Author’s Accepted Manuscript Multi-color quantum DoT-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk Erqun Song, Mengqun Yu, Yunyun Wang, Weihua Hu, Dan Cheng, Mark T. Swihart, Yang Song www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(15)30114-7 http://dx.doi.org/10.1016/j.bios.2015.05.018 BIOS7680
To appear in: Biosensors and Bioelectronic Received date: 28 January 2015 Revised date: 4 May 2015 Accepted date: 6 May 2015 Cite this article as: Erqun Song, Mengqun Yu, Yunyun Wang, Weihua Hu, Dan Cheng, Mark T. Swihart and Yang Song, Multi-color quantum DoT-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multi-color quantum dot-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk Erqun Songa, ⃰, Mengqun Yua, Yunyun Wanga, Weihua Hub, Dan Chenga, Mark T. Swihartc, Yang Songa a
Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China. Fax: +862368251225; Tel: +862368251225. E-mail: [email protected] b
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy,
Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Southwest University, Chongqing 400715, People’s Republic of China. c
Department of Chemical and Biological Engineering, University at Buffalo, State
University of New York, Buffalo, NY 14260, USA.
Abstract: Antibiotic residues, which are among the most common contaminants in animal-based food products such as milk, have become a significant public health concern.
Here,
we
combine
a
multicolor
quantum
dot
(QD)-based
immunofluorescence assay and an array analysis method to achieve simultaneous, sensitive and visual detection of streptomycin (SM), tetracycline (TC), and penicillin G (PC-G) in milk. Antibodies (Abs) for SM, TC and PC-G were conjugated to QDs with different emission wavelengths (QD520nm, QD565nm and QD610nm) to serve as 1
detection probes (QD-Ab). Then a direct competitive fluorescent immunoassay was performed in antigen-coated microtiter plate wells for simultaneous qualitative and quantitative detection of SM, TC, and PC-G residues, based on fluorescence of the QD-Ab probes. The linear ranges for SM, TC and PC-G were 0.01-25 ng/mL, 0.01-25 ng/mL and 0.01-10 ng/mL, respectively, with detection limit of 5 pg/mL for each of them. Based on fluorescence of the QD-Ab probes, residues of the three antibiotics were determined visually and simultaneously. Compared with a commercial enzyme-linked immunosorbent assay kit, our method could achieve simultaneous analysis of multiple target antibiotics in multiple samples in a single run (high-throughput analysis) and improved accuracy and sensitivity for analysis of residues of the three antibiotics in authentic milk samples. This new analytical tool can play an important role in ameliorating the negative impact of the residual antibiotics on human health and the ecosystem. . Keywords: fluorescence immunoassay; array; multicolor; quantum dot; antibiotics; residues
1. Introduction Various food safety incidents in recent years have increased the need for food safety monitoring for contaminants such as drug residues and illegal additives. Among various possible contaminants in animal-based food products, antibiotics residues are of particular interest, due to their frequent use in animal husbandry, not 2
only for treatment of diseases but also for prophylactic and prevention purposes and to improve agricultural productivity. Consumption of animal products contaminated with antibiotic residues can cause allergic reactions in humans and reduce the efficacy of antibiotics for treatment human infections. The resulting increase in antibiotic-resistant bacteria is a public health concern. Moreover, residues of antibiotics are the most frequently detected contaminants in milk and other dairy products (Chafer-Pericas et al. 2010). These developments have increased the demand for accurate, sensitive and high-throughput analytical methods for the determination of antibiotic residues in foodstuffs. Several methods for detection of antibiotic residues are currently available, including microbiological assays (Aureli et al. 1996; Lourenco et al. 2013), instrumental assays (Bohm et al. 2012; Wang et al. 2006) and immunoassays (Bang-Ce et al. 2008; Meng and Xi 2011; Zhao et al. 2007a). However, microbiological assays are time consuming and have relatively poor sensitivity and specificity. Instrumental assays require complex sample preparation and expensive equipment. Thus, these approaches cannot fulfill the demand for fast, easy and simultaneous detection of antibiotic residues in milk. Immunoassays can offer higher sensitivity and specificity, along with greater convenience. However, the most common
enzyme-linked
immunosorbent
assay
(ELISA)
and
conventional
fluorescence immunoassay (FIA) does not provide high-throughput quantification of multiple analytes (e.g. several different antibiotics). Therefore, the development of new methods for the accurate, sensitive, rapid, and convenient detection of multiple antibiotics residues in milk remains an important need in food safety monitoring. 3
Photoluminescent semiconductor nanocrystals, such as quantum dots (QDs), have excellent fluorescence characteristics (e.g., stable, narrow, and tunable emission spectra with broad excitation spectra) compared with conventional organic fluorescent dyes (Alivisatos 1996; Bruchez et al. 1998; Medintz et al. 2005). As a result, they have been increasingly used as fluorescent labels in FIA (Esteve-Turrillas and Abad-Fuentes 2013; Goldman et al. 2002; Hu et al. 2010; Nichkova et al. 2007; Yang and Li 2006; Zhu et al. 2011) for the detection of pathogens (Wang et al. 2011; Yang and Li 2006; Zhao et al. 2009), proteins (Cao et al. 2011; Hu et al. 2010; Wang et al. 2007a; Wang and Mountziaris 2013), and small molecules (Esteve-Turrillas and Abad-Fuentes 2013; Garcia-Fernandez et al. 2014; Peng et al. 2009; Zhu et al. 2011). Zhu and coworkers developed an immunoassay using dual-color QDs and enzymes to achieve simultaneous detection of multiple chemical residues in milk. (Zhu et al. 2011). However, their method provided only qualitative results. Recently, Dzantiev et.al developed an immunochromatographic test for detection of several antibiotics based on multicolor QD, which shows a great potential convenient strategy for daily food safety control without aid of experiment instrument. However, the minimum detected values of visual qualitative analysis in their work is higher than the maximum
residue
limits
(MRLs)
required
by
the
EU
Commission
Regulation.(Taranova et al. 2015) The aim of this work is to achieve simultaneous, sensitive and visual detection of residues of multiple antibiotics in milk by combining multicolor QD-based competitive
fluorescence
immunoassay 4
(mQD-cFIA)
and
array
analysis.
Streptomycin (SM), tetracycline (TC) and penicillin G (PC-G) are the most frequently used among the aminoglycoside, tetracycline, and penicillin families of antibiotics, and are employed to treat a variety of infectious diseases of cows. Thus, they were selected as the model targets in this study. Their structures are shown in Fig. S1. Their corresponding monoclonal antibodies (mAbs) were covalently coupled to QDs with peak emission wavelengths of 520 nm, 565 nm and 610 nm, respectively, to construct the detecting probes (QD-Ab). As shown in Scheme 1, three coating antigens of SM, TC and PC-G were fixed in different areas of a microtiter plate, and the corresponding three kinds of antibiotics were sensitively detected based on cFIA using the QD-Ab probes.
Scheme1.
2. Experimental 2.1 Materials and instruments 5
Streptomycin sulfate (SM), tetracycline hydrochloride (TC), penicillin G (PC-G), kanamycin (KM), doxycycline (DC), penbritin (PB) and albumin bovine V (BSA) was purchased from Gen-view Scientific Inc. (USA). Streptomycin-BSA (SM-BSA), tetracycline-BSA (TC-BSA), penicillin G-BSA (PC-G-BSA), anti-SM antibody, anti-TC antibody and anti-PC-G antibody (all are polyclonal al antibodies) were purchased from Abcam. ELISA kits for SM, TC and PC-G were purchased from R&D Systems (USA). Thioglycolic acid (TGA), tellurium (Te), cadmium chloride hemipentahydrate(CdCl2·2.5H2O), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl) and N-Hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). NaBH4, NaOH, HCl, NaH2PO4, Na2HPO4, Boric acid, Tris-HCl and ethanol were all analytical grade. Black polystyrene microtiter plates and black polystyrene microtiter plates with transparent bottom were purchased from Corning Incorporated (USA). Milli-Q purified water (18.2 m, Elga, England) was used in all experiments. Phosphate buffer(PB, containing NaH2PO4 and Na2HPO4). Phosphate buffer saline(PBS, containing Na2HPO4、KH2PO4、NaCl and KCl). The Fluorescence spectra were obtained using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Fluorescence images were obtained under an inverted fluorescence microscope (Olympus IX71, Japan). A microplate reader from Tecan Company (Infinite M200 Pro, Switerland) was used. Photos of electrophoresis gels were taken using a gel imaging system (Vilber Lourmat, France).
2.2 Preparation of QD-Ab detecting probes 6
The TGA stabilized multicolor CdTe QDs used in this work were synthesized following previous reports (Gaponik et al. 2002; Zhao et al. 2007b). See supporting information for further details of the synthetic protocol and the spectra of the resulting QDs (Fig. S2). The QDs were conjugated with mAbs for each corresponding antibiotic to produce QD-Ab probes using EDC and NHS as coupling reagents (So et al. 2006; Wang et al. 2007b). To do so, QDs (2 nmol/mL) were first activated with EDC (1.6 mg/mL) and NHS (0.2 mg/mL) for 0.5 h at room temperature, and then mixed with mAbs (4 μg/mL) with shaking for 2 h at room temperature. Finally, the reaction was blocked by centrifugation using a millipore amicon (MW cutoff: 10KD) ultra-0.5mL centrifugal filter and the product was stored in a refrigerator at 4ºC.
2.3 Preparation of antigen coated microtiter plate The three coating antigens (BSA-SM, BSA-TC and BSA-PC-G) were embedded in each well in the different sections of the black microtiter plate (Scheme 1) by the following procedure. Firstly, the microtiter plate was exposed under UV light (254 nm) for 1h, followed by adding coating antigens (with concentration of 10 μg/mL in 0.01 M PBS, pH 7.4) and incubating overnight at 4ºC. Then excess binding sites were blocked with 5% (w/v) BSA in 0.01 M PBS (pH 7.4) for 2 h at 37ºC after removing excess coating antigens by washing several times with washing buffer (PBS containing 0.05% Tween-20, PBST). The microtiter plate modified with coating antigen was then ready for further use.
7
2.4 Quantification of three kinds of antibiotics by mQD-cFIA assay The three model antibiotics (SM, TC and PC-G) were determined by mQD-cFIA as follows, with all measurements done in triplicate. Firstly, 100 μL of standard free target antigens at a series of concentrations and 100 μL of the corresponding QD-Ab probes (200 ng/mL) were added into the wells of microtiter plate and were incubated for 1 h at 37ºC. After three washes with PBST, the fluorescence intensity of each micro-well in the plates was automatically recorded by a fluorescence spectrophotometer under 370 nm excitation.
2.5 Visual detection of antibiotics based on fluorescent imaging array A microtiter plate with transparent bottom was used for this experiment. The experiment was carried out as described in section of 2.4 except the fluorescence spectrophotometer recording was replaced by fluorescence imaging using an inverted fluorescence microscope. After fully incubation and wash, the microtiter plate was imaged using a inverted fluorescence microscope (Olympus IX71, Japan) with blue light excitation for 1 s, and then the images were captured with a color CCD camera (DP72, Olympus Corporation) plus a software named of DP72-TWAIN from Olympus Corporation. Finally, the color cards for the three antibiotics were generated by matching each fluorescence image with its corresponding concentration of each target antibiotic in the microtiter plate.
2.6 Sample preparation and analysis of authentic milk samples 8
Commercial milk samples were purchased from a local supermarket and treated with ethyl acetate to prepare the defatted milk samples according to the following procedure. That is: 10 mL of milk was transferred to a 50-mLcentrifuge tube containing 10 mL of ethyl acetate. The sample was fully mixed by treating with ultrasonic. After oscillation for 30 min, and then the sample was centrifuged at 1000 r/min for 10 min to remove the fat and protein in upper layer. The rest sample in the tube was dried under nitrogen flow, following suspended in PBS buffer for further usage. Then these treated commercial milk samples were analyzed for residues of the three antibiotics (SM, TC and PC-G) using the mQD-cFIA strategy. Commercial ELISA kits were also used to quantify residues of the three antibiotics as a reference standard.
3. Results and discussion 3.1 Characterization of QD-Ab probes QDs with fluorescence emission wavelength of 520 nm (QD520nm, forest green), 565 nm (QD565nm, chartreuse) and 610 nm (QD610nm, vermillion) were prepared, with the fluorescence spectra shown in Fig. S2. The QD-Ab probes for the three antibiotics were prepared by conjugating each type of QD with the corresponding mAbs through the reaction between carboxyl groups on QDs and amino groups on mAb. The formation of QD-Ab probes was characterized by fluorescence spectra and agarose gel electrophoresis (So et al. 2006). As shown in Fig. S3A, after modification with mAb, the fluorescence intensity of QDs was enhanced without any change of peak 9
position or peak shape. Agarose gel electrophoresis showed slower migration of the QD-Ab probes compared to the unmodified QDs (Fig. S3B), which we attribute to the larger size of the QDs after conjugation with mAbs, suggesting the successful formation of QD-Ab. Then the effects of different buffers on the fluorescence stability of mAb-QDs and their storage stability were studied by measuring the fluorescence spectra of QD-Ab in different buffers over time. Fig. S4A and B show that QD-Abs showed high fluorescence intensity in PB buffer (0.01 M, pH7.4) which was then chosen as the reaction buffer for subsequent experiments. The fluorescence intensity decreased by only 5.5 % during storage for more than three months (Fig. S5).
3.2 Standard curves plotting for the determination of three kinds of antibiotics Before developing the standard curves for the three antibiotics (SM, TC and PC-G), the assay conditions (the concentration of QD-Ab probes, reaction temperature and time) for detection of the three antibiotics by mQD-cFIA were optimized. Results are presented in Fig. S6 and S7 (taking SM as an example). Then the optimized mQD-cFIA method was applied to determine three antibiotics (SM, TC and PC-G) respectively. In the assay, QD-Ab probes and each corresponding standard antigen solution were simultaneously added into the coating antigen pre-modified wells of microtiter plates. After incubation and washing, the fluorescence intensity in the wells was measured. As shown in Fig. 1(A1, B1, and C1), with increasing concentration of the standard antigen, the fluorescence intensity from the wells gradually decreased. Standard curves for the three antibiotics were constructed by 10
plotting the fluorescence intensity (y) against the logarithm concentration(x) of analytes (Fig. 1(A2, B2, and C2)).The fitting linear equations, correlation coefficients (r), linear range, and limit of detection (LOD) for each of the three antibiotics are presented in Fig. 1(A2, B2, and C2). We found that the mQD-cFIA method could achieve quantitative detection of antibiotics of SM, TC and PC-G over a linear range of 0.01-25, 0.01-25 and 0.01-10 ng/mL respectively. The LOD for each of the three antibiotics was 0.005 ng/mL, showing comparable or higher sensitivity than conventional immunoassay methods (Garcia-Fernandez et al. 2014; Liu et al. 2014; Que et al. 2013; Thavarungkul et al. 2007; Wu et al. 2014).
Fig. 1.
11
3.3 Visual fluorescence imaging array detection of three antibiotics Due to the broad absorption properties of QDs, the simultaneous excitation of several QD populations below the band edge location of the smallest population while generating distinct emission spectrum of each population could be easily achieved. The QD520 nm, QD565 nm and QD610 nm used in this study could present hunter green, chartreuse, and vermillion fluorescence colors, respectively, under excitation with blue light. Based on this, the three different antibiotics could be determined by different fluorescence colors of QDs, and the quantities of each kind of antibiotic could be determined based on the different shades of each color image (the color cards for the three antibiotics is shown in Fig. S8). As shown in Fig. 2A, the color of images gradually faded with the increasing concentration of antibiotics, and moreover the image color of antibiotics with low concentration of 0.05 ng/mL could be distinguished from the control image of a sample that did not contain any of the antibiotics. This QD-based fluorescence visual assay for antibiotics improved upon the sensitivity of previous colorimetric-based visual assay (Bohm et al. 2012; Kang et al. 2012; Kim et al. 2010). In order to examine the feasibility of visual array analysis for multiple-analytes, the three different antibiotics were analyzed by the multicolor QD-based fluorescence visual assay. Fig.2B shows that the specific fluorescence color response could be obtained for the samples containing single or multiple antibiotics.
12
Fig. 2.
3.4 Specificity of the mQD-cFIA assay Three structural analogues (KM, DC and PB) of the three tested antibiotics (SM, TC and PC-G) were selected to evaluate the specificity of the mQD-cFIA strategy. As shown in Fig. S9, significantly reduced fluorescence intensity, ∆F (∆F=F0-F, F0 and F represent fluorescence intensity before and after adding analyte, respectively), was obtained only for the target analytes. Minimal responses were obtained using these structural analogues. Additionally, the mutual interference between SM, TC and PC-G was also investigated and the results were shown in Fig.S9. The results suggested that 13
the assay for a target analyte was not affected by the presence of other antibiotics. All the results from Fig. S9 indicate the specificity of the mQD-cFIA assay for multiple antibiotics was acceptable.
3.5 Accuracy of QD-based cFIA multiplex analyte assay To evaluate the accuracy of this method, the recoveries of the three kinds of antibiotics were measured in the milk with known concentrations of 0.5 ng/mL, 2.5 ng/mL and 5.0 ng/mL, respectively. The results were compared with standard ELISA methods. Table 1 shows the average recoveries from mQD-cFIA and ELISA. The aver-age recoveries of the two methods for three kinds of antibiotics in the milk samples were 80.21-109.2 %, and the RSDs were 2.48-9.96%. The results from these
Table 1. Recoveries of three kinds of antibiotics in milk analyzed with mQD-cFIA and ELISA Antibiotics
Concentration (ng/mL)
SM
TC
PC-G
mQD-cFIA (n=3) Rate of recovery(%)
ELISA(n=3)
RSD(%)
Rate of recovery (%)
RSD(%)
0.5
92.93
10.08
80.21
13.16
2.5
97.10
8.94
95.42
10.42
5.0
98.50
8.81
108.3
10.21
0.5
88.10
9.80
80.5
8.92
2.5
91.03
9.96
109.2
9.21
5.0
96.83
6.73
97.3
7.89
0.5
93.00
8.44
82.4
8.34
2.5
95.33
5.05
89.3
6.22
5.0
96.37
3.32
101.4
2.48
two methods were coincident. This confirms that the mQD-cFIA method was an accurate, sensitive and effective detection method, which could be used for the
14
detection of three antibiotic residues in milk samples.
3.6 Analysis of residues of three antibiotics in authentic milk samples Eight different brands of milk samples (A, B, C, D, E, F, G and H) were purchased from local supermarkets, and two methods (mQD-cFIA and ELISA) were used to detect and quantify residues of the three target antibiotics. Results are shown in Table 2. The residues of these three antibiotics in the eight milk samples were much lower than MRLs requested by the European Union and other organizations (Beltran et al. 2013; Karageorgou et al. 2014; Thavarungkul et al. 2007; van Bruijnsvoort et al. 2004; Wan et al. 2006). The associated values of RSD were acceptable (5.57%-12.45 for SM, 5.64%-13.21% for TC and 3.24%-10.52% for PC-G), which meant the assay results for the three antibiotics had good precision. The results of mQD-cFIA were consistent with the results from commercial ELISA kits, suggesting this method could be used for the detection of the three kinds of antibiotics residues in authentic milk samples. However, the mQD-cFIA strategy could achieve simultaneous detection of the three antibiotics in one 96-well microtiter plate for more than 30 samples in a single run. Moreover, the three antibiotics in the eight actual milk samples were analyzed based on the visual fluorescence imaging array. As shown, Fig. 3 presented the visual detection results of antibiotics in real milk samples. Compared with the standard color card (Fig. 2A), it could be found that residues of three antibiotics were close to or lower than 0.5 ng/mL, which was consistent with the results in Table 2. These results proved the feasibility of visual detection with mQD-cFIA. 15
Table 2. Comparison results for three kinds of antibiotics levels in eight different brand milk actual samples obtained with mQD-cFIA and ELISA from three successive tests of different batches. Sample
A B C D E F G H
SM (ng/mL)/RSD% mQD-cFIA 0.45/12.45 0.49/12.44 0.50/5.57 0.56/7.39 0.49/5.64 0.38/6.58 0.35/7.92 0.33/6.74
ELISA 0.51/13.21 0.55/12.12 0.61/10.14 0.51/9.83 0.48/6.12 0.45/9.75 0.41/8.38 0.33/7.39
TC (ng/mL)/RSD% mQD-cFIA 0.25/9.74 0.32/13.21 0.28/11.00 0.36/5.68 0.45/5.64 0.35/8.86 0.42/9.45 0.42/7.92
ELISA 0.31/8.33 0.29/8.96 0.31/9.15 0.32/8.17 0.47/10.66 0.37/8.41 0.41/11.42 0.46/6.02
PC-G (ng/mL)/RSD% mQD-cFIA 0.41/4.90 0.46/3.24 0.43/6.53 0.49/4.86 0.47/7.43 0.40/7.98 0.36/8.2 0.38/9.43
ELISA 0.52/8.32 0.63/9.21 0.49/8.56 0.57/9.81 0.43/10.52 0.46/5.05 0.40/5.57 0.41/8.47
Fig. 3.
4. Conclusion This study demonstrated a new strategy of multi-color QD-based direct competitive fluorescence immunoassay arrays for simultaneous and visual detection of residues of multiple antibiotics in milk. In this strategy, QDs with different fluorescent emission wavelengths were coupled with antibodies (corresponding to each kind of antibiotic) to serve as the detection probes. The assay could be completed in 90 minutes, and provided specific responses for each antibiotic and each 16
sample on different sections of one 96-well plate. This strategy shows good sensitivity (5 fg/mL), accuracy and specificity for the target analytes even when the sample contains a mixture of other potential interferents. Moreover, this strategy shows good performance for both semiquantitative and quantitative assays of target antibiotics in real milk samples by visual fluorescence imaging with a standard color chart, and could achieve high throughput analysis (analysis of multiple target antibiotics in multiple samples) in a single run. The convenient operation, good sensitivity, high-throughput, and visual detection of the proposed mQD-cFIA method make it a promising approach for developing multi-analyte immunosensor systems for daily food safety control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21477098), Science and Technology Talent Cultivation Project of Chongqing (cstc2014kjrc-qnrc00001), Fundamental Research Funds for the Central Universities (XDJK2013B009, XDJK2014A020).
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Figure captions Scheme1. A schematic illustration of the multi-analyte assay for three kinds of antibiotics by mQD-cFIA.
Fig. 1. Photoluminescence spectra from mQD-cFIA assays for determination of individual antibiotic residues. The fluorescence spectra (A1, B1 and C1) and standard curves (A2, B2 and C2) are shown for SM (A1 and A2), TC (B1 and B2 and PC-G (C1 and C2), respectively. And the data of linear equation, range and LOD for each antibiotics was also shown in A2, B2 and C2.
Fig. 2. (A) Fluorescence images of microtiter plate wells with different concentrations of each of the three kinds of antibiotics imaged using an inverted fluorescence microscope with blue illumination. (B) Visual detection of SM, TC and PC-G in various combinations using the fluorescence imaging array.
Fig. 3. Visual fluorescence imaging array detection of three antibiotics in actual milk samples. ►A new strategy of multicolor QD-based fluorescence immunoassay arrays was developed. ►The strategy could achieve visual and high-throughput analysis of multiple antibiotics ► The strategy could provide rapid and specific assay for residues of three antibiotics. 21
► The strategy provided low detection limit of 5fg/mL for analysis of target antibiotic.
22
PII: DOI: Reference:
S0956-5663(15)30114-7 http://dx.doi.org/10.1016/j.bios.2015.05.018 BIOS7680
To appear in: Biosensors and Bioelectronic Received date: 28 January 2015 Revised date: 4 May 2015 Accepted date: 6 May 2015 Cite this article as: Erqun Song, Mengqun Yu, Yunyun Wang, Weihua Hu, Dan Cheng, Mark T. Swihart and Yang Song, Multi-color quantum DoT-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Multi-color quantum dot-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk Erqun Songa, ⃰, Mengqun Yua, Yunyun Wanga, Weihua Hub, Dan Chenga, Mark T. Swihartc, Yang Songa a
Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China. Fax: +862368251225; Tel: +862368251225. E-mail: [email protected] b
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy,
Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Southwest University, Chongqing 400715, People’s Republic of China. c
Department of Chemical and Biological Engineering, University at Buffalo, State
University of New York, Buffalo, NY 14260, USA.
Abstract: Antibiotic residues, which are among the most common contaminants in animal-based food products such as milk, have become a significant public health concern.
Here,
we
combine
a
multicolor
quantum
dot
(QD)-based
immunofluorescence assay and an array analysis method to achieve simultaneous, sensitive and visual detection of streptomycin (SM), tetracycline (TC), and penicillin G (PC-G) in milk. Antibodies (Abs) for SM, TC and PC-G were conjugated to QDs with different emission wavelengths (QD520nm, QD565nm and QD610nm) to serve as 1
detection probes (QD-Ab). Then a direct competitive fluorescent immunoassay was performed in antigen-coated microtiter plate wells for simultaneous qualitative and quantitative detection of SM, TC, and PC-G residues, based on fluorescence of the QD-Ab probes. The linear ranges for SM, TC and PC-G were 0.01-25 ng/mL, 0.01-25 ng/mL and 0.01-10 ng/mL, respectively, with detection limit of 5 pg/mL for each of them. Based on fluorescence of the QD-Ab probes, residues of the three antibiotics were determined visually and simultaneously. Compared with a commercial enzyme-linked immunosorbent assay kit, our method could achieve simultaneous analysis of multiple target antibiotics in multiple samples in a single run (high-throughput analysis) and improved accuracy and sensitivity for analysis of residues of the three antibiotics in authentic milk samples. This new analytical tool can play an important role in ameliorating the negative impact of the residual antibiotics on human health and the ecosystem. . Keywords: fluorescence immunoassay; array; multicolor; quantum dot; antibiotics; residues
1. Introduction Various food safety incidents in recent years have increased the need for food safety monitoring for contaminants such as drug residues and illegal additives. Among various possible contaminants in animal-based food products, antibiotics residues are of particular interest, due to their frequent use in animal husbandry, not 2
only for treatment of diseases but also for prophylactic and prevention purposes and to improve agricultural productivity. Consumption of animal products contaminated with antibiotic residues can cause allergic reactions in humans and reduce the efficacy of antibiotics for treatment human infections. The resulting increase in antibiotic-resistant bacteria is a public health concern. Moreover, residues of antibiotics are the most frequently detected contaminants in milk and other dairy products (Chafer-Pericas et al. 2010). These developments have increased the demand for accurate, sensitive and high-throughput analytical methods for the determination of antibiotic residues in foodstuffs. Several methods for detection of antibiotic residues are currently available, including microbiological assays (Aureli et al. 1996; Lourenco et al. 2013), instrumental assays (Bohm et al. 2012; Wang et al. 2006) and immunoassays (Bang-Ce et al. 2008; Meng and Xi 2011; Zhao et al. 2007a). However, microbiological assays are time consuming and have relatively poor sensitivity and specificity. Instrumental assays require complex sample preparation and expensive equipment. Thus, these approaches cannot fulfill the demand for fast, easy and simultaneous detection of antibiotic residues in milk. Immunoassays can offer higher sensitivity and specificity, along with greater convenience. However, the most common
enzyme-linked
immunosorbent
assay
(ELISA)
and
conventional
fluorescence immunoassay (FIA) does not provide high-throughput quantification of multiple analytes (e.g. several different antibiotics). Therefore, the development of new methods for the accurate, sensitive, rapid, and convenient detection of multiple antibiotics residues in milk remains an important need in food safety monitoring. 3
Photoluminescent semiconductor nanocrystals, such as quantum dots (QDs), have excellent fluorescence characteristics (e.g., stable, narrow, and tunable emission spectra with broad excitation spectra) compared with conventional organic fluorescent dyes (Alivisatos 1996; Bruchez et al. 1998; Medintz et al. 2005). As a result, they have been increasingly used as fluorescent labels in FIA (Esteve-Turrillas and Abad-Fuentes 2013; Goldman et al. 2002; Hu et al. 2010; Nichkova et al. 2007; Yang and Li 2006; Zhu et al. 2011) for the detection of pathogens (Wang et al. 2011; Yang and Li 2006; Zhao et al. 2009), proteins (Cao et al. 2011; Hu et al. 2010; Wang et al. 2007a; Wang and Mountziaris 2013), and small molecules (Esteve-Turrillas and Abad-Fuentes 2013; Garcia-Fernandez et al. 2014; Peng et al. 2009; Zhu et al. 2011). Zhu and coworkers developed an immunoassay using dual-color QDs and enzymes to achieve simultaneous detection of multiple chemical residues in milk. (Zhu et al. 2011). However, their method provided only qualitative results. Recently, Dzantiev et.al developed an immunochromatographic test for detection of several antibiotics based on multicolor QD, which shows a great potential convenient strategy for daily food safety control without aid of experiment instrument. However, the minimum detected values of visual qualitative analysis in their work is higher than the maximum
residue
limits
(MRLs)
required
by
the
EU
Commission
Regulation.(Taranova et al. 2015) The aim of this work is to achieve simultaneous, sensitive and visual detection of residues of multiple antibiotics in milk by combining multicolor QD-based competitive
fluorescence
immunoassay 4
(mQD-cFIA)
and
array
analysis.
Streptomycin (SM), tetracycline (TC) and penicillin G (PC-G) are the most frequently used among the aminoglycoside, tetracycline, and penicillin families of antibiotics, and are employed to treat a variety of infectious diseases of cows. Thus, they were selected as the model targets in this study. Their structures are shown in Fig. S1. Their corresponding monoclonal antibodies (mAbs) were covalently coupled to QDs with peak emission wavelengths of 520 nm, 565 nm and 610 nm, respectively, to construct the detecting probes (QD-Ab). As shown in Scheme 1, three coating antigens of SM, TC and PC-G were fixed in different areas of a microtiter plate, and the corresponding three kinds of antibiotics were sensitively detected based on cFIA using the QD-Ab probes.
Scheme1.
2. Experimental 2.1 Materials and instruments 5
Streptomycin sulfate (SM), tetracycline hydrochloride (TC), penicillin G (PC-G), kanamycin (KM), doxycycline (DC), penbritin (PB) and albumin bovine V (BSA) was purchased from Gen-view Scientific Inc. (USA). Streptomycin-BSA (SM-BSA), tetracycline-BSA (TC-BSA), penicillin G-BSA (PC-G-BSA), anti-SM antibody, anti-TC antibody and anti-PC-G antibody (all are polyclonal al antibodies) were purchased from Abcam. ELISA kits for SM, TC and PC-G were purchased from R&D Systems (USA). Thioglycolic acid (TGA), tellurium (Te), cadmium chloride hemipentahydrate(CdCl2·2.5H2O), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl) and N-Hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). NaBH4, NaOH, HCl, NaH2PO4, Na2HPO4, Boric acid, Tris-HCl and ethanol were all analytical grade. Black polystyrene microtiter plates and black polystyrene microtiter plates with transparent bottom were purchased from Corning Incorporated (USA). Milli-Q purified water (18.2 m, Elga, England) was used in all experiments. Phosphate buffer(PB, containing NaH2PO4 and Na2HPO4). Phosphate buffer saline(PBS, containing Na2HPO4、KH2PO4、NaCl and KCl). The Fluorescence spectra were obtained using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Fluorescence images were obtained under an inverted fluorescence microscope (Olympus IX71, Japan). A microplate reader from Tecan Company (Infinite M200 Pro, Switerland) was used. Photos of electrophoresis gels were taken using a gel imaging system (Vilber Lourmat, France).
2.2 Preparation of QD-Ab detecting probes 6
The TGA stabilized multicolor CdTe QDs used in this work were synthesized following previous reports (Gaponik et al. 2002; Zhao et al. 2007b). See supporting information for further details of the synthetic protocol and the spectra of the resulting QDs (Fig. S2). The QDs were conjugated with mAbs for each corresponding antibiotic to produce QD-Ab probes using EDC and NHS as coupling reagents (So et al. 2006; Wang et al. 2007b). To do so, QDs (2 nmol/mL) were first activated with EDC (1.6 mg/mL) and NHS (0.2 mg/mL) for 0.5 h at room temperature, and then mixed with mAbs (4 μg/mL) with shaking for 2 h at room temperature. Finally, the reaction was blocked by centrifugation using a millipore amicon (MW cutoff: 10KD) ultra-0.5mL centrifugal filter and the product was stored in a refrigerator at 4ºC.
2.3 Preparation of antigen coated microtiter plate The three coating antigens (BSA-SM, BSA-TC and BSA-PC-G) were embedded in each well in the different sections of the black microtiter plate (Scheme 1) by the following procedure. Firstly, the microtiter plate was exposed under UV light (254 nm) for 1h, followed by adding coating antigens (with concentration of 10 μg/mL in 0.01 M PBS, pH 7.4) and incubating overnight at 4ºC. Then excess binding sites were blocked with 5% (w/v) BSA in 0.01 M PBS (pH 7.4) for 2 h at 37ºC after removing excess coating antigens by washing several times with washing buffer (PBS containing 0.05% Tween-20, PBST). The microtiter plate modified with coating antigen was then ready for further use.
7
2.4 Quantification of three kinds of antibiotics by mQD-cFIA assay The three model antibiotics (SM, TC and PC-G) were determined by mQD-cFIA as follows, with all measurements done in triplicate. Firstly, 100 μL of standard free target antigens at a series of concentrations and 100 μL of the corresponding QD-Ab probes (200 ng/mL) were added into the wells of microtiter plate and were incubated for 1 h at 37ºC. After three washes with PBST, the fluorescence intensity of each micro-well in the plates was automatically recorded by a fluorescence spectrophotometer under 370 nm excitation.
2.5 Visual detection of antibiotics based on fluorescent imaging array A microtiter plate with transparent bottom was used for this experiment. The experiment was carried out as described in section of 2.4 except the fluorescence spectrophotometer recording was replaced by fluorescence imaging using an inverted fluorescence microscope. After fully incubation and wash, the microtiter plate was imaged using a inverted fluorescence microscope (Olympus IX71, Japan) with blue light excitation for 1 s, and then the images were captured with a color CCD camera (DP72, Olympus Corporation) plus a software named of DP72-TWAIN from Olympus Corporation. Finally, the color cards for the three antibiotics were generated by matching each fluorescence image with its corresponding concentration of each target antibiotic in the microtiter plate.
2.6 Sample preparation and analysis of authentic milk samples 8
Commercial milk samples were purchased from a local supermarket and treated with ethyl acetate to prepare the defatted milk samples according to the following procedure. That is: 10 mL of milk was transferred to a 50-mLcentrifuge tube containing 10 mL of ethyl acetate. The sample was fully mixed by treating with ultrasonic. After oscillation for 30 min, and then the sample was centrifuged at 1000 r/min for 10 min to remove the fat and protein in upper layer. The rest sample in the tube was dried under nitrogen flow, following suspended in PBS buffer for further usage. Then these treated commercial milk samples were analyzed for residues of the three antibiotics (SM, TC and PC-G) using the mQD-cFIA strategy. Commercial ELISA kits were also used to quantify residues of the three antibiotics as a reference standard.
3. Results and discussion 3.1 Characterization of QD-Ab probes QDs with fluorescence emission wavelength of 520 nm (QD520nm, forest green), 565 nm (QD565nm, chartreuse) and 610 nm (QD610nm, vermillion) were prepared, with the fluorescence spectra shown in Fig. S2. The QD-Ab probes for the three antibiotics were prepared by conjugating each type of QD with the corresponding mAbs through the reaction between carboxyl groups on QDs and amino groups on mAb. The formation of QD-Ab probes was characterized by fluorescence spectra and agarose gel electrophoresis (So et al. 2006). As shown in Fig. S3A, after modification with mAb, the fluorescence intensity of QDs was enhanced without any change of peak 9
position or peak shape. Agarose gel electrophoresis showed slower migration of the QD-Ab probes compared to the unmodified QDs (Fig. S3B), which we attribute to the larger size of the QDs after conjugation with mAbs, suggesting the successful formation of QD-Ab. Then the effects of different buffers on the fluorescence stability of mAb-QDs and their storage stability were studied by measuring the fluorescence spectra of QD-Ab in different buffers over time. Fig. S4A and B show that QD-Abs showed high fluorescence intensity in PB buffer (0.01 M, pH7.4) which was then chosen as the reaction buffer for subsequent experiments. The fluorescence intensity decreased by only 5.5 % during storage for more than three months (Fig. S5).
3.2 Standard curves plotting for the determination of three kinds of antibiotics Before developing the standard curves for the three antibiotics (SM, TC and PC-G), the assay conditions (the concentration of QD-Ab probes, reaction temperature and time) for detection of the three antibiotics by mQD-cFIA were optimized. Results are presented in Fig. S6 and S7 (taking SM as an example). Then the optimized mQD-cFIA method was applied to determine three antibiotics (SM, TC and PC-G) respectively. In the assay, QD-Ab probes and each corresponding standard antigen solution were simultaneously added into the coating antigen pre-modified wells of microtiter plates. After incubation and washing, the fluorescence intensity in the wells was measured. As shown in Fig. 1(A1, B1, and C1), with increasing concentration of the standard antigen, the fluorescence intensity from the wells gradually decreased. Standard curves for the three antibiotics were constructed by 10
plotting the fluorescence intensity (y) against the logarithm concentration(x) of analytes (Fig. 1(A2, B2, and C2)).The fitting linear equations, correlation coefficients (r), linear range, and limit of detection (LOD) for each of the three antibiotics are presented in Fig. 1(A2, B2, and C2). We found that the mQD-cFIA method could achieve quantitative detection of antibiotics of SM, TC and PC-G over a linear range of 0.01-25, 0.01-25 and 0.01-10 ng/mL respectively. The LOD for each of the three antibiotics was 0.005 ng/mL, showing comparable or higher sensitivity than conventional immunoassay methods (Garcia-Fernandez et al. 2014; Liu et al. 2014; Que et al. 2013; Thavarungkul et al. 2007; Wu et al. 2014).
Fig. 1.
11
3.3 Visual fluorescence imaging array detection of three antibiotics Due to the broad absorption properties of QDs, the simultaneous excitation of several QD populations below the band edge location of the smallest population while generating distinct emission spectrum of each population could be easily achieved. The QD520 nm, QD565 nm and QD610 nm used in this study could present hunter green, chartreuse, and vermillion fluorescence colors, respectively, under excitation with blue light. Based on this, the three different antibiotics could be determined by different fluorescence colors of QDs, and the quantities of each kind of antibiotic could be determined based on the different shades of each color image (the color cards for the three antibiotics is shown in Fig. S8). As shown in Fig. 2A, the color of images gradually faded with the increasing concentration of antibiotics, and moreover the image color of antibiotics with low concentration of 0.05 ng/mL could be distinguished from the control image of a sample that did not contain any of the antibiotics. This QD-based fluorescence visual assay for antibiotics improved upon the sensitivity of previous colorimetric-based visual assay (Bohm et al. 2012; Kang et al. 2012; Kim et al. 2010). In order to examine the feasibility of visual array analysis for multiple-analytes, the three different antibiotics were analyzed by the multicolor QD-based fluorescence visual assay. Fig.2B shows that the specific fluorescence color response could be obtained for the samples containing single or multiple antibiotics.
12
Fig. 2.
3.4 Specificity of the mQD-cFIA assay Three structural analogues (KM, DC and PB) of the three tested antibiotics (SM, TC and PC-G) were selected to evaluate the specificity of the mQD-cFIA strategy. As shown in Fig. S9, significantly reduced fluorescence intensity, ∆F (∆F=F0-F, F0 and F represent fluorescence intensity before and after adding analyte, respectively), was obtained only for the target analytes. Minimal responses were obtained using these structural analogues. Additionally, the mutual interference between SM, TC and PC-G was also investigated and the results were shown in Fig.S9. The results suggested that 13
the assay for a target analyte was not affected by the presence of other antibiotics. All the results from Fig. S9 indicate the specificity of the mQD-cFIA assay for multiple antibiotics was acceptable.
3.5 Accuracy of QD-based cFIA multiplex analyte assay To evaluate the accuracy of this method, the recoveries of the three kinds of antibiotics were measured in the milk with known concentrations of 0.5 ng/mL, 2.5 ng/mL and 5.0 ng/mL, respectively. The results were compared with standard ELISA methods. Table 1 shows the average recoveries from mQD-cFIA and ELISA. The aver-age recoveries of the two methods for three kinds of antibiotics in the milk samples were 80.21-109.2 %, and the RSDs were 2.48-9.96%. The results from these
Table 1. Recoveries of three kinds of antibiotics in milk analyzed with mQD-cFIA and ELISA Antibiotics
Concentration (ng/mL)
SM
TC
PC-G
mQD-cFIA (n=3) Rate of recovery(%)
ELISA(n=3)
RSD(%)
Rate of recovery (%)
RSD(%)
0.5
92.93
10.08
80.21
13.16
2.5
97.10
8.94
95.42
10.42
5.0
98.50
8.81
108.3
10.21
0.5
88.10
9.80
80.5
8.92
2.5
91.03
9.96
109.2
9.21
5.0
96.83
6.73
97.3
7.89
0.5
93.00
8.44
82.4
8.34
2.5
95.33
5.05
89.3
6.22
5.0
96.37
3.32
101.4
2.48
two methods were coincident. This confirms that the mQD-cFIA method was an accurate, sensitive and effective detection method, which could be used for the
14
detection of three antibiotic residues in milk samples.
3.6 Analysis of residues of three antibiotics in authentic milk samples Eight different brands of milk samples (A, B, C, D, E, F, G and H) were purchased from local supermarkets, and two methods (mQD-cFIA and ELISA) were used to detect and quantify residues of the three target antibiotics. Results are shown in Table 2. The residues of these three antibiotics in the eight milk samples were much lower than MRLs requested by the European Union and other organizations (Beltran et al. 2013; Karageorgou et al. 2014; Thavarungkul et al. 2007; van Bruijnsvoort et al. 2004; Wan et al. 2006). The associated values of RSD were acceptable (5.57%-12.45 for SM, 5.64%-13.21% for TC and 3.24%-10.52% for PC-G), which meant the assay results for the three antibiotics had good precision. The results of mQD-cFIA were consistent with the results from commercial ELISA kits, suggesting this method could be used for the detection of the three kinds of antibiotics residues in authentic milk samples. However, the mQD-cFIA strategy could achieve simultaneous detection of the three antibiotics in one 96-well microtiter plate for more than 30 samples in a single run. Moreover, the three antibiotics in the eight actual milk samples were analyzed based on the visual fluorescence imaging array. As shown, Fig. 3 presented the visual detection results of antibiotics in real milk samples. Compared with the standard color card (Fig. 2A), it could be found that residues of three antibiotics were close to or lower than 0.5 ng/mL, which was consistent with the results in Table 2. These results proved the feasibility of visual detection with mQD-cFIA. 15
Table 2. Comparison results for three kinds of antibiotics levels in eight different brand milk actual samples obtained with mQD-cFIA and ELISA from three successive tests of different batches. Sample
A B C D E F G H
SM (ng/mL)/RSD% mQD-cFIA 0.45/12.45 0.49/12.44 0.50/5.57 0.56/7.39 0.49/5.64 0.38/6.58 0.35/7.92 0.33/6.74
ELISA 0.51/13.21 0.55/12.12 0.61/10.14 0.51/9.83 0.48/6.12 0.45/9.75 0.41/8.38 0.33/7.39
TC (ng/mL)/RSD% mQD-cFIA 0.25/9.74 0.32/13.21 0.28/11.00 0.36/5.68 0.45/5.64 0.35/8.86 0.42/9.45 0.42/7.92
ELISA 0.31/8.33 0.29/8.96 0.31/9.15 0.32/8.17 0.47/10.66 0.37/8.41 0.41/11.42 0.46/6.02
PC-G (ng/mL)/RSD% mQD-cFIA 0.41/4.90 0.46/3.24 0.43/6.53 0.49/4.86 0.47/7.43 0.40/7.98 0.36/8.2 0.38/9.43
ELISA 0.52/8.32 0.63/9.21 0.49/8.56 0.57/9.81 0.43/10.52 0.46/5.05 0.40/5.57 0.41/8.47
Fig. 3.
4. Conclusion This study demonstrated a new strategy of multi-color QD-based direct competitive fluorescence immunoassay arrays for simultaneous and visual detection of residues of multiple antibiotics in milk. In this strategy, QDs with different fluorescent emission wavelengths were coupled with antibodies (corresponding to each kind of antibiotic) to serve as the detection probes. The assay could be completed in 90 minutes, and provided specific responses for each antibiotic and each 16
sample on different sections of one 96-well plate. This strategy shows good sensitivity (5 fg/mL), accuracy and specificity for the target analytes even when the sample contains a mixture of other potential interferents. Moreover, this strategy shows good performance for both semiquantitative and quantitative assays of target antibiotics in real milk samples by visual fluorescence imaging with a standard color chart, and could achieve high throughput analysis (analysis of multiple target antibiotics in multiple samples) in a single run. The convenient operation, good sensitivity, high-throughput, and visual detection of the proposed mQD-cFIA method make it a promising approach for developing multi-analyte immunosensor systems for daily food safety control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21477098), Science and Technology Talent Cultivation Project of Chongqing (cstc2014kjrc-qnrc00001), Fundamental Research Funds for the Central Universities (XDJK2013B009, XDJK2014A020).
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Figure captions Scheme1. A schematic illustration of the multi-analyte assay for three kinds of antibiotics by mQD-cFIA.
Fig. 1. Photoluminescence spectra from mQD-cFIA assays for determination of individual antibiotic residues. The fluorescence spectra (A1, B1 and C1) and standard curves (A2, B2 and C2) are shown for SM (A1 and A2), TC (B1 and B2 and PC-G (C1 and C2), respectively. And the data of linear equation, range and LOD for each antibiotics was also shown in A2, B2 and C2.
Fig. 2. (A) Fluorescence images of microtiter plate wells with different concentrations of each of the three kinds of antibiotics imaged using an inverted fluorescence microscope with blue illumination. (B) Visual detection of SM, TC and PC-G in various combinations using the fluorescence imaging array.
Fig. 3. Visual fluorescence imaging array detection of three antibiotics in actual milk samples. ►A new strategy of multicolor QD-based fluorescence immunoassay arrays was developed. ►The strategy could achieve visual and high-throughput analysis of multiple antibiotics ► The strategy could provide rapid and specific assay for residues of three antibiotics. 21
► The strategy provided low detection limit of 5fg/mL for analysis of target antibiotic.
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