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Microchim ActaDOI 10.1007/s00604-011-0548-9 Simultaneous determination of four different antibioticresidues in honey by chemiluminescence multianalytechip immunoassays Klaus Wutz & Reinhard Niessner & Michael Seidel Received: 9 November 2010 / Accepted: 14 January 2011 # Springer-Verlag 2011 Abstract We are presenting the first method for identifi- Keywords Antibiotic microarray . Chemiluminescence cation and quantification of antibiotic derivatives in honey detection . Regenerable biochip . Automated flow-injection samples using regenerable antigen microarrays in combi- system . Microarray image evaluation nation with an automated flow injection system. Thescheme is based on an indirect competitive immunoassayformat using monoclonal antibodies bound to the surface of the microarray. The surface of glass slides was coated withepoxy-activated poly(ethylene glycol) and enables direct Honey is generally considered as natural and healthy immobilization of the antibiotic derivatives. The antigen/ product of animal origin. However, in the recent years antibody interaction on the surface of the chip can be there were some publications dealing with the determina- detected by chemiluminescence (CL) read-out via CCD tion of antimicrobial contaminants in bee products. Anti- camera. The method allows for fast analysis of the four biotics are used in apiculture for the treatment of bacterial analytes simultaneously and without purification or extrac- diseases, in particular American and European foulbrood tion. An effective data evaluation method also was For the effective abatement of the causers Paenibacillus developed to warrant unambiguous identification of the larvae and Melissoccocus pluton, respectively, drugs based spots and to establish grey levels of CL intensities. The on antibiotic derivatives, e.g. sulfonamides and tetracy- software developed enables fast and automated processing clines, have been approved All sulfonamides inhibit of the CL images. Dose–response curves were obtained for the bacterial synthesis of folic acid due to their structural the derivatives of enrofloxacin, sulfadiazine, sulfametha- analogy to p-aminobenzoic acid, whereas the group of zine and streptomycin. Spiking experiments revealed tetracylines interferes with the ribosomal protein synthesis.
adequate recoveries within the dynamic ranges of the The broad activity spectrum of these antimicrobials has led calibration curves of enrofloxacin (92%±6%), sulfametha- to widely use in veterinary practice since the 1950s zine (130%±21%), sulfadiazine (89%±20%) and strepto- implicating the appearance of bacterial resistance. Hence, mycin (93% ±4%).
new synthetic agents, e.g. the class of quinolones, havebeen developed to replace effectless antibiotics. The use ofantibiotics for the treatment of honey bees is illegal in theEuropean Union, but due to the high import quota from Electronic supplementary material The online version of this article other countries, contaminated honey products can be found contains supplementary material, on the European markets In the last years another which is available to authorized users.
source for the contamination of honey with antibiotic K. Wutz : R. Niessner : M. Seidel (*) residues has been attracting notice. The plant disease Institute of Hydrochemistry and Chair for Analytical Chemistry, fireblight has become a serious problem for fruit-growers.
Technische Universität München, Fireblight is caused by the bacterium Erwinia amylovora Marchioninistraße 17, which affects plants from the family of rosaceae, e.g. apple 81377 München, Germanye-mail: [email protected] and pear. This infection disease leads to dieback of the K. Wutz et al.
blossoms and even can cause death of the entire tree [].
flow injection system []. Analytical microarrays are a The associated great economic loss for fruit-growers powerful tool for the simultaneous detection of multiple requires effective abatement of Erwinia amylovora. Up to analytes in a single measurement due to separated affinity- now, agents based on the aminoglycoside streptomycin are binding events at the surface interface, which reduces time the most common treatment of affected plants and orchards and costs for the analysis of contaminations in food [ Thus, antibiotic residues can be found in honey bee They have been approved for the detection of DNA target products due to contaminated collected pollen.
molecules [as well as for e.g. microorganisms , As contamination of food with antibiotic residues poses and toxins [or pharmaceuticals [in food and water risks for human health particularly with regard to increasing samples. For analytical microarray read-out purposes formation of resistance in bacteria strains to antimicrobials, fluorescence []- and chemiluminescence (CL) [ there is a need for monitoring of antibiotic contamination in detection methods are often used [, ]. CL microarrays honey. Most of the screening methods which have been measure the light emitted by an enzyme-assisted chemical published are using chromatographic techniques. In partic- reaction. In contrast to fluorescence, there is no background ular, liquid chromatography with fluorescence detection or signal, neither from the light source nor from light combined with coupled mass spectrometry detection (LC- scattering from the matrix. Therefore, CL is the most sen- MS/MS) is used for the determination of antibiotics in food sitive read-out principle for microarrays , matrices. In most cases, multi-analyte screening methods Since microarray-immunoassay methods require several are only applicable for antimicrobials of similar molecular incubation and washing steps, miniaturized bioanalysis structures. There were methods published for the determi- platforms have been developed which combine microarray nation of several macrolides sulfonamides [] and assays with fluidic microsystems , ]. Using flow- tetracyclines in honey. Some chromatographic multi- through microarrays, the microarray is part of a flow cell class methods have been established in the past years which through which samples and reagents are pumped. In allow identification [and also quantification [] of a comparison with conventional micro-well plates, flow- broad spectrum of antibiotics in honey. All HPLC based through microarrays present thinner diffusion layers en- methods require liquid-liquid or solid-phase extraction of abling efficient mass transport [This reduces the time the matrix followed by pre-concentration or clean-up of the needed to perform a multianalyte assay. Thus, automated extract. For multiclass screening there is the additional need flow-through microarrays allow the analysis of a sample of several extraction steps in dependency on polarity and within minutes and ensure reproducible and easy operating.
solubility of the various analyte structures. This sample The design of the platform MCR 3 (Microarray Chip preparation is time-consuming as well as laborious and can Reader 3), which is used in this study, has been published lead to loss of analytes along the extraction procedure. The for the determination of antibiotic residues in milk [ development of an adequate chromatographic separation of The specific antibody/antigen interactions on the regener- a broad variety of antibiotic residues is also a challenge able microarray chip surface allow for determination of which requires optimization. Additionally, in case of honey different analytes in parallel without extraction and separa- strong matrix effects due to the floral ingredients can cause tion steps before analysis. The detection is carried out by interferences which have to be minimized for chromato- sensitive CL read-out using horseradish peroxidase (HRP)- graphic analysis ].
Thus, chromatographic methods are not preferable for fast and cost-efficient high-throughput monitoring.
The advantage of immunochemical methods is the usage Materials and methods of specific antibodies to the analyte, which is dedicated formultianalyte screening. Various studies have been Chemicals and reagents concerned with the immunological screening for antimicro-bial substances in honey and other food matrices [].
The water used for all aqueous buffer solutions was Otherwise, as heterogeneous immunochemical methods are deionized and treated by a Milli-Q plus 185 system based on the formation of the antigen/antibody complex on (Millipore, Schwalbach, Germany, solid phase, time-consuming incubation steps are necessary.
All standard chemicals for the production of buffer Because of this, methods based on ELISA formats executed solutions were obtained from Sigma-Aldrich (Taufkirchen, in standard micro-well plates are not applicable for fast Germany, ). The antibiotic deriva- screening of high sample amounts. Therefore, at our tives streptomycin sulfate, sulfadiazine (SDZ) sodium salt, institute we have been focusing on microarray technique sulfamethazine (SMZ) sodium salt and enrofloxacin were based on multianalyte immunoassays (MIA) in combination purchased from Sigma-Aldrich. Clinafloxacin hydrochlo- with a flow-through principle realized by construction of a ride was obtained from Axxora (Lörrach, Germany, Simultaneous determination of four antibiotic residues in honey ). The positive control N-(2,4-dinitrophenyl)- slides were functionalized with 0.6 mL molten diamino- ethylene diamine (DNPEDA) was purchased from Chem- PEG for one sandwich in smelter at 98 °C for 15 h. The Pur (Karlsruhe, Germany, resulting diamino-PEG-coated glass slides were washed For surface modification 3-glycidyloxypropyltrimethoxysilane with water and dried under a nitrogen flow. The diamino- (GOPTS) and poly(ethylene glycol)diglycidyl ether PEG chips were stored at room conditions for a maximum (diepoxy-PEG) were obtained from Sigma-Aldrich.
Diamino-poly(ethylene glycol) (diamino-PEG) was pro- Diepoxy-PEG glass slides were prepared by dispensing vided by Huntsman Holland (Rozenburg, The Netherlands, 0.6 mL diepoxy-PEG on one diamino-PEG glass slide and covering it with another diamino-PEG glass slide. The The monoclonal primary antibodies (mAb) used for the slides were incubated in a sandwich format for 15 h at detection of norfloxacin (mAb 1F7, reactive with enroflox- 100 °C. After cleaning with methanol and drying under acin), streptomycin (mAb 4E2), sulfamethazine (mAb 4D9) nitrogen the produced diepoxy-PEG-coated glass slides and sulfadiazine (mAb 2G6) were produced at the Chair of were directly used for the spotting process.
Hygiene and Technology of Milk (LMU München) ].
For spotting, the antibiotic derivatives were dissolved in The mouse monoclonal antibody to trinitrotoluene (mAb mixtures of DMSO and carbonate buffer. The carbonate A1) was obtained from Strategic Diagnostics Inc. (Newark, buffer (pH 9.6) contained 15 mM disodium carbonate, USA, The horseradish peroxidase-labelled 35 mM sodium hydrogen carbonate and 3 mM sodium anti-mouse IgG produced in horse was purchased from azide in 1 L of water. For SMZ sodium salt, SDZ sodium Axxora. The chemiluminescence substrates Westar Super- salt, clinafloxacin hydrochloride (used for immobilization nova ELISA Luminol solution and Westar Supernova instead of enrofloxacin) and the positive control DNPEDA ELISA Peroxide solution were obtained from Cyanagen a 1:1 mixture of DMSO and carbonate buffer was used, for (Bologna, Italy, ).
streptomycin sulfate a 2:3 mixture was prepared. Each ofthe four antibiotics was spotted in different concentrations ranging between 0.01 mg mL−1 and 10 mg mL−1. Thepositive control was immobilized with a concentration of CL-MIA measurements were performed with the automated 0.1 mg mL−1. As negative control a 1:1 mixture of DMSO microarray chip read-out platform MCR 3 (GWK Präzi- and carbonate buffer without any additives was used. 200 sionstechnik, München, Germany, ).
μL of each spotting solution were given into the cavities of For microarray chip production conventional microscope a 96-well PP microtiter plate and spotted on the diepoxy- glass slides (26×76×1 mm) were purchased from Carl PEG glass slides. The spotting process was carried out with Roth (Karlsruhe, Germany, The car- a BioOdyssey Calligrapher Miniarrayer from Bio-Rad riers for the microarray flow cells were fabricated from Laboratories (München, Germany) using the Stealth Solid black poly(methyl methacrylate) at the Institute of Hydro- Pin SNS 9 from ArrayIt (Sunnyvale, USA). Two 14×5 chemistry (TU München). The double-sided adhesive foil clusters were set on one microarray glass chip with a grid ARcare 90106 was supplied by Adhesive Research Ireland spacing of 1,100 μm for the columns and 1,300 μm for the Ltd. (Limerick, Ireland, ). The rows, respectively. During the spotting process the chips production of the laser cuts with the microfluidic measuring were cooled to 20 °C and the humidity in the spotting channels was carried out by A.L.L. Lasertechnik GmbH chamber was set to 35%. After spotting the microarray (München, Germany, chips were incubated for 15 h at 25 °C and 50% humidity.
96-well polypropylene (PP) microtiter plates were The deactivation of free binding sides was carried out by obtained from Greiner Bio-One (Frickenhausen, Germany, sonicating the chips in 1 M Tris–HCl-buffer (pH 8.5) for 15 min. Further, the chips were cleaned by sonicating inwater and methanol for 5 min. After drying under a Microarray surface chemistry continuous nitrogen flow, the microarray glass slides wereconnected with plastic carriers by use of a double-sided The fabrication of microarray chips was carried out adhesive foil forming the microfluidic measuring channels.
following the standard procedure published by our groupformerly , Briefly, for cleaning and activation the Sample preparation glass slides were immersed first in methanol/hydrochloricacid (1:1), then in fuming sulfuric acid. The activated glass For measurement on the MCR 3 platform the honey slides were silanized by dispensing 0.6 mL GOPTS on one samples were diluted with phosphate buffered saline slide and covering it with a second slide ("sandwich (PBS, pH 7.6) consisting of 145 mM sodium chloride, format") for 1 h at room temperature. The silanized glass 10 mM potassium dihydrogen phosphate and 70 mM K. Wutz et al.
dipotassium hydrogen phosphate. To get a low viscous, SMA, anti-SDA, anti-Streptomycin and anti-Norfloxacin homogeneous liquid sample, 1 g honey was dissolved in and the anti-trinitrotoluene antibody with a concentration of 9 g PBS and the solution was vortexed vigorously. The 1 : 0.5 mg L−1 (diluted in running buffer). The mixture of 10 (w/w) solution of all honey samples with PBS ensures sample and primary antibody solution was pumped over the consistent pH of 7.6 for the analysis, which minimizes chip at a flow rate of 0.6 mL min−1. After a washing step possible matrix influences associated with acidic pH values with 2 mL of running buffer, 1 mL of the secondary and formation of interfering polymeric phenolic compounds antibody solution with a concentration of 1 mg L−1 (diluted in running buffer) was given over the chip at a flow rate of The samples for calibration and determination of the 6 mL min−1 for the first 0.2 mL and 0.6 mL min−1 for the recovery rates were spiked with the four antibiotic remaining 0.8 mL. Afterwards a second washing step was derivatives. The antibiotic concentrations of the samples executed. For the detection of bound antibodies, 0.2 mL of were calculated on the honey content, so no dilution factor a luminol respectively peroxide solution were mixed and has to be taken in consideration for the determination of the pumped over the fluidic cell at a flow rate of 9 mL min−1.
recovery. For calibration, samples with 0.01, 0.1, 1, 10, Then the flow was stopped and a picture was taken with an 100, 1,000 and 10,000 μg kg−1 of each antibiotic were exposure time of 60 s by a highly sensitive cooled CCD prepared. The honey used for the measurements was camera. All immunochemical assay steps including the obtained from Bayerisches Landesamt für Gesundheit und chemiluminescence reaction took 8 min. Afterwards, an Lebensmittelsicherheit (Erlangen, Germany) and was tested extended rinsing program was carried out to remove sugar negative for streptomycin (<2.5 μg kg−1).
and antibiotic residues in the fluidic system and on the chip.
After intensive rinsing of the tubes and the sample syringe with a total volume of 30 mL (running buffer) the chip was treated with 4 mL of the regeneration buffer at a flow rateof 15 mL min−1 (3 mL), respectively 0.6 mL min−1 (1 mL).
The Chemiluminescence-Microarray-Immunoassay (CL- Finally, 2 mL of running buffer were pumped over the chip MIA) measurements on the MCR 3 platform are based on at a flow rate of 30 mL min−1. The overall assay time the specific antigen/antibody interaction. The assay format including rinsing and regeneration steps was less than is an indirect competitive immunoassay on a heterogeneous phase. There is a competition between immobilizedanalytes on the microarray chip surface and the free analytes in the sample. The more antibiotic contaminantsare in the sample, the less specific antibodies can bind to The 2D images of the CCD camera were automatically the immobilized analytes. The bound antibodies can be saved as text-files. Before the measurements with one chip detected via HRP-labelled secondary antibodies by chemi- were carried out, a background picture was taken. This background noise of the camera was subtracted from the One characteristic of the MCR 3 measurement is the measuring images using LabVIEW 8.2 (National Instru- flow-through principle. All reagent solutions are pumped ments, USA). These pictures were evaluated with a new over the microarray chip without time consuming image evaluation software MCRImageAnalyzer developed incubation steps known from classical microtiter plate for the automated data-processing for CL microarrays in cooperation with GWK Präzionstechnik GmbH, Munich.
The second characteristic of the MCR 3 is the use of The calculated chemiluminescence data were transferred to regenerable microarray chips. This implicates the removal of Origin 7.0 (MicroCal Software Inc., Newark, USA) for the highly affine antibodies from the chip surface, so one graphical evaluation. The data of the calibrations were chip can be used for several repeated measurements. For fitted by use of a 4-parameter logistic (4-PL) function, chip regeneration a buffer solution consisting of 10 mM which gave sigmoidal-shaped semi-logarithmic calibration glycin, 100 mM sodium chloride and 0.1% (w/v) sodium curves For determination of the recoveries, the dodecyl sulfate in 1 L of water adjusted to pH 3 with obtained CL signal of the sample measurement (SCLsample) hydrochloric acid was used. As running buffer PBS contain- was corrected by referencing the blank measurement ing 0.5% (w/v) casein was used for all measurements.
directly before (SCLreference) with the blank measurement, The measurement steps were as following: 0.5 mL which was done before calibration (SCLblank). This correc- sample and 0.5 mL of primary antibody solution were tion method is expressed by Eq. injected simultaneously in an incubation loop at a flow rateof 3.6 mL min−1. The primary antibody solution was a SCLsample;referenced ¼ SCLblank  SCLsample cocktail of the four specific monoclonal antibodies anti-

Simultaneous determination of four antibiotic residues in honey The corrected CL signals of the spiked samples were As each analyte was spotted in five replicates, the generated normalized and set in the 4-PL calibration function.
CL signal of one analyte is represented by the mean value(MV) of the five single spot signal values and its standarddeviation.
Results and discussion The calculation of the signal intensity based on the ten brightest pixels was chosen instead of integration of all Data evaluation of chemiluminescence microarrays pixels within the grid cell, because we found this methodmore robust regarding the spot morphology. Slight defi- The measurements on the MCR 3 platform result in 2D ciencies in the printing process or non-uniform evaporation images (2×2 pixel binning mode, 696×520 pixels) of the can lead to deviations in the detailed shapes of individual chip surface obtained by a 16-bit CCD camera. The spots from the ideal circular form. In cases with inhomo- resolution of one pixel is 41 μm. The immobilized analyte geneous morphologies the summation of the grey level molecules are visible as bright spots where the CL reaction intensities within the corresponding grid cells leads to has taken place. The brightness (CL intensity) is described bigger variances of the mean CL signal for one analyte than by grey level intensities of single pixels ranging between 0 the evaluation of the ten brightest pixels (see Supp. Fig. and 65535 a.u. (saturation). The camera background noise and Supp. Table ).
of ca. 2000 a.u. was subtracted from the measurement CL Since the quantification of the analyte molecules in the signal of each spot.
sample is based on the indirect proportional CL intensity, Following the indirect competitive immunoassay format, the brightness of the spots is the decisive factor for the the CL intensity is depending on the amount of free analysis. In consequence, the established method is prefer- analytes in the sample. For simple and fast quantification of able for analytical quantitative CL data evaluation, because these antibiotic residues, an efficient method for the the relative standard deviation of the analyte CL intensity is evaluation of the CL intensities is needed.
clearly minimized.
Thus, we developed the software MCRImageAnalyzer The second aim of the data processing was to establish which enables easy and automated processing of the an automated outlier control. In some cases, there are spots measurement raw data. For recognition of the spots we with significantly decreased CL intensities compared with used a grid pattern (see Fig. ), where size and distances of the other spots of the same analyte or missing of the whole the quadratic grid cells are adjustable to the spotting array.
spot. This phenomenon can be explained by the existence For our investigations we created squares with a size of of small bubbles in the measurement flow channel during 25×25 pixels (equivalent to an area of ca. 1 mm2). In light exposure. The occurrence of air bubbles in the flow- relation to the spot diameters, which vary in dependency on injection system could not be completely excluded, since in the analyte between 7 and 13 pixels, these cells are large- this study we did not use any air trap (see Supp. Fig. scaled. The advantage of the big sized grid cells is the Thus, an efficient detection of outlier spots before evalua- entire registration of each spot even if there are drifts in the tion of the analyte CL intensities is needed to minimize spotting array, what means that the spots are not immobi- irregular influences on the dose–response measurements.
lized in perfect horizontal or vertical lines.
For this purpose, the algorithm implemented works on basis For calculation of the CL signal of one spot the software of the standard deviations. The first calculation step is the detects the ten brightest pixels within the corresponding determination of the overall relative standard deviation (R.
square. The average value represents the CL signal of one S.D.). Outliers are defined by a certain R.S.D. limit value.
single spot. Defective pixels with grey level intensities in For our investigations this limit was set to 20%. Exceeding the saturation region are filtered by an implemented of the limit value indicates the occurrence of one or more threshold to exclude artificial influences on the mean value.
outlier spots in the measured analyte column. Because of Fig. 1 Grid pattern for theevaluation of the chemilumines-cence intensities (differentanalytes in x-direction, repli-cates of the same analyte iny-direction). Size of the gridcells is 25×25 pixels

K. Wutz et al.
Fig. 2 Characteristic imageof the antibiotic microarray.
Immobilization in three differentspotting concentrations perderivative this, the second calculation step is the determination of the With this method outlier spots can be effectively R.S.D. for the possible combinations x following Eq. eliminated by choosing the combination of spots without the outlier. The working principle of the algorithm is explained in Supp. Table The algorithm developed allows the elimination of a second outlier per analyte in In this equation n represents the number of spots per rare cases of exceeding the limit value though. All analyte and k is the number of chosen spots for the R.S.D combinations of three spots (n=5, k=3) are evaluated by their corresponding R.S.D. and the combination with Fig. 3 Dose–response curvesfor the four antibiotic analytesenrofloxacin (a), SDZ (b), SMZ(c) and streptomycin (d) inhoney samples. Standard devia-tion is represented by error bars(m=5) Simultaneous determination of four antibiotic residues in honey Table 1 Characteristics of the dose–response curves lization process. For SMZ and SDZ all spotting concen-trations were applicable for the further experiments regarding IC50 [μg kg−1] the obtained SNR, but regeneration studies showed strongdetaching effects of excessive molecules on the spot surface in case of high spotting concentrations (data not shown).
Thus, the spotting concentrations of the antibiotic derivatives selected for calibration were 0.1 mg mL−1 for SMZ and clinafloxacin and 1.0 mg mL−1 for SDZ and streptomycin,respectively.
minimal deviation is chosen for calculation of the analyte Simultaneous dose–response measurements and determination of recovery Spotting adjustment for antibiotic microarrays Dose–response measurements were carried out for thesulfonamides sulfadiazine (SDZ) and sulfamethazine The establishment of an appropriate microarray immunoas- (SMZ), the aminoglycoside streptomycin and the fluoro- say for the detection of antibiotic contaminants requires quinolone enrofloxacin. The multianalyte ELISA assay on adjustment of the spotting concentrations. On the one hand, the microarray chips was performed in an indirect compet- the amount of analyte molecules immobilized on the spot itive format, which is most applicable for small analytes.
surface affects the obtained CL signal and has to be The first two measurements with one chip were used for optimized to achieve high signal-to-noise ratios (SNR). On complete loading of reagents to obtain maximal CL signal, the other hand, the spotting concentration influences the IC50 the third measurement was used for the blank measurement values of the standard calibration curves and for this reason with a honey sample without antimicrobial additives.
the working ranges. Furthermore, choosing to high spotting Afterwards, the standard solutions were measured along concentrations can lead to an excess of analyte molecules on increasing analyte concentration. Figure illustrates the the spot surface detectable by a decrease of the CL signal resulting dose–response curves. The characteristic standard during the first measurement cycles due to detaching effects.
calibration data for the four determined analytes are shown Therefore, different concentrations of the four antibiotics in Table The working ranges (WR) were defined as examined were tested for immobilization carrying out blank 10%–80% of the maximum CL signal in case of enroflox- measurements. The resulting microarray image is depicted in acin, for the other analytes as 20%–80%.
Fig. the spotting sizes and SNR values are shown in Supp.
For determination of the recovery rates, spiking experi- ments were carried out. Honey samples were prepared For all immobilized antibiotic derivatives similar trends with various contents of the four antibiotics. Spiked were obtained. With increasing spotting concentrations samples were directly measured after calibration on the enhanced CL signal values could be observed. In case of microarray chip. Due to an observed signal decrease along streptomycin, the spotting concentration of 0.1 mg mL−1 chip regeneration cycles a blank measurement was done showed low SNR of 16 : 1 for the blank measurement and before analysis of each spiked sample. The obtained CL inhomogeneous surface covering densities, whereas higher signal (SCL) of the sample measurement was corrected amounts of streptomycin lead to sufficient CL signals of the following Eq. To confirm the feasibility of this spots. The spots measured for the clinafloxacin spotting correction method the determination of the recovery rates solution with 0.01 mg mL−1 gave small spot sizes was performed as duplicates for each sample on the same compared with the other tested solutions for the immobi- chip. In addition, a contaminated honey sample that was Table 2 Recoveries of the spiking experiments Contaminated sample 1.0 μg kg−1 [μg kg−1] 10 μg kg−1 [μg kg−1] 100 μg kg−1 [μg kg−1] (25.5 μg kg−1 streptomycin) [μg kg−1] K. Wutz et al.
determined to 25.5 μg kg−1 streptomycin by LC-MS-MS milk which need no dilution steps. Due to the regenerability analysis was examined. The results of the spiking experi- of the antibiotics microarray each chip could be individu- ments are presented in Table ally calibrated before the analysis is performed and more The dose–response curve for enrofloxacin showed good than 40 analyses could be done per chip which reduces the sensitivity with distinct differences of the CL signal in the costs per analysis and achieve an automated work flow in range between 1 and 29 μg kg−1, so precise analysis of routine laboratories. Spiking experiments showed with a samples within this contamination level is possible. The recovery rate of ±10% a high accuracy of enrofloxacin calibrations for SDZ, SMZ and streptomycin dynamic down to a concentration of 1 μg kg−1. Sulfamethazine, ranges allow determination of antibiotic contamination sulfadiazine and streptomycin have a recovery rate between within a broader span.
75% and 146% at 100 μg kg−1 as this concentration lies in A high precision of the calibration for enrofloxacin could the dynamic ranges of their calibration curves in honey.
be confirmed by recovery for the samples spiked with Finally, the multianalyte immunoassay has successfully 1.0 μg kg−1 and 10 μg kg−1, respectively. The spiked identified a streptomycin contaminated honey product enrofloxacin sample of 10 μg kg−1 was in the working which was approved through LC-MS/MS.
range of the multianalyte immunoassay. A recovery of Additionally, a new data processing method for CL 92%±6% was achieved. The calculated detection limit was microarrays images was examined in this study for rapid 4.2 μg kg−1 although a concentration of 1.0 μg kg−1 microarray analysis in routine laboratories. Each spot was enrofloxacin could be precisely quantified with recovery of automatically evaluated; outlier could be identified and 95%±7%. This indicates that the precision of this system were excluded for the analysis. This algorithm is important depends mainly on the spot quality of the microarray. These to reduce the variances in the CL-MIA and allows an differences between the LOD and the analytical sensitivity automated data processing for analysis.
are also known in other immunoassay assay platforms [ We have shown with this study that a multianalyte . A concentration of 100 μg kg−1 enrofloxacin was immunossay based on an automated flow-through chemi- outside of the working range and therefore, the two luminescence microarray technique is suitable for the determined recoveries were 56% and 76%. Thus, samples quality control of food sample even for such difficult with high enrofloxacin contamination levels have to be matrices like honey. Further investigations have to be diluted before analysis. SDZ and streptomycin could be focused on the analytical requirements associated with quantified precisely at 10 μg kg−1 and 100 μg kg−1 with 2002/657/EC for validation as effective screening method recoveries between 75% and 114%. SMZ shows an over- for routine residue analysis. In particular, more samples estimation. The measurement of a honey sample spiked have to be analyzed to determine the false non-compliant with 100 μg kg−1 sulfamethazine showed recoveries rate (α-error) and the false compliant rate (β-error).
of 130% ±21%. Streptomycin had a detection limit of15.9 μg kg−1. Nevertheless, a spiked concentration of This work was supported by the Bayerische Forschungsstiftung (BFS AZ-842-08). We would also thank Prof. E.
10 μg kg−1 could be quantified with a recovery of 103%± Märtlbauer and Dr. R. Dietrich (Lehrstuhl für Hygiene und 9%. The recovery of streptomycin in a contaminated real Technologie der Milch, LMU München, Oberschleißheim, Germany) honey sample was 130.4% ± 0.6% referring to the result of for providing the monoclonal antibodies to enrofloxacin, SDZ, SMZ the LC-MS/MS analysis which is comparable to other and streptomycin. Further, we thank Dr. C. Hinkel (Bayerisches Land-esamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany) for the honey samples and Huntsman Corporation (Rotterdam, TheNetherlands) for the free DAPEG samples.
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