A New, Versatile Field Immunosensor for Environmental Pollutants:: Development and Proof of Principle With TNT, Diuron, and Atrazine

A New, Versatile Field Immunosensor for Environmental Pollutants:: Development and Proof of Principle With TNT, Diuron, and Atrazine
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  Biosensors and Bioelectronics 21 (2005) 354–364 A new, versatile field immunosensor for environmental pollutantsDevelopment and proof of principle with TNT, diuron, and atrazine Ioan M. Ciumasu a , b , Petra M. Kr¨amer a , b , ∗ , Cristina M. Weber b , Gunther Kolb c ,David Tiemann c , Stefan Windisch d , Ines Frese c , Antonius A. Kettrup a a Technical University Munich, Chair of Ecological Chemistry and Environmental Analysis, Weihenstephaner Steig 23,85350 Freising-Weihenstephan, Germany b GSF, National Research Centre for Environment and Health, Institute for Ecological Chemistry, Ingolst¨ adter Landstrasse 1,85764 Neuherberg, Germany c  Institut f¨ ur Mikrotechnik Mainz GmbH, Carl-Zeiss-Strasse 18-20, 55129 Mainz, Germany d  Evolution Technologies GmbH, Carl-Zeiss-Strasse 18-20, 55129 Mainz, Germany Received 30 August 2004; received in revised form 15 October 2004; accepted 18 October 2004Available online 8 December 2004 Abstract This paper presents a new, versatile, portable miniaturized flow-injection immunosensor which is designed for field analysis. Thetemperature-controlled field prototype can run for 6h without external power supply. The bio-recognition element is an analyte-specificantibody immobilized on a gold surface of pyramidal structures inside an exchangeable single-use chip, which hosts also the enzyme-tracerand the sample reservoirs. The competition between the enzyme-tracer and the analyte for the antigen-binding sites of the antibodies yieldsin the final step a chemiluminescence signal that is inversely proportional to the concentration of analyte in the given range of detection. Aproof of principle is shown for nitroaromatics and pesticides. The detection limits (DL; IC 20 ) reached with the field prototype in the laboratorywas below 0.1  gl − 1 for 2,4,6-trinitrotoluene (TNT), and about 0.2  gl − 1 for diuron and atrazine, respectively. Important aspects in thisdevelopment were the design of the competition between analyte and enzyme-tracer, the unspecific signal due to unspecific binding and/orluminescence background signal, and the flow pattern inside the chip.© 2004 Elsevier B.V. All rights reserved. Keywords: Flow-injection immunosensor; Chemiluminescence; Nitroaromatic compounds; Pesticides; TNT; Atrazine; Diuron; Isoproturon 1. Introduction Reviewing the multitude of techniques and methods thatare applied in the field of analytical chemistry and biochem-istryisdaunting,particularlywithbiosensors.Inawideprac-tical perspective many approaches look promising, but onegeneral conclusion that emerged with time is the need of ver-satility. There is a trade between different analytical perfor-mances, notably sensitivity and precision versus simplicityand speed of analysis. Essentially, the best compromise mustbe found between the basic performances of a method and ∗ Corresponding author. Tel.: +49 89 3187 2772; fax: +49 89 3187 3371.  E-mail address: (P.M. Kr¨amer). themanypracticalrequirementslikemeasurementtime,tem-perature control or sensors’ autonomy. The trick is to designflexible analytical techniques.Earlier physico-chemical approaches for the field screen-ing of explosives were colorimetric methods, like detectionof2,4,6-trinitrotoluene(TNT),2,4-dinitrotoluene(2,4-DNT)and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by usingsodium/potassium hydroxide or sodium disulfide (Medary,1992; Jenkins and Walsh, 1992).Currently,theconventionalandacceptedlaboratorymeth-ods for the quantitative analysis of explosives are basedon GC/MS or GC/ECD (Psillakis et al., 2000; U.S. EPA,2000) and HPLC (Godejohann et al., 1997; U.S. EPA,1994). 0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2004.10.027   I.M. Ciumasu et al. / Biosensors and Bioelectronics 21 (2005) 354–364 355Fig. 1. Details (left) and portable box (right) of the temperature-controlled field prototype. (A) Details of the temperature-controlled part: (1) Peltier elementsfor temperature control; reservoirs for buffer (2) and substrate solutions (3); (4) black PMMA ground plate with valves, hosting the fluidic part and connectionsto the chip; (5) single-use chip (upside down) with sample reservoir (6), enzyme-tracer, and gold structures (the last two are not visible); (7) USB stick for datastorage. (B) The whole instrument is within a box suitable to be carried into the field: (a) touch screen of computer, (b) temperature-controlled part. Syringepump with step motor is also incorporated, but not visible. Immunochemical techniques are also widespread, mainlydue to their flexibility and affordability. For nitroaromaticcompounds, numerous systems were developed, includingimmunoassays and various immunosensors with differenttransducer principles (e.g.,Bart et al., 1997; Buttner et al.,1997; Narang et al., 1997; Dosch et al., 1998; Heiss et al.,1999; Balkatcheva et al., 1999; Rabbany et al., 1998, 2000;Green et al., 2002; Holt et al., 2002; Sapsford et al., 2002;Wilson et al., 2003a, 2003b; Shriver-Lake et al., 2003).In addition, there are a number of different immunoas-say test-kit formats, which have already been validated(Eikenberg et al., 1997; Kr¨amer, 1998).Most of these immunochemical methods developed so farhad poor trade-offs between the analytical-practical require-ments mentioned earlier. The big challenge still remains ahigh-performance, cost-effective field analysis. One majortendency in environmental analysis is to combine an effec-tive field screening with a high-performance laboratory tech-nique. Nowadays, while HPLC and GC/MS techniques arethe best analytical tools in the laboratory, immunochemicalmethods emerge as a top option for field screening.First commercial types of field-use immunosensors wereengineered by Research International (Woodinville, WA,USA) starting in 1997, albeit they did not report that therewas a temperature-control build in. FAST 2000 (flow assaysensing and testing) system (Kusterbeck and Charles, 1998;Charles et al., 2000; Kusterbeck and Shriver-Lake, 2000;Dindal et al., 2000; Gauger et al., 2001; Shriver-Lake et al.,2003)and FAST 6000 are based on fluorescence displace-ment immunoassays (on permeable membranes containingthe tagged analyte), and reach detection limits for explosives(TNTandRDX)between1and10ppb.  Analyte2000 isafibreoptic fluorometer based on competitive fluoroimmunoassay,and reach detection limits for explosives (TNT and RDX)between 5 and 10  gl − 1 in water and 50 and 100mgkg − 1 insoil (Van Bergen et al., 2000; Kusterbeck and Shriver-Lake,2000), however, significant problems were encountered withrespect to accuracy and precision in environmental samplemeasurements.  RAPTOR isafibreopticportableimmunosen-sor for bacteria toxins (Anderson et al., 2000; Anderson andRowe-Taitt, 2001).Likewithexplosives,determinationofpesticidesintheen-vironment (a much wider field than analysis of explosives) isperformed in laboratories with established chromatographicmethods like LC–MS or GC–MS, as well as some immuno-chemical methods, such as immunoassays and immunosen-sors (Kr¨amer, 1996; Mallat et al., 2001).In this paper we describe the new development and theproof of principle of a portable, temperature-controlled,power-supply autonomous field immunosensor for environ-mental applications (Fig. 1),inspired by earlier developed immunochemical detection systems (Kr¨amer et al., 1997;Meusel et al., 1998), and built by IMM GmbH, Mainz, Ger-many (Kolb et al., 2004). This instrument was developed andoptimized using TNT as the key target. In addition, the pesti-cidesdiuronandatrazinewereusedtodemonstratetheversa-tilityofthisplatform.Finally,anewlydevelopedisoproturonassay (Kr¨amer et al., 2004a) was adapted towards the futureusage of this field immunosensor.Monoclonal antibodies (mAbs) are immobilized via ad-sorption on a gold surface with numerous pyramidal struc-tures. The recognition reaction is enhanced in three ways:(1) via the enzymatic reaction, (2) via the pyramidal struc-tures with the gold surface cover, and (3) via the detectionof the chemiluminescence of the product through a very sen-sitive photomultiplier. The latter is situated directly abovethe pyramid tips. Immunoreagents (enzyme-tracer and anti-body) together with the environmental sample are located ina single-use chip, which is replaced after each measurement.This chip is the key to the versatility of the analytical system.  356 I.M. Ciumasu et al. / Biosensors and Bioelectronics 21 (2005) 354–364 Transportofreagentsisperformedwithanautomated,minia-turizedflow-injectionsystem,whichistheconsistentpartandapplicable for all analytes. The chemiluminescence signal isinversely proportional to the amount of analyte present in thesample. 2. Materials and methods 2.1. Chemicals, standards, immunoreagents, and  proteins Conjugation reagents (  N  -hydroxysulfosuccinimidesodium salt and 1,3-dicyclohexyl carbodiimide) and3,3  ,5,5  -tetramethylbenzidine (TMB) were purchasedfrom Sigma–Aldrich Chemie GmbH, Schnelldorf, Ger-many. Solvents N  ,  N  -dimethyl formamide, 99% (DMF)and dimethylsulfoxide, 99% (DMSO) were from Fluka(now Sigma–Aldrich, Schnelldorf, Germany). Glycerol,SuperSignal ® ELISA Femto Maximum Sensitivity Substrate(luminescence), and SuperFreeze TM Peroxidase ConjugateStabilizer were from Pierce, Rockford, IL, USA. Micro-O-protect was obtained from Roche Diagnostics, Penzberg,Germany. Buffer salts and Tween 20 were purchased fromMerck-Schuchardt, Darmstadt, Germany.TNP-glycylglycine (2,4,6-trinitrophenyl-glycylglycine,100%), DNP-glycine (  N  -(2,4-dinitrophenyl)-glycine,97%), TNP-glycine, DNP-  -aminocaproic acid, DNP-  -aminobutyric acid, TNP-  -aminobutyric acid, andTNP-  -aminocaproic acid were purchased from ResearchOrganics (Cleveland, OH, USA).TNT was purchased either from Dr. Ehrenstorfer(Augsburg, Germany), Cerilliant Corporation (Austin, TX,USA), or Institute of Industrial Organic Chemistry (War-saw, Poland). 2-Amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were purchased either from Dr. Ehrenstorfer(Augsburg,Germany)orCerilliantCorporation(Austin,TX,USA).Isoproturon,diuron,andatrazinewerepurchasedfromRiedel-de Ha¨en, Seelze, Germany.Goat anti-mouse IgG unconjugated, Protein A,Purified ® Protein A/G, and Purified ® Protein G werepurchased from Pierce, Rockford, IL, USA. Casein frombovine milk (powder) and skim milk powder were purchasedfrom Fluka (now Sigma–Aldrich, Schnelldorf, Germany).Mouse anti-rat IgG (TIB 172,  -specific) is an in-houseclone (E. Kremmer, IMI, GSF); it is commercially avail-able through ATTC (American Type Culture Collection,Manassas, VA, USA). Anti-TNT mAb (mouse, A1.1.1,10.6mgml − 1 ) was purchased from Strategic BioSolutionsInc. (SBS), Newark, DE, USA. The mAb (rat, IOC 7E1)against isoproturon was developed in-house (Kr¨amer etal., 2004a). Anti-atrazine mAb (mouse, AM5D1-3) andanti-diuron mAb (mouse, 481.3) have been developed andwere described byKaru et al. (1991, 1994).The enzyme-tracer for the atrazine assay (‘Atrazine–HRPconjugate’, 65-IA01) was purchased from Fitzgerald Indus-tries International Inc. (Concord, MA, USA).Bovine serum albumin fraction V (BSA, powder) wasfrom Sigma–Aldrich Chemie GmbH, Schnelldorf, Ger-many. Peroxidase from horseradish (HRP; ca. 1000Umg − 1 )was from SERVA Electrophoresis GmbH, Heidelberg,Germany. 2.2. Materials and instruments Slide-A-Lyser ® dialysis cassettes, needles and syringeswere purchased from Pierce, Rockford, IL, USA. Dem-ineralised water was prepared by a Milli-Q (MQ) filtrationsystem (Millipore, Eschborn, Germany), and was used forthe preparation of all standard solutions and buffers. Mi-crotiter plates (NUNC F-96 certified MaxiSorp TM ) and lidswere purchased from NUNC, Wiesbaden, Germany. Wash-ing steps in microtiter plates were carried out with a NUNCImmunowash (Roskilde, Denmark) connected to a vacuumpump (KNF Neuberger, Laboport, K&K Laborservice, Mu-nich, Germany). Absorbance was read with a ThermoMaxor SpectraMax microtiter plate reader (Molecular Devices,Palo Alto, CA, USA). During the preparation of conjugates,centrifugations were carried out with a Heraeus SepatechBiofuge15(Heraeus,Hanau,Germany).Duringincubations,usuallysolutionswerecoveredbyParafilm(PechineyPlasticPackaging, Menasha, WI, USA). 2.2.1. Solid supports for antibody immobilization,single-use chip, and the prototype Three types of solid supports and set-ups (correspondingto three distinct steps of ELISA transfer to the immunosen-sor) were employed for antibody immobilization (Table 1):(a)microtiterplates,(b)‘batchstructures’,whicharethegoldcovered structures later incorporated into the disposable chip(one structure per glass vial; each structure consisted in acertain number of micro-pyramids (with square base) carvedonto the upper face of a PMMA (poly(methyl methacrylate))plastic disc, and covered with a thin layer of gold (IMMGmbH, Mainz, Germany), and (c) structures incorporatedinto a disposable, but reusable chip (technical details, seeTable1).Thesingle-usechipfitsontothegroundplate(black PMMA;Fig. 1A), which hosts the fluidic system (channelsand µ -valves).Thefinalversionofthewholestructurewithpyramidshasthe following dimensions: basic disc of 8mm diameter and3mm height; the upper surface bears a gold layer covering314 pyramids (intermediate height, ‘aspect 1’, see also Sec-tion3.2),each pyramid with an ‘active optical surface’ of  0.277mm 2 (total active surface per structure: 86.978mm 2 ).Thesingle-usechip(Table1)sheltersontheonesideanin- cubation/measurement part with pyramidal structures (Freseet al., 2003), as well as the enzyme-tracer reservoir with ameandering channel (ca. 0.7ml), and on the other side (topside for the user) the sample reservoir (ca. 1ml). In the in-cubation/measurement part, the volume above pyramids is9.478  l,givingavolumepersurfacevalueof0.11  lmm − 2 .Bycomparison,thecorrespondingratioinamicrotiterplateis   I.M. Ciumasu et al. / Biosensors and Bioelectronics 21 (2005) 354–364 357Table 1Comparison of three different immunochemical platforms and their characteristics, shown for the example of TNT analysisConventional ELISA Batch-ELISA Single-use ChipSet-up and adsorption surfaceSurface area (mm 2 ) 139 (for 150  l) 87 87Maximum volume (  l) 150 1000 9.5Volume per surface area (  lmm − 2 ) 1.08 11.5 0.11Antibody dilution 1:35000 1:10000 1:1000Antibody volume (  l) 150 1000 9.5Enzyme-tracer dilution 1:2000 1:2000 1:6000Enzyme-tracer volume (  l) 50 300 1/3 of 9.5  lAnalyte volume (  l) 100 600 2/3 of 9.5  lSubstrate TMB/H 2 O 2 TMB/H 2 O 2 SuperSignal ® ELISA (luminol-enhancer/H 2 O 2 )Substrate volume (  l) 150 1000 9.5Signal Absorbance Absorbance Chemiluminescence ca. 10 × higher (1.08  lmm − 2 ), while in a glass vial (batch)this ratio is ca. 100 × higher (11.5  lmm − 2 ), because 1mlof volume is needed in vials). The total volume of the in-cubation/measurement cell in the chip comprises, in addi-tion to the volume (strictly) above the pyramids surface, twoother volumes (in the direction of flow, in front of and be-hind the pyramids), which insure the fluids transport and theeven flow over the pyramids. Thus, the total volume of theincubation/measurementcellisca.30  l.Thismayinfluencethe speed of the chemical reactions to a certain extent (lim-ited by the diffusion speed and the incubation time of thereagent molecules), but cannot be a serious source of vari-ation, because the fluidics and the process temperature areautomatically controlled.The single-use chip and the ground plate represent theheart of the instrument. The instrument itself incorporatesalso an electric step-motor syringe pump, a chemilumines-cencedetector(photomultiplier,HamamatsuPhotonicsK.K.,HamamatsuPhotonicsDeutschlandGmbH,Herrsching,Ger-many), reagent reservoirs (for carrier buffer and substrate),and a computer control unit with a touch-screen user inter-face (Fig. 1Aand B). In addition, the prototype is a one-box instrument, portable, autonomous from external power sup-ply (6h), and temperature-controlled (IMM GmbH, Mainz,Germany;Kolb et al., 2004).As such, the instrument is a miniaturized and automatedflow-injection system, employing the analyte detection viathe antibody-recognition reaction within a single-use chip,and uses chemiluminescence derived from an enzymatic re-action as transducer. 2.3. Methods2.3.1. Synthesis of enzyme-tracers A battery of six enzyme-tracers for nitroaromatic com-pounds as haptens were produced by hapten conjugation toHRP via N  -hydroxysuccinimide active esters (for details, seeKr¨amer et al., 2004b).In total, six conjugates were producedby this method and tested as enzyme-tracers: TNP-glycine-HRP; TNP-glycylglycine-HRP; TNP-  -aminobutyric acid-HRP; DNP-glycine-HRP; DNP-  -aminobutyric acid-HRP;DNP-  -aminocaproic acid-HRP.The enzyme-tracer for the isoproturon assay (III-HRP)wasdescribedbyKr¨ameretal.(2004a).Thehaptenforthedi-uron assay (1-(5-carboxypentyl)-3-(3,4-dichlorophenyl)-1-methylthiourea), synthesized by M.H. Goodrow, formerlyUCDavis,Davis,CA,USA,wasconjugatedin-housetoHRPusing also the same conjugation method. 2.3.2. Competitive ELISA on microtiter plates Thecoatingantibodysolution(anti-speciesmAb)wasim-mobilized via physical adsorption. The solution of coatingantibody (usually 2  gml − 1 ) in 50mM carbonate buffer, pH9.6, was incubated on microtiter plates, 200  l/well, eitherovernightat3 ◦ Cor2hatroomtemperature.Microtiterplatesincubatedwithcaptureantibodieswerebroughttoroomtem-perature, washed (3 times), and dried. After washing with4mM PBST, pH 7.6 (phosphate buffered saline, including0.05% (v/v) Tween 20), the analyte-specific mAb (1:35,000or 303ngml − 1 in 40mM PBS, pH 7.6) was incubated on thecapture antibody at room temperature, for 2h (except whenotherwise stated and justified). After another washing (3times) and drying, the plate was ready for the inhibition step.Standard solutions (different concentrations: 100  l/well)and the corresponding enzyme-tracer (50  l/well; e.g., op-timized dilution for TNP-glycylglycine 1:2000) were incu-bated together on the anti-analyte mAb immobilized on themicrotiterplates,between5and30min,atroomtemperature.Plates were washed again, and the substrate for the enzymereaction was added (150  l/well; TMB/H 2 O 2 in 100mMsodium acetate buffer, pH 5.5), and incubated for 10–30min.  358 I.M. Ciumasu et al. / Biosensors and Bioelectronics 21 (2005) 354–364 The enzymatic reaction was stopped with 50  l/well of 2MH 2 SO 4 . Plates were read with an ELISA absorbance reader(SpectraMax or ThermoMax) at 450nm (reference 650nm).Using the commercial anti-TNT mAb A1.1.1 and our in-house produced enzyme-tracers, the competitive ELISA wasdescribed and optimized for sensitivity, selectivity, pH, sol-vent tolerance, incubation times and enzyme-tracer stability.The standard curves were described using commercial soft-ware for ELISAs (SOFTmax ® Pro, Molecular Devices, PaloAlto, CA, USA).Similarly, different ELISAs were performed and furtheroptimized using mAbs against atrazine (Karu et al., 1991), diuron (Karu et al., 1994), and isoproturon (Kr ¨amer et al.,2004a). For these assays, coating proteins were Protein A,ProteinGorProteinA/G,2  gml − 1 ,goatanti-mouse(GAM)ormouseanti-rat(TIP-172),2.4or2.0  gml − 1 ,respectively.The dilutions of mAb were 1:4000 (600ngml − 1 ) in theatrazine ELISA, 1:4000 (from the culture supernatant) in thediuron ELISA, and 1:20,000 (75ngml − 1 ) in the isoproturonELISA. The dilutions of the corresponding enzyme-tracerswere 1:2000 in the atrazine and isoproturon ELISAs, respec-tively, and 1:4000 in the diuron ELISA. 2.3.3. Competitive ELISA on solid structures —batch-ELISA After optimization on the microtiter plates, the ELISAprocedure for each target analyte was adapted to the batch-ELISA format, where the wells of the microtiter plate werereplaced by the gold covered discs with numerous pyramidstructures, which were placed and treated individually inglass vials (Table 1).Thegeneralprocedureofthebatch-ELISAwasasfollows.Coating antibodies or proteins (anti-species mAbs or ProteinA/G; 1ml solution per vial) were set-up in 50mM carbonatebuffer,pH9.6,andincubatedat3 ◦ C,overnight(alternatively,2h at room temperature). The next day, structures were al-lowedtocometoroomtemperature(15min),thecoatingpro-tein solution was removed, and thestructures washed 3 timeswith1mlofwashingbuffer(4mMPBST,pH7.6).Afterthis,the analyte-specific mAb (in 40mM PBS, pH 7.6) was incu-bated (usually 2h) at room temperature then, this antibodysolution was removed (washing not needed) and replaced bya solution made from skimmed milk powder, 1% (w/v, in40mM PBS, pH 7.6), 30min at room temperature (block-ing step). Following another washing, for the competitionstep, the analyte standards (in 40mM PBS) were added ontothe immobilized antibody before the enzyme-tracer (40mMPBS,pH7.6).Duringcompetition,thevialswereincubatedatroom temperature and slowly moved with a microtiter plateshaker. After competition and another washing, the signalwas obtained by incubating the structures in 1ml per vialof substrate/chromogen solution for HRP (TMB/H 2 O 2 ) in100mM sodium acetate buffer, pH 5.5, 30min (alternatively,up to 90min) at room temperature, in the dark. In order tominimize unspecific binding, the structures can be quickly(but gently) transferred to clean glass vials before substrateincubation. After substrate incubation, 150  l of incubatedsubstrate/chromogen solution was transferred from each vialto a microtiter plate and the enzyme reaction was stoppedwith 2M H 2 SO 4 , 50  l/well. The microtiter plate was readat 450nm (reference 650nm).After reading, the structures were rinsed with water, andtheirsurfacewasregeneratedbywashingfirst3timeswithre-generationbuffer(100mMsodiumcitrate,pH2.5),1ml/vial,then 3 times with Milli-Q water, 1ml/vial. After the firstwashing with water, the structures were transferred to cleanglass vials and washed again 2 times with Milli-Q water. Theregenerated structures were either stored in air at room tem-peratureorusedforanotherexperiment.Thestructurescouldbe regenerated for at least 50 times.Specific batch-ELISA conditions for the different ana-lytes tested were as follows. For TNT assays, the coat-ing protein was Protein A/G (alternatively, goat anti-mouseIgG) for mouse mAb A1.1.1; coating concentration was4  gml − 1 . The recognition antibody was anti-TNT mAbA1.1.1, 2  gml − 1 , 2h at room temperature (alternatively,down to 10min). After the blocking step, the TNT stan-dards and the enzyme-tracer solution (TNP-glycylglycine-HRP, 1:2000) were incubated together on the structures,10min.Diuron batch-ELISA used as coating antibody goat anti-mouse IgG, 2  gml − 1 . The recognition antibody was anti-diuron mAb 481.1 (cell culture supernatant 1:2500); it wasincubated for 2h at room temperature. After the blockingstep, the diuron standards and the enzyme-tracer solutions(1:4000) were incubated together on structures, 10min.Batch-ELISA for atrazine used as coating antibodygoat anti-mouse IgG, 4  gml − 1 . The recognition antibodywas anti-atrazine mAb AM5D1-3 (cell culture supernatant1:2000),2hatroomtemperature.Aftertheblockingstep,theatrazine standards and the enzyme-tracer solutions (1:2000)were incubated together on structures, 10min.The isoproturon batch-ELISA used as coating anti-body mouse anti-rat TIB 172, 2  gml − 1 . The analyte-selectiveantibodywastheanti-isoproturonratmAbIOC7E1(75ngml − 1 ), and it was incubated for 2h at room tempera-ture. After the blocking step, the isoproturon standards andthe enzyme-tracer solutions (III-HRP 1:200) were incubatedtogether on structures, 30min. 2.3.4. Competitive ELISA by the immunosensor  prototype (demonstrator) Off-line preparation of the chips. All off-line fluidhandling on the chips was carried out with a simple single-usesyringe,withput-onneedle(standards)orwithoutneedle(all the rest). The chips (incubation cell) were incubated: (1)with the appropriate catching protein at room temperature,(2)withthecorrespondinganalyte-specificmAb,and(3)with1%(w/v)skimmedmilkpowderdissolvedin40mMPBS.Fi-nally, the chips were washed with 4mM PBST, 1ml/chip. Atthis point, the chips were either used immediately for mea-surements, or they were labelled and stored in the freezer


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