurements with 0.1 mg/mL Bovine Serum Albumin (BSA) crystal surface was evaluated by sensor measurements with 0.1 mg/mL Bovine Serum Albumin (BSA) in phosphate buffer saline (PBS) (0.1 M KH2PO4, 150 mM NaCl at pH:7.2). The analysis was conducted in phosphate buffer saline (0.1 MofKH PO4 , 150Frequency mM NaClchanges at pH:7.2). The analysis was conducted at room temperature with(PBS) a flow rate 502μL/min. resulting from protein binding at room temperature with a flow rate of 50 µL/min. Frequency changes resulting from protein binding were recorded with in-house developed software. Experiments were conducted with at least 3 were recorded with in-house developed software. Experiments were conducted with at least 3 replicas. replicas.

2.4.2.4. AFB1 Immobilization AFB1 Immobilizationtotothe theQuartz QuartzCrystal Crystal ForFor thethe experiment, mg carboxymethyl carboxymethylhydroxylamine hydroxylamine HCl experiment,1 1mg mgAFB1 AFB1in in400 400 µL μL methanol, methanol, 22 mg HCl ˝ C for 3 h (CMO) in 100 µL dH O and 100 µL pyridine were mixed. The reaction proceeded at 100 2 2O and 100 μL pyridine were mixed. The reaction proceeded at 100 °C for 3 h (CMO) in 100 μL dH followed byby 2424 hh incubation wereevaporated evaporatedjust justtotodryness dryness under followed incubationatatroom roomtemperature. temperature. Solvents Solvents were under gentle N2Nflow [34]. inPBS PBStotoget getaa1010mM mMconcentration. concentration. gentle 2 flow [34].Resulting ResultingAFB1-oxime AFB1-oxime was was dissolved dissolved in In In addition, solutionforfor1010min min with addition,1010mM mMAFB1-Oxime AFB1-Oxime was was activated activated by EDC/NHS EDC/NHS solution with 1:51:5 AFB1-oxime:EDC ratio. Activated AFB1-oximewas wasincubated incubatedononthe theamine aminefunctionalized functionalizedsurface surfacefor AFB1-oxime:EDC ratio. Activated AFB1-oxime 15 The min.surface The surface blocked with 1M acetatebuffer, buffer,pH:4.8 pH:4.8for for 10 min. 15 for min. waswas blocked with 1M acetate min.The TheAF AFimmobilization immobilization strategy presented in the current study is summarized in Figure 1. strategy presented in the current study is summarized in Figure 1.

Figure 1. 1. Schematic illustration of AF immobilization strategy to gold coated crystalcrystal surface. Figure Schematic illustration of AF immobilization strategy to gold coated surface.

AFB1 MeasurementProcedure Procedure 2.5.2.5. AFB1 Measurement AF specific IgA isotype MAM-D12E2 (IC50AFB1: 0.86 ng/mL) and IgG isotype MAM-8G8 AF specific IgA isotype MAM-D12E2 (IC50AFB1 : 0.86 ng/mL) and IgG isotype MAM-8G8 (IC50AFB1: 0.86 ng/mL) antibodies were used in inhibitory immunoassay with the prepared crystals. (IC50AFB1 : 0.86 ng/mL) antibodies were used in inhibitory immunoassay with the prepared crystals. 0.1 mg/mL purified MAM-D12E2 or MAM-8G8 antibody was competitively interacted with changing 0.1 mg/mL purified MAM-D12E2 or MAM-8G8 antibody was competitively interacted with changing concentrations of AFB1 in PBS for comparative evaluation. MAM-D12E2 antibody was used for the concentrations of AFB1 in PBS for comparative evaluation. MAM-D12E2 antibody was used for the establishment of standard curve for AFB1 in PBS. Antibody-AFB1 mixtures were delivered to the establishment of standard curve for AFB1 in PBS. Antibody-AFB1 mixtures were delivered to the

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prepared sensor surface at room temperature with 50 µL/min flow rate. Differential frequency shifts resulting from competition of free AFB1 at different concentrations, and surface immobilized AF to bind the antibody were evaluated. The x-axis of the sensogams were normalized for alignment using BIAevaluation Software Version 4.1 (Biacore, Buckinghamshire, UK). The experiments were conducted with at least 4 replicas. Limit of detection (LOD) for the developed sensor was calculated with the formula LOD = (k * sb)/m, where k = 2 for 92.1 confident level, sb is standard deviation of blank, and m is the slope of the linear response curve [35]. 2.6. Surface Regeneration Different solutions were used for the regeneration of MAM-D12E2 from sensor surface. HPLC grade water, 0.1 M HCl, 50% Methanol, 50 mM NaOH + 30% ACN, 100 mM Glycine HCl, pH:2, 50 mM NaOH + 0.5% SDS, 50 mM NaOH + 1% SDS and 1.2 mM NaOH + 30 mM EDTA were injected to the sensor surface after antibody binding with 50 µL/min flow rate. Regeneration efficiencies were evaluated by frequency changes in PBS. 2.7. Safety Considerations AFB1 is a class-1 human carcinogen. Therefore, all experiments should be conducted at a biosafety level-2 laboratory with proper protection. Preparation of AF sensors requires extensive handling of the toxin. Liquid waste created during the process was incubated overnight in 1% hypochloride solution and sent to incineration. AF immobilized crystals were cleaned with 1:1:5 solution of hydrogen peroxide (30%), ammonia (25%) and deionized water heated to a temperature of about 75 ˝ C for 3 min [36] and treated with argon plasma cleaner for 3 min at 40 mV prior to use. 3. Results and Discussion 3.1. Preparation of SAM Functionalized Surface and Blocking Optimization of Free Amine Groups Reactive groups were introduced to the gold surface by the use of 11-MUA SAM. Carboxylic acid groups of 11-MUA were converted to amine in order to provide binding sites for carboxyl bearing AFB1-Oxime. The reaction involved activation of the carboxyl groups with EDC and NHS prior to the binding of EDA. The surface was activated with EDC/NHS solution for either 10 or 15 min to evaluate the effect of duration on nonspecific binding of proteins to the prepared sensor surface. The frequency changes upon application of 0.1 mg/mL BSA were 4 Hz on 10 min of activated surface, and 9.75 Hz on 15 min activated surface (Table 1). It was shown that EDC/NHS solution, which was used for activation of the carboxyl bearing surface, was incubated on the crystal for 15 min instead of 10 min, nonspecific binding increased two fold. Table 1. Effect of different durations of EDC/NHS activation on non-specific protein binding to the MUA coated QCM crystals. EDC/NHS Application Time (min)

Frequency Change upon 0.1 mg/mL BSA Application (Hz)

10 15

4.0 ˘ 1.4 9.75 ˘ 1.8

EDC/NHS coupling chemistry is one of the most frequently used immobilization methods due to its high coupling yields and robustness. In this reaction, carboxyl groups form an NHS-ester intermediate, which will, in turn, react with the amine groups of the ligand (Figure 1). Despite its high efficiency, this coupling method may result in side-reactions such as esterification of hydroxyl groups as well as cross-activation of ligand carboxyl groups and buffer components with carboxyl groups [37]. Furthermore, NHS-ester intermediate is stable up to several hours at pH:4–5 [38], where our working pH is 4.8 to increase efficiency. One to twenty minutes of activation is recommended for the carboxyl

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bearing sensor surface, and the duration is a factor affecting the activation level [37]. Limiting the duration ofthe activation decreasesboth thedecreases possibility ofpossibility side-reactions, and prevents the formation Limiting durationboth of activation the of side-reactions, and prevents the of excess number of NHS-esters, may lead to nonspecific formation of excess number ofwhich NHS-esters, which may lead tobinding. nonspecific binding. Free amine wereblocked blockedwith withcarboxylic carboxylic acid acetate buffer. Free aminegroups groupson onthe theprepared prepared surface surface were acid in in acetate buffer. Frequency BSA solution solution was wasused usedtotodemonstrate demonstratethethe blocking Frequencyshift shiftresulting resultingfrom from0.1 0.1 mg/mL mg/mL BSA blocking efficiency surface. Amine activated surfacewas wastreated treatedwith with6060mM mMacetate acetatebuffer, buffer,pH:4 pH:4oror efficiency of of thethe surface. Amine activated surface 1M 1 M acetate pH:4.8 to evaluate blocking efficiencies. Additionally,carboxylic carboxylicacid acid groups groups of acetate buffer,buffer, pH:4.8 to evaluate theirtheir blocking efficiencies. Additionally, 60 mM acetate buffer activated with EDC/NHS, andused usedasasblocking blockingagent. agent. 60 of mM acetate buffer werewere activated with EDC/NHS, and Figure 2 presents nonspecific protein binding after different blocking conditions. In control Figure 2 presents nonspecific protein binding after different blocking conditions. In control crystal, surface many unblocked highly reactive amine groups. Application of acetate thecrystal, surfacethe bears manybears unblocked highly reactive amine groups. Application of acetate buffer, either buffer, either 60 mM, pH:4 or 1 M pH:4.8, significantly reduced nonspecific binding to the crystals. 60 mM, pH:4 or 1 M pH:4.8, significantly reduced nonspecific binding to the crystals. Nevertheless, Nevertheless, when a carboxyl group of acetate was activated with EDC/NHS solution in order to when a carboxyl group of acetate was activated with EDC/NHS solution in order to improve the improve the blocking efficiency, the blocking property of the solution completely vanished. This blocking efficiency, the blocking property of the solution completely vanished. This result was strongly result was strongly correlated with the previous finding that the duration of EDC/NHS activation of correlated with the previous finding that the duration of EDC/NHS activation of MUA increased the MUA increased the nonspecific reactivity of the surface. We consider the reason for the extreme nonspecific reactivity of the surface. We consider the reason for the extreme reactivity is the retreatment reactivity is the retreatment of the surface with EDC/NHS solution. of the surface with EDC/NHS solution.

Figure 2. 2. Blocking surface.Non-specific Non-specificprotein protein binding sensor Figure Blockingoptimization optimizationon onamine amine activated activated surface. binding to to sensor surface after 60 mM mM acetate acetatebuffer, buffer,pH:4 pH:4ororEDC/NHS EDC/NHS surface afterblocking blockingwith with11M Macetate acetate buffer, buffer, pH:4.8, pH:4.8, 60 activated 6060 mM errors. activated mMacetate acetatebuffer. buffer.Error Errorbars barsrepresent represent standard standard errors.

Applicationofofactivated activatedacetate acetate can can be be considered considered as binding, so so wewe Application asaasimulation simulationofofAF-oxime AF-oxime binding, cannot avoid the exposure of the surface to EDC/NHS solution. Considering the better cannot avoid the exposure of the surface to EDC/NHS solution. Considering the better reproducibility reproducibility and stability of the surface blocked with 1 M Acetate buffer, pH:4.8, this condition and stability of the surface blocked with 1 M Acetate buffer, pH:4.8, this condition was chosen for was chosen for blocking of AFB1-oxime bound crystals. blocking of AFB1-oxime bound crystals. InhibitoryImmunoassay ImmunoassayUsing UsingIgA IgAor or IgG IgG Antibodies Antibodies 3.2.3.2. Inhibitory Changing concentrations of AFB1 (5 ng/mL–40 ng/mL) was mixed with equal volume of 0.1 Changing concentrations of AFB1 (5 ng/mL–40 ng/mL) was mixed with equal volume of mg/mL IgA isotype MAM-D12E2 antibody or IgG isotype MAM-8G8 antibody with similar affinity 0.1 mg/mL IgA isotype MAM-D12E2 antibody or IgG isotype MAM-8G8 antibody with similar to AFB1. The mixture was delivered to the AF immobilized sensor surface, and frequency shifts were affinity to AFB1. The mixture was delivered to the AF immobilized sensor surface, and frequency recorded (Figure 3). Maximal frequency shifts observed in control samples with no AFB1 competition shifts were recorded (Figure 3). Maximal frequency shifts observed in control samples with no AFB1 were different in two antibodies as an indication of antibody structure. IgG isotype antibody (150 competition were32 different two antibodies as an indication of antibody structure. IgG isotype kDa) produced Hz and in IgA isotype MAM-D12E2 antibody (340 kDa) produced 55 Hz control antibody (150 kDa) produced 32 Hz and IgA isotype MAM-D12E2 antibody (340 kDa) produced 55 Hz sensor response. When we examined the frequency changes in AFB1 competition samples with control sensor response. When we examined the frequency changes in AFB1 competition samples with respect to control samples, we showed that higher control sensor response was correlated with respect to sensor controlsignals samples, we 2). showed that higher control sensor was correlated with with greater greater (Table MAM-D12E2 showed three timesresponse as much frequency difference

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sensor signals (Table 2). MAM-D12E2 showed three times as much frequency difference with respect Sensors 2016, 16, 1274 of 12 to control ng/mL AFB1 competition, and twice much difference at 407 at ng/mL respect at to 5–20 control at 5–20 ng/mL AFB1 competition, andastwice as frequency much frequency difference 40 AFB1 competition when compared to MAM-8G8. The sensor was developed using the IgAthe antibody ng/mL AFB1 competition when compared to MAM-8G8. The sensor was developed using IgA respect to control at 5–20 ng/mL AFB1 competition, and twice as much frequency difference at 40 dueantibody to its broader competition range. to MAM-8G8. due competition toAFB1 its broader AFB1compared competition range. ng/mL AFB1 when The sensor was developed using the IgA antibody due to its broader AFB1 competition range.

Figure 3. Inhibitory AFB1 analysis with MAM-8G8 and MAM-D12E2 antibodies using AFB1

Figure 3. Inhibitory AFB1 analysis with MAM-8G8 and MAM-D12E2 antibodies using AFB1 immobilized sensor chip (0–40 ng/mL AFB1). Error bars and represent the standard errors. using AFB1 Figure 3. Inhibitory AFB1 analysis with MAM-8G8 MAM-D12E2 antibodies immobilized sensor chip (0–40 ng/mL AFB1). Error bars represent the standard errors.

immobilized sensor chip (0–40 ng/mL AFB1). Error bars represent the standard errors. Table 2. The frequency difference between control and competition samples for MAM-8G8 and Table 2. The frequency difference between control and competition samples for MAM-8G8 MAM-D12E2. Table 2. The frequency difference between control and competition samples for MAM-8G8 and

and MAM-D12E2. MAM-D12E2. AFB1 MAM-8G8 MAM-D12E2 Fold Difference (ng/mL) (0.1 mg/mL) MAM-D12E2 (0.1 mg/mL) (MAM-D12E2/MAM-8G8) AFB1 MAM-8G8 Fold AFB1 MAM-8G8 MAM-D12E2 FoldDifference Difference (ng/mL) (0.1 mg/mL) (0.1 mg/mL) (MAM-D12E2/MAM-8G8) (ng/mL) (0.1 mg/mL) (0.1 mg/mL) (MAM-D12E2/MAM-8G8) 5 3.2 Hz 9.4 Hz 3.0 5 3.2 Hz Hz 9.4 3.0 3.5 10.2 2.9 510 3.2 9.4Hz Hz 3.0 10 3.5 10.2Hz Hz 2.9 10 3.5 Hz Hz 10.2 2.9 20 3.8 11.7 Hz 3.0 20 3.8 11.7 Hz 3.0 20 3.8 Hz Hz 11.7 3.0 40 8.5 18.1 Hz Hz 2.1 40 8.5 Hz 18.1 Hz 2.1 40 8.5 Hz 18.1 Hz 2.1 3.3. Optimization of Sensor Surface Regeneration 3.3. 3.3. Optimization of Sensor Surface Optimization of Sensor SurfaceRegeneration Regeneration Recovery of MAM-D12E2 antibody from sensor surface using different regeneration solutions ofinMAM-D12E2 antibody from surface different regeneration solutions Recovery of MAM-D12E2 antibody fromsensor sensor surface using different regeneration solutions isRecovery presented Figure 4. MAM-D12E2 antibody binding can using be fully recovered with 50 mM NaOH is presented Figure 4.antibody MAM-D12E2 antibody binding can besolutions fully recovered with 5050mM NaOH is presented in Figure 4. MAM-D12E2 antibody binding can be fully recovered withionic mM NaOH and 1%inSDS. The could not be regenerated using that disrupt bonds, butand 1% SDS. TheSDS. antibody could not regenerated using solutions that ionic bonds, but efficiently and 1% The antibody could notionic be regenerated usingresult solutions disrupt ionic bonds, but efficiently regenerated with a be strong detergent. This is disrupt thethat indication of a hydrophobic efficientlywith regenerated with adetergent. strong detergent. is the of indication of a hydrophobic regenerated a strong ionic This result isThis the result indication a hydrophobic interaction of interaction of the antibody with AF. ionic interaction of the antibody with AF. the antibody with AF.

Figure 4. Regeneration of AFB1 immobilized sensor surface after MAM-D12E2 application using Figure 4. solutions. Regeneration AFB1 immobilized sensor surface MAM-D12E2 application using Figure 4. Regeneration ofofAFB1 sensor surfaceafter after MAM-D12E2 application using different Results are immobilized presented as the percentage of the antibody recovered from the different solutions. Results are presented as the percentage of the antibody recovered from the surface, calculated with binding and regeneration frequency different solutions. Results are presented as the percentage ofshifts. the antibody recovered from the surface, surface,with calculated with binding and regeneration frequency calculated binding and regeneration frequency shifts. shifts.

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Reproducibility of regeneration MAM-D12E2 Reproducibility regeneration was was tested tested with with sequential sequential injections injections of of 0.1 0.1 mg/mL mg/mL MAM-D12E2 The surface surface could could be be regenerated regenerated at at least nine times without antibody to the regenerated surface. The 5).5). Similar surfaces were shown to betoregenerated up toup 75 times significant loss lossof ofperformance performance(Figure (Figure Similar surfaces were shown be regenerated to 75 with that can regenerated at milder conditions such such as 10as mM application [31]. timesantibodies with antibodies thatbecan be regenerated at milder conditions 10 NaOH mM NaOH application However, MAM-D12E2 is a very high affinity antibody that can only be regenerated using strong [31]. However, MAM-D12E2 is a very high affinity antibody that can only be regenerated using ionic detergents in addition to high pH, andpH, at least sequential regenerations could be achieved strong ionic detergents in addition to high andnine at least nine sequential regenerations could be through the use of the surface.sensor We observed that theobserved number ofthat regeneration cycles achieved through theprotein-free use of thesensor protein-free surface. We the number of could be improved the surface water after the application regeneration solution, regeneration cycleswhen couldwe be wash improved when with we wash the surface with waterofafter the application of before PBS injection. regeneration solution, before PBS injection.

Figure 5. Regeneration Regeneration of AF immobilized immobilized sensor Figure 5. of AF sensor surface. surface. (A) (A) Sequential Sequential regeneration regeneration of of the the sensor sensor surface SDS. Graph Graph shows shows sensor sensor response response as as frequency frequency change change upon upon surface with with 50 50 mM mM NaOH NaOH and and 1% 1% SDS. delivery of0.1 0.1mg/mL mg/mLMAM-D12E2 MAM-D12E2 antibody after regeneration; (B) Sensogram showing a delivery of antibody after eacheach regeneration; (B) Sensogram showing a typical typical regeneration cycle with MAM-D12E2 antibody. regeneration cycle with MAM-D12E2 antibody.

3.4. 3.4. QCM QCM Based Based Detection Detection of of AFB1 AFB1 After After the the determination determination of of the the antibody antibody to to be be used used as as aa sensing sensing element element and and optimization optimization of of the the assay conditions including regeneration and competition, we generated a standard curve for AFB1 assay conditions including regeneration and competition, we generated a standard curve for AFB1 detection antibody. In In the the work work conducted conducted for for the detection using using MAM-D12E2 MAM-D12E2 antibody. the choice choice of of sensing sensing element, element, high high concentrations be able able to to evaluate evaluate the the working working competition competition range range concentrations of of AFB1 AFB1 were were chosen chosen in in order order to to be for for each each antibody. antibody. The The sensograms sensograms showing showing the the frequency frequency shifts shifts upon upon application application of of different different AFB1 AFB1 concentrations in inhibitory immunoassays is presented in Figure 6A. Dose response curve concentrations in inhibitory immunoassays is presented in Figure 6A. Dose response curve showing showing the is shown the sensor sensor response response to to different different AFB1 AFB1 concentrations concentrations is shown in in Figure Figure 6B. 6B. The The results results show show that that the the surface is responsive to the inhibition assay conducted with IgA isotype MAM-D12E2 antibody surface is responsive to the inhibition assay conducted with IgA isotype MAM-D12E2 antibody and and AFB1. limit of of detection detection was was shown shownto tobe be1.625 1.625ng/mL ng/mLwith with1.25 1.25ng/mL–10 ng/mL–10 ng/mL ng/mL detection detection AFB1. The The limit 2 range. (R(R=20.998). Sensitivity calculated from the range. The Thelinear linearrange rangeof ofthe thecurve curvewas was0.625–2.5 0.625–2.5ng/mL ng/mL = 0.998). Sensitivity calculated from slope of the linear curve indicated a 9.6% change for 1 ng/mL AF. We presume two possible causes the slope of the linear curve indicated a 9.6% change for 1 ng/mL AF. We presume two possible about source of variation in theindata. The The initial reason forfor this variation causes the about the source of variation the data. initial reason this variationmay maybe bethe thelack lack of of temperature control of the experimental setup, where temperature fluctuations affect A/T cut crystals temperature control of the experimental setup, where temperature fluctuations affect A/T cut crystals 88 Hz/1 ˝ Cin Hz/1°C inaqueous aqueousphase phase[39]. [39].The Thesecond secondreason reasonwe wepropose propose is is the the fact fact that that the the data data could could not not be be normalized according to the nonspecific binding since the experiments were conducted in a single normalized according to the nonspecific binding since the experiments were conducted in a single oscillator allow parallel parallel experiments. experiments. oscillator device device that that did did not not allow Different maximum allowable AF levels Different maximum allowable AF levels for for different different feed feed products products established established by by different different countries and organizations. The lowest maximum allowable AFB1 levels set by EU go down countries and organizations. The lowest maximum allowable AFB1 levels set by EU go down to to 22 µg/kg μg/kg for for products products intended intended for for the the consumption consumption of of infants. infants. The The highest highest limits limits are are applied applied for for foodstuff as pistachio pistachio and and almonds, almonds, which which is is 12 12 µg/kg μg/kg [3]. foodstuff such such as [3]. Therefore, Therefore, the the developed developed sensor sensor has has the potential to be used for the detection of AF in foodstuff. the potential to be used for the detection of AF in foodstuff.

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Figure6.6.AFB1 AFB1sensor sensorresponse responsewith withIgA IgAantibody antibody(A) (A)Sensograms Sensogramsshowing showingthe thesensor sensorresponse responseatat Figure differentconcentrations concentrationsofofAFB1; AFB1;(B) (B)Sensor Sensorresponse responsecurve curvefor forAFB1 AFB1detection. detection.Error Errorbars barsrepresent represent different standard errors. standard errors.

To the authors’ knowledge, there are two previous studies using AF immobilized surface in To the authors’ knowledge, there are two previous studies using AF immobilized surface in QCM immunosensor development for AF detection, both of which utilized murine monoclonal IgG QCM immunosensor development for AF detection, both of which utilized murine monoclonal antibodies. In these studies, AFB1-BSA conjugate was immobilized to the gold surface, and detection IgG antibodies. In these studies, AFB1-BSA conjugate was immobilized to the gold surface, and required the use of secondary antibodies. The first study used an enzyme labeled secondary antibody detection required the use of secondary antibodies. The first study used an enzyme labeled secondary for electrochemical QCM measurement, and had a detection range of 0.01–10 ng/mL [30]. The second antibody for electrochemical QCM measurement, and had a detection range of 0.01–10 ng/mL [30]. study used gold nanoparticle labeled secondary antibodies, and achieved a detection range of 0.1– The second study used gold nanoparticle labeled secondary antibodies, and achieved a detection 100 ng/mL [40]. Other work employing a competitive immunoassay strategy used either monoclonal range of 0.1–100 ng/mL [40]. Other work employing a competitive immunoassay strategy used IgG antibodies or polyclonal antibodies, and utilized optical or electrochemical transducers. Among either monoclonal IgG antibodies or polyclonal antibodies, and utilized optical or electrochemical the label-free optical sensors developed with the same strategy, there are two SPR studies using a transducers. Among the label-free optical sensors developed with the same strategy, there are two SPR similar surface preparation approach. One study used monoclonal IgG antibodies, and could achieve studies using a similar surface preparation approach. One study used monoclonal IgG antibodies, and a detection range of 0.2–10 ng/g with prior concentration of AFs using IACs [5], and the other used could achieve a detection range of 0.2–10 ng/g with prior concentration of AFs using IACs [5], and the multimeric recombinant antibody fragments, and had 0.2–24 ng/mL detection range [31]. Other other used multimeric recombinant antibody fragments, and had 0.2–24 ng/mL detection range [31]. optical AF sensors utilized either fluorescent [25,41] or chemiluminescent [27] labels for limits of Other optical AF sensors utilized either fluorescent [25,41] or chemiluminescent [27] labels for limits detection ranging from 0.1 to 1.5 ng/mL. Therefore, the present study was the first development of of detection ranging from 0.1 to 1.5 ng/mL. Therefore, the present study was the first development competitive AF immunosensor development without using any kind of labels or tags, which was of competitive AF immunosensor development without using any kind of labels or tags, which was possible with the use of an IgA isotype antibody. possible with the use of an IgA isotype antibody. 4. Conclusions 4. Conclusions AF was immobilized on to gold coated QCM crystal functionalized via SAM as sensing layer for AF was immobilized on to gold coated QCM crystal functionalized via SAM as sensing layer the direct determination of AF in samples. Our results indicated that nonspecific reactivity of the for the direct determination of AF in samples. Our results indicated that nonspecific reactivity of surface increases with increasing duration of EDC/NHS application to the surface. EDC/NHS the surface increases with increasing duration of EDC/NHS application to the surface. EDC/NHS activation is commonly used in sensor development studies, and the duration of activation should activation is commonly used in sensor development studies, and the duration of activation should carefully be optimized to obtain minimal exposure of the surface. On the other hand, nonspecific carefully be optimized to obtain minimal exposure of the surface. On the other hand, nonspecific binding to the surface can be minimized by careful choice of blocking reagents. We used a carboxyl binding to the surface can be minimized by careful choice of blocking reagents. We used a carboxyl group bearing acetate buffer to block unreacted amine groups on the surface. Our results showed group bearing acetate buffer to block unreacted amine groups on the surface. Our results showed that that 1 M acetate buffer, pH:4.8 provided a good blocking efficiency. The prepared surface was 1 M acetate buffer, pH:4.8 provided a good blocking efficiency. The prepared surface was regenerated regenerated for at least nine sequential analyses with strong detergent containing regeneration for at least nine sequential analyses with strong detergent containing regeneration solution, which solution, which would disrupt conventionally used protein containing surfaces. would disrupt conventionally used protein containing surfaces. We developed a QCM immunosensor for AFB1 detection using monoclonal IgA antibody, and We developed a QCM immunosensor for AFB1 detection using monoclonal IgA antibody, and showed its advantage over a conventional AF specific IgG antibody with similar affinity to AFB1. In showed its advantage over a conventional AF specific IgG antibody with similar affinity to AFB1. this hitherto first study of sensor development with IgA antibodies, we compared frequency shifts In this hitherto first study of sensor development with IgA antibodies, we compared frequency shifts observed upon AFB1 competition for both antibodies. A three times wider working frequency range observed upon AFB1 competition for both antibodies. A three times wider working frequency range was observed with MAM-D12E2, a monoclonal IgA antibody which has twice molecular weight was observed with MAM-D12E2, a monoclonal IgA antibody which has twice molecular weight when when compared to IgG isotype MAM-8G8, in the inhibitory immunoassay. In the immunoassay compared to IgG isotype MAM-8G8, in the inhibitory immunoassay. In the immunoassay development, development, we did not use any labels like magnetic beads or dendrimeric material for signal we did not use any labels like magnetic beads or dendrimeric material for signal enrichment. AF was enrichment. AF was detected with monoclonal IgA antibody based inhibitory immunoassay in only one step. We showed that higher molecular weight of IgA antibodies contributed to a higher

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detected with monoclonal IgA antibody based inhibitory immunoassay in only one step. We showed that higher molecular weight of IgA antibodies contributed a higher sensitivity, and provided sensitivity, and provided a natural signal enrichment fortoQCM immunosensor development a(Figure natural7).signal enrichment for QCM immunosensor development (Figure 7).

Figure 7. 7. Use Use of of different different antibody antibody isotypes isotypes in in sensor sensor development. development. Figure

The results showed that the IgA antibodies provide sufficient affinity and signal enrichment in results showed that the IgAand antibodies provide sufficient affinity and signal target enrichment in QCMThe immunosensor development, this concept can be implemented for different analytes QCM immunosensor development, and this concept can be implemented for different target analytes for the development of mass sensitive sensors. for the development of mass sensitive sensors. Acknowledgments: We are grateful to Dr. Esin Aslankaraoğu Akçael and Şerife Şeyda Pirinçci who kindly provided MAM-8G8We antibody and Harun for his technical assistance. This work was supported by Acknowledgments: are grateful to Dr. Kocaağa Esin Aslankarao gu ˘ Akçael and S¸ erife S¸ eyda Pirinçci who kindly provided antibody and and Harun Kocaaga ˘ Marmara for his technical Thisnumber work was supported by TÜBİTAKMAM-8G8 project number 110G139 TÜBİTAK Research assistance. Center project 5133101. ˙ ˙ TÜBITAK project number 110G139 and TÜBITAK Marmara Research Center project number 5133101. Author Contributions: Özlem Ertekin, Selma Öztürk and Zafer Ziya Öztürk conceived and designed the Author Contributions: Özlem Ertekin, the Selma Öztürk and Zafer Ziya Öztürk conceived the experiments. Özlem Ertekin performed experiments. Özlem Ertekin, Selma Öztürk and and Zaferdesigned Ziya Öztürk experiments. Özlem Ertekin performed the experiments. Özlem Ertekin, Selma Öztürk and Zafer Ziya Öztürk analyzed the data and wrote the paper. analyzed the data and wrote the paper. Conflictsof ofInterest: Interest: The The authors authorsdeclare declareno noconflict conflictof ofinterest. interest. Conflicts

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Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal M...
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