Talanta 140 (2015) 183–188

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Chemiluminescence immunoassay using magnetic nanoparticles with targeted inhibition for the determination of ochratoxin A Sumin Kim, H.B. Lim n Department of Chemistry, Dankook University, Yongin-si, Gyeonggi-do, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 29 January 2015 Received in revised form 19 March 2015 Accepted 21 March 2015 Available online 30 March 2015

In this work, a chemiluminescence (CL) immunoassay with targeted inhibition was developed for the determination of toxins in food products. For sample treatment, amine-functionalized magnetic nanoparticles (MNPs) were synthesized to extract target molecules, and horseradish peroxidase (HRP) tagged on an antibody was used as a label for CL reaction. In particular, amine-targeted inhibition using aldehyde, i.e., specifically capping the amine with an alkyl group, was developed for a non-specific extraction platform to lower background and improve signal-to-background ratio. For demonstration, ochratoxin A (OTA) was determined in rice using a lab-built drop-type chemiluminescence (DCL) system with luminol–H2O2 reagent. The obtained limit of detection was 1.39 pg mL  1, which was about 7.3 times better than that of ELISA. Recovery of the method in the range of 87–99% was observed, which was compared with ELISA. & 2015 Elsevier B.V. All rights reserved.

Keywords: Determination of ochrotoxin A Chemiluminescence Magnetic nanoparticles Inhibition Immunoassay

1. Introduction Recently, various detection methods combined with nanotech nologies have shown potential, and promise to advance bioassay to higher levels. Among them, sample preparation platforms through immunoreaction using nanoparticles have been successfully utilized for target extraction and tagging, based on non-specific and sandwich-type. Both platforms often employ surface modified magnetic nanoparticles (MNPs) for simple separation and purification [1,2], and several types of nanoparticles as a detection probe, i.e., quantum dots [3,4] and dye-doped core–shell silica nanoparticles for fluorescence [5,6], gold or silver nanoparticles for Raman spectrometry [7,8], and metal-doped nanoparticles for ICP-MS [9–11]. When the nanoparticles were combined with immunoassay, these methods revealed unique merits of sensitivity and selectivity owing to signal amplification and selective immunoreaction. While a sandwich-type platform has intensively been used for biomarkers (28–200 kDa), a nonspecific extraction platform was preferred for the determination of small antibiotics (about o1 kDa), such as enrofloxacin and salinomycin, in which the targets were directly bound to MNPs through formation of an amide bond [5,6]. Compared to the sandwich-type, the non-specific platform has a relative risk of high background due to non-specific adsorption or binding of proteins, which can be critical in the determination of bio-targets requiring high sensitivity. In order n

Corresponding author. Tel.: þ 82 31 8005 3151. E-mail address: [email protected] (H.B. Lim).

http://dx.doi.org/10.1016/j.talanta.2015.03.044 0039-9140/& 2015 Elsevier B.V. All rights reserved.

to reduce the background in immunoassay, typical samples are treated to remove the fats and proteins prior to magnetic extraction. In addition, functional groups on the MNPs are blocked using BSA or skimmed milk after the extraction. Such inhibition proved greatly effective for relatively large molecules, including biomarkers and cells, but not for small molecules such as antibiotics. It is likely that the inhibitors, generally much larger than the antibiotics, weakened the activity of the small target molecules to react with the incoming antibodies in immunoreaction. For this reason, along with incomplete matrix removal, inhibition has rarely been employed in non-specific platforms. Therefore, platforms for the determination of small molecules like toxins require a new type of inhibition method for suppression of the background. In this work, a targeted inhibition technique using an aldehyde compound, propanal, was developed for specific capping of amine groups, not only on the MNPs, but also on the non-specifically adsorbed molecules. This targeted inhibition of amine capping with alkyl groups can alleviate the interaction forces of the MNP surface with incoming antibodies through change of the chemical and physical properties, including H-bonding and polarity. For demonstration, a toxin in food products was determined by chemiliminescence (CL) immunoassay with the non-specific platform, in which chemiliminescence from horseradish peroxidase (HRP) tagged on a secondary antibody was detected by a lab-built drop-type chemiluminescence (DCL) system [12]. Even though the probe signal was not amplified, the DCL system revealed excellent sensitivity with small sample consumption owing to inherently low spectral

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background, and its analytical performance has already been proven in various applications of the semiconductor industry. As a target, detection of ochratoxin A (OTA) in a rice sample was selected because it shows severe toxicity to humans as a carcinogen and neurotoxin, which caused the European Commission to establish concentration limits of OTA in several goods. Due to its relatively high stability, OTA can resist food processing operations such as cooking, roasting and fermenting [13,14]. Because it may be present in all kinds of raw agricultural materials, including commodities and beverages [15,16], the detection of OTA is of great importance for food safety and to prevent the risk of human exposure. Currently, the most sensitive analytical techniques is liquid chromatography– mass spectrometry (LC–MS), for which the reported limit of detection (LOD) was in the range of 1 pg mL  1 to 0.5 ng mL  1 [17–21]. Since the DCL system used in this work has portability with small sample consumption [22], the analytical results, including recovery, were compared with those of ELISA for method validation.

2. Experimental 2.1. Experimental schematic for the detection of ochratoxin A using DCL A schematic diagram of the experimental procedure for the determination of OTA using DCL is displayed in Fig. 1. For sample treatment, the OTA target was extracted using amine-functionalized MNPs, and the unreacted amine groups were capped for inhibition (step 1). Then, OTA was tagged with antibody (step 2), followed by immunoreaction with HRP-tagged antibody to the primary antibody for CL detection (step 3). Finally, the concentration of OTA was determined by measuring CL through a luminol–H2O2 system catalyzed by HRP. 2.2. Synthesis of amine-functionalized magnetic nanoparticles (MNPs) For the magnetic extraction of OTA target, Fe3O4 MNPs were synthesized by the co-precipitation method, and the surface was

modified as described in our previous articles [1,10]. Firstly, 1.35 g of FeCl3  6H2O (Iron(III) chloride hexahydrate, 97%, Sigma-Aldrich, Korea) and 0.5 g of FeCl2  4H2O (Iron(II) chloride tetrahydrate, 99%, Sigma-Aldrich, Korea) were dissolved in 25 mL of deionized water (DW, 18.2 MΩ, Millipore-Q, USA), and the mixture was heated to 80 °C under Ar gas flow. Next, 12.5 mL of ammonia hydroxide (28– 30%, Sigma-Aldrich, Korea) was added slowly until the solution turned black. After stirring for 20 min at 350 rpm, the Fe3O4 MNPs formed were washed three times with DIW and ethanol, and then stored in anhydrous ethanol (99.9%, Duksan, Korea). For the silica coating, 350 mg of the synthesized MNPs was mixed with 520 μL of ammonia and DIW, and then 3.3 mL of TEOS (Tetraethyl orthosilicate, 98%, Sigma-Aldrich, Korea) was added. Dark brown silica-coated MNPs were formed by heating the solution to 40 °C for 30 min and to 70 °C for 60 min under Ar gas, after which they were collected using a permanent magnet for washing three times with DIW and anhydrous ethanol, alternatively. They were then stored in anhydrous ethanol until the next step. In order to functionalize the amine groups, 50 mg of MNPs was dispersed in a mixture of 10 mL DMF (dimethylformamide, extra pure, Acros, Korea) and 5 mL toluene (anhydrous, Sigma-Aldrich, Korea), and then reacted with 5 mL of APTES (3-aminopropyl triethoxysiliane, 99%, Sigma-Aldrich, Korea) under inert conditions at 80 °C for 180 min. After washing with toluene and ethanol three times, the amine-functionalized Fe3O4 MNPs were stored in anhydrous ethanol until use. 2.3. Extraction of OTA and capping of unreacted amine-functional groups Before the extraction of OTA, the amine-functionalized MNPs were washed three times with phosphate buffered saline (20  , PBS, Tech & Innovation, Korea) solution, diluted 1/20. After washing, 500 μL of the amine-functionalized MNPs in 1  PBS were added to 200 μL of solution spiked with 25–350 pg mL  1 OTA. Activation of the carboxylic acid in OTA followed the process reported in our previous article [1,10]. 500 μL of 5 mM EDC (N-(3-dimethyl amino propyl)-N-ethyl carbodiimide hydrochloride, 99%, Sigma-Aldrich, Korea) and 5 mM NHS (N-hydroxysuccinimide, 98%, Sigma-Aldrich,

Fig. 1. Experimental schematic for the detection of ochratoxin A (OTA) using a lab-built drop-type chemiluminescence (DCL) system. After extraction of OTA from sample (step 1) using MNPs, primary antibody and HRP tagged antibody of primary antibody were tagged on the target (step 2 and 3). Then, CL emission was observed for the determination of OTA (step 4, a: de-ionized water (DI), b: sample, c: 10 mM luminolþ 100 mM boric acid þ1.5 mM p-Iodophenol, d: 100 mM H2O2).

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Fig. 2. Schematic of aldehyde inhibition procedure of unreacted amine groups on MNPs.

Korea) in 1  PBS were added as zero length cross-linkers and reacted for 2 h. The OTA-MNPs formed were washed three times and dispersed in 1 mL of 1  PBS solution for the next step. After OTA extraction, the unreacted amine-functional groups were capped using propanal (99%, Acros, Korea) for inhibition. To accomplish this, the OTA-MNPs were dispersed in 1 mL of 0.836 mM propanal solution and reacted for 30 min at room temperature, as shown in Fig. 2. The MNP products obtained were washed three times with 1  PBS with the aid of magnetic separation and dispersed in 1 mL of 1  PBS.

40 mL DIW. For calibration, standard solutions were prepared with the final concentration in the range of 25–350 pg mL  1. For quantification by standard addition, the rice samples purchased in local markets were spiked with a stock solution of 10 μg mL  1, prepared by dissolving 0.1 mg of OTA (99.5%, Sigma-Aldrich, USA) in 10 mL of 60% methanol. Since OTA was not found in the commercial rice samples, the spiked OTA samples were used for recovery tests.

2.4. Immunoreaction to tag HRP-conjugated antibody to primary antibody for CL detection

Chemiluminescence reaction was carried out using a luminol– H2O2 system. A luminol solution at pH 9.5 was prepared from a mixture of 10 mM luminol (3-aminophthalhydrizide, 97%, SigmaAldrich Chem. Co., USA), 100 mM boric acid (99.999%, Sigma-Aldrich Chem. Co., USA), and 1.5 mM p-iodophenol (99%, Sigma-Aldrich Chem. Co., USA) in DW (18.2 MΩ, Millipore-Q, USA). For the CL reaction, a mixture (1þ1) of luminol and 100 mM of H2O2 in the cell was reacted with a droplet of the prepared sample solutions, diluted 1/10. The sample blank was carried throughout this experiment, for which the results were subtracted from the experimental signal. For method validation, an OTA ELISA Kit (MBS706966, Mybiosource, USA) was used in accordance with the manufacturer’s instruction manual (Supplementary Fig. S4).

Before tagging of the HRP-conjugated polyclonal secondary antibody (ab6728, abcam) for CL detection, 80 mL of monoclonal primary antibody (ab23965, abcam) was reacted with 0.3 mg of the capped OTA-MNPs at room temperature for 30 min. The unbound antibodies were then removed by washing twice with 0.1% PBST (Tween 20 in 1  PBS). The reaction products were then washed twice with 1  PBS and dispersed in 1 mL 1  PBS. For the reaction with HRP-conjugated secondary antibody, 80 mL of the secondary antibody was added to the solution and reacted for 30 min. The complexes were then washed in the same manner and diluted with 10 mL 1  PBS.

2.7. Chemiluminescence measurement and method validation

2.5. Drop-type chemiluminescence (DCL) system

3. Results and discussion

A unique DCL system designed in our lab was used to detect luminescence from the reaction between HRP and the luminol–H2O2 mixture (step 4 of Fig. 1) [12]. For CL measurement, a 0.4 mL drop of sample solution supplied through the PFA tubing was reacted with a luminal–H2O2 mixture filled in a polymer reaction cell of 1.0 mL. The reacted solution was drained out by a peristaltic pump for the next measurement. The resulting CL emission was collected by the optical fiber surrounding the PFA tubing coaxially and transferred into a photomultiplier tube (PMT; R955, Hamamatsu, Japan) with a thermoelectric cooler ( 25 °C, C9144, Hamamatsu) through an interference filter (420–460 nm, FF01-435/460, Semrock, USA). The output of the PMT was fed into a photon counting unit (C3866, Hamamatsu) coupled with a photon counting board (M8784, Hamamatsu) of 10 Hz acquisition frequency.

3.1. Extraction of OTA by non-specific platform

2.6. Application to rice sample For analysis of a real sample, the sample treatment process to remove fats and proteins followed the method in the ELISA instruction manual for the determination of OTA in a rice sample. Following the manual, 5 g of rice purchased from a local market was ground and added to 25 mL 60% methanol and 2 mL petroleum ether, and then vortexed vigorously for 5 min. The solution was centrifuged for 5 min at 4000 rpm, and then 20 mL of methanol (ACROS Organics, USA) was taken and diluted with

For the extraction of OTA, the non-specific extraction platform was employed, as shown in Fig. 1, in which the amine groups of the MNPs were directly reacted with the carboxylic group of OTA, forming an amide bond. Such formation of a chemical bond stabilized the reaction product and greatly improved the extraction efficiency, compared to adsorption. Since the treated sample solution still had the potential to contain biomaterials which can interact with the amine groups due to incomplete removal of fats and proteins, MNPs were added in excess with respect to the target. However, the excess addition raised another issue of high background. Typically, the antigen‐antibody reaction of an immunoassay proceeds through the formation of a strong bond of similar strength to a covalent bond by multiple interactions of hydrogen bonding, ionic force, van der Waals force, and hydrophobic force. Even though those concurrent interactions are significantly alleviated in non-immunoreactions, incoming antibodies containing thiol, amine or carboxylic groups can still bind to the excess MNPs through interaction with the amine groups, causing enhancement of the background [23]. Therefore, if the surface of the MNPs was not properly inhibited, the non-specific platform would suffer from high background and poor sensitivity. For inhibition, inhibitors such as BSA, skimmed milk, and casein have typically been used to block the surface from the approach of

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antibodies [24,25]. Among them, BSA is the most popular, owing to its cheap price and moderate purification; however, it reportedly has the potential to react with OTA [26,27]. Therefore, an aldehyde was used for inhibition in this work, which specifically reacted with amines. This amine-targeted inhibition capped all the amine groups of the excess MNPs and the non-specifically adsorbed molecules. 3.2. Preparation of MNPs and optimization of extraction The presence of functionalized amino groups on the MNPs synthesized was confirmed by electron microscopy and FT-IR, for which the TEM images are shown in Fig. 3. The amine layer of the MNP was clearly shown in Fig. 3(b). The core size in Fig. 3(a) was 9.95 nm (72.08 nm), which increased to 19.07 nm (73.08 nm) due to the coating. Furthermore, the presence of amine groups was confirmed by FT-IR, as shown in Supplementary Fig. S1. Assuming that the added APTES (5 mL) was completely used up to make the shell of the core particles, each MNP was roughly estimated to contain about 7.9  104 amine groups. The optimized amount of MNPs used in this work was 0.3 mg when the rice sample was spiked with 3.5 ng OTA (Supplementary Fig. S2(a)), which corresponded to an excess of about 1.4 times with respect to the OTA molecules. Noticeably, the added MNPs were sufficient to cover the unexpected adsorption or reaction of the proteins because fats and proteins were mostly removed by the treatment process (Supplementary Fig. S3). Under these conditions, the reaction time was optimized to be 1 h, as shown in Supplementary Fig. S2(b).

capping, shown in Fig. 4(b), because the –NH2 was changed to an imine group. In addition, the appearance of a peak at 2983 cm  1 indicated the presence of a –CH3 stretching mode from the propanal used for capping. The increase of absorption intensity at 1640 cm  1 can also provide strong evidence of the presence of an imine group. The stability of the coated propanal layer was also demonstrated by monitoring the peaks after 1.5 h and 3 h had passed. As shown in Fig. 4(c) and (d), the absorption peaks related to the imine and –CH3 groups still appeared after the washings, which confirmed that the inhibited layer on the MNPs was stable enough for the next process. 3.4. Optimization of the inhibition process Optimization of the inhibition process by changing the reaction time (a) and molar ratio of aldehyde to amine groups (b) is illustrated in Fig. 5. For the reaction time, the characteristic peaks of the C–N and C˭N bonds, 1334 cm  1 and 1641 cm  1, respectively, were monitored using FT-IR. As the time increased, the C–N peak disappeared, while an abrupt increase of the C˭N peak was observed, due to the formation of an imine group. After 30 min, almost no change of the peak was observed, which indicated the end of the reaction. Therefore, the reaction time for inhibition was fixed at 30 min. Secondly, the amount of propanal with respect to the MNPs was also tuned by monitoring the background intensity

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3.3. Aldehyde inhibition for amine capping

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As discussed, the inhibition of unbound amine groups is very important to lower the background and enhance the signal-tobackground ratio. For inhibition, the selected propanal contained a functional group that can specifically react with amine groups through the formation of an imine bond, as shown in Fig. 2. In addition, it has less hydrophobicity among various aldehydes, while its small size allows efficient dispersion and it has almost no reactivity with OTA. Although an acetaldehyde may be another candidate, there would be difficulty in experimental application due to its high volatility (b.p. of 21 °C at 1 atm). The presence of an aldehyde layer was confirmed by FT-IR, as shown in Fig. 4. From Fig. 4(a), the product showed the absorption peaks of –NH2 bending at 1563 cm  1 and C–N at 1324 cm  1 in the finger print range of 1300–1700 cm  1. These peaks disappeared after

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Fig. 4. Confirmation of imine group formed by aldehyde inhibition and its stability test: FT-IR spectra of (a) before amine capping and (b) after capping. The spectra of (c) and (d) were obtained at 1.5 h and 3 h later after capping, respectively.

Fig. 3. TEM images of MNPs (a) and amine-coated MNPs (b).

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1.6 1334cm , C-N -1 1641cm , C=N

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3.5. Optimization of HRP tagging The immunoreactions for HRP tagging in Fig. 1 were also examined, i.e., reaction of the primary antibody with the OTA target (step 2) and the secondary antibody with the primary antibody (step 3). Experimentally, sufficient primary antibody was added, at about 1.5 times higher than the maximum concentration of OTA, and the excess antibody was washed away. The amount of HRP-tagged secondary antibody was tuned with respect to 3.5 ng of OTA, as shown in Supplementary Fig. S2(c). As the molar ratio of secondary antibody to OTA was increased from 1:1 to 10:1, the net signal intensity remained almost unchanged after 2:1; however, the S/B ratio continuously decreased. Noticeably, too much excess addition ruined the S/B ratio due to non-specific adsorption. 3.6. Comparison of aldehyde inhibition with BSA The effects of inhibition using 3% BSA and aldehyde on the signal intensity of chemiluminescence are illustrated in Fig. 6. As shown in the figure, the background levels for both inhibition methods were significantly lower compared to no inhibition. In particular, inhibition with BSA showed the lowest background level, i.e., about 11 times and 2.3 times lower than no inhibition and aldehyde inhibition, respectively. In contrast, the aldehyde inhibition showed higher signal-to-background (S/B) ratio. This result provided evidence that BSA inhibited non-specific adsorption effectively, but also blocked the reactivity of OTA as reported [26,27], which suppressed the net signal intensity stronger than the background. Similarly, the aldehyde inhibition showed the signal-to-noise (S/N) ratio of 21.1, which was about 7 times higher than the BSA. The evidence of steric hindrance by BSA was unclear at this moment because aldehydes with large alkyl group cannot be tested due to insufficient solubility and specificity. As a conclusion, the amine-targeted inhibition using propanal exhibited higher S/B and S/N ratios compared to BSA, with excellent stability against washing, but it was not valid for non-specific adsorption, producing higher background.

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of chemiluminescence when the ratio of propanal to MNPs was varied from 3.5  104:1 to 7.0  106:1 for 1.0 ng of OTA. As shown in Fig. 5(b), the signal to background ratio (S/B) was maximized at the molar ratio of 3.5  104:1, after which it remained almost unchanged. Therefore, an excess of propanal of about 10 times was added for the complete inhibition of amine groups.

Chemiluminescence intensity of blank (counts)

Fig. 5. Optimization of aldehyde inhibition for (a) reaction time and (b) the ratio of aldehyde with respect to magnetic nanoparticle.

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Fig. 6. Effect of inhibition on background luminescence and signal-to-background ratio.

3.7. Application on rice samples The developed analytical platform was applied to rice samples purchased from a local market (Nonghyup, Korea). Since no OTA was detected in the samples, the samples were spiked with standard OTA in the range of 25–350 pg mL  1 for demonstration. The spiked rice samples prepared for calibration showed a linear calibration curve in standard addition with a regression coefficient (R2) of 0.993, as shown in Fig. 7(a). The averaged limit of detection (LOD, 3s/m) of 1.39 pg mL  1 was obtained through four sets of replicate measurements. For method validation, a calibration curve of ELISA with a linear regression coefficient (R2) of 0.988 was also obtained through four replicates, as shown in Fig. 7(b). The obtained LOD was 10.16 pg mL  1, which was about 7.3 times higher than the DCL method developed in this work. For the recovery test, three different rice samples containing 50, 250, and 350 pg mL  1 of OTA were analyzed by standard addition. The results of three repeated measurements are shown in Table 1. Excellent recoveries of the developed DCL method were obtained in the range of 87.2–98.7%, with relative standard deviations in the range of 4.1–11.9%. ELISA also showed recoveries of 99.7–113.6%, which were 12–14% higher than for DCL. The reason for the low recovery was unclear, but sample loss during the washing steps for magnetic extraction and separation may be one of the candidates, which can be minimized by a ratiometric measurement monitoring the concentration of MNPs.

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yscale(Y) = A + B * xscale(X) Parameter Value Error -----------------------------------------------------A -3612.56455 360.4419 m 3426.85564 176.3907 -----------------------------------------------------R SD N P -----------------------------------------------------0.99655 0.80929 6

Chemiluminescence immunoassay using magnetic nanoparticles with targeted inhibition for the determination of ochratoxin A.

In this work, a chemiluminescence (CL) immunoassay with targeted inhibition was developed for the determination of toxins in food products. For sample...
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