Article pubs.acs.org/est
Time-Resolved Fluoroimmunoassay as an Advantageous Analytical Method for Assessing the Total Concentration and Environmental Risk of Fluoroquinolones in Surface Waters Zhen Zhang,†,‡ Jing-fu Liu,*,† Ting-ting Feng,§ Yan Yao,§ Li-hong Gao,† and Gui-bin Jiang† †
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China ‡ School of the Environment, Jiangsu University, Zhenjiang 212013, China § Department of Isotope, China Institute of Atomic Energy, Beijing 102413, China S Supporting Information *
ABSTRACT: Due to the widespread occurrence in the environment and potential risk toward organisms of fluoroquinolones (FQs), it is of importance to develop high efficient methods for assessing their occurrence and environmental risk. A monoclonal antibody (Mab) with broad cross-reactivity to FQs was produced by immunizing BALB/c mice with a synthesized immunogen prepared by conjugating ciprofloxacin with bovine serum albumin. This developed Mab (C2F3C2) showed broad and high cross-reactivity (40.3−116%) to 12 out of the 13 studied FQs. Using this Mab and norfloxacin conjugated with carrier protein ovalbumin as coating antigen, a timeresolved fluoroimmunoassay (TRFIA) method was developed for determining the total concentration of at least 12 FQs in environmental waters. The respective detection limit (LOD) and IC50 calculated from the standard curve were 0.053 μg/L and 1.83 μg/L for enrofloxacin (ENR). The LODs of the other FQs, estimated based on the corresponding cross-reactivity and the LOD of ENR, were in the range of 0.051−0.10 μg/L. The developed TRFIA method showed good tolerance to various interfering substances present in environmental matrix at relevant levels, such as humic acids (0−10 mg/L DOC), water hardness (0−2% Ca2+ and Mg2+, w/v), and heavy metals (0−1 mg/L). The spiked recoveries estimated by spiking 0.5, 1, and 2 μg/L of five representative FQs into various water samples including paddy water, tap water, pond water, and river water were in the range of 63−120%. The measured total FQ concentration by TRFIA agreed well with that of liquid chromatography−tandem mass spectrometry and was applied to directly evaluate the occurrence and environmental risk of FQs in the surface water of a case area. TRFIA showed high efficiency and great potential in environmental risk assessment as it measures directly the total concentration of a class of pollutants.
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INTRODUCTION Fluoroquinolones (FQs), as one of the most important classes of synthetic antibiotics, are widely used as prophylactic agents and medicine for treatment of diseases such as pulmonary, urinary, and digestive infections in human and domestic animals.1−7 The extensive use of FQs has resulted in their widespread occurrence in environment and therefore risk toward organisms. FQ residues enter the environment mainly as a result of their incomplete removal in excreta of humans and animals in wastewater treatment plant (WWTP) and the use of animal excreta as agricultural fertilizers as well as the direct discharge from aquaculture treatments. FQ residues have been detected in a wide range of environmental waters including wastewater, surface and groundwater, and drinking water.1,6−10 Ciprofloxacin was found to interfere with the photosynthesis and inhibit the growth of spinach plants,11 while significant toxicity of FQs to microorganisms and aquatic vertebrates were also reported.12−14 Furthermore, the exposure of organisms to © 2012 American Chemical Society
FQs in environment increases the incidence of antimicrobial resistant bacteria, which disturbs the balance of aquatic ecosystems and minimizes efficacy of FQs as an important class of human and veterinary medicine.15−18 Therefore, it is of great importance to monitor the occurrences and assess the exposure and risk of FQs in environmental waters. Currently available methods for the determination of FQs in environmental samples are mainly based on preconcentration with solid-phase extraction (SPE) followed by high performance liquid chromatography (HPLC) analysis with fluorescence, UV, or mass spectrometry (MS) detection.5,6 Although these methods are very sensitive and selective, they required sample enrichment and cleanup procedures that are timeReceived: Revised: Accepted: Published: 454
April 1, 2012 December 6, 2012 December 7, 2012 December 7, 2012 dx.doi.org/10.1021/es303858a | Environ. Sci. Technol. 2013, 47, 454−462
Environmental Science & Technology
Article
(St. Louis, MO, USA). N′-[p-Isothiocyanatobenzyl]diethylenetriamine-N1, N2, N3, N4-tetraacetate-Eu3+ (DTTAEu3+) was obtained from Tianjin Radio-Medical Institute (Tianjin, China). G-50 and Sephadex 6B were purchased from Pharmacia (Uppsala, Sweden). Other chemicals were of analytical grade or above and were supplied by Beijing Reagent Corporation (Beijing, China). The chemical structures of the 13 FQs are shown in Figure S1. Buffers and solutions used in this study were as follows: dilution buffer, 0.05 mol/L Tris-HCl buffer (pH 8.0) containing 0.9% NaCl; coating buffer, 0.1 mol/L carbonate buffer (pH 7.8) containing 0.9% NaCl and 0.05% NaN3; blocking buffer, 0.05 mol/L Tris-HCl buffer (pH 8.0) containing 0.5% BSA, 0.9% NaCl, and 0.04% NaN3; washing buffer, 0.05 mol/L Tris-HCl buffer (pH 8.0) containing 0.9% NaCl and 0.04% Tween 20; and the enhancement solution, 0.1 mol/L potassium biphthalate-acetic acid buffer containing 15 μmol/L β-naphthoyltrifluoroacetone and 0.1% Triton X-100. Hapten Conjugation. The hapten conjugate was synthesized by the carbodiimide-modified active ester method as shown in Figure S2.33,34 The carrier protein (BSA) was activated by adding 100 μL of 10% EDA, 5 mg of NHS, and 5 mg of EDC into the 10 mg carrier protein dissolved in 0.05 mol/L phosphate buffer (PB, pH 8), and then the solution was stirred for 6 h at room temperature and exhaustively dialyzed against 0.05 mol/L PB (pH 8.0). In order to attach the hapten onto the activated carrier proteins, into 4 mL of 0.05 mol/L PB (pH 8.0) dissolved with 10 mg of the activated carrier protein were added a solution of 2 mL of DMF dissolved with 5 mg of CIP (or ENR or NOR), 10 mg of EDC, and 10 mg of NHS. After having been stirred overnight at 4 °C in the dark, the solution was dialyzed against 0.02 mol/L PB (pH 7.4). Antibody Production. The antibody was prepared by a procedure modified from the literature.35 Female BALB/c mice of 6−8 weeks old were immunized subcutaneously on days 1, 14, and 28 with 100 μg of synthesized immunogens prepared in 0.2 mL of physiological saline with Freund’s complete adjuvant. Two and four weeks after the initial injection, the mice were boosted with the same 100 μg of immunogens in 0.2 mL of physiological saline with Freund’s incomplete adjuvant, respectively. Four weeks after the initial injection, mice were tail-bled, and the serum were tested by competitive indirect ELISA (ciELISA)36 to identify the antibody against hapten. After a rest period of 1 month, the mouse was immunized intraperitoneally at a final time with 100 μg of immunogens in 0.2 mL of physiological saline. Four days later, the mouse was sacrificed, the splenocytes were fused with SP2/0 myeloma cells with polyethylene glycol, and the fused cells were propagated in HAT-selection medium and distributed over 96-well microculture plates. Hybridomas producing hapten-specific antibodies were cloned twice by limiting dilution. Cross-Reactivity Determination. The cross-reactivity (CR) of the antibodies was evaluated by determining the respective IC50 values toward 13 structurally related quinolones using ciELISA,36 calculated by the equation CR% = [(IC50 value of hapten)/(IC50 value of competitor)] × 100. Preparation and Purification of Europium Chelate Labeled Mab. Europium-labeled Mab (tracers) was prepared as described in our previous study.37 In brief, after being dialyzed against carbonate buffer, 0.5 mg of Mab was added into a little amber bottle containing 1 mg DTTA-Eu3+, and the mixture was kept at 4 °C for 24 h with stirring. Then the solution was purified by a Sephadex 6B/G-50 column, self-
consuming, solvent intensive, and costly. These drawbacks limited their application in routine monitoring analysis and screening of large numbers of environmental samples. Furthermore, while these methods cost a lot to separate and quantify the individual FQs in environmental samples, it is preferred to conduct the environmental exposure and risk assessment based on the total concentration of FQs due to the potential additive toxicity of FQs.8,19 Therefore, it is of great interest to develop analytical methods to directly determine the total FQ concentrations for evaluating the exposure and risk, which is more direct, cost-effective, and efficient. Immunoassay is an ideal choice for direct determination of total FQs by using broad-specific antibodies, as it has the inherent properties of high throughput, low sample consumption (down to microliter of water samples), very limited sample pretreatment, and low cost. Because of their low sensitivity and high matrix interferences, however, common immunoassay techniques like enzyme-linked immunosorbent assays (ELISA) cannot meet the demands of determining trace contaminants in aquatic system. As an ultrasensitive method, time-resolved fluoroimmunoassay (TRFIA) possess the merits of high sensitivity and effective elimination of background fluorescence from the coexisting sample matrixes.20−24 Like other immunoassay techniques, the successful use of TRFIA in assaying FQs as a class of antibiotics relies on the availability of broad-specific antibodies for FQs. So far, however, most antibodies have limited cross-reactivity to the large number of FQs. Although both polyclonal25−28 and monoclonal29−32 antibodies capable of detecting 7−15 FQs simultaneously in various animal origin samples have been described, no method has been reported for assaying the total concentration of FQs in environmental samples. Given the low concentration level and the broad-spectrum of FQs in environmental waters, it remains a great challenge to develop monoclonal antibodies with high cross-reactivity for quinolone analogs to screen trace FQs in environmental waters. In this study, a monoclonal antibody (Mab) with high crossreactivity for quinolone analogs was engineered by using a synthesized immunogen prepared by conjugating bovine serum albumin with ciprofloxacin, which is a typical FQ with relatively high toxicity and wide occurrence in environment. Then, a TRFIA method based on this produced antibody was developed for high throughput analysis of the total concentration of at least twelve FQs in environmental waters. The established method was further applied to determine the total concentration and therefore evaluate the occurrences and environmental risk of FQs at Neijiang River region located at Zhenjiang, Jiangsu Province.
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EXPERIMENTAL SECTION Chemicals and Solutions. Enrofloxacin (ENR, 99.9%), ciprofloxacin (CIP) hydrochloride (99.9%), sarafloxacin (SAR, 99.6%), pefloxacin methanesulfonate (PEF, 99.9%), oxolinic acid (OXO, 99.9%) fleroxacin (FEL), and enoxacin (ENO, 99.6%) were purchased from the China Institute of Veterinary Drug Control (Beijing, China). Norfloxacin (NOR, ≥99.0%), difloxacin (DIF, >99.0%), ofloxacin (OFL, ≥99.0%), lomefloxacin (LOM, >99.5%), danofloxacin (DAN, ≥99.0%), nalidixic acid (NAL, >99.0%), marbofloxacin (MAR, ≥99.0%), ethylenediamine (EDA), N-hydroxysulfosuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA), and ovalbumin (OVA) were obtained from Sigma Chemical Co. 455
dx.doi.org/10.1021/es303858a | Environ. Sci. Technol. 2013, 47, 454−462
Environmental Science & Technology
Article
Table 1. IC50 (μg/L) Value and Cross-Reactivity (CR, %) of the Developed 6 Antibodies to 13 FQs Evaluated by ciELISAa E12H 6B11
N4E5A1
N5C7D11
C2F3C2
C5F6D5
C7A2B9
FQs
IC50
CR
IC50
CR
IC50
CR
IC50
CR
IC50
CR
IC50
CR
CIP ENR NAL DIF SAR OXO OFL NOR MAR LOM DAN PEF ENO
>2000 6.4 >2000 >2000 >2000 >2000 >2000 >2000 >1000 >2000 >2000 489 >2000
