Simultaneous Detection of Forbidden Chemical Residues in Milk Using Dual-Label Time-Resolved Reverse Competitive Chemiluminescent Immunoassay Based on Amine Group Functionalized Surface

In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) in chemiluminesecence (CL) systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP) for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP) and CLE (ALP-CLE). The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 105) of CLE as background for HRP CL signal value (about 107) of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC50) values of CAP and CLE in milk samples were 0.00501 µg L−1 and 0.0128 µg L−1, with the ranges from 0.0003 µg L−1 to 0.0912 µg L−1 and from 0.00385 µg L−1 to 0.125 µg L−1, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE.


Introduction
Feeding of drugs and chemicals to food animals (pigs, cattle, and goats) can leave residues in meat or milk, offal and other parts of the animals. With food poisoning cases and toxic effects arising from consumption of these residues and the emergence of drug resistant bacteria over recent years, there is a need to control the feeding of drugs and chemicals to the food animals [1][2][3]. A variety of analytical methods to detect and qualify drugs and chemicals in food matrices have been reported, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC) with an iron trap detector and LC-MS, enzyme linked immunoassay (ELISA), surface plasmon resonance-based biosensor, immunoassay based on gold nanoparticles and magnetic beads, quantum dot-based lateral flow immunoassay, electrochemical biosensor, amperometric immunosensor and piezoelectric immunosensor [4][5][6][7][8][9][10][11][12][13]. Briefly, current trends for detection chemicals are divided to two directions: confirmatory methods such as GC-MS or LC-MS which could differentiate the chemicals and make multi-component detection, and rapid screening methods which satisfied the requirement of rapid and 'Yes/NO' at the level of interest for in-field controls. Monitoring the growing number of antibiotics, hormones, endocrine disrupting chemicals, toxins in animal-derived food samples places a high demand on rapid, cheap and reliable screening methods. Recently, increasing attention has been paid to the development of various multiplexed immunoassay (MIA) in a single run to achieve multi-residue determination and serve specific analytical purposes, which has been demonstrated to be promising in food safety, clinical diagnosis and environmental monitoring [14][15][16][17]. In this study, the goat anti-rabbit and goat anti-mouse immunoglobulins were bound covalently to the 3-aminopropyltriethoxysilane (APTES)functionalized microtiter plates (MTP) in a leach-proof fashion using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (SNHS) to enhance the sensitivity of the MIA and reduce assay duration [18][19][20].
Chloramphenicol (CAP), a broad spectrum antibiotic, is frequently employed in animal production for its excellent antibacterial and pharmacokinetic properties. However, in humans it leads to hematotoxic side effects [2], in particular CAPinduced aplastic anaemia for which a dosage-effect relationship has not yet been established, leading to a prohibition of CAP for the treatment of food-producing animals [21]. Clenbuterol (CLE), a representative of the class of synthesized b 2 -adrenergic agonists, is abused illegally for food-producing animals as ''lean meat agent'', which have potential hazard to human health. Hence, the development of an immunoassay format with enhanced analytical performance is essential for the precise simultaneous detection of trace CAP and CLE.
In this study, we have developed a sensitive rapid dual-label time-resolved reverse competitive chemiluminescent immunoassay (DLTRRC-CIA) for simultaneous determination of trace CAP and CLE applicable in milk. Figure 1 introduces the new assay design, which incorporates the advantages of different properties of chemiluminescence (CL) reaction (horseradish peroxidase (HRP) and alkaline phosphatase (ALP)) and sensitivity improvement of covalent binding. To date, the DLTRRC-CIA is the most sensitive assay with the least assay time for the simultaneous determination of trace CAP and CLE. (e) Buffers and solutions were prepared in Milli-Q deionised water (DIW). Coating buffer (CB, pH 9.6) was made with 1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 in 1 L of purified water. Blocking  Each well of the 96-well MTP was treated with 100 mL of 1.0% (w/v) KOH at 37uC for 10 min followed by five DIW washes (300 mL/well). The KOH-treated wells were then functionalized with amino groups by incubating with 100 mL of 2% (v/v) APTES at room temperature (RT) for 1 h inside the fume cabinet. The amine-functionalized MTP was subsequently washed five times with DIW in order to remove excess unbound APTES from the surface. The 990 mL of goat anti-rabbit (2 mg mL 21 ) and goat antimouse immunoglobulins (2 mg mL 21 ) mixture (capture antibodies) was incubated with 10 mL of a pre-mixed solution of EDC (4 mg mL 21 ) and SNHS (11 mg mL 21 ) for 15 min at 37uC. The 100 mL of resulting EDC-SNHS cross-linked goat anti-rabbit and goat anti-mouse immunoglobulins solution was added to each of the APTES-functionalized wells and incubated for 1 h at 37uC. The goat anti-rabbit and goat anti-mouse immunoglobulins -bound wells were then washed five times with PBS (300 mL/well).

The Procedure of DLTRRC-CIA for CAP and CLE
The goat anti-rabbit and goat anti-mouse immunoglobulinsbound MTP wells were blocked with 200 mL/well of blocking buffer at 37uC for 0.5 h, subsequently washed five times with PBST. The goat anti-rabbit and goat anti-mouse immunoglobulins -bound MTP wells were incubated with mixture of anti-CAP PAbs (1:40000 dilution in PBST) and anti-CLE MAb (1:40000 dilution in PBST) for 30 min at 37uC, and subsequently washed five times with PBST. Then 100 mL/well of standard (cocktail of CAP and CLE) in 0.02 M PB or sample solution was added, followed 50 mL/well mixture of CAP-HRP (1:40000 dilution in PBST) and CLE-ALP (1:10000 dilution in PBST). The competitive reaction was allowed to take place for 30 min at RT. After washing five times, the CL signal was measured using a chemiluminesence reader at 2 min after automatic addition of 100 mL/well Visiglo Plus AP Chemiluminescent Substrate in injector A and the results were expressed in relative light units (RLU). At 2.5 min, the HRP activity was revealed by automatically adding 100 mL/well of a freshly prepared substrate mixture of Super Signal substrate solution in injector B. The CL signal was measured using a chemiluminesence reader at 4 min and the results were expressed in RLU.

Data Analysis
Standards and samples were run in quadruplicate wells, and mean chemiluminescence intensity values were divided by RLU max (chemiluminescence intensity in the absence of analyte).The ratio is defined as B/B 0 . Standard curves were obtained by plotting B/B 0 against the logarithm of analyte concentration and fitted to a four-parameter logistic equation using Origin (version 8.0, Microcal, Northampton, MA, USA) software packages where A is the asymptotic maximum 1, B is the curve slope at the inflection point, C is the x value at the inflection point (corresponding to the analyte concentration that reduces RLU max to 50%), and D is the asymptotic minimum (RLU background signal / RLU max ).

Analysis of Field Milk Samples
Forty whole cow milk samples with pakages were purchased from retail outlets in Beijing. Each sample was divided into three portions in brown polystyrene bottle, which would be analyzed by developed DLTRRC-CIA, conventional ELISA kits. All of the samples were stored at 220uC until use.

Sample Preparation
For extraction of CAP and CLE from milk, added 500 mL Carrez A and 500 mL Carrez B to 10 mL milk, mixed thoroughly, then centrifuged for 10 min at 4000 g in 4uC. Transferred 4.4 mL of aqueous supernatant to a new tube, adjusted the pH to 11 with 1 M NaOH and thoroughly mixed with 8.0 mL ethyl acetate for 10 min in a new tube. Centrifuged at 4000 g for 10 min, 4 mL of organic supernatant was transferred to a new tube and dried by nitrogen at 60uC. The residue was dissolved in 2 mL of 0.02 M PB. The sample solution could be used for determination.

DLTRRC-CIA Strategy Based on Time-Resolved CL and Covalent Binding of Capture Antibodies
The DLTRRC-CIA for CAP and CLE based on time-resolved CL and covalent binding of capture antibodies is illustrated in Figure 1. HRP and ALP were adopted as the signal probes to tag CAP and CLE, respectively, due to their different CL kinetic characteristics. With a competitive immunoassay format, the HRP-and ALP-tagged immunocomplexes (goat anti-rabbit immunoglobulins-anti-CAP PAbs-CAP-HRP and goat antimouse immunoglobulins-anti-CLE MAb-CLE-ALP) were formed in the well of the MTP. The two CL signals were successively triggered by adding the two different CL substrates (ALP and HRP CL solutions were prepared in different injections in the automatic luminometer).
The CL signal of ALP-tagged immunocomplex reached the maximum value at 2 min after injection of ALP CL substrate in injector A, and showed a steady plateau in the whole measured period (Figure 2). The CL signal of the HRP-tagged immunocomplex reached the maximum value at 4 min after injection of HRP CL substrate in injector B (at 2.5 min), and showed a steady plateau in the whole measured period. Then the signal for CLE was detected at 2 min after adding ALP CL substrate (injector A) into the well. The signal for CAP was collected at 4 min after adding HRP CL substrate (injector B) into the wells at 2.5 min. When the chemiluminesence reader collected the CL signal at 4 min, there were two chemiluminescent systems-HRP CL system for CAP and ALP CL system for CLE, and the CL value was the sum of HRP CL for CAP and ALP CL for CLE. But the ALP value of RLU max-CLE was only 1/61 of RLU max-CAP and the effect of ALP CL signal for CLE (0.65 million) on HRP CL signal for CAP (40 million) was negligible ( Figure S1a). Thus we detected the signals of CAP at 2 min and CLE at 4 min, after the automatic injection of ALP CL substrate at 0 min and HRP CL substrate at 2.5 min. When CAP and CLE were both absent, the maximum HRP CL signal for CAP was 61 times of maximum ALP CL signal for CLE. Due to the distinguishable gap for HRP and ALP signals, the cross-talk resulting from the mixed CL reaction systems was effectively avoided (Materials S1 and Materials S2).
The APTES-functionalized bioanalytical platforms have been widely used for the leach-proof immobilization of biomolecules as they enable high immobilization density, long-term stability, high reproducibility, and less biofouling [19,20,22,23]. In the present study, the covalent crosslinking of antibodies in a leach-proof manner on APTES-functionalized bioanalytical platform leads to high functional antibody immobilization density, which results in highly sensitive analyte detection and saving the assay duration ( Table 1).

Performance of DLTRRC-CIA in Standard Solutions
Under the optimal conditions, the CL responses decreased linearly with the increase in the concentrations of CAP and CLE since a competitive immunoassay mode was employed. The developed DLTRRC-CIA detected CAP and CLE in the range of 0.0003-0.0912 mg L 21 (R 2 = 0.9971) and 0.00385-0.125 mg L 21 (R 2 = 0.9974), with 50% inhibition concentration (IC 50 ) values of 0.00501 and 0.0128 mg L 21 , respectively (Figure 3). The sensitivity represented by IC 50 of the developed DLTRRC-CIA for CAP were more than 9.2 times and 93 times greater than reverse competitive CL-ELISA (IC 50 = 0.046 mg L 21 ) and traditional ELISA method with colorimetric detector (IC 50 = 0.47 mg L 21 ) developed by our own, respectively (Figure 3a). Meanwhile, the sensitivity of the developed DLTRRC-CIA for CLE were more than 8 times and 96 times greater than reverse competitive CL-ELISA (IC 50 = 0.11 mg L 21 ) and traditional ELISA method with colorimetric detector (IC 50 = 1.25 mg L 21 ) developed by our own, respectively (Figure 3b). The detection limits represented by 10% inhibition concentration (IC 10 ) values for CAP and CLE were 0.0003 and 0.001 mg L 21 , respectively.
TAP, FF, FFA, SAL and RAC, structurally related with CAP or CLE, were selected for cross-reactivity (CR) experiments to evaluate the specificity of anti-CAP PAbs and anti-CLE MAb. No significant CR of anti-CAP PAbs to other amphenicols and badrenergic agonist was observed (Table 2). Meanwhile, no amphenicols and b-adrenergic agonists at concentration up to more than 1000 mg L 21 showed binding with the anti-CLE MAb. Furthermore, to help define the specificity of the two antibodies, structurally unrelated drugs including SUL, CIP and PEN were also tested. No CR was observed.

Comparison of the Developed DLTRRC-CIA for CAP and CLE with Other Immunoassays in Standard Solutions
The analytical comparison of various immunoassays for CAP and CLE detection is important to analyze their suitability for screening the residue in field samples. The developed DLTRRC-CIA decreased the overall assay duration significantly by more  Moreover, it has better analytical performance, i.e. better analytical sensitivity with lower limit of detection (LOD), than our previously developed competitive direct CL-ELISA formats for CAP [24] and newly reported time-resolved chemiluminescence immunoassay for CLE [17], where the anti-CAP PAbs and anti-CLE MAb were passively adsorbed to the MTP, respectively.

Matrix Effect
To apply a new method in real sample analysis, a matrix effect is an important issue to be considered, especially in animal tissues due to the complicated matrix. In this study, the established DLTRRC-CIA was used to determine CAP and CLE in field milk samples. When determining the matrix effects, interferences are quantified by comparing a standard inhibition curve in buffer with that generated in the milk extract matrix known to be free of CAP and CLE. Extracting with ethyl acetate, drying by nitrogen and dissolving in the same buffer with the standard curve buffer were adopted to overcome matrix interference. The two group curves for either of CAP and CLE are superposable, indicating that the matrix effect is not significant (Figure 4). Then the samples can be analyzed using the standard curve instead of the matrix curve.

Application in Real Samples
Precision and recovery. Some spiked milk samples were detected using the developed DLTRRC-CIA method to evaluate the application potential. Five blank samples were spiked with CAP and CLE standards at different known amounts, prior to the recovery tests. As seen in Table 3, the recoveries of the spiked CAP and CLE were in the range of 85.5-91.5% and 83.7-89.6%, respectively. The coefficients of variation (CVs) for the detections of the two analytes were all less than 8.9%, indicating acceptable accuracy of the proposed method.
Analysis of CAP and CLE in Field Milk Samples. To evaluate the determination capability of the developed DLTRRC-CIA in milk samples, 40 field samples were analyzed by the developed DLTRRC-CIA, conventional ELISA kits ( Table 4). The results demonstrated that the developed DLTRRC-CIA could simultaneously screen CAP and CLE in the incurred samples as the ELISA kits did. Thereafter, the developed DLTRRC-CIA was reliable for the simultaneous screening of trace CAP and CLE residues in milk samples.

Conclusion
A novel sensitive DLTRRC-CIA was developed for simultaneous detection of CAP and CLE in milk based on distinguishable CL kinetic characteristics and covalent binding of capture antibodies. Since the adding substrate and detect time windows for HRP and ALP were different, and CL values for CAP (about 10 7 ) and CLE (about 10 5 ) were not in the same orders, the differentiation of CAP and CLE can be easily achieved. Goat antirabbit and goat anti-mouse immunoglobulins were covalently bound to the APTES-functionalized MTP, enhancing the sensitivity of the CIA for CAP and CLE, and reducing the assay duration. There was almost no cross-interaction resulting from the mixed CL reaction systems, testified by the recovery test. This strategy shows outstanding advantages such as low cost, high sensitivity, short assay duration and high efficiency. The recovery test and analysis in field samples demonstrates potential utility of

Supporting Information
Figure S1 Gross standard inhibition curves and revised standard inhibition curves in Figure 1.