Research article Received: 26 December 2014,

Revised: 9 March 2015,

Accepted: 21 April 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2940

Direct competitive chemiluminescence immunoassays based on gold-coated magnetic particles for detection of chloramphenicol Xiaohui Liang,a Xiangyi Fang,a* Manwen Yao,c Yucong Yang,b Junfeng Li,a Hongjun Liud and Linyu Wangd ABSTRACT: Direct competitive chemiluminescence immunoassays (CLIA) based on gold-coated magnetic nanospheres (Au-MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au-MNPs were modified with carboxyl groups and amino groups by 11-mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). NSP-DMAE-NHS, a new and effective luminescence reagent, was employed to label antiCAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a ‘homemade’ luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC50) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme-linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSPDMAE-NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: chemiluminescent immunoassays (CLIA); gold-coated magnetic nanoparticles (Au-MNPs); CAP detection; NSP-DMAE-NHS

Introduction Chloramphenicol (CAP), a cheap and effective bacteriostatic broad-spectrum antibiotic, has been widely used in both human and poultry medicines. However, recent research found that CAP has many side-effects in humans e.g., it may produce severe or fatal bone-marrow depression and aplastic anaemia, with a syndrome of cyanosis and cardiovascular collapse known as the ‘grey syndrome’ which may occur particularly in neonates. Thus, its use has been banned in all kinds of food (1–3). In 2003, the minimum detection limit (MRPL) of CAP in milk, egg, meat, shrimp, honey and other animal foods was set at 0.3 μg/kg in the European Union (4). The US Food and Drug Administration (USFDA) has been conducting research on more sensitive methods to reduce the detection limit from 0.3 ppb to 0.1 ppb. Even though there is prohibition of CAP usage, the ban is still abused for food animals and its residues are often found in various food samples, such as meat, shrimp, milk and honey (5). In order to effectively monitor the CAP illegal use, a series of analytical methods have been developed to rapidly detect CAP in food for screening purposes. Microbiological assays are generally used for preliminary screening (6,7), but their specificity is poor. Gas/liquid chromatography (GC/LC) (8,9) and gas/liquid chromatography-mass spectrometry (10,11) are of high sensitivity. These methods, however, are expensive, complicated, and not suitable for the analysis of a large number of samples for screening purposes. Immunoassays have lately gained increasing interest as alternative methods for large monitoring programs, such as radio immunoassay (RIA) (12) and enzyme-linked immunosorbent assay (ELISA) (13,14). However,

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they have some shortcomings such as radioactive waste and a higher limit of detection (LOD) respectively. In recent years, chemiluminescence (CL) reactions have drawn increased attention in many sectors of analytical chemistry including immunoassays, immunosensor measurement, DNA probebased assay and biochemical analysis (15). CL is generally defined as the emission of light (ultraviolet, visible, or infrared) during the process of a chemical reaction (16). The CL reactions possess high sensitivity, wide dynamic range, short analysis time and low-cost apparatus. However, some drawbacks exist such as poor selectivity, as any species already present in the solution can alter the analytical signals. The relatively low emission intensity of some

* Correspondence to: X. Fang, Physics Department, School of Science, Xi’an Jiaotong University, Xi’an, People’s Republic of China. E-mail: [email protected] xjtu.edu.cn a

School of Science, Xi’an Jiaotong University, Xi’an, People’s Republic of China

b

Department of Clinical Laboratory The First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, People’s Republic of China

c

Tongji University, Shanghai, People’s Republic of China

d

School of Electronic and Information Engineering, Xi’an Jiaotong University, Xi’an, People’s Republic of China Abbreviation: AE, acridinium ester; ELISA, enzyme-linked immunosorbent assay; FDA, Food and Drug Administration; LOD, limits of detection; MP, magnetic particles; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline.

Copyright © 2015 John Wiley & Sons, Ltd.

L. Xiaohui et al. CL reactions, due to very low efficiency in transforming chemical energy into light, is another possible shortcoming of the CL-detection systems (17). So, much effort has been made to enhance emission intensity and improve selectivity for CL-based quantitative analysis. Current CL reactions that generally exploit luminol or ABEI [N-(4-aminobutyl)-N-(ethylisoluminol)] as the luminescence reagent and H2O2 as an enhancer in the presence of horseradish peroxidase (HRP) are called chemiluminescence enzyme immunoassays (CLEIA) (18–20). Compared with CLEIA, CLIA is an easier luminescence measurement using chemiluminescent reagents such as acridinium ester (AE) or, particularly, NSP-DMAENHS (2′,6′-dimethylcarbonylphenyl-10-sulfopropyl acridinium-9carboxylate 4′-NHS ester). NSP-DMAE-NHS is a new and effective luminescence reagent with higher luminous efficiency than luminol and with the better thermal stability than AE to directly label the antigen or antibody, without the participation of an enzyme (21). NSP-DMAE-NHS has the potential to emit light in 2 sec and allow full automation and, therefore, it is a promising option to apply to rapid and sensitive detection of low-molecularweight contaminants such as antibiotics and pesticides. Magnetic particles (MPs) have found many applications in the fields of bioassay, bio-separation and drug delivery due to their remarkable merits including large ratio of surface area to volume, low cost of synthesis, magnetic susceptibility, low toxicity and compatibility with biomaterials (22). It has been demonstrated that MPs-CLIA needs less reagents than conventional CLIA without MPs and, moreover MPs can be easily separated from the sample matrix after target capture for further analysis, thus reducing matrix interference (23). Gold-coated magnetic nanoparticles (Au-MNPs), a kind of new magnetic composite particle, have outstanding characteristics as they combine the superparamagnetism of magnetic nanoparticles and the ability to be modified by biomolecules on a gold surface. In addition, Au-MNPs are excellent biological markers because they are capable of conjugating with biomolecules, such as proteins and DNA, without hindering their biochemical activity (24). It has been reported that the exploitation of gold-coated nanoparticles as an anti-AFP–HRP carrier has led to improvements in detection sensitivity, and it was observed that the LOD of MP-CLIA using gold-coated nanoparticles was about 100 times fewer than that of MP-CLIA without gold-coated nanoparticles (25). To our knowledge, there have been no previous report of CLIA based on Au-MNPs using the NSP-DMAE-NHS system to detect CAP. In this study, competitive CLIAs based on Au-MNPs for detecting CAP have been developed. CAP base and CAP succinate molecules were respectively immobilized on Au-MNPs. The anti-CAP antibodies were labeled by NSP-DMAE-NHS. Certain amounts of labeled antibodies were mixed with the target analyst at various concentrations and then specifically reacted with antigens on the Au-MNPs. After the Au-MNPs were washed, the chemiluminescent emission light intensity was measured with a ‘home-built’ luminescent measurement system.

N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N,N-dimethylformamide (DMF) were purchased from Sigma Company (China). Monodisperse gold-coated magnetic nanoparticles (40 nm, 5% (m/v)) were purchased from Nanodot Co., Ltd (Xiamen, China). l-Lysine and Sephadex G-50 was purchased from Seebio (Shanghai, China).

Construction of luminescent measurement system The measurement system was composed of a photomultiplier tube (PMT), a photon counting unit (H8259–01, Hamamatsu) and a dark box, with the counting unit linked to a computer. The dark box was homemade, could block outside light and had a transparent glass tube (5.5 cm×1.2 cm) inside with a volume capacity of 5 mL. The reaction tube was fixed in the correct position of an upright straight slot inside the box and the photocathode of a PMT tube was placed in front of it to receive the emitted light. The optimized detection wavelength of the PMT corresponded to the chemiluminescence emission wavelength (430 nm). A tube was connected to the reaction tube through a needle in the upper part of the dark box, with the inlet part of it connected to an injector and a peristaltic pump for pumping trigger solution into the tube. The chemiluminescent emission photons from the analyte in the black box were counted and the data were monitored and handled in real time by a computer (Fig 1).

Preparation of NSP-DMAE-NHS labeled CAP mAb Purified CAP mAb (0.1 mg dissolved in 100 μL of carbonate buffer solution, 0.1 M, pH 10.1) was labeled with 20 μg of NSP-DMAE-NHS dissolved in 10 μL of DMF. The mixture was gently shaken for 30 min at room temperature and incubated at 4°C overnight, followed by the addition of 10 μL of l-lysine (10 mg/mL of distilled water) for 10 min. The labeled antibody was separated from unbound NSP-DMAE-NHS by gel filtration on a Sephadex G-50 column (1×25 cm) and equilibrated phosphate-buffered saline (PBS) (0.1 M, pH 6.3, 0.9% NaCl). The eluted solution was collected in dozens of bottles and detected by an ultraviolet (UV) light spectrometer at 280 nm. In addition, the luminescent intensity of the eluted solution was measured. The high absorbance fraction and high luminescent intensity fraction were collected in single-dose vials. The procedure was carried out in accordance with a previous report but with partial modification (26).

Immobilization of CAP molecules on Au-MNPs Two kinds of CAP molecules were immobilized on the surface of Au-MNPs as described below.

Experimental Reagent The monoclonal antibody against CAP (mAb) was purchased from Bioss (Beijing, China). NSP-DMAE-NHS(2′,6′-dimethylcarbonylphenyl10-sulfopropylacridinium-9-carboxylate 4′-NHS ester), CAP succinate, CAP base were purchased from MaterWin New Materials Co., Ltd (Shanghai, China). 11-Mercaptoundecanoicacid (MUA), cysteamine,

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Figure 1. Luminescent measurement system. (a) Substrate solution, (b) peristaltic pump, (c) black box, (d) reaction tube, (e) photomultiplier tube, (f ) photon counting unit and ( g) computer.

Copyright © 2015 John Wiley & Sons, Ltd.

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Magnetic particle based chemiluminescence immunoassays A 100 μL volume of carboxyl-(Au-MNPs) formed by immersing AuMNPs (50 nm, 50 mg/mL) in a solution of MUA (5 mM) in ethanol for 16 h at room temperature was washed twice with 2-(Nmorpholino)ethanesulfonicacid (MES) buffer (25 mM, pH 5.12). Afterwards, the carboxyl-(Au-MNPs) were resuspended in 1 mL MES buffer and then the carboxyl groups on the spheres were activated by incubation with 100 μL of a 1:1 ratio mixture of 200 mM EDC (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 50 mM NHS (N-hydroxysulfosuccinimide) in MES buffer at 37°C for 30 min. After washing, the Au-MNPs were incubated with 100 μL of CAP base (1 mg/mL in borate buffer (0.05 M, pH 8.5)) at 37°C for 2 h. After incubation, the supernatant was removed and washed three times with PBS (0.01 M, pH 7.4) in a magnetic field. Ethanolamine (1 M, pH 8.5) was added and allowed to react for 30 min. Finally, the Au-MNPs with CAP base immobilized on the surface were washed and stored in PBS for standby.

Method I: Immobilization of CAP base on carboxyl-(Au-MNPs).

A 100 μL volume of amino-(Au-MNPs) formed by immersing AuMNPs (50 nm, 50 mg/mL) in a solution of cysteamine in distilled water for 4 h at room temperature was washed twice with MES buffer (25 mM, pH 5.12). Afterwards, the amino-(Au-MNPs) were resuspended in 1 mL MES buffer. A volume of 100 μL CAP succinate (20 mg/mL in DMF) was activated by incubation with 100 μL of a 1:1 ratio mixture of 200 mM EDC and 50 mM NHS in MES buffer at 37°C for 30 min. The activated CAP succinate solution was added to the amino-(Au-MNPs) suspension, and then the mixture was incubated at 37°C for 2 h. After a thorough wash with PBS, the AuMNPs with CAP succinate immobilized on the surface were stored in PBS for standby. Method II: Immobilization of CAP succinate on amino-(Au-MNPs).

was added to the particles by a peristaltic pump to induce chemiluminescence. The instantly emitted light was collected with the PMT tube and measured with a photon counter. As a result, the sensitivities of both methods were obtained and compared with each other. Figure 2 shows the flow chart of the complete procedures.

Results and discussion Determination of optimized amount of NSP-DMAE-NHS labeled mAb Different amounts (10, 20, 25, 30 or 40 μL) of NSP-DMAE-NHS labeled mAb with a concentration of 0.01 mg/mL were added into 100 μL of the CAP immobilized Au-MNPs (for the two methods) and were incubated at 37°C for 2 h. The particles were washed in a magnetic field, and then the chemiluminescent emissions were measured. Figure 3 shows the chemiluminescent emission intensities based on different amounts of NSP-DMAE-NHS labeled mAb in

Competitive chemiluminescence assays based on Au-MNPs for CAP detection. Next, 100 μL of the target CAP succinate sodium solution

(0, 0.001, 0.01, 0.1, 1, 10 and 100 ng/mL) were mixed with the optimized amount of NSP-DMAE-NHS labeled mAb solutions and then reacted separately with the above prepared two types of CAP immobilized Au-MNPs. The mixtures were incubated at 37°C for 2 h with gently shaking. Then the particles were washed with PBS in a magnetic field. In this study, H2O2 and NaOH acted as chemiluminescence substrate. A volume of 100 μL of the substrate

Figure 3. Chemiluminescence intensity versus the amount of NSP-DMAE-NHS labeled mAb with a concentration of 0.01 mg/mL by two methods. Measurements were done in duplicate and the average values were taken.

Figure 2. The flow chart of the two methods to detect CAP in competitive chemiluminescence assays. (A) The Au-MNPs were coated with CAP antigen (CAP base and CAP succinate). (B) A mixture of 100 μL of CAP succinate sodium standard solution and optimized amount of NSP-DMAE-NHS labeled mAb solution (0.01 mg/mL) was added into the Au-MNPs and reacted with the CAP antigens on Au-MNPs. (C) Chemiluminescence substrate solution was added, and light intensity was detected.

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Copyright © 2015 John Wiley & Sons, Ltd.

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L. Xiaohui et al. Method I and Method II. It could be clearly seen that the chemiluminescent emission intensities increased with the increasing concentration of NSP-DMAE-NHS labeled mAb. The results indicated that both of the two CAP antigens (CAP base and CAP succinate) had been successfully immobilized on the Au-MNPs’ surfaces. As shown in Fig. 3, the maximum response by Method I was obtained with the amount of 40 μL. By compromising sensor response and tracer consumption, we used 30 μL of NSP-DMAE-NHS labeled mAb as the fixed volume in later competitive reactions. In the same way, the amount of NSP-DMAE-NHS labeled mAb by Method II was also chosen as 30 μL. Calibration curves for CAP succinate sodium Different concentrations of CAP succinate sodium (0, 0.001, 0.01, 0.1, 1, 10 or 100 ng/mL) were mixed with 30 μL NSP-DMAE-NHS labeled mAb solutions and were incubated at 37°C for 1 h, and the mixture was then reacted separately with 100 μL of either the CAP base immobilized Au-MNPs (Method I) or CAP succinate immobilized Au-MNPs (Method II), and then the mixtures were incubated overnight at 37°C for 2 h. The particles were washed with PBS in magnetic field, and then the chemiluminescence trigger solution containing NaOH and H2O2 was introduced into the washed Au-MNPs. Figure 4 shows the calibration curves of CAP succinate sodium by the two methods, in which the peak heights of the chemiluminescence intensity were plotted against the logarithm of the concentration of CAP succinate sodium. The IC50 values (the competitor concentration that causes 50% growth inhibition) of Method I and Method II were about 0.044 ng/mL and 0.072 ng/mL respectively, both of which indicated good sensitivity. From Fig. 4, the LOD of Method I and Method II can also be obtained approximately as 0.001 ng/mL and 0.006 ng/mL respectively. Comparing these two methods, Method I has advantages due to its sensitivity. The higher sensitivity for Method I might be caused because the CAP base provides a simple one-step coupling to the activated carboxylated particle surface, resulting in a relatively strong resilient chemical bond. In contrast, CAP succinate, which is an ester, is unstable when subjected to acid or alkaline conditions (27). Compared with the reported ELISA immunoassay based on direct hapten-coated format and the biotin–streptavidin system (IC50 of 10.5 ng/mL and LOD of 0.2 ng/mL) (28), both methods

Figure 4. Calibration curves for detection of CAP succinate sodium by Method I and Method II.

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tested here were more sensitive. The better sensitivity might have two causes, firstly that the introduction of Au-MNPs increased the binding ratio of antigen with antibody, and secondly the application of NSP-DMAE-NHS, which is a relatively effective luminescence reagent.

Conclusion We describe the use of a new luminescence reagent NSP-DMAENHS to conjugate with the anti-CAP antibody as a tracer in two direct competitive CLIAs for detection of CAP based on Au-MNPs. The Au-MNPs were modified with carboxyl and amino groups respectively by MUA and cysteamine, such that the two CAP antigens (CAP base and CAP succinate) were immobilized on their surfaces. IC50 of 0.044 ng/mL and 0.072 ng/mL were obtained for Method I and Method II, respectively. The LODs of both the methods were much less than that of traditional ELISA. Method I was the more sensitive of the two methods. The results also indicated that NSPDMAE-NHS labeling had not affected the bio-activity of the antibodies, thereby offering an excellent way to detect CAP molecules. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 81371642) and the Fundamental Research Funds for the Central Universities of China.

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Direct competitive chemiluminescence immunoassays based on gold-coated magnetic particles for detection of chloramphenicol.

Direct competitive chemiluminescence immunoassays (CLIA) based on gold-coated magnetic nanospheres (Au-MNPs) were developed for rapid analysis of chlo...
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