Development of a new immunosensor for pesticide detection: a disposable system with liposome-enhancement and amperometric detection

Biosensors & Bioelectronics 13 (1998) 519–529

Development of a new immunosensor for pesticide detection: adisposable system with liposome-enhancement and amperometric

detection

Antje J. Baumner, Rolf D. SchmidInstitute for Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

Received 8 July 1997; received in revised form 31 October 1997; accepted 4 November 1997

Abstract

The development of a disposable amperometric immunomigration sensor for the detection of triazine pesticides in real samplesis presented. Thick film electrodes printed on PVC were used as strip-type transducers. Monoclonal antibodies against atrazine andterbutylazine served as biorecognition element. For the generation and amplification of the signal, hapten-tagged liposomes entrap-ping ascorbic acid as a marker molecule were chosen. A capillary gap and a wicking filter membrane strip served as migrationzone. For signal detection on a graphite electrode, liposomes were lysed by Triton X-100 and the released ascorbic acid wasquantified at a potential of+ 300 mV vs printed Ag/AgCl. Signal response time was 1–3 min, and sensitivity of measurements intap water was below 1mg l 2 1 of atrazine and terbutylazine. Atrazine determinations in soil extracts correlated well with standardprocedures based on ELISA and HPLC. 1998 Elsevier Science S.A. All rights reserved.

Keywords:Immunosensor; Triazine pesticides; Liposomes; Thick film electrodes; Amperometry; Monoclonal antibodies

1. Introduction

Pesticides are widely used in agriculture to protectseed and crop. As their persistence in the geosphere maycause problems in the biosphere, their presence in soiland water is continuously monitored. According to EUregulations, concentration limits in drinking water are0·1mg l21 for a single pesticide and 0·5mg l 2 1 for mix-tures. Current standard procedures for pesticide determi-nation in soil and water samples (HPLC and GC/MS)are accurate but rather time-consuming and beyond theanalytical capacities of smaller water works as theyrequire expensive instrumentation. Consequently, immu-nochemical methods are being widely investigated as analternative analytical procedure and in fact in some caseshave already been successfully adapted to environmentalanalyses (Sadik and Van Emon, 1996; Hock, 1989).Enzyme-linked immunosorbent assay (ELISA) kits arenow commercially available for a wide range of environ-mentally important compounds such as pesticides, polya-romatic hydrocarbons (PAH), halogenated aromaticcompounds, and explosives (Meulenberg et al., 1995;Mouvet et al., 1995; Ohmicron, 1995). While these tests

0956-5663/98/$19.00 1998 Elsevier Science S.A. All rights reserved.PII: S0956-5663 (97)00131-0

are promising for use in the laboratory, only few aresuitable for application in the field, and disposableimmunosensors and immunotest strips might constitutean interesting solution to this problem. Considerablework has been done on the development of immunoteststrips for clinical diagnosis and several systems arealready commercially available. In most of them, animmunochromatographic step is applied, and reporterenzymes, liposomes, fluorescent labels, or gold and latexparticles are used as signal-generating and -amplifyingsystems. These commercial systems mostly providequalitative results (yes/no answers) but some of themallow for a semiquantitative detection based on visualcomparison to a reference panel or by a CCD camera.

For the analysis of xenobiotics in the environment, anumber of disposable immunostrips have been proposed.Thus, dip stick assays (Giersch, 1993; Wittman et al.,1996; Rubtsova, 1997; Schneider et al., 1995) and animmunomigration test strip with optical readout (Reeveset al., 1995b) were described for quantitative or semi-quantitative measurements. Detection limits were usu-ally very low: thus Reeveset al. determined 1mg l21 ofalachlor with a system based on liposomes, whereas the

520 A.J. Baumner, R.D. Schmid/Biosensors & Bioelectronics 13 (1998) 519–529

dip-stick assays of Giersch, Wittmannet al. and Rubt-sovaet al. permitted detection of 0·5mg l21 of atrazineor 0·06mg l21 of 2,4-dichlorophenoxyacetic acid (2,4-D). Major drawbacks of the latter systems, however, arethe rather long time required for one test (up to 20 min)and a tedious assay procedure which requires at leastfour steps of consecutive dipping and rinsing. In com-parison, the liposomal immunomigration system is moreadvantageous as it requires a shorter assay time of 8 minand just two steps.

A disposable amperometric immunosensor for 2,4-Dwas described by Kala´b and Skla´dal (1995), who couldmeasure concentrations in the range of 0·1mg l21 butrequired an assay time of 60 min per test due to a longpreincubation step.

Compared to optical detection systems, amperometricsensors have several advantages. They are very sensitiveand usually exhibit a wide linear range. The transducersare easily prepared and quite inexpensive; portabledevices are also available. We thus set out to combinethe advantages of optical immunomigration sensors withthe sensitivity of amperometric measurements andpresent in this publication the development of a dispos-able amperometric immunomigration sensor, based on aliposomal enhancement system (Fig. 1). We have studiedits applicability for measurements of real samples anddetermined the atrazine contents of soil extracts.

Fig. 1. Format of the amperometric immunosensor using a screen printed electrode system with a migration format and liposome amplification sys-tem.

2. Materials and methods

2.1. Materials

All lipids were purchased from Lipoid (Ludwigshafen,FRG), organic solvents and pesticides were obtainedfrom Riedel de Hae¨n (Seelze, FRG). 3,39,5,59 Tetrame-thylbenzidene (TMB), Dichlorophenoindophenol(DCPIP), Potassium ferrocyanide, 7,7,8,8-Tetracyano-quino-methane (TCNQ) and rabbit anti mouse antibodieswere from Sigma (Deisenhofen, FRG). Horseradish per-oxidase (for EIA) was from Boehringer Mannheim(Mannheim, FRG). Polyvinyl chloride substrate (glasklar500mm) was obtained from SKK (Denzlingen, FRG).Silver (Electrodag 477SSRFU), silver/silver chloride(AV 458) and graphite based P.T.F. (Electrodag 423SS) inks were purchased from Acheson (Scheemda,Netherlands), the insulating ink Matt Vinyl (MV 027)from Apollo Colours Limited (London, GB) and the var-nish from Marabou (Tamm, FRG). Ashless filter paper589/1 was bought from Schleicher and Schu¨ll (Dassel,FRG). Microtiter plates (F96 Maxisorp) were from NuncCompany. Atrazine and terbutylazine specific mono-clonal antibodies K4E7 and P6A7, respectively, werekindly provided from Dr. Th. Giersch, Technical Univer-sity of Munich, Freising, FRG.

521A.J. Baumner, R.D. Schmid/Biosensors & Bioelectronics 13 (1998) 519–529

2.2. Preparation of transducers

The thick-film transducers were prepared as describedbefore (Baumner et al., 1996). The layout was optimizedby varying the working electrode material and size, theinsulation layer and the support material. A 2-electrodesystem was compared to a 3-electrode system. As aresult of these studies, we chose an electrode format asshown in Fig. 2. Electrodes of this type were printed asfollows: electrical contacts between the working elec-trode (WE) and the counter electrode (CE) were pro-vided by silver tracks. The WE and CE pads (2× 8 mm)were positioned at one end of the silver tracks ensuringcontact by a 1 mm overlapping region. Two insulationlayers, a dye and a varnish, with openings allowing bothfor external electrical contact to one end of the strip andfor access of analyte to the WE and CE at the other,were printed over the whole strip. Between each printingstep the layers were dried for 30 min at 80°C. Finally,the transducers were stored at 4°C.

2.3. Preparation and characterization of liposomes

Liposomes were prepared following the previouslypublished protocol (Baümner et al., 1996). They weretagged with an atrazine hapten based on a C6-alkylspacer and contained either one of two markers: sulfo-rhodamine B for photometric detection and ascorbic acidfor electrochemical detection. After preparation andprior to dialysis the liposome solution was extruded ninetimes at 70°C through polycarbonate filters with 200 nmpore size in order to homogenize the vesicle population.

Liposomes were characterized by their size, con-ditions for lysis and storage stability. For size determi-nation, the vesicles were negatively stained for 2 minwith 10% uranyl acetate solution and investigated withelectron microscopy (TEM, Zeiss EM 9). Additionally,the size distribution of the vesicle population was studiedwith light scattering (Zeta Sizer 3, Malvern, Herrenberg,Germany) using a 5 mW He–Ne-laser (l= 632·8 nm).

Fig. 2. Layout of optimized thick film electrodes used as transducerin the immunosensor: (1) PVC; (2) silver conducting paths; (3) insulat-ing layer; (4) graphite working electrode; (5) Ag/AgCl reference elec-trode.

Lysis was monitored photometrically (Biochrom 4060,Pharmacia, Upsalla, Sweden) using liposomes entrap-ping sulforhodamine B. Due to a hypsochromic effect,the absorption spectrum of the free and the entrappeddye were different (Reeves and Durst, 1995). Lysis ofthe vesicle solution was investigated in the presence ofvarious concentrations of Triton X-100, Brij 35 andSDS, and the addition of 0·01% Triton X-100 waschosen for the experiments with the immunomigrationsensor. Storage stability was determined electrochemi-cally via oxidation of leaked ascorbic acid in 20 mM Trisbuffer solution pH 7·2.

2.4. Antibody immobilization and liposome binding

Antibodies (1mg ml21) were immobilized via adsorp-tion on PVC sheets. They were dissolved in carbonatebuffer pH 9·2 and incubated with the PVC for 12 h at4°C. Residual binding sites on the solid support wereblocked with PVP (polyvinyl pyrrolidone) or BSA. Theimmobilization of antibodies was proven by incubationwith horseradish peroxidase coupled to atrazine hapten(‘enzyme tracer’). Bound enzyme and thus immobilizedantibodies was detected visually by the formation of acoloured product from TMB in the presence of H2O2. Asimilar approach was used to determine the time requiredfor liposome binding. Liposomes (1:1000 dilution) andenzyme tracer (1:10000 dilution) were incubated for 1,2 or 5 min with the antibodies. Subsequently, the PVCsheets were washed and bound enzyme was detected. Adecrease in colour intensity formed on the PVC sheetswhen compared to control experiments proved liposomebinding to the antibodies. In control experiments all anti-bodies were bound by enzyme as they were carried outwithout vesicles or with untagged vesicles.

2.5. Electrochemical measurements

The transducer strips were used in two measurementtechniques: dipped in large buffer volumes or withmigration of the buffer solution. Additionally, they werecharacterized by cyclic voltammetry.

2.5.1. Amperometric detection in large volumesExperiments were carried out in beakers with 5–10 ml

volume. The solution was stirred with an overheadstirrer. Ascorbic acid was measured at a potential of+300 mV (641-VA-Detector, Metrohm, Filderstadt,FRG) against a printed Ag/AgCl reference electrode.Experiments were performed in 20 mM Tris buffer sol-ution pH 7·2 containing 0·01% Triton X-100. The cur-rent signal output was recorded on an x,t-printer(Linseis, Munchen, FRG). The storage stability experi-ments were done without addition of detergent.


2.5.2. Cyclic voltammetryExperiments were performed in beakers with 5 ml

volume. Cyclic voltammograms were carried out usinga signal generator and a potentiostat (Voltage ScannerWenking MVS 87, MCP 94 Potentiostat, Bank, Claus-thal-Zellerfeld, FRG). They were monitored with anA/D-signal converter (IBS Schaber, Bad Krozingen,FRG) and a PC.

2.5.3. Measurements with the sensor migration formatIn Fig. 3 different formats for the migration zone of

the immunosensor are shown. Formats A and B form themigration zone by a capillary gap, formats C and D byusing a membrane. In format C the electrodes aredirectly printed on a filter paper, in format D the elec-

Fig. 3. Investigated formats of the amperometric immunomigration sensor. (1) Electrodes; (2) PVC sheets; (3) absorbing filter paper; (4) applicationof sample; (5) wicking filter paper; (6) immobilized antibodies; (7) detergent; (8) buffer containing liposomes and standard or sample; (9) buffercontaining antibodies, liposomes and pesticide standard or sample; (10) capillary gap; (11) migration zone of the test strip; (12) insulation area;(13) nitrocellulose membrane; (14) plug for secure connection to potentiostat; arrows indicate the flow direction.

trodes are printed on PVC and a nitrocellulose mem-brane is mounted on top. For the analysis of ascorbicacid and liposomes the filter paper or nitrocellulose weredipped into the solution and the solution allowed tomigrate. In formats A and B the solution (120ml persample) is applied manually or is drawn in by a filterpaper and guided into a capillary gap (10–120mm),respectively. By means of capillary forces the solutionis transported through the gap, passing the electrodes andwicked away by a second filter paper. This set up allowsa constant flow of solutions (. 120ml) for about 30 min.A constant potential of+ 300 mV vs printed Ag/AgClis applied and the current recorded as described abovein the amperometric experiments in large volume.

The sensor was equilibrated dipping the wicking filter


Table 1Summary of liposome preparation and characterization

steps methods and purpose data

preparation reversed phase evaporation and extrusion 2 h(200 nm)

purification dialysis 24 hsize determination electronmicroscopy

dynamic light scattering before extrusion: 818·8± 194·5 nmafter extrusion: 206·7± 21·7 nm

lysis photometrical detection 0·01% Triton X-100storage stability electrochemical detection . 3 months, 4°Centrapment of:sulforhodamine B photometrical detectionascorbic acid electrochemical detectionbuffer control experiments

tagging of membrane surface atrazine hapten with C6 spacer binding to atrazine antibodiespermethric acid binding to allethrin antibodies

determination of time required for binding dip stick assay with competition between in 2–5 min 40–50% of antibodies areenzyme tracer and liposomes bound to liposomes

paper into buffer solution or tap water. For the followingmeasurements the buffer was replaced by standard sol-utions or samples. Standard curves for ascorbic acid andliposomes were carried out applying standard solutionsto the migration format. Pesticides were detected bymixing a pesticide standard solution with antibodies andliposomes and incubating the mixture between 4 and12 min prior to measurements with the sensor. Up tofour different solutions were applied sequentially to thesensor within one measurement. Optimization experi-ments, standard curves and measurements of realsamples were performed with this sequential detectionmode.

2.6. Pesticide standards and real samples

Stock solutions of pesticides were prepared as1 mg ml21 ethanol. Standard solutions were diluted inTris buffer 20 mM pH 7·2 or in tap water.

Soil samples and data for the HPLC analysis werekindly provided from Dr Andrea Dankwardt, TUMunchen-Weihenstephan, FRG (Dankwardt et al.,1995). Spiking of the sample was carried out afterextraction prior to the detection with the immunosensorand the ELISA.

2.7. ELISA analysis of real samples

Rabbit anti mouse antibodies (1mg l21) in 50 mMsodium carbonate buffer (pH 9·2) were incubated in amicrotiter plate (300ml per well) for 12 h at 4°C. Theplates were washed three times with a washing buffer(8 mM phosphate buffer containing 15 mM NaCl and0·5% Tween 20). Subsequently, 300ml per well of atraz-ine antibodies (0·1mg l 2 1) in 50 mM sodium carbonatebuffer (pH 9·2) were incubated for 2 h at room tempera-

ture. The plates were washed again as described pre-viously. For the assay 200ml of standard or sample and50 ml of enzyme tracer solution (1:10000 diluted in80 mM phosphate buffer pH 7·2 containing 150 mMNaCl) were pipetted into each well. After 1 h the plateswere washed again and 200ml of substrate solution(400ml of 6% TMB in DMSO, 25 ml 0·1 M acetatebuffer, pH 5·5, 100ml 1% H2O2) were pipetted into eachwell. The reaction was stopped with 50ml of 2N H2SO4.Absorbance was detected at 450 nm with a microtiterplate reader (Titertek Plus MS 212 ELISA Reader, ICN).

3. Results and discussion

3.1. Characterization of liposomes

Results are summarized in Table 1. For the prep-aration of vesicles of uniform size, a combination ofreversed phase evaporation, extrusion through polycar-bonate filters and subsequent dialysis proved best andresulted in a liposome population with an average diam-eter of about 200 nm (Fig. 4). As described in a previouspublication, vesicles can be conveniently tagged with aphospholipid–hapten conjugate and bind quantitativelyto the immobilized antibody within 2–5 min if an optim-ized spacer length is used (Baümner et al., 1996). Thestorage stability of the tagged liposomes at 4°C was bet-ter than 3 months, in agreement with the findings ofReeves et al., 1995b. An analogous procedure was suc-cessfully investigated with liposomes tagged with per-methric acid–phospholipid conjugate and allethrin-spe-cific antibodies (data not presented).


Fig. 4. Electronmicrocopy of liposomes entrapping ascorbic acidafter extrusion. Magnification (a) 29 000 times (1:10 dilution) and (b)65 000 times (1:100 dilution).

3.2. Electrochemical measurements

Results are summarized in Table 2. Additionally a 3-electrode and a 2-electrode system were compared. Con-cerning reproducibility of signals, equilibration time andstability of the baseline, the 3-electrode system showedno advantage over the 2-electrode system. Due to itssimpler preparation, the 2-electrode system was chosenas the transducer.

Transducer–analyte combinations were characterizedby cyclic voltammetry. Among DCPIP, potassium ferro-cyanide and ascorbic acid, ascorbic acid proved the bestmarker in terms of reproducibility and oxidation at lowpotential. It was irreversibly oxidized at an oxidationpotential of 300 mV vs Ag/AgCl (printed). Contrary toDCPIP, which is known to polymerize upon electro-chemical oxidation, ascorbic acid did not irreversiblymodify the electrode surface.

Standard curves for the oxidation of free and lipo-some-trapped ascorbic acid in our system have alreadybeen reported (Baümner et al., 1996).

All detergents (Tween 20, Brij 35 and SDS) tested forthe lysis of vesicles had a negative effect on the electro-chemical measurements by reducing the signal height.Triton X-100 was the most efficient surfactant even atvery low concentration (0·01%), thus had the least nega-tive effect and was used in our further experiments. Weassume that the electroinactive detergent adsorbs to thehydrophobic surface of the electrode hindering elec-troactive molecules in their approach, as described forelectrochemical processes (Koryta et al., 1993). In ouroptimized system, however, we could reproduciblydetect very low concentrations of liposomes.

3.3. The immunomigration format

In Table 3 investigations of the different migrationformats are summarized. Set ups C and D allowed a verysimple migration of the buffer solutions. However, thedetection of ascorbic acid and liposomes was found tobe unreproducible. Therefore, further experiments werecarried out using formats A and B in which the solutionis flowing lateral to the electrodes through a capillarygap. As set up A needs, in contrast to set up B, a manualapplication of the sample, all experiments were finallyperformed using format B.

Standard curves with the immunomigration formatwere obtained separately for ascorbic acid and liposomes(Fig. 5), and the detection limit for ascorbic acid was0·5mM. The standards were applied via a wicking filterpaper dipping into the solution (see Fig. 3). Solvent frontmovement over time was constant without externalmanipulations (visual detection). As the electrochemicalsignal for ascorbic acid entrapped in liposomes was con-stant for solutions with the same concentration measuredover at least 30 min, it can be concluded that the amountof detergent immobilized in the capillary gap was suf-ficient for lysis during the entire course of measurement.A series of four measurements could be carried out usingjust one sensor. Thus it was possible to measure eithera standard curve of four different concentrations or asample within a standard curve of three concentrations.Standard deviations were below 30%. The main featuresof the immunomigration format are summarized inTable 4.


Table 2Features of the electrochemical detections using thick film electrodes. Measurements at+ 300 mV vs printed Ag/AgCl

measurements results

equilibration time in buffer 3–6 minsignal response 30 scyclic voltammograms measuring. 5 cycles with no difference in the cyclic

voltammogramdetection of ascorbic acid irreversible oxidation

detection at+ 300 mV vs Ag/AgClno improvement by modification of the working electrode with

TCNQstandard curvesascorbic acid 10–150mM and 0·025–0·5mMliposomes 1:100–1:20 000

reproducibilitycomparison of five electrodes maximal standard deviation: 18%multi detection with one electrode maximal standard deviation: 12%detection of liposomes maximal standard deviation: 14%

storage stability of the transducer . 6 months, 4°C, in the darkeffect of detergent (0·01% Triton X-100) decrease of basis signal

50% decrease in ascorbic acid signalresulting from this low current

Table 3Comparison of the investigated formats

formats maximal standard sensitivity disadvantagesdeviation

(a) Capillary with manual addition of sample 10% (1mM aa) 0·1 mM aa manual work

(b) Capillary with sample application via filter paper 11% (0·5 mM aa) 0·1mM aa slow flow30% (1:8000 liposomes) 1:20 000 liposomes

(c) Electrodes printed on paper/membranes 25mM aa low sensitivity preparation

(d) Nitrocellulose-PVC format , 10% 2mM aa lower sensitivity1:1000 liposomes lower signals (40%)

aa = Ascorbic acid.

3.4. Triazine measurements with the immunomigrationsensor

3.4.1. Standard samplesThe sensor was first used to determine standard con-

centrations of atrazine and terbutylazine in buffer. Theratio of antibody and tagged liposomes in the preincu-bation mixture was optimized for the detection of lowpesticide concentrations (Fig. 6). Sensitivity was highestwith low amounts of both antibodies and liposomes. Pre-incubation (4–12 min) of the sample with antibodiesprior to the addition of liposomes had only a small effecton the sensitivity. As indicated by experiments E and Fin Fig. 7 0·1mg l21 atrazine could be detected. However,standard deviations for the measurements of one concen-tration with different sensors were 13–20%. Substitutionof buffer by tap water yielded similar results (Figs 7and 8). The standard curves shown are carried out under

different conditions (e.g. antibody concentration) andthus vary in the sensitivity.

The time required for performing one full measure-ment cycle (standard curve plus sample) was 20–30 min.If no standard curve were needed for each analysis theassay time could even be reduced to 8 min which is fav-orable.

3.4.2. Soil extractsIn order to investigate the applicability of the sensor

for the detection of real samples atrazine concentrationsin soil extracts were measured in the same format asstandard samples. Experiments were carried out using atleast three sensors for one sample, and the pesticide con-tent was determined separately for each sensor from theconcomitantly recorded standard curve. Compared withELISA and HPLC analyses (Table 5) the data obtainedwith the immunosensor correlated good (± 25%) with


Fig. 5. Standard curves for ascorbic acid and liposomes in 20 mM Tris buffer pH 7·2 applying the migration format (n = 3). Insert is a typicalrecorded signal that shows the oxidation of different ascorbic acid concentrations.

Table 4Features of the measurements with the migration format of the transducer

measurement result

equilibration time in buffer 4–6 minsignal response time 1–3 minsequential detection mode measurement conditions are stable up to 30 mindetection limit for ascorbic acid 0·5mMmaximal standard deviationsascorbic acid 11%liposomes 30%

Fig. 6. Optimization of antibody and liposome concentrations. 6 min incubation time, measurements in 20 mM Tris buffer pH 7·2 at+ 300 mVvs Ag/AgCl (A–F indicate the numbers of experiments with different optimization conditions). The current signals of the highest concentrationwas set to 100% in each experiment separately. L= liposomes; A= antibodies.


Fig. 7. Standard curves for atrazine in 20 mM Tris buffer pH 7·2, 6 min incubation time,+ 300 mV vs Ag/AgCl. 1:2000 liposome dilution and0·1mg l21 atrazine antibody (K4E7).

Fig. 8. Standard curves for terbuthylazine in tap water, 10 min incubation time,+ 300 mV vs Ag/AgCl. 1:12500 liposome dilution and0·08mg l21 terbuthylazine antibody (P6A7).

Table 5Detection of atrazine in soil extracts. Comparison of data of the immunosensor (n = 3), ELISA (n = 6) and HPLC

sample immunosensor (mg l21) ELISA (mg l21) HPLC (mg l21)

no. 1 (1:50) 0·50± 0·19 0·54± 0·028 0·532no. 2 (1:100) 0·37 0·28± 0·014 0·266

(1:50) 0·88no. 3 (1:100)

+ 0 mg l 2 1 atrazine 0·27± 0·04 0·212± 0·026 0·11+ 0·5mg l 2 1 atrazine 0·81± 0·18+ 0·3mg l 2 1 atrazine 0·511± 0·005


the ELISA. However, correlation with HPLC analysiswas not sufficient and standard deviations of up to 39%were observed.

Various dilution and spiking experiments were carriedout in order to account for matrix effects. As the signaldecreased for 58% at 50% dilution of the sample (sample2), and in spiked samples 108% of atrazine was reco-vered, no major matrix effects could be stipulated. Base-line stability was influenced by only one sample, render-ing measurement impossible.

These experiments proved that even though measure-ments in real samples were possible, the sensor stillneeds improvement to be used for real sample analysis.

4. Summary and conclusion

We here present the development of an amperometricimmunomigration sensor for the rapid and sensitivedetection of pesticides in real samples. The main featuresof our prototype are summarized in Table 6, and advan-tages and disadvantages of the format are listed. If onecompares the characteristics of our homogeneous immu-nomigration format with immuno test strips and ampero-metric immunosensors proposed for pesticide detection(Table 7), the format described here exhibits advantagesas to the time required for measurement. However, itstill needs improvement in order to serve as a precise,robust system for field tests.

Table 6Features, advantages, disadvantages and future aspects of the amperometric immunosensor

featuressingle use system with immunomigration format2 step process of measurements: equilibration and measurementequilibration time: 3–6 minsingle measurement: 8 minone sequence of measurements: 20–30 minmaximal standard deviation for pesticide standard solutions: 20%maximal standard deviation for pesticide in a real sample: 39%detection of concentrations below 1mg l21

advantagesflexible systemsimple adaptation to new analytesmeasurements in the low ppb-rangestorage of transducer is independent from biological compounds

disadvantageslow reproducibilitycausing long duration of measurementstorage of diluted antibody and liposome solution for the application as field device

future work and aspectsoptimization of the capillary systeminvestigation of different lysis systemsentrapment of higher concentrations of ascorbic acidwith higher reproducibility of measurementsapplication of more than one sample within one standard curve using the sensor for multi analyte detection

Table 7Comparison of the amperometric immunosensor with immuno teststrips and amperometric immunosensors for pesticide detection inthe literature

amperometric immunosensor immuno test strips andamperometric

immunosensors in theliterature

detection of pesticides in the lower ppb similar to *†‡

rangeprototype for field tests similar to †

system * is alreadyapplied

immuno reagents have to be added to the similar to *†‡

sample (an additional pipetting step isrequired)short incubation time similar to *

shorter than †‡

migration format less accurate than *

* Durst and Roberts, 1996†Skladal and Kala´b, 1995‡Rubtsova, 1997

Acknowledgements

We thank Thomas Giersch und Bertold Hock, Techni-sche Universita¨t Munchen-Weihenstephan, for providingus with the antibodies used in this study and AlfredBlume, Kaiserslautern, for the possibility to carry outthe light scattering measurements on liposomes. Electron


microscopy could be carried out by support of WilhelmOppermann, University of Stuttgart, and is gratefullyacknowledged. This project was carried out under con-tract EV5V-CT94-0358 with the EU Environment andClimate Programme.

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Development of a new immunosensor for pesticide detection: a disposable system with liposome-enhancement and amperometric detection