A Quantum Dot-Based Immunoassay for Screening of Tetracyclines in

Jan 20, 2014 - tetracycline, chlortetracycline, and doxycycline) in contaminated bovine muscle tissues. First, the synthesis and characterization of a...
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A Quantum Dot-Based Immunoassay for Screening of Tetracyclines in Bovine Muscle Jenifer García-Fernández, Laura Trapiella-Alfonso, José M. Costa-Fernández, Rosario Pereiro, and Alfredo Sanz-Medel* Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, 33006 Oviedo, Spain S Supporting Information *

ABSTRACT: A reliable and robust direct screening methodology based on a quantum dot (QD) fluorescent immunoassay has been developed to detect trace levels of different antibiotic species from the family of the tetracyclines (e.g., oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in contaminated bovine muscle tissues. First, the synthesis and characterization of a new immunoprobe (oxytetracycline-bovine serum albumin-QD) has been carried out for its further application in the development of a competitive fluorescent QD-immunoassay. The developed fluoroimmunoassay provides sensitive and binary “yes/no” responses being appropriate for the screening of this family of antibiotics above or below a preset concentration threshold. The detection limit achieved with this strategy, 1 μg/L in aqueous media and 10 μg/kg in bovine muscle samples, is 10-fold lower than the maximum level concentration allowed by International Legislation in muscle tissue, enabling suitable and efficient screening of the antibiotics. KEYWORDS: quantum dots, immunoassay, screening, tetracyclines, fluorescence

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INTRODUCTION Tetracyclines (TCCs) are antibiotics with a broad antibacterial spectrum and bacteriostatic activity. Moreover, their low cost, availability, easy administration, and efficiency for the treatment of bacterial diseases make them the most frequently used in human health, animal husbandry, and some agricultural areas.1,2 Oxytetracycline (Oxy), tetracycline (TC), chlortetracycline (Chlor), and doxycycline (Doxy) are the most representative compounds (see Figure 1) among all the members of the TCC family. However, they can be considered a risk to human health not only due to their increasing abuse for therapeutic use but also because they can be employed as fraudulent promoters of accelerated growth in food-producing animals. Residues of these TCCs remain even in cooked animal tissues and could

have harmful effects on human consumers, including allergic reactions, liver damage, yellowing of teeth, and gastrointestinal disturbance. In addition, long-term exposure to such species can lead to an increased drug-resistance of microbial strains.3 Therefore, in order to preserve safety and food control, different worldwide governmental organizations (e.g., FAO/ WHO,4 US FDA,5 and Japanese Ministry of Health, Welfare, and Labour6) have established maximum residue limits (MRLs) allowed to be present in different foodstuff. Particularly, one of the most complete and restrictive legislations has been elaborated by the European Commission, which has set MRLs for TCCs in bovine muscle (100 μg/kg), kidney (600 μg/kg), and liver (300 μg/kg).7 Several methods of TCC residues analyses in food have been developed. Some of them are based on microbiological assays as they are easy to perform. Unfortunately such methods are time-consuming (usually require 2−3 days for microbe growth) and lack the needed specificity and sensitivity.8−11 Capillary electrophoresis has been evaluated also as a possible alternative,12 but it presents important drawbacks including laborious pretreatment steps and difficulty to determine low concentrations of those antibiotics. Due to their high sensitivity and specificity, chromatographic techniques have been also evaluated13 (especially HPLC-based methods). However, such methodologies require high-cost instrumentation and samples usually need extensive and time-consuming processing (extraction and purification) before the analysis.14 Therefore, the development of sensitive, rapid, selective, and cost-effective Received: Revised: Accepted: Published:

Figure 1. Chemical structure of TCCs. © 2014 American Chemical Society

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Oxy−BSA conjugate was stored in a 28 mM Na3PO4, 300 mM NaCl, and 33 mM sorbitol buffer (PBS), a previous cleanup step was carried out (sample desalting by Zip Tips). A solution of 1 mg/mL of BSA standard in water was used as reference. The MALDI plate was loaded with standards, matrix, and/or samples, and when the spots had been dried (at least after 30 min), the MALDI-MS analysis was performed. Finally, following a strategy previously developed in our lab25 based on the combination of the information provided by the Bradford test and the MALDI analysis, the final concentration of Oxy in the conjugate can be calculated. Such information is necessary to further optimize the bioconjugation of Oxy−BSA to QDs. Synthesis and Characterization of the Immunoprobe: Oxy− BSA−QD Bioconjugate. The fluorescent labels used for labeling the Oxy−BSA conjugate giving rise to the tracer of the immunoassay are the CdSe/ZnS QDs. A brief description about their synthesis and solubilization are presented in the Supporting Information. The bioconjugation strategy used to synthesize the fluorescent tracer was based on a coupling procedure between −COOH groups from outer polymeric layers of QDs and −NH2 groups from the BSA of the Oxy− BSA conjugate owing to the catalytic activity of EDC.26 After stirring of the mixture of reagents during 2 h at room temperature, a purification step is needed to separate the bioconjugate from the rest of undesirable products (e.g., excess of reagents). The purification step is based on differences in molecular weights between the Oxy−BSA conjugate (71.2 kDa) and the Oxy−BSA−QD bioconjugate (>150 kDa). So, it can be carried out by ultrafiltration (UHF) using a 100 kDa membrane filter. Experimental conditions used for UHF were 3000 g, 5 min, making 3 washes with 100 mM PBS buffer at pH 7.4 at 4 °C. The characterization of this bioconjugate consists on the evaluation of the fluorescent properties of QDs after the reaction and the stability in time of the tracer. These studies were carried out using fluorescent measurements. Competitive Immunoassay Protocol. In a conventional immunoassay analysis the following experimental procedure was performed. First, the microtiter plate is coated with 100 μL/well of a 1 μg/mL antibody solution and is incubated at 37 °C for 2 h. In this step the antibody is immobilized on the microtiter plate wells by absorption over the surface of the support of polystyrene. Antibody solution is then removed and a blocking step is performed by adding 200 μL/well of 3% casein solution in water, in order to minimize further unspecific bindings. The microtiter plate is then incubated overnight at 4 °C. In a further step, the microtiter plate is washed three times using a washing solution (200 μL/well of 10 mM PBS pH 7.4 + 0.05% Tween20) to remove the excess of reagents. Then, the competition is established by the addition of a mixture of the standard (or the sample) and a known amount of the tracer (Oxy−BSA−QD) in a total volume of 150 μL/ well. The reaction is incubated at 37 °C for 2 h. After the corresponding washing step, the fluorescence emission of the QDs from the Oxy−BSA−QD recognized by the antibody is measured. Cross Reactivity Calculation. Cross reactivity (CR) is generally defined as the necessary amount of mass or concentration of interference able to produce an equal signal as when the analyte is assayed to provoke a signal inhibition of 50%.27 Therefore, in this work CR rates, in terms of percentage (%), were calculated according to the expression (eq 1)

methods for TCC residue detection and screening in routine assays is still a research demand. Immunochemical assays could be an alternative to meet all those requirements, as detection of low concentrations of TCC residues in many samples in a short time and in some cases avoiding long and tedious sample pretreatment steps may be possible. In fact, several approaches of competitive ELISA format assays have been already developed for the analysis of TCCs, being applied in most of the cases to milk samples.1,15−17 Assuming that a majority of samples analyzed may not be contaminated by any of the TCCs, sample screening methods providing a reliable “yes/no” binary response related to a preset concentration threshold are of increasing interest in food control. Screening systems give mainly qualitative or semiquantitative information as they are focused on identifying and selecting a group of samples that contain one or more analytes above a preset concentration or threshold level.18,19 In this context, the high selectivity and sensitivity typically provided by immunological methods are particularly suitable for the development of such screening strategies. The use of quantum dots (QDs) as fluorescent labels in bioassays has been increasing due to their advantageous features, including excellent photostability and chemical stability, long fluorescence lifetimes, intense and narrow emission spectra, and size- and composition-tunable properties.20 Furthermore, the possibility of attaching QDs to biomolecules keeping their functionality has allowed development of different formats of immunoassays such as microarrays21 or Western blotting22 as well as luminescent sensors for the analysis of health-risk environmental pollutants (e.g., based on molecular imprinted polymers23). However, the development of fluorescent immunoassay can report an additional benefit over the ELISA immunoassays, that is, saving time and costs in avoiding the final step of enzymatic reaction. In this context, a QD-based immunoassay for direct screening of four TCCs in bovine muscle tissue is here described. The fluorescent probe consists of QDs bioconjugated to oxytetracycline−BSA conjugate (Oxy−BSA−QD), prepared following the carbodiimide chemistry. Due to the small size of the TCCs (haptens with only one site for binding to the antibody), the immunoassay design is restricted to a competitive format. So, competition for antibody binding sites between free antigen of the sample (unlabeled TCC) and the bioconjugates (Oxy−BSA−QD) is established.

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MATERIALS AND METHODS

Reagents, materials, and instrumentation used through the work are available in the Supporting Information. Characterization of Oxy−BSA conjugates. The synthesis and purification of Oxy−BSA conjugates are based on a commercial kit, and a detailed description about the procedure is collected in the Supporting Information. Once Oxy−BSA conjugates are obtained, an exhaustive characterization step is mandatory. A Bradford test24 was used to determine the BSA concentration in the Oxy−BSA conjugate. The test correlates the ratio between absorbance of the samples measured at 465 and 595 nm (595/465) with the BSA (protein) concentration. According to a linear regression, BSA concentration in the conjugate was then calculated. Molecular weight and stoichiometry of the conjugate were evaluated by MALDI-MS. A solution of 5 mg/mL of sinapinic acid in 30% of acetonitrile, containing 0.1% of trifluoroacetic acid, was prepared. An amount of 1−10 pmol of protein in the sample was needed, being 2.42 pmol in this case. As the sample has to be ideally clean of salts and the

CR = [(IC50(analyte)/IC50(interference))] × 100

(1)

where IC50 is the necessary concentration of species (analyte or interference) to induce a signal inhibition of 50%. Screening Theory and Determination of the Unreliability Region. A screening system provides mainly qualitative or semiquantitative information. In this type of analysis it is not possible to express uncertainty in the same way as in quantitative analysis. For that reason it is necessary to define new concepts that characterize screening methods. Thus, “reliability” of a screening test is defined as the “proportion of correct yes/no responses from a large number of tests carried out on ‘n’ aliquots of the same sample”. On the other hand, the term “uncertainty” should be replaced by “unreliability”, that is the concentration range of the analyte around the limit where false 1734

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Figure 2. Work-flow diagram of the TCC screening in bovine muscle samples by fluorescent competitive QD-based immunoassay.

Figure 3. MALDI-MS characterization of the synthesized Oxy−BSA conjugate. The mass spectra were obtained using sinapic acid as matrix. BSA standard (black line) and Oxy−BSA conjugate (gray line) mass spectra are represented, showing clearly the mass shift between both (ΔM). positives, false negatives, and dubious results are obtained.18,28 In order to determine the unreliability region (UR) in the screening procedures, the cutoff level must be defined. The UR is finally calculated from the probability−concentration curve established for the screening test. In this work the limits defining the UR were determined as the levels of false positives (probability that a negative sample is classified as positive, also called α error) and false negatives (probability that a positive sample is classified as negative, also called β error).29 In this work, to obtain the probability vs concentration graph, 20 assays (random samples containing variable concentrations of the individual TCC but ensuring the same total antibiotic concentration) were carried out for each point of the curve, and the relative proportion (in terms of percentage) of “yes/no” answers was determined. In all cases, the binary “yes/no” response was obtained from the analytical signal (fluorescent intensity measured at the final step of the immunoassay) and was classified as “negative” or “positive” after its comparison with the fluorescence intensity signal registered for the corresponding cutoff concentration (I0).

Analysis of Bovine Muscle Samples. Bovine muscle samples from noncontaminated cows were extracted following the Oka et al. procedure,30 blending successively the meat samples (5 g) with three successive portions of 20, 20, and 10 mL of 0.1 M Na2EDTAMcIlvaine buffer pH 4.0, centrifuging the mixture (at 850g for 5 min), and collecting and combining the supernatants by decanting. As no detectable levels of TCC residues were found in the meat samples, 5 g of chopped meat was spiked with the appropriate amount of TCC mixtures (see Table S1 in the Supporting Information for details of the concentrations of each TCC) and carefully stirred manually during 5 min. The samples were kept at 4 °C for 1 h. Afterward, the McIlvaine buffer was added and the Oka et al. procedure30 was then followed. However, additional ultrafiltration steps were needed to remove the color that remains in the extract, which can interfere in the fluorescence measurements. For that reason, ultrafiltration with Amicon membrane filter of 10 kDa followed by ultrafiltration with Amicon membrane filter of 3 kDa (centrifuged at 12000g for 15 min in both cases) was performed, collecting the filtered solution. Twohundred microliter aliquots of such solution were then used in the 1735

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immunoassays. A work-flow diagram of the complete process for the analysis of TCCs in bovine muscle samples is shown in Figure 2.

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RESULTS AND DISCUSSION Characterization of the Oxy−BSA Conjugate. As it is not possible to purchase commercial Oxy−BSA conjugate, it is necessary to synthesize it as described in Materials and Methods. A complete characterization of synthesized Oxy− BSA conjugate was performed following the strategy developed by Trapiella-Alfonso et al.25 The molecular weight of the conjugate, the stoichiometry, and the concentration of Oxy and BSA in the conjugate are obtained. Based on results obtained from a Bradford assay, BSA concentration was calculated to 335 ± 9 μg/mL. Molecular weight and stoichiometry were investigated by MALDI-MS. Results from MALDI experiments were used to calculate the molecular weight of the conjugate, being 71223 ± 99 Da (n = 15). Comparing the mass spectrum of BSA standard with the Oxy−BSA spectrum (see Figure 3), a mass shift (ΔM) is noticed, and this can only be attributed to the incorporation of the Oxy in the BSA structure. If Oxy molecular weight is considered as 460.434 Da, dividing the ΔM observed by the Oxy molecular weight, the number of Oxy per BSA can be estimated. For this case, the found value was close to 10, leading to a ratio of Oxy:BSA of 10:1. Finally, applying eq 2 the final concentration of Oxy in the conjugate can be estimated.25 COxy = 10C BSA(MWOxy /MWBSA)

Figure 4. QDs (gray line) and Oxy−BSA−QD bioconjugate (black line) fluorescence emission spectra in buffer 10 mM PBS (pH = 7.4).

Development of the Competitive Fluorescent Immunoassay and Analytical Performance Characteristics. The immunoassay format consists of a competitive fluorescent immunoassay where nanoparticles are used as antigen labels. In this format, sample antigen and tracer (Oxy−BSA−QD) compete for the limited binding sites of the immobilized antibody. First, the concentration of anti-Oxy antibody used for coating the well-plate and immunoprobe concentration added in the competition step were optimized for the development of the immunoassay. Optimum conditions (referring to the highest fluorescence intensity, the lowest signal deviation, and the best inhibition curve) were achieved with 1 μg/mL of the antibody solution and 10 μg/mL of the tracer (Oxy−BSA−QD) solution, ensuring a 1:1 volume ratio between the sample and the immunoprobe involved in the competition (see Table 1).

(2)

where COxy is the concentration of oxytetracycline in the conjugate, 10 is the Oxy:BSA ratio, CBSA is the concentration of BSA in the conjugate obtained by the Bradford test, and MWOxy and MWBSA are the molecular weights of oxytetracycline and BSA respectively. Synthesis and Characterization of Oxy−BSA−QD Bioconjugate. The molar ratios used to carry out the bioconjugation reaction were optimized by varying the ratios QDs:Oxy−BSA from 1:1 to 3:1 (always with an excess of EDC in a ratio of 1500 mol per mol of biomolecule to ensure that bioconjugation is completed). Optimum molar ratio values were observed for 2:1:1500 (QDs:Oxy−BSA:EDC) attending to the highest fluorescence intensity signals measured, indicating a high efficiency on the labeling of the Oxy−BSA conjugate with the nanoparticles. According to TrapiellaAlfonso et al.31 the concentration of Oxy and BSA in the tracer and the bioconjugation yield can be calculated. In order to characterize the purified bioconjugate, fluorescence measurements were performed to check that QD emission is not interfered with the bioconjugation reaction. As can be seen in Figure 4, the Oxy−BSA−QD bioconjugate does not modify significantly QD emission wavelength or fluorescence intensity; therefore, it is possible to develop a fluorescent immunoassay with this tracer. Finally, the stability of the immunoprobe (utility valid period of the tracer for immunoassays) was evaluated attending to the stability of the fluorescence emission and the ability of the antibody to recognize the tracer. It was observed that 10 days was established as the maximum period where the Oxy−BSA− QD is stable and suitable for immunosensing, being stored at 4 °C. In the Supporting Information, Figure S1 shows data from a stability study. It is observed that the fluorescence emission of the immunoprobe is reduced more than 50% after 10 days, limiting its utility in the immunoassay.

Table 1. Optimized Experimental Conditions of the Immunoassay Based on QDs step/condition

optimized value

coating blocking [Oxy] in the bioconjugate sample:bioconjugate ratio

100 μL/well Aboxy (1 μg/mL) 200 μL/well 3% casein 10 μg/mL 1:1

The analytical performance characteristics are assessed by the analysis of a series of Oxy standard solutions at different concentration levels, using the optimized QD-based immunoassay. The corresponding inhibition curve was fitted using a four-parameter equation with SOFTmax Pro software (see Figure 5). The principal parameters (see Table 2) are defined by different inhibitory concentration (IC) values calculated from the inhibition curve. Thus, a linear relationship was achieved ranging from 0.26 to 3.53 μg/L (IC20−IC80) enabling Oxy trace determination in bovine muscle. The detection limit (IC10) was 0.12 μg/L of Oxy and the sensitivity of the assay (IC50) was 0.96 μg/L of Oxy in aqueous media. 1736

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Table 3. Cross Reactivity Test Using as Antibody Rabbit Anti-Oxytetracycline IgG

Table 2. Analytical Characteristics of the Inhibition Curve for the Analysis of TCCs in Bovine Muscle analytical parameter

obtained value 0.12 μg/L 0.26 μg/L 0.26−3.53 μg/L 0.96 μg/L 0.999

CR (%)

tetracycline chlortetracycline doxytetracycline

89 90 82

total TCCs in the samples over a preset concentration level (e.g., the maximum residue level authorized for such family of antibiotics). Therefore, fluorescence signals from the different TCCs that could be present in the sample should be expected as additive. To evaluate whether the analytical signals from the TCCs are additive, sets of aqueous samples containing only a single type of tetracyclines, as well as binary, ternary, and quaternary mixtures of the TCCs under study, were prepared and analyzed by the QD-based immunoassay. In all cases solutions contained 2 μg/L of TCC total concentration. The samples were analyzed according to the general procedure described above, and results are shown in Figure 6. A good agreement between experimental concentrations extracted from immunoassay fluorescence measurements and expected concentrations were observed. This is a proof of the analytical potential of the immunochemical method proposed for the development of the intended screening system of TCCs. Characterization of the Screening System for TCC Detection. In order to characterize the here-developed screening test, and thus obtain the UR region, a screening curve for TCC detection was designed and characterized in aqueous media. For this purpose, a probability versus concentration graph was elaborated selecting a cutoff level of 1 μg/L total concentration of TCCs, according to the maximum sensitivity reached by the immunoassay methodology (IC50 factor extracted from the inhibition curve was 0.96 μg/L). The limits defining the UR of our method were determined by calculating the concentrations of TCCs that produce a probability of 5% to obtain a false positive and a probability of having a 95% of positive response (α, β errors) as it is explained in Materials and Methods. According to these patterns, the experimental unreliability region obtained was in the range between 0.56 and 1.25 μg/L total TCC concentration (see Figure 7). This means that the presence of TCCs in aqueous media can be confirmed reliably by the proposed screening system for concentrations higher than 1.25 μg/L and, on the other hand, samples giving total concentration of TCCs below 0.56 μg/L can be reliably considered as noncontaminated. Application to Bovine Muscle Samples. The applicability of this QD-based immunochemical method for the screening of TCC residues in contaminated bovine meat samples was finally evaluated. As no TCCs were detected by immunoassay in the scrutinized samples, they were spiked with known amounts of TCCs and then screened following the general procedure above-described. In order to get a validation of the used methodology for the extraction of analytes from bovine muscle, a high-performance liquid chromatography (HPLC) with spectrophotometric detection for the determination of TCCs in bovine tissues was carried out. Bovine meat samples were extracted and then analyzed following the Cinquina et al. procedure.34 The analyses were performed using a mobile phase of 0.01 M oxalic acid:acetonitrile:methanol (60:25:15 in volume) on a

Figure 5. Fluorescent inhibition curve obtained for the analysis of TCCs in bovine muscle samples using oxytetracycline labeled with QDs as tracer in the competitive immunoassay. All the points that plot the curve exhibit a RSD < 5%.

limit of detection (IC10) limit of quantification (IC20) linear range (IC20−IC80) sensitivity (IC50) R2

antigens assayed (TCC family)

The here developed competitive QD-based fluoroimmunoassay shows some attractive features that make it advantageous over the established conventional immunoassays for the analysis of TCCs in bovine meat (in general competitive ELISA formats). Such advantages include a lower detection limit (