- Email: [email protected]
Optimal liquidation of financial derivatives
Analytical Biochemistry 577 (2019) 45–51
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Integrated immunochromatographic assay for qualitative and quantitative detection of clenbuterol
T
Yuan Chena, Zhen Huanga, Song Hua, Ganggang Zhanga, Juan Penga, Jun Xiab,**, Weihua Laia,* a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China Jiangxi Province Institute of Veterinary Drug and Food Control, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Colloidal gold Time-resolved fluorescent nanobeads Clenbuterol Immunochromatographic assay Integrated detection
In this study, colloidal gold (CG) and time-resolved fluorescent nanobead (TRFN) probes were used to establish an integrated immunochromatographic assay (ICA) to qualitatively and quantitatively detect clenbuterol (CLE). The best experimental conditions for the two probes in separate ICAs were obtained by optimizing the antibody labeling concentration, the amount of antigen, and the concentration of probe. When the CG and TRFN probes co-existed in the ICA, the latter had no effect on the sensitivity of qualitative detection of the CG probe-based ICA. However, the CG probe optimized the linear range of quantitative detection in the TRFN probe-based ICA. The integrated test strip can be used for qualitative and quantitative detection of CLE in one step. When the amount of antigen reached 0.4 mg/mL, the CG probe concentration reached 1.2 μg/mL, and the TRFN probe concentration reached 0.68 μg/mL. The qualitative sensitivity of the integrated ICA was 0.5 ng/mL and its quantitative limit of detection was 0.04 ng/mL with a detection range of 0.1–2.7 ng/mL. This developed method is of great significance for large-scale samples screening and positive monitoring in the field of food safety testing.
1. Introduction Clenbuterol (CLE), a β-adrenergic receptor stimulant, is used to treat muscle spasms and asthma [1,2]. In the early 1980s, CLE significantly promoted animal growth, and increased the proportion of lean meat in animals and reduced fat proportion [3]. Thus, CLE has been used by some countries as a feed additive in animal breeding. Subsequent research on the use of CLE to promote animal growth, however, has raised concerns about its safety and toxicity [4–6]. Therefore, adding CLE to animal feeds and drinking water has been forbidden by most governments. The limit of detection (LOD) of the Chinese National Standard GB/T5009.192–2003 for detecting CLE in animal foods is 0.5 μg/kg. The methods for detecting CLE residues are generally divided into two categories: the confirmation method, which is based on molecular structural analyses, such as gas chromatography–tandem mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) [7–9]; and the screening method, which includes enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) [10–12]. Among these techniques, ICA is widely used for large-scale on-site screening of CLE because it is
*
simple, rapid, and inexpensive [13–16]. To date, qualitative or quantitative detection of CLE is usually achieved through ICA. Qualitative detection distinguishes a negative or positive result by the naked eye, whereas quantitative detection calculates the concentration of the analyte by using a portable instrument. These methods present advantages and disadvantages in terms of practical application. Qualitative detection is simple and time saving but cannot precisely determine the concentration of CLE in the sample. Quantitative detection can determine the accurate concentration of CLE in the sample but requires additional time to read the signal on the test line. In the present study, an integrated ICA for qualitative and quantitative detection of CLE is first developed. Colloidal gold (CG), which is characterized by stable preparation, high molar extinction coefficient, and obvious color [17], was selected as a label for qualitative detection. Time-resolved fluorescent nanobeads (TRFN), which feature high fluorescence intensity, narrow emission bands, large Stoke shifts, and a long fluorescence lifetime [18], were selected as a label for quantitative detection. In the integrated ICA, negative samples can be rapidly excluded by the naked eye with qualitative detection. If the sample screened positive, further quantitative detection is performed in the
Corresponding author. No. 235 Nanjing East Road, Nanchang, 330047, China. Corresponding author. No. 698 Jingdong Road, Nanchang, 330096, China. E-mail addresses: [email protected] (J. Xia), [email protected] (W. Lai).
**
https://doi.org/10.1016/j.ab.2019.04.013 Received 27 August 2018; Received in revised form 25 January 2019; Accepted 16 April 2019 Available online 22 April 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Scheme 1. Schematic of the integrated ICA method for detecting clenbuterol (CLE).
were purchased from Hangzhou Feng hang Technology Co., Ltd. (Hangzhou, China). The light-emitting diodes in the TRFN reader served as the excitation source (365 nm), and the detection wavelength of this reader was 615 nm. The size distributions and surface morphologies of CG and TRFN were determined by using a high-resolution transmission electron microscope (JEOL JEM 2100, Tokyo, Japan). The UV–vis absorption spectra of CG were obtained by a UV–vis spectrophotometer (Shimadzu, UV-2300, Japan).
same test strip to obtain precise concentrations and avoid false positive results. CLE detection is efficiently and precisely achieved using the developed ICA. 2. Materials and methods 2.1. Materials and equipment CG (25 nm) was prepared in our laboratory. TRFN (1%, solid content, w/v; carboxylate-modified Eu (III)-chelate-doped polystyrene nanobeads; excitation = 365 nm, emission = 615 nm) was purchased from Nanjing Weice Biotech Co., Ltd. (Nanjing, China). Nitrocellulose membrane (CN140; NC membrane) was supplied by Sartorius Co., Ltd. (Gottingen, Germany). Polyvinylchloride and backing, absorbent, filter, sample, and conjugate pads were purchased from Shanghai Jinbiao Tech Co., Ltd. (Shanghai, China). Mouse anti-CLE monoclonal antibody (CLE-m Ab), goat anti-mouse IgG, and CLE-bovine serum albumin (BSA) conjugate antigen were provided by Jiangxi Zodolabs Biotech Co., Ltd. (Jiangxi, China). BSA and N-(3-dimethylaminopropyl)-N-ethylcarbodimide hydrochloride (EDC·HCL) were acquired from Sigma Co., Ltd. (St. Louis, MO, USA). CLE, ractopamine (RAC), salbutamol (SAL), bambuterol (BAM), mabuterol (MAB), penbutolol (PEN), and terbutaline (TER) were obtained from Dr. Ehretorfer, GmbH (Augsburg, Germany). The 20 pork samples were purchased from a supermarket. All solvents and other chemicals used in the experiments were of analytical-reagent grade. XYZ-3050 Platform was purchased from Bio Dot Co., Ltd. (Irvine, USA). TRFN test strip readers and an automatic programmable cutter
2.2. Preparation of the mAb probes 2.2.1. Preparation of the CG-mAb probe The CG-labeled anti-CLE mAb (CG-mAb) probe was prepared according to the reported method with a few modifications [19]. A total of 1 mL CG solution was adjusted to pH 6.5 with 0.2 M K2CO3. With gentle stirring, 0.1 mL of anti-CLE mAb (30 μg/mL) was added dropwise to the CG solution. The mixed solution was incubated at room temperature for 60 min and then blocked with 0.1 mL of 1% (w/v) polyethylene glycol solution for 30 min and 0.1 mL of BSA (10%, w/v) for 30 min. Finally, the mixture was centrifuged at 9000 rpm at 4 °C for 30 min. The pellet was suspended in 0.05 mL of 0.01 M phosphatebuffered saline (PBS, pH 7.4). 2.2.2. Preparation of the TRFN-mAb probe The TRFN-labeled anti-CLE mAb (TRFN-mAb) probe was prepared as previously reported with some modifications [20]. TRFN (20 μL, 10 mg/mL, w/v) was added to 2 mL of 0.05 M boric acid buffer and mixed in a vortex mixer. Thereafter, 20 μL of fresh EDC (0.5 mg/mL) 46
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
probes flowed on the nitrocellulose membrane with the sample and bound to the coating antigens on the test line. Visually, the color of the test line (T line) was darker than or as dark as the control line (C line) in the CG probe-based ICA. When the CLE concentration in the sample was equal to or greater than 0.5 ng/mL, most probes combined with the CLE in the sample and cannot bind to the coating antigen on the T line. Thus, the signal of the probes was reduced on the T line. The color of the T line was shallower than that of the control line in the CG probebased ICA. The TRFN probe demonstrated the same principle, and its signal value was obtained by using a fluorescence reader. (B) Detection diagram: CG probe-based ICA was used for the qualitative analysis of CLE. If the sample was positive, further quantitative analysis was carried out by using the TRFN probe-based ICA.
was added to the mixture, which was then stirred for 15 min. Next, 30 μL of antianti-CLE mAb (150 μg/mL) was added dropwise to the mixed solution, which was incubated at room temperature for 2 h, and blocked with 200 μL of blocking buffer containing BSA (10%, w/v). The mixture was centrifuged at 14,000 rpm at 4 °C for 20 min. The pellet was suspended in 2 mL of boric acid buffer (0.05 M, pH 7.4) with 0.2% BSA and 0.5% Tween-20. 2.3. Determination of the optimal concentration of the coating antigen and volume of the probe in a single system 2.3.1. Selection of the optimal antigen concentration and preparation of test strips Coating antigen (CLE-BSA), which was diluted with 0.01 M PBS to concentrations of 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL, was sprayed onto the NC membrane with XYZ 3050 Platform as the T line (0.74 μL/cm). Goat anti-mouse IgG was diluted with 0.01 M PBS to 0.6 mg/mL and sprayed onto the NC membrane with XYZ3050 Platform as the C line (0.74 μL/cm). The prepared NC membrane was placed in a drying oven at 37 °C for 8 h. As shown in Scheme 1, the sample pad, conjugate pad, NC membrane, and absorbent pad were assembled as a test strip. The optimal concentration of CLE-BSA was based on the signal intensity of the T line in the negative sample and the competitive inhibition ratio of the positive sample. The competitive inhibition ratio was obtained by using the equation (B0−B)/B0 × 100%. For the TRFN probe-based ICA, B0 and B denoted the fluorescence intensities of the T line in the negative and positive samples (0.5 ng/mL), respectively. For the CG probe-based ICA, B0 and B denoted the signal ratios of the T line to the C line (AT/AC) in the negative and positive samples (0.5 ng/mL), respectively.
2.5. Determination of interactions between the TRFN and CG systems 2.5.1. Effect of TRFN-mAb on the CG system 0.6 μg/mL of the optimal CG-mAb was mixed with 0, 0.23, 0.45, 0.68, and 0.90 μg/mL of TRFN-mAb. The CLE standard solutions (0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL) were detected with the test strip thrice, and the optimal concentration of the TRFN probe was selected according to the linear range and detection sensitivity of the CG probebased ICA. 2.5.2. Effect of CG-mAb on the TRFN system 0.68 μg/mL of the optimal TRFN-mAb was mixed with 0, 0.3, 0.6, 1.2, and 1.8 μg/mL of CG-mAb, and the CLE standard solutions (0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL) were detected thrice by using the test strip. 2.6. Establishment of the standard curve of the quantitative system A series of CLE solutions was prepared by adding the CLE standard solution to 0.01 M PBS to obtain concentrations of 0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL. A standard curve was established by plotting the fluorescence intensity of the T line against the logarithm of the CLE concentration. LOD was defined as the mean plus three times the standard deviation of 20 negative samples [21]. Half-maximal inhibitory concentration (IC50) was defined as the CLE concentration in 0.01 M PBS that caused a 50% decrease in B/B0 compared with that produced by PBS. Each test was repeated thrice.
2.3.2. Optimal concentration of the TRFN probe The prepared TRFN-mAb probe (0.11, 0.23, 0.45, 0.68, and 0.90 μg/ mL) was added to an ELISA well, which was then dried at 30 °C for 1.5 h. 2.3.3. Optimal concentration of the CG probe The prepared CG-mAb probe (0.06, 0.30, 0.60, 0.9, and 1.2 μg/mL) was added to an ELISA well, which was then dried at 30 °C for 1.5 h. The optimal concentration of the CG probe was based on the signal intensity of AT/AC of the negative sample and the competitive inhibition ratio of the positive sample (0.5 ng/mL).
2.7. Accuracy and precision assay 2.4. Detection procedure Three positive samples (0.5, 1.0, and 1.5 ng/mL) were prepared by spiking CLE into pork. This developed method was used to detect the concentrations of the three positive samples and calculate the recovery and standard deviation (SD). The experiments were repeated thrice.
PBS buffer (0.01 M) was used as the negative sample. CLE standard solutions were prepared by adding CLE to the negative sample to obtain concentrations of 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL. One hundred and 10 μL of each sample was separately added to an ELISA well containing the probe and incubated for 5 min. All samples in the ELISA wells were added to the test strip for 5 min, and qualitative results were obtained by observing changes in the coloration of the T and C lines. After 20 min, quantitative results were read by using a fluorescent microsphere reader. The particle sizes of CG and TRFN were quite different, and the mobility of the two labels after coupling the antibody on the NC membrane was different. The CG particle size was small, the moving speed was fast on the NC membrane, the color development of the T line and the C line were rapid, and the judgment time was short. Therefore, the time of qualitative test was 5 min. TRFN showed large particle size and slow color development and required longer judgment time than CG. Thus, the time of the quantitative test was 20 min. The detection principle is shown in Scheme 1 (A) The integrated test strip developed in this study was based on the principle of competitive immune analysis. When no CLE was present in the sample, the two
2.8. Specificity assay The specificity of the quantitative TRFN test strip was evaluated using seven β-adrenergic agonists (CLE, RAC, SAL, MAB, BAM, PEN, and TER; 10 μg/mL; Fig. S4) in 0.01 M PBS. Each test was repeated thrice. 2.9. Validation assay The 20 samples of pork used in the test were purchased from a supermarket. Pretreatment of samples and LC–MS/MS were performed according to Standard Method 1025-18-2008 (Announcements of the Ministry of Agriculture, China). Briefly, the 2 g sample was accurately weighed and added in a 50 mL centrifuge tube. Then, 8 mL of ammonium acetate solution (0.2 mol/L, pH 5.2) and 40 μL of arylsulfatase were added. After shaking at 37 °C in a dark water bath for 16 h for 47
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
successfully conjugated on the surface of the two labels. The UV–vis spectrum of CG is shown in Fig. 1E, and the characteristic absorption peak of CG is observable at 520 nm. This peak slightly red-shifted to 526 nm after coupling with the antibody, indicating that the antibody was successfully coupled to the CG ICA system.
enzymatic hydrolysis, the mixture was centrifuged at 10,000 rpm for 10 min. All the supernatant was transferred to a 50 mL centrifuge tube, and 5 mL of 0.1 mol/L perchloric acid solution was added to adjust the pH to 1.0. After centrifuging at 10,000 rpm for 10 min, the supernatant was transferred to a new 50 mL centrifuge tube. The pH was adjusted to 9.5 with 10 mol/L NaOH, 15 mL of ethyl acetate was added, and the upper organic phase was removed by centrifugation. 10 mL of Tertbutanol/tert-butyl methyl ether (60:40, v/v) was added to the lower aqueous phase, which was then adequately oscillated. After centrifugation at 5000 rpm for 5 min, the organic phases were combined and concentrated by using a nitrogen evaporator. The residue was dissolved in 5 mL of 0.2% aqueous formic acid (v/ v). The reconstituted solution was separated and purified via solid phase extraction (using Mixed-mode Cation Exchanger SPE Column), and the purified solution was filtered through a 0.22 μm cellulose membrane for LC–MS/MS analysis. The LC system for CLE detection included a BEH-C18 column (2.1 × 50 mm, 1.7 μm), which was kept at 30 °C for chromatographic separation. The flow rate of the mobile phase was 0.30 mL/min, and the injection volume of the sample solution was 10 μL. The mobile phase consisted of aqueous formic acid (acetonitrile:0.1% formic = 50:50, v/v). An electrospray positive-ion source was used, and MS acquisition was performed in multiple-reaction monitoring mode. The monitoring ion pairs were CLE m/z 277.11/ 202.78 (quantitative ion) and 277.11/258.94 (qualitative ion). The detection results of 20 pork samples by the LC–MS/MS and the developed method were compared.
As shown in Fig. S1A, the T line signal intensity of the TRFN probebased ICA increased with increasing concentration of CLE-BSA on the T line, and the strongest intensity (10793 a.u.) was obtained at 0.8 mg/ mL CLE-BSA. A maximum competitive inhibition ratio of 82.2% and a relatively strong T line signal intensity of 6418 a.u. were observed with 0.4 mg/mL CLE-BSA. Thus, 0.4 mg/mL was regarded as the optimal concentration of CLE-BSA on the T line. The concentration of TRFN-mAb is a key consideration in preparing the test strips. As shown in Fig. S1B, the T line signal intensity of the TRFN probe-based ICA gradually increased with increasing concentration of TRFN-mAb, and a maximum intensity of 9002 a.u. was observed at a TRFN concentration of 0.9 μg/mL. However, when 0.68 μg/mL of TRFN-mAb was applied, a maximum competitive inhibition ratio of 79% and a strong T line signal intensity of 6575 a.u. were observed. Considering the intensity of the T line (negative sample) and the maximum inhibition ratio (0.5 ng/mL, positive sample), 0.68 μg/mL was regarded as the optimal concentration of TRFN-mAb for TRFN probe-based ICA.
3. Results and discussion
3.3. Optimal conditions for CG probe-based ICA
3.1. Characteristics of free labels and label-mAb probes
As shown in Fig. S2A, the T line signal intensity of the CG probebased ICA increased with increasing concentration of CLE-BSA on the T line, and maximum intensity (13.19) was obtained with 0.8 mg/mL CG. However, a maximum competitive inhibition ratio of 87% and a strong T line signal intensity of 2.67 were observed with 0.4 mg/mL CLE-BSA. Therefore, 0.4 mg/mL was regarded as the optimal concentration of CLE-BSA on the T line. The concentration of CG-mAb is another key consideration in preparing the test strips. As shown in Fig. S2B, the inhibition ratio of the
3.2. Optimal conditions for the TRFN probe-based ICA
Transmission electron microscopy (TEM, Fig. 1A–B) indicated that the size distributions of TRFN and CG were relatively uniform, and the probes had average particle diameters of 210 and 25 nm, respectively. The hydration particle sizes of TRFN and CG (Fig. 1C–D) were 225.1 and 20 nm, respectively. After modification with the antibody, the hydration particle sizes of TRFN and CG (Fig. 1C–D) increased to 279.8 and 49.3 nm, respectively, thereby indicating that the antibody was
Fig. 1. TEM images of (A) CG and (B) TRFN. Dynamic light-scattering spectra of (C) the TRFN label and TRFN-mAb probe and (D) the CG label and CG-mAb probe. (E) UV–vis absorption spectra of CG label and CG-mAb probe. 48
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Fig. 2. Effect of the TRFN-mAb probe on the CG-mAb probe. (A) Results of the negative and 0.5 ng/mL positive samples. (B) Standard curve of the CG-mAb probe.
probe are 1.2 and 0.68 μg/mL, respectively, the qualitative and quantitative results match. The CG-mAb probe affects the TRFN-mAb probe because the excitation wavelength of TRFN is 365 nm, and CG partly adsorbs light at 365 nm [22]. Therefore, the CG probe can absorb some of the excitation light of TRFN and affect the fluorescence intensity of the TRFN-mAb probe on the T line. When the concentrations of the CGmAb probe were 0, 0.3, 0.6, 1.2, and 1.8 μg/mL, the linear ranges of the TRFN probe-based ICA were 0.01–0.9, 0.1–0.9, 0.1–0.9, 0.1–2.7, and 0.1–2.7 ng/mL, respectively (Table S1).
CG probe-based ICA gradually decreased with increasing concentration of CG-mAb, and a maximum ratio of 95% was obtained at 0.3 μg/mL of CG-mAb. However, the intensity of C line color development was too low at 0.06 and 0.3 μg/mL of CG-mAb. Considering that the best conditions should be selected in combination with the visual results and a reasonably high ratio of 88% was observed at 0.6 μg/mL of CG-mAb, this concentration was regarded as the optimal concentration of the CGmAb probe for the CG probe-based ICA. 3.4. Interaction between probes
3.5. Establishment of the standard curve 3.4.1. Effect of the TRFN-mAb probe on the CG-mAb probe As shown in Fig. 2A, the detection results of the CG probe-based ICA did not change significantly with addition of TRFN-mAb. AT/AC ranged from 2.0 to 2.5 for all negative samples and from 0.30 to 0.38 for positive samples (0.5 ng/mL). No significant difference was observed among the results. The AT/AC intensity of the GC probe-based ICA is the signal of gray value of the GC probe. Fig. 2B compares the CG probe-based ICA obtained by mixing the CG probe with five different volumes of TRFN-mAb and the CG probe-based ICA without the TRFN probe. The T line intensity of negative samples in the standard curve ranged from 1985 a.u. to 2096 a.u., and the inhibition rates of the six spiking concentrations (0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL TRFN) exhibited no significant difference. Therefore, addition of the TRFNmAb probe does not affect the detection results of the CG probe-based ICA. This finding may be attributed to several reasons. First, the TRFNmAb probe does not emit light in the absence of excitation. Second, the TRFN label presents high fluorescence intensity and low use of antibody. Calculation of the molar amounts of antibody coupled to the two probes revealed that the amount of antibody coupled to the TRFN label is 1/10 that coupled to the CG label. Thus, the TRFN probe does not affect the color development of the CG-mAb probe.
Using the logarithm of CLE concentration as the x-coordinate and the T line fluorescence intensity of the TRFN-mAb probe as the y-coordinate, the standard competitive inhibition curve was established (Fig. 4A). The linear regression equation of the standard curve was y = −834.6log(x) + 1102.6 with R2 = 0.997. The quantitative detection range, LOD, and IC50 were 0.1–2.7, 0.04, and 0.33 ng/mL, respectively. Fig. 4B shows an image of the TRFN-mAb probe-based test strip under 365 nm excitation. The qualitative result of the CG-mAb probe is shown in Fig. S3. 3.6. Accuracy and precision Recovery experiments were conducted to evaluate the accuracy and precision of the developed method by using CLE-spiked samples. The average intra-assay recoveries of the developed method were from 91.1% to 95.6%, with coefficients of variation of 2.1%–7.6%. The average inter-assay recoveries of the developed method were from 85.3% to 93.1%, with coefficients of variation of 7.7%–10.9% (Table 1). 3.7. Specificity
3.4.2. Effect of the CG-mAb probe on the TRFN-mAb probe The results in Fig. 3A show that, when the two probes are mixed, the TRFN-mAb fluorescence intensity gradually decreases with increasing concentration of the CG-mAb probe. The T line intensity also decreases by 25% from 7318 a.u. to 5431 a.u. When a positive sample was detected, the fluorescence intensity of the T line gradually decreased as the concentration of the positive sample increased (Fig. 3B). When the CLE concentration in the positive sample was 8.1 ng/mL, the T line fluorescence intensity remained basically the same. However, as the strength of the negative T line decreased, IC50 of the standard curve changed (Table S1). Based on the standard curve data, we determined the best combination concentration of CG-mAb and TRFN-mAb probes. When the concentrations of the CG-mAb probe and the TRFN-mAb
The developed method exhibited no cross-reaction to RAC, MAB, BAM, PEN, and TER. Its cross-reaction to SAL was less than 0.25% (Table S2). 3.8. Accuracy of actual sample Twenty samples of pork were tested by the developed method, and the results were consistent with the LC–MS/MS method (Table 2). 4. Conclusions CLE can be qualitatively and quantitatively detected using the 49
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Fig. 3. Effect of the CG-mAb probe on the TRFN-mAb probe. (A) Results of the negative samples. (B) Standard curve of the TRFN-mAb probe. Table 2 Detection result of real pork samples by LC-MS/MS and the developed method. sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fig. 4. Qualitative and quantitative results of the TRFN probe-based test strips: (A) Standard curve of TRFN-mAb. (B) Image of the TRFN probe-based test strip under 365 nm excitation (1, 2, 3, 4, 5, and 6 represent probe concentrations of 0, 0.1, 0.3, 0.5, 0.9, and 2.7 ng/mL, respectively).
Table 1 The accuracy and precision of the TRFN ICA in CLE-spiked pork samples. (n = 3). Spiked CLE (ng/mL)
0.5 1.0 1.5 a
intra-assay
a
LC-MS/MS (ng/mL)
Na 0.07 N N N N 0.11 N N N N N 0.18 N N N N N N N
SD
0.010
0.015
0.006
Developed method qualitative result
quantitative result (ng/mL)
N N N N N N N N N N N N N N N N N N N N
N 0.10 N N 0.03 N 0.15 N 0.01 N 0.05 N 0.15 N N N 0.04 0.02 N N
SD
0.016
0.006 0.023 0.002 0.009 0.017
0.009 0.003
N = not detected.
inter-assay
Recovery %a
SD
CV %
Recovery %a
SD
CV %
91.1 92.7 95.6
0.02 0.07 0.03
4.5 7.6 2.1
87.3 85.3 93.1
0.05 0.08 0.11
10.9 9.4 7.7
Acknowledgement This work was supported by earmarked fund for Jiangxi agriculture research system (JXARS-03). Appendix A. Supplementary data
Recovery = (detection concentration/spiked concentration) *100%.
Supplementary data related to this article can be found at https:// doi.org/10.1016/j.ab.2019.04.013.
integrated test strip developed in this work. The visual sensitivity of the test strip was 0.5 ng/mL, and its quantitative detection range was 0.1–2.7 ng/mL. Based on the effect of CG on the negative fluorescence intensity of TRFN, IC50 and linear range of the TRFN probe were increased from 0.03 ng/mL to 0.33 ng/mL and from 0.01 to 0.9 ng/mL to 0.1–2.7 ng/mL, respectively. The linear range of quantitative detection matched the sensitivity of qualitative detection. In practice, the developed method can greatly reduce the detection time of batch samples and quantitatively measure the positive results. In addition, this developed method is convenient to use, provides accurate results, and presents obvious advantages and broad application prospects in food safety detection.
Conflicts of interest The authors declare that they have no conflict of interest. References [1] O. Bohorov, P.J. Buttery, J. Correia, J.B. Soar, The effect of the beta-2-adrenergic agonist clenbuterol or implantation with estradiol plus trenbolone acetate on protein-metabolism in wether lambs, Br. J. Nutr. 57 (1987) 99–107. [2] M. Pairet, P. Engelmann, H. VonNicolai, P. Champeroux, S. Richard, G. Rauber, G. Engelhardt, Ambroxol improves the broncho-spasmolytic activity of clenbuterol in the Guinea-pig, J. Pharm. Pharmacol. 49 (1997) 184–186. [3] P.J. Reeds, S.M. Hay, P.M. Dorwood, R.M. Palmer, Stimulation of muscle growth by
50
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
[4] [5] [6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14] S. Gao, C. Nie, J. Wang, J. Wang, L. Kang, Y. Zhou, J.-L. Wang, Colloidal gold-based immunochromatographic test strip for rapid detection of abrin in food samples, J. Food Prot. 75 (2012) 112–117. [15] Y. Niu, D. Wang, L. Cui, B. Wang, X. Pang, P. Yu, Monoclonal antibody-based colloid gold immunochromatographic strip for the rapid detection of Tomato zonate spot tospovirus, Virol. J. 15 (2018) 56–61. [16] I. Nurulfiza, M. Hair-Bejo, A.R. Omar, I. Aini, Immunochromatographic gold-based test strip for rapid detection of Infectious bursal disease virus antibodies, J. Vet. Diagn. Investig. 23 (2011) 320–324. [17] X. Huang, Z.P. Aguilar, H. Xu, W. Lai, Y. Xiong, Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: a review, Biosens. Bioelectron. 75 (2016) 166–180. [18] X. Tang, Z. Zhang, P. Li, Q. Zhang, J. Jiang, D. Wang, J. Lei, Sample-pretreatmentfree based high sensitive determination of aflatoxin M1 in raw milk using a timeresolved fluorescent competitive immunochromatographic assay, RSC Adv. 5 (2015) 558–564. [19] T. Peng, F.S. Zhang, W.C. Yang, D.X. Li, Y. Chen, Y.H. Xiong, H. Wei, W.H. Lai, Lateral-flow assay for rapid quantitative detection of clorprenaline residue in swine urine, J. Food Prot. 77 (2014) 1824–1829. [20] E. Juntunen, T. Myyryläinen, T. Salminen, T. Soukka, K. Pettersson, Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay, Anal. Biochem. 428 (2012) 31–39. [21] L. Rivas, A. de la Escosura-Muñiz, L. Serrano, L. Altet, O. Francino, A. Sánchez, A. Merkoçi, Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control, Nano Res. 8 (2015) 3704–3714. [22] L.-M. Hu, K. Luo, J. Xia, G.-M. Xu, C.-H. Wu, J.-J. Han, G.-G. Zhang, M. Liu, W.H. Lai, Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine, Biosens. Bioelectron. 91 (2017) 95–103.
clenbuterol-lack of effect on muscle protein-biosynthesis, Br. J. Nutr. 56 (1986) 249–258. G.S. Lynch, J.G. Ryall, Role of beta-adrenoceptor signaling in skeletal muscle: implications for muscle wasting and disease, Physiol. Rev. 88 (2008) 729–767. C.F. Kearns, K.H. McKeever, Clenbuterol and the horse revisited, Vet. J. 182 (2009) 384–391. F. Ramos, A. Cristino, P. Carrola, T. Eloy, J.M. Silva, M.D. Castilho, M.I.N. da Silveira, Clenbuterol food poisoning diagnosis by gas chromatography-mass spectrometric serum analysis, Anal. Chim. Acta 483 (2003) 207–213. W.J. Blanchflower, S.A. Hewitt, A. Cannavan, C.T. Elliott, D.G. Kennedy, Detection of clenbuterol residues in bovine liver, muscle, retina and urine using gas-chromatography mass-spectrometry, Biol. Mass Spectrom. 22 (1993) 326–330. D. Courtheyn, C. Desaever, R. Verhe, High-performance liquid-chromatographic determination of clenbuterol and cimaterol using postcolumn derivatization, J. Chromatogr. Biomed. Appl. 564 (1991) 537–549. P.R. Kootstra, C. Kuijpers, K.L. Wubs, D. van Doorn, S.S. Sterk, L.A. van Ginkel, R.W. Stephany, The analysis of beta-agonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening, Anal. Chim. Acta 529 (2005) 75–81. C.T. Elliott, W.J. McCaughey, H.D. Shortt, Residues of the beta-agonist clenbuterol in tissues of medicated farm-animals, Food Addit. Contam. 10 (1993) 231–244. Q. Huang, T. Bu, W. Zhang, L. Yan, M. Zhang, Q. Yang, L. Huang, B. Yang, N. Hu, Y. Suo, J. Wang, D. Zhang, An improved clenbuterol detection by immunochromatographic assay with bacteria@Au composite as signal amplifier, Food Chem. 262 (2018) 48–55. T. Huo, C. Peng, C. Xu, L. Liu, Development of colloidal gold-based immunochromatographic assay for the rapid detection of medroxyprogesterone acetate residues, Food Agric. Immunol. 17 (2006) 183–190. B.B. Dzantiev, N.A. Byzova, A.E. Urusov, A.V. Zherdev, Immunochromatographic methods in food analysis, Trac. Trends Anal. Chem. 55 (2014) 81–93.
51
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Integrated immunochromatographic assay for qualitative and quantitative detection of clenbuterol
T
Yuan Chena, Zhen Huanga, Song Hua, Ganggang Zhanga, Juan Penga, Jun Xiab,**, Weihua Laia,* a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China Jiangxi Province Institute of Veterinary Drug and Food Control, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Colloidal gold Time-resolved fluorescent nanobeads Clenbuterol Immunochromatographic assay Integrated detection
In this study, colloidal gold (CG) and time-resolved fluorescent nanobead (TRFN) probes were used to establish an integrated immunochromatographic assay (ICA) to qualitatively and quantitatively detect clenbuterol (CLE). The best experimental conditions for the two probes in separate ICAs were obtained by optimizing the antibody labeling concentration, the amount of antigen, and the concentration of probe. When the CG and TRFN probes co-existed in the ICA, the latter had no effect on the sensitivity of qualitative detection of the CG probe-based ICA. However, the CG probe optimized the linear range of quantitative detection in the TRFN probe-based ICA. The integrated test strip can be used for qualitative and quantitative detection of CLE in one step. When the amount of antigen reached 0.4 mg/mL, the CG probe concentration reached 1.2 μg/mL, and the TRFN probe concentration reached 0.68 μg/mL. The qualitative sensitivity of the integrated ICA was 0.5 ng/mL and its quantitative limit of detection was 0.04 ng/mL with a detection range of 0.1–2.7 ng/mL. This developed method is of great significance for large-scale samples screening and positive monitoring in the field of food safety testing.
1. Introduction Clenbuterol (CLE), a β-adrenergic receptor stimulant, is used to treat muscle spasms and asthma [1,2]. In the early 1980s, CLE significantly promoted animal growth, and increased the proportion of lean meat in animals and reduced fat proportion [3]. Thus, CLE has been used by some countries as a feed additive in animal breeding. Subsequent research on the use of CLE to promote animal growth, however, has raised concerns about its safety and toxicity [4–6]. Therefore, adding CLE to animal feeds and drinking water has been forbidden by most governments. The limit of detection (LOD) of the Chinese National Standard GB/T5009.192–2003 for detecting CLE in animal foods is 0.5 μg/kg. The methods for detecting CLE residues are generally divided into two categories: the confirmation method, which is based on molecular structural analyses, such as gas chromatography–tandem mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) [7–9]; and the screening method, which includes enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) [10–12]. Among these techniques, ICA is widely used for large-scale on-site screening of CLE because it is
*
simple, rapid, and inexpensive [13–16]. To date, qualitative or quantitative detection of CLE is usually achieved through ICA. Qualitative detection distinguishes a negative or positive result by the naked eye, whereas quantitative detection calculates the concentration of the analyte by using a portable instrument. These methods present advantages and disadvantages in terms of practical application. Qualitative detection is simple and time saving but cannot precisely determine the concentration of CLE in the sample. Quantitative detection can determine the accurate concentration of CLE in the sample but requires additional time to read the signal on the test line. In the present study, an integrated ICA for qualitative and quantitative detection of CLE is first developed. Colloidal gold (CG), which is characterized by stable preparation, high molar extinction coefficient, and obvious color [17], was selected as a label for qualitative detection. Time-resolved fluorescent nanobeads (TRFN), which feature high fluorescence intensity, narrow emission bands, large Stoke shifts, and a long fluorescence lifetime [18], were selected as a label for quantitative detection. In the integrated ICA, negative samples can be rapidly excluded by the naked eye with qualitative detection. If the sample screened positive, further quantitative detection is performed in the
Corresponding author. No. 235 Nanjing East Road, Nanchang, 330047, China. Corresponding author. No. 698 Jingdong Road, Nanchang, 330096, China. E-mail addresses: [email protected] (J. Xia), [email protected] (W. Lai).
**
https://doi.org/10.1016/j.ab.2019.04.013 Received 27 August 2018; Received in revised form 25 January 2019; Accepted 16 April 2019 Available online 22 April 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Scheme 1. Schematic of the integrated ICA method for detecting clenbuterol (CLE).
were purchased from Hangzhou Feng hang Technology Co., Ltd. (Hangzhou, China). The light-emitting diodes in the TRFN reader served as the excitation source (365 nm), and the detection wavelength of this reader was 615 nm. The size distributions and surface morphologies of CG and TRFN were determined by using a high-resolution transmission electron microscope (JEOL JEM 2100, Tokyo, Japan). The UV–vis absorption spectra of CG were obtained by a UV–vis spectrophotometer (Shimadzu, UV-2300, Japan).
same test strip to obtain precise concentrations and avoid false positive results. CLE detection is efficiently and precisely achieved using the developed ICA. 2. Materials and methods 2.1. Materials and equipment CG (25 nm) was prepared in our laboratory. TRFN (1%, solid content, w/v; carboxylate-modified Eu (III)-chelate-doped polystyrene nanobeads; excitation = 365 nm, emission = 615 nm) was purchased from Nanjing Weice Biotech Co., Ltd. (Nanjing, China). Nitrocellulose membrane (CN140; NC membrane) was supplied by Sartorius Co., Ltd. (Gottingen, Germany). Polyvinylchloride and backing, absorbent, filter, sample, and conjugate pads were purchased from Shanghai Jinbiao Tech Co., Ltd. (Shanghai, China). Mouse anti-CLE monoclonal antibody (CLE-m Ab), goat anti-mouse IgG, and CLE-bovine serum albumin (BSA) conjugate antigen were provided by Jiangxi Zodolabs Biotech Co., Ltd. (Jiangxi, China). BSA and N-(3-dimethylaminopropyl)-N-ethylcarbodimide hydrochloride (EDC·HCL) were acquired from Sigma Co., Ltd. (St. Louis, MO, USA). CLE, ractopamine (RAC), salbutamol (SAL), bambuterol (BAM), mabuterol (MAB), penbutolol (PEN), and terbutaline (TER) were obtained from Dr. Ehretorfer, GmbH (Augsburg, Germany). The 20 pork samples were purchased from a supermarket. All solvents and other chemicals used in the experiments were of analytical-reagent grade. XYZ-3050 Platform was purchased from Bio Dot Co., Ltd. (Irvine, USA). TRFN test strip readers and an automatic programmable cutter
2.2. Preparation of the mAb probes 2.2.1. Preparation of the CG-mAb probe The CG-labeled anti-CLE mAb (CG-mAb) probe was prepared according to the reported method with a few modifications [19]. A total of 1 mL CG solution was adjusted to pH 6.5 with 0.2 M K2CO3. With gentle stirring, 0.1 mL of anti-CLE mAb (30 μg/mL) was added dropwise to the CG solution. The mixed solution was incubated at room temperature for 60 min and then blocked with 0.1 mL of 1% (w/v) polyethylene glycol solution for 30 min and 0.1 mL of BSA (10%, w/v) for 30 min. Finally, the mixture was centrifuged at 9000 rpm at 4 °C for 30 min. The pellet was suspended in 0.05 mL of 0.01 M phosphatebuffered saline (PBS, pH 7.4). 2.2.2. Preparation of the TRFN-mAb probe The TRFN-labeled anti-CLE mAb (TRFN-mAb) probe was prepared as previously reported with some modifications [20]. TRFN (20 μL, 10 mg/mL, w/v) was added to 2 mL of 0.05 M boric acid buffer and mixed in a vortex mixer. Thereafter, 20 μL of fresh EDC (0.5 mg/mL) 46
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
probes flowed on the nitrocellulose membrane with the sample and bound to the coating antigens on the test line. Visually, the color of the test line (T line) was darker than or as dark as the control line (C line) in the CG probe-based ICA. When the CLE concentration in the sample was equal to or greater than 0.5 ng/mL, most probes combined with the CLE in the sample and cannot bind to the coating antigen on the T line. Thus, the signal of the probes was reduced on the T line. The color of the T line was shallower than that of the control line in the CG probebased ICA. The TRFN probe demonstrated the same principle, and its signal value was obtained by using a fluorescence reader. (B) Detection diagram: CG probe-based ICA was used for the qualitative analysis of CLE. If the sample was positive, further quantitative analysis was carried out by using the TRFN probe-based ICA.
was added to the mixture, which was then stirred for 15 min. Next, 30 μL of antianti-CLE mAb (150 μg/mL) was added dropwise to the mixed solution, which was incubated at room temperature for 2 h, and blocked with 200 μL of blocking buffer containing BSA (10%, w/v). The mixture was centrifuged at 14,000 rpm at 4 °C for 20 min. The pellet was suspended in 2 mL of boric acid buffer (0.05 M, pH 7.4) with 0.2% BSA and 0.5% Tween-20. 2.3. Determination of the optimal concentration of the coating antigen and volume of the probe in a single system 2.3.1. Selection of the optimal antigen concentration and preparation of test strips Coating antigen (CLE-BSA), which was diluted with 0.01 M PBS to concentrations of 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL, was sprayed onto the NC membrane with XYZ 3050 Platform as the T line (0.74 μL/cm). Goat anti-mouse IgG was diluted with 0.01 M PBS to 0.6 mg/mL and sprayed onto the NC membrane with XYZ3050 Platform as the C line (0.74 μL/cm). The prepared NC membrane was placed in a drying oven at 37 °C for 8 h. As shown in Scheme 1, the sample pad, conjugate pad, NC membrane, and absorbent pad were assembled as a test strip. The optimal concentration of CLE-BSA was based on the signal intensity of the T line in the negative sample and the competitive inhibition ratio of the positive sample. The competitive inhibition ratio was obtained by using the equation (B0−B)/B0 × 100%. For the TRFN probe-based ICA, B0 and B denoted the fluorescence intensities of the T line in the negative and positive samples (0.5 ng/mL), respectively. For the CG probe-based ICA, B0 and B denoted the signal ratios of the T line to the C line (AT/AC) in the negative and positive samples (0.5 ng/mL), respectively.
2.5. Determination of interactions between the TRFN and CG systems 2.5.1. Effect of TRFN-mAb on the CG system 0.6 μg/mL of the optimal CG-mAb was mixed with 0, 0.23, 0.45, 0.68, and 0.90 μg/mL of TRFN-mAb. The CLE standard solutions (0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL) were detected with the test strip thrice, and the optimal concentration of the TRFN probe was selected according to the linear range and detection sensitivity of the CG probebased ICA. 2.5.2. Effect of CG-mAb on the TRFN system 0.68 μg/mL of the optimal TRFN-mAb was mixed with 0, 0.3, 0.6, 1.2, and 1.8 μg/mL of CG-mAb, and the CLE standard solutions (0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL) were detected thrice by using the test strip. 2.6. Establishment of the standard curve of the quantitative system A series of CLE solutions was prepared by adding the CLE standard solution to 0.01 M PBS to obtain concentrations of 0, 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL. A standard curve was established by plotting the fluorescence intensity of the T line against the logarithm of the CLE concentration. LOD was defined as the mean plus three times the standard deviation of 20 negative samples [21]. Half-maximal inhibitory concentration (IC50) was defined as the CLE concentration in 0.01 M PBS that caused a 50% decrease in B/B0 compared with that produced by PBS. Each test was repeated thrice.
2.3.2. Optimal concentration of the TRFN probe The prepared TRFN-mAb probe (0.11, 0.23, 0.45, 0.68, and 0.90 μg/ mL) was added to an ELISA well, which was then dried at 30 °C for 1.5 h. 2.3.3. Optimal concentration of the CG probe The prepared CG-mAb probe (0.06, 0.30, 0.60, 0.9, and 1.2 μg/mL) was added to an ELISA well, which was then dried at 30 °C for 1.5 h. The optimal concentration of the CG probe was based on the signal intensity of AT/AC of the negative sample and the competitive inhibition ratio of the positive sample (0.5 ng/mL).
2.7. Accuracy and precision assay 2.4. Detection procedure Three positive samples (0.5, 1.0, and 1.5 ng/mL) were prepared by spiking CLE into pork. This developed method was used to detect the concentrations of the three positive samples and calculate the recovery and standard deviation (SD). The experiments were repeated thrice.
PBS buffer (0.01 M) was used as the negative sample. CLE standard solutions were prepared by adding CLE to the negative sample to obtain concentrations of 0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL. One hundred and 10 μL of each sample was separately added to an ELISA well containing the probe and incubated for 5 min. All samples in the ELISA wells were added to the test strip for 5 min, and qualitative results were obtained by observing changes in the coloration of the T and C lines. After 20 min, quantitative results were read by using a fluorescent microsphere reader. The particle sizes of CG and TRFN were quite different, and the mobility of the two labels after coupling the antibody on the NC membrane was different. The CG particle size was small, the moving speed was fast on the NC membrane, the color development of the T line and the C line were rapid, and the judgment time was short. Therefore, the time of qualitative test was 5 min. TRFN showed large particle size and slow color development and required longer judgment time than CG. Thus, the time of the quantitative test was 20 min. The detection principle is shown in Scheme 1 (A) The integrated test strip developed in this study was based on the principle of competitive immune analysis. When no CLE was present in the sample, the two
2.8. Specificity assay The specificity of the quantitative TRFN test strip was evaluated using seven β-adrenergic agonists (CLE, RAC, SAL, MAB, BAM, PEN, and TER; 10 μg/mL; Fig. S4) in 0.01 M PBS. Each test was repeated thrice. 2.9. Validation assay The 20 samples of pork used in the test were purchased from a supermarket. Pretreatment of samples and LC–MS/MS were performed according to Standard Method 1025-18-2008 (Announcements of the Ministry of Agriculture, China). Briefly, the 2 g sample was accurately weighed and added in a 50 mL centrifuge tube. Then, 8 mL of ammonium acetate solution (0.2 mol/L, pH 5.2) and 40 μL of arylsulfatase were added. After shaking at 37 °C in a dark water bath for 16 h for 47
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
successfully conjugated on the surface of the two labels. The UV–vis spectrum of CG is shown in Fig. 1E, and the characteristic absorption peak of CG is observable at 520 nm. This peak slightly red-shifted to 526 nm after coupling with the antibody, indicating that the antibody was successfully coupled to the CG ICA system.
enzymatic hydrolysis, the mixture was centrifuged at 10,000 rpm for 10 min. All the supernatant was transferred to a 50 mL centrifuge tube, and 5 mL of 0.1 mol/L perchloric acid solution was added to adjust the pH to 1.0. After centrifuging at 10,000 rpm for 10 min, the supernatant was transferred to a new 50 mL centrifuge tube. The pH was adjusted to 9.5 with 10 mol/L NaOH, 15 mL of ethyl acetate was added, and the upper organic phase was removed by centrifugation. 10 mL of Tertbutanol/tert-butyl methyl ether (60:40, v/v) was added to the lower aqueous phase, which was then adequately oscillated. After centrifugation at 5000 rpm for 5 min, the organic phases were combined and concentrated by using a nitrogen evaporator. The residue was dissolved in 5 mL of 0.2% aqueous formic acid (v/ v). The reconstituted solution was separated and purified via solid phase extraction (using Mixed-mode Cation Exchanger SPE Column), and the purified solution was filtered through a 0.22 μm cellulose membrane for LC–MS/MS analysis. The LC system for CLE detection included a BEH-C18 column (2.1 × 50 mm, 1.7 μm), which was kept at 30 °C for chromatographic separation. The flow rate of the mobile phase was 0.30 mL/min, and the injection volume of the sample solution was 10 μL. The mobile phase consisted of aqueous formic acid (acetonitrile:0.1% formic = 50:50, v/v). An electrospray positive-ion source was used, and MS acquisition was performed in multiple-reaction monitoring mode. The monitoring ion pairs were CLE m/z 277.11/ 202.78 (quantitative ion) and 277.11/258.94 (qualitative ion). The detection results of 20 pork samples by the LC–MS/MS and the developed method were compared.
As shown in Fig. S1A, the T line signal intensity of the TRFN probebased ICA increased with increasing concentration of CLE-BSA on the T line, and the strongest intensity (10793 a.u.) was obtained at 0.8 mg/ mL CLE-BSA. A maximum competitive inhibition ratio of 82.2% and a relatively strong T line signal intensity of 6418 a.u. were observed with 0.4 mg/mL CLE-BSA. Thus, 0.4 mg/mL was regarded as the optimal concentration of CLE-BSA on the T line. The concentration of TRFN-mAb is a key consideration in preparing the test strips. As shown in Fig. S1B, the T line signal intensity of the TRFN probe-based ICA gradually increased with increasing concentration of TRFN-mAb, and a maximum intensity of 9002 a.u. was observed at a TRFN concentration of 0.9 μg/mL. However, when 0.68 μg/mL of TRFN-mAb was applied, a maximum competitive inhibition ratio of 79% and a strong T line signal intensity of 6575 a.u. were observed. Considering the intensity of the T line (negative sample) and the maximum inhibition ratio (0.5 ng/mL, positive sample), 0.68 μg/mL was regarded as the optimal concentration of TRFN-mAb for TRFN probe-based ICA.
3. Results and discussion
3.3. Optimal conditions for CG probe-based ICA
3.1. Characteristics of free labels and label-mAb probes
As shown in Fig. S2A, the T line signal intensity of the CG probebased ICA increased with increasing concentration of CLE-BSA on the T line, and maximum intensity (13.19) was obtained with 0.8 mg/mL CG. However, a maximum competitive inhibition ratio of 87% and a strong T line signal intensity of 2.67 were observed with 0.4 mg/mL CLE-BSA. Therefore, 0.4 mg/mL was regarded as the optimal concentration of CLE-BSA on the T line. The concentration of CG-mAb is another key consideration in preparing the test strips. As shown in Fig. S2B, the inhibition ratio of the
3.2. Optimal conditions for the TRFN probe-based ICA
Transmission electron microscopy (TEM, Fig. 1A–B) indicated that the size distributions of TRFN and CG were relatively uniform, and the probes had average particle diameters of 210 and 25 nm, respectively. The hydration particle sizes of TRFN and CG (Fig. 1C–D) were 225.1 and 20 nm, respectively. After modification with the antibody, the hydration particle sizes of TRFN and CG (Fig. 1C–D) increased to 279.8 and 49.3 nm, respectively, thereby indicating that the antibody was
Fig. 1. TEM images of (A) CG and (B) TRFN. Dynamic light-scattering spectra of (C) the TRFN label and TRFN-mAb probe and (D) the CG label and CG-mAb probe. (E) UV–vis absorption spectra of CG label and CG-mAb probe. 48
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Fig. 2. Effect of the TRFN-mAb probe on the CG-mAb probe. (A) Results of the negative and 0.5 ng/mL positive samples. (B) Standard curve of the CG-mAb probe.
probe are 1.2 and 0.68 μg/mL, respectively, the qualitative and quantitative results match. The CG-mAb probe affects the TRFN-mAb probe because the excitation wavelength of TRFN is 365 nm, and CG partly adsorbs light at 365 nm [22]. Therefore, the CG probe can absorb some of the excitation light of TRFN and affect the fluorescence intensity of the TRFN-mAb probe on the T line. When the concentrations of the CGmAb probe were 0, 0.3, 0.6, 1.2, and 1.8 μg/mL, the linear ranges of the TRFN probe-based ICA were 0.01–0.9, 0.1–0.9, 0.1–0.9, 0.1–2.7, and 0.1–2.7 ng/mL, respectively (Table S1).
CG probe-based ICA gradually decreased with increasing concentration of CG-mAb, and a maximum ratio of 95% was obtained at 0.3 μg/mL of CG-mAb. However, the intensity of C line color development was too low at 0.06 and 0.3 μg/mL of CG-mAb. Considering that the best conditions should be selected in combination with the visual results and a reasonably high ratio of 88% was observed at 0.6 μg/mL of CG-mAb, this concentration was regarded as the optimal concentration of the CGmAb probe for the CG probe-based ICA. 3.4. Interaction between probes
3.5. Establishment of the standard curve 3.4.1. Effect of the TRFN-mAb probe on the CG-mAb probe As shown in Fig. 2A, the detection results of the CG probe-based ICA did not change significantly with addition of TRFN-mAb. AT/AC ranged from 2.0 to 2.5 for all negative samples and from 0.30 to 0.38 for positive samples (0.5 ng/mL). No significant difference was observed among the results. The AT/AC intensity of the GC probe-based ICA is the signal of gray value of the GC probe. Fig. 2B compares the CG probe-based ICA obtained by mixing the CG probe with five different volumes of TRFN-mAb and the CG probe-based ICA without the TRFN probe. The T line intensity of negative samples in the standard curve ranged from 1985 a.u. to 2096 a.u., and the inhibition rates of the six spiking concentrations (0.1, 0.3, 0.5, 0.9, 2.7, and 8.1 ng/mL TRFN) exhibited no significant difference. Therefore, addition of the TRFNmAb probe does not affect the detection results of the CG probe-based ICA. This finding may be attributed to several reasons. First, the TRFNmAb probe does not emit light in the absence of excitation. Second, the TRFN label presents high fluorescence intensity and low use of antibody. Calculation of the molar amounts of antibody coupled to the two probes revealed that the amount of antibody coupled to the TRFN label is 1/10 that coupled to the CG label. Thus, the TRFN probe does not affect the color development of the CG-mAb probe.
Using the logarithm of CLE concentration as the x-coordinate and the T line fluorescence intensity of the TRFN-mAb probe as the y-coordinate, the standard competitive inhibition curve was established (Fig. 4A). The linear regression equation of the standard curve was y = −834.6log(x) + 1102.6 with R2 = 0.997. The quantitative detection range, LOD, and IC50 were 0.1–2.7, 0.04, and 0.33 ng/mL, respectively. Fig. 4B shows an image of the TRFN-mAb probe-based test strip under 365 nm excitation. The qualitative result of the CG-mAb probe is shown in Fig. S3. 3.6. Accuracy and precision Recovery experiments were conducted to evaluate the accuracy and precision of the developed method by using CLE-spiked samples. The average intra-assay recoveries of the developed method were from 91.1% to 95.6%, with coefficients of variation of 2.1%–7.6%. The average inter-assay recoveries of the developed method were from 85.3% to 93.1%, with coefficients of variation of 7.7%–10.9% (Table 1). 3.7. Specificity
3.4.2. Effect of the CG-mAb probe on the TRFN-mAb probe The results in Fig. 3A show that, when the two probes are mixed, the TRFN-mAb fluorescence intensity gradually decreases with increasing concentration of the CG-mAb probe. The T line intensity also decreases by 25% from 7318 a.u. to 5431 a.u. When a positive sample was detected, the fluorescence intensity of the T line gradually decreased as the concentration of the positive sample increased (Fig. 3B). When the CLE concentration in the positive sample was 8.1 ng/mL, the T line fluorescence intensity remained basically the same. However, as the strength of the negative T line decreased, IC50 of the standard curve changed (Table S1). Based on the standard curve data, we determined the best combination concentration of CG-mAb and TRFN-mAb probes. When the concentrations of the CG-mAb probe and the TRFN-mAb
The developed method exhibited no cross-reaction to RAC, MAB, BAM, PEN, and TER. Its cross-reaction to SAL was less than 0.25% (Table S2). 3.8. Accuracy of actual sample Twenty samples of pork were tested by the developed method, and the results were consistent with the LC–MS/MS method (Table 2). 4. Conclusions CLE can be qualitatively and quantitatively detected using the 49
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
Fig. 3. Effect of the CG-mAb probe on the TRFN-mAb probe. (A) Results of the negative samples. (B) Standard curve of the TRFN-mAb probe. Table 2 Detection result of real pork samples by LC-MS/MS and the developed method. sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fig. 4. Qualitative and quantitative results of the TRFN probe-based test strips: (A) Standard curve of TRFN-mAb. (B) Image of the TRFN probe-based test strip under 365 nm excitation (1, 2, 3, 4, 5, and 6 represent probe concentrations of 0, 0.1, 0.3, 0.5, 0.9, and 2.7 ng/mL, respectively).
Table 1 The accuracy and precision of the TRFN ICA in CLE-spiked pork samples. (n = 3). Spiked CLE (ng/mL)
0.5 1.0 1.5 a
intra-assay
a
LC-MS/MS (ng/mL)
Na 0.07 N N N N 0.11 N N N N N 0.18 N N N N N N N
SD
0.010
0.015
0.006
Developed method qualitative result
quantitative result (ng/mL)
N N N N N N N N N N N N N N N N N N N N
N 0.10 N N 0.03 N 0.15 N 0.01 N 0.05 N 0.15 N N N 0.04 0.02 N N
SD
0.016
0.006 0.023 0.002 0.009 0.017
0.009 0.003
N = not detected.
inter-assay
Recovery %a
SD
CV %
Recovery %a
SD
CV %
91.1 92.7 95.6
0.02 0.07 0.03
4.5 7.6 2.1
87.3 85.3 93.1
0.05 0.08 0.11
10.9 9.4 7.7
Acknowledgement This work was supported by earmarked fund for Jiangxi agriculture research system (JXARS-03). Appendix A. Supplementary data
Recovery = (detection concentration/spiked concentration) *100%.
Supplementary data related to this article can be found at https:// doi.org/10.1016/j.ab.2019.04.013.
integrated test strip developed in this work. The visual sensitivity of the test strip was 0.5 ng/mL, and its quantitative detection range was 0.1–2.7 ng/mL. Based on the effect of CG on the negative fluorescence intensity of TRFN, IC50 and linear range of the TRFN probe were increased from 0.03 ng/mL to 0.33 ng/mL and from 0.01 to 0.9 ng/mL to 0.1–2.7 ng/mL, respectively. The linear range of quantitative detection matched the sensitivity of qualitative detection. In practice, the developed method can greatly reduce the detection time of batch samples and quantitatively measure the positive results. In addition, this developed method is convenient to use, provides accurate results, and presents obvious advantages and broad application prospects in food safety detection.
Conflicts of interest The authors declare that they have no conflict of interest. References [1] O. Bohorov, P.J. Buttery, J. Correia, J.B. Soar, The effect of the beta-2-adrenergic agonist clenbuterol or implantation with estradiol plus trenbolone acetate on protein-metabolism in wether lambs, Br. J. Nutr. 57 (1987) 99–107. [2] M. Pairet, P. Engelmann, H. VonNicolai, P. Champeroux, S. Richard, G. Rauber, G. Engelhardt, Ambroxol improves the broncho-spasmolytic activity of clenbuterol in the Guinea-pig, J. Pharm. Pharmacol. 49 (1997) 184–186. [3] P.J. Reeds, S.M. Hay, P.M. Dorwood, R.M. Palmer, Stimulation of muscle growth by
50
Analytical Biochemistry 577 (2019) 45–51
Y. Chen, et al.
[4] [5] [6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14] S. Gao, C. Nie, J. Wang, J. Wang, L. Kang, Y. Zhou, J.-L. Wang, Colloidal gold-based immunochromatographic test strip for rapid detection of abrin in food samples, J. Food Prot. 75 (2012) 112–117. [15] Y. Niu, D. Wang, L. Cui, B. Wang, X. Pang, P. Yu, Monoclonal antibody-based colloid gold immunochromatographic strip for the rapid detection of Tomato zonate spot tospovirus, Virol. J. 15 (2018) 56–61. [16] I. Nurulfiza, M. Hair-Bejo, A.R. Omar, I. Aini, Immunochromatographic gold-based test strip for rapid detection of Infectious bursal disease virus antibodies, J. Vet. Diagn. Investig. 23 (2011) 320–324. [17] X. Huang, Z.P. Aguilar, H. Xu, W. Lai, Y. Xiong, Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: a review, Biosens. Bioelectron. 75 (2016) 166–180. [18] X. Tang, Z. Zhang, P. Li, Q. Zhang, J. Jiang, D. Wang, J. Lei, Sample-pretreatmentfree based high sensitive determination of aflatoxin M1 in raw milk using a timeresolved fluorescent competitive immunochromatographic assay, RSC Adv. 5 (2015) 558–564. [19] T. Peng, F.S. Zhang, W.C. Yang, D.X. Li, Y. Chen, Y.H. Xiong, H. Wei, W.H. Lai, Lateral-flow assay for rapid quantitative detection of clorprenaline residue in swine urine, J. Food Prot. 77 (2014) 1824–1829. [20] E. Juntunen, T. Myyryläinen, T. Salminen, T. Soukka, K. Pettersson, Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay, Anal. Biochem. 428 (2012) 31–39. [21] L. Rivas, A. de la Escosura-Muñiz, L. Serrano, L. Altet, O. Francino, A. Sánchez, A. Merkoçi, Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control, Nano Res. 8 (2015) 3704–3714. [22] L.-M. Hu, K. Luo, J. Xia, G.-M. Xu, C.-H. Wu, J.-J. Han, G.-G. Zhang, M. Liu, W.H. Lai, Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine, Biosens. Bioelectron. 91 (2017) 95–103.
clenbuterol-lack of effect on muscle protein-biosynthesis, Br. J. Nutr. 56 (1986) 249–258. G.S. Lynch, J.G. Ryall, Role of beta-adrenoceptor signaling in skeletal muscle: implications for muscle wasting and disease, Physiol. Rev. 88 (2008) 729–767. C.F. Kearns, K.H. McKeever, Clenbuterol and the horse revisited, Vet. J. 182 (2009) 384–391. F. Ramos, A. Cristino, P. Carrola, T. Eloy, J.M. Silva, M.D. Castilho, M.I.N. da Silveira, Clenbuterol food poisoning diagnosis by gas chromatography-mass spectrometric serum analysis, Anal. Chim. Acta 483 (2003) 207–213. W.J. Blanchflower, S.A. Hewitt, A. Cannavan, C.T. Elliott, D.G. Kennedy, Detection of clenbuterol residues in bovine liver, muscle, retina and urine using gas-chromatography mass-spectrometry, Biol. Mass Spectrom. 22 (1993) 326–330. D. Courtheyn, C. Desaever, R. Verhe, High-performance liquid-chromatographic determination of clenbuterol and cimaterol using postcolumn derivatization, J. Chromatogr. Biomed. Appl. 564 (1991) 537–549. P.R. Kootstra, C. Kuijpers, K.L. Wubs, D. van Doorn, S.S. Sterk, L.A. van Ginkel, R.W. Stephany, The analysis of beta-agonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening, Anal. Chim. Acta 529 (2005) 75–81. C.T. Elliott, W.J. McCaughey, H.D. Shortt, Residues of the beta-agonist clenbuterol in tissues of medicated farm-animals, Food Addit. Contam. 10 (1993) 231–244. Q. Huang, T. Bu, W. Zhang, L. Yan, M. Zhang, Q. Yang, L. Huang, B. Yang, N. Hu, Y. Suo, J. Wang, D. Zhang, An improved clenbuterol detection by immunochromatographic assay with bacteria@Au composite as signal amplifier, Food Chem. 262 (2018) 48–55. T. Huo, C. Peng, C. Xu, L. Liu, Development of colloidal gold-based immunochromatographic assay for the rapid detection of medroxyprogesterone acetate residues, Food Agric. Immunol. 17 (2006) 183–190. B.B. Dzantiev, N.A. Byzova, A.E. Urusov, A.V. Zherdev, Immunochromatographic methods in food analysis, Trac. Trends Anal. Chem. 55 (2014) 81–93.
51