Immunoanalytical techniques for analyzing pesticides in the environment

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Immunoanalytical techniquesfor analyzing pesticidesin the environmentC. Raman Suri, Robin Boro, Yogesh Nangia, Sonu Gandhi, Priyanka Sharma,

Nishima Wangoo, Kumar Rajesh, G.S. Shekhawat

Affinity-based immunosensing techniques for pesticide analysis have attracted intense interest and have found many applications

in environmental monitoring. This article reflects recent advances in immunochemical techniques for environmental analysis and

monitoring of pesticides.

We highlight development of bioreceptors (e.g., antibodies) against low-molecular-weight organic pollutants, and the use of

different transduction elements for detecting physico-chemical, optical or mass changes produced by biological interactions. We

give details of their applications in developing real-time measurement devices and systems.

We also describe the fundamentals of transducer technologies used in immunosensor development. We cite examples of the

immunochemical analysis of pesticides using immunochromatography-based, rapid screening, optical detection and cantilever-

based, micro-mechanical immunosensing, currently being developed in our laboratory.

We especially emphasize the use of nanotechnology in developing immunosensors, including their miniaturization.

ª 2008 Elsevier Ltd. All rights reserved.

Keywords: Environmental monitoring; Immunoanalysis; Immunoassay; Immunochemistry; Immunochromatography; Immunosensor;

Miniaturization; Nanotechnology; Pesticide; Transducer

1. Introduction

Pesticides derived from chemicals areessential inputs in increasing agriculturalproduction by preventing crop lossesbefore and after harvesting. Dependingupon the species of pest, these chemicalshave been divided into groups (e.g., her-bicides, insecticides, fungicides, rodenti-cides, avicides, and nematocides).However, their indiscriminate use, apartfrom being an operational hazard, isposing a serious threat to human health[1]. Pesticides are intended to be biologi-cally active. These organic toxins enteranimal and human bodies directly orindirectly through the food chain ordrinking water. Pesticides, dependingupon their water solubility can eitherremain in the soil and are broken downby action of microorganisms, or washedoff, eventually into surface waters andgroundwater [2]. As they are degraded,these chemicals slowly leave behind resi-dues in food and water sources, which

can become more concentrated as theymove up the food chain.

Because of their severe toxicity, even attrace levels, it is essential to monitor thelevels of pesticides in the environment,foodstuffs and soil. A European UnionDirective (1980) set the maximum per-missible limit for any individual pesticidein groundwater as around 0.1 ng/ml. Thislow level is currently monitored by variousanalytical techniques [i.e. gas chroma-tography (GC), high-pressure liquidchromatography (HPLC), capillary elec-trophoresis (CE) and mass spectrometry(MS)] [3–7].

However, these conventional methodsfor pesticide monitoring have disadvan-tages and suffer from drawbacks. Mostare complex and time consuming, andrequire costly, bulky instrumentation.Sample preparation for these techniquesis tedious and prolonged, and requiresskilled personnel, so they are unsuitablefor field studies and in situ monitoring ofsamples.

C. Raman Suri*, Robin Boro,

Yogesh Nangia, Sonu Gandhi,

Priyanka Sharma,

Nishima Wangoo,

Kumar Rajesh

Institute of Microbial

Technology (CSIR), Sector

39-A, Chandigarh 160036,

India

G.S. Shekhawat

Institute for Nanotechnology,

Northwestern University,

Evanston, IL 60208, USA

*Corresponding author.

Tel.: +91 172 2636680;

Fax: +91 172 2690632;

E-mail: [email protected]

Trends in Analytical Chemistry, Vol. 28, No. 1, 2009 Trends

0165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.09.017 290165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.09.017 29


There has therefore been a great demand fordeveloping inexpensive, reliable assay techniques foreffective field monitoring of these toxic molecules.Modern techniques to eliminate some of these diffi-culties include immunochemical techniques, whichare based on the specificity of the antibody–antigen(Ab–Ag) reaction. Immunosensing is becomingattractive because Abs can be produced against pes-ticide molecules with low molecular mass [8–10].Most immunosensors can obtain quantitative resultswith similar or greater sensitivity, accuracy and pre-cision than other analytical methods because of theavailability of high-quality Abs against target ana-lytes. Immunosensor technology is therefore importantfor analysis because it complements existing analyticalmethods and it provides a low-cost confirmatoryplatform for many compounds, including pharmaceu-ticals and pesticides.

2. Immunosensors for pesticide analysis

Immunosensors are based on the binding interactionsbetween immobilized biomolecules (Ab or Ag) on thetransducer surface with the analyte of interest (Ag orAb), resulting in a detectable signal. The sensor systemtakes advantage of the high selectivity provided by themolecular recognition characteristics of an Ab, whichbinds reversibly with a specific Ag. In solution phase, Abmolecules interact specifically and reversibly with an Agto form an immune complex (Ab–Ag) according to thefollowing equilibrium equation:

Abþ Ag$Ka

Kd

Ab� Ag

where Ka and Kd are the rate constants for associationand dissociation, respectively. The equilibrium constant(or the affinity constant) of the reaction is expressed asfollows:

K ¼ Ka=Kd ¼ ½Ab–Ag�=½Ab�½Ag�The equilibrium kinetics of Ab binding with Ag insolution suggests that both association and dissociationare relatively rapid. The direction of equilibrium dependson the overall affinity, which is basically the summationof both attractive and repulsive non-covalent forces. Animmune complex usually shows low Kd values (in therange 10�6–10�12) and also displays higher affinity (i.e.K value typically 104). Immunosensors, which usuallyincorporate Abs immobilized on solid matrix do not showthe same kinetics of reaction established for those reac-tants in solution. Immobilization of biomolecules on asolid surface can alter the properties of the reactants(measured in reaction-rate constants) or the surfacecharacteristics (e.g., surface charge and hydrophobicity)of the support itself.

Until the mid-1980s, immunosensors were mainlyused for clinical diagnostics, mostly for the detection ofmacromolecules [11]. Not much work had been reportedin environmental monitoring using immunosensors.Development of immunosensors for environmental pol-lutants, mainly for pesticide analysis was delayed forseveral reasons. First, getting Abs against pesticidemolecules has been a tedious task, primarily because ofthe low molecular weight of pesticide molecules (usuallyless than 1 kD), as is evident from the fact that not manyAbs against pesticides are available. Second, repeatedmeasurements are less efficient, especially with high-affinity Abs (and unlike enzymes). However, develop-ment of Ab technologies and advancement in thetransducer system have provided further impetus[10,12,13]. Table 1 shows recent developments inimmunoanalysis using transduction devices.

3. Biomolecular aspects of immunosensors

Pesticides, organic compounds of molecular mass, areusually non-immunogenic, so they will not elicit an im-mune response unless coupled with some macromolecules(e.g., proteins). It is therefore necessary to modify these

Table 1. Recent developments in immunosensors for pesticidesdetection

Transduction system (analytes) Limit of detection Ref.

Optical biosensorAtrazine 0.155 ng/ml [48]Isoproturon 0.046 ng/ml [48]Herbicide subclasses 3 nM [49]Diuron and linuron 0.01 and 0.14 ng/ml [50]DDT 15 pg/ml [51]

Surface-plasmon resonanceAtrazine 1 lg/ml [52]2,4-D 0.1 ng/ml [53]

Fluorescence/chemiluminescence2,4-D, linuron and simazine 0.02 ng/ml [54]2,4-D 4 ng/ml [55]

Quartz-crystal microbalance2,4-D 0.5 nM [34]Organophosphorus and carbamate 1 ng/ml [56]

Electrochemical2,4-D 50 ng/ml [35]Simazine 5–175 ng/ml [57]Monocrotophos 0.3 ng/ml [58]

Micromechanical cantileverAtrazine 0.1 ng/ml [15]

Rapid screening (dipstick)Atrazine <1.0 ng/ml [46]Sulfamethazine 50–100 ng/ml [59]

Trends Trends in Analytical Chemistry, Vol. 28, No. 1, 2009

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small substances (haptens) to couple them withmacromolecules (carriers) so as to make a stable car-rier-hapten complex. The carrier-hapten conjugatedeveloped can be used to generate Ab againstpesticides. This section highlights Ab generation andoptimization of assay techniques for the detection ofpesticide molecules.

Abs are specialized proteins capable of recognizing Agswith high specificity. The quality of the Ab employedgreatly influences the specificity and the sensitivity of animmunosensor. Different kinds of Ab have been used invarious immunoassays. Polyclonal Abs (pAbs), conve-niently prepared from sera, comprise a wide variety ofAb molecules of different specificities and affinity [17].Hybridoma technology provides an excellent alternative,whereby monoclonal Abs (mAbs) can be produced inunlimited quantities with constant characteristics. Be-sides, mAbs of desired affinities can also be selected.Recombinant DNA technology has been exploited toengineer recombinant Abs (rAbs), whereby Ab genescan be cloned and expressed in various host systems,thus further improving the sensitivity and the repro-ducibility of immunoassay [14]. The choice betweenpAbs, mAbs or rAbs for development of an immuno-sensor depends upon the various aspects of the assay(e.g., cost of development, relative specificity and sensi-tivity of the Abs, ease of screening and selection of Abs)[15].

3.1. Hapten design and bioconjugationHapten, a derivative of the target molecule, contains anappropriate group for attachment to carrier protein.Direct coupling of haptens with carrier proteins is pos-sible if the target compound contains functional groups(e.g., –NH2, –COOH, –CHO, or –SH). However, it isnecessary to derivatize the pesticide with such func-tionality before conjugation with carrier protein.

Design of haptens for linking with carrier proteins isthe most important aspect of specific Ab generationagainst pesticide molecules. Hapten molecules are syn-thesized so that they:� mimic the structure of the compound; and,� contain a reactive group that can form a covalent

linkage with the carrier proteins.Minimal structure alteration to the hapten ensures

that the Ab recognizes the unmodified hapten in theassay system. The hapten should also preserve to a greatextent the chemical structure and the spatial confor-mation of the target compound. Careful hapten selectionmight be important for generating class-specific orcompound-specific Abs for the immunosensor system.Class specificity of the Abs developed could be achievedby preserving Ag determinants common among relatedcompounds. Similarly, masking these common determi-nants and targeting a distinct molecular entity couldobtain compound specificity. Specific strategies for

hapten design for Ab production have been discussedextensively elsewhere [16].

For the bioconjugation of hapten with carrier-proteinmolecules, the functional group of the hapten governsthe selection of the conjugation method to be employed.However, the stability of the hapten while conjugatingwith the carrier protein is an important factor. Theproblem of hapten stability during conjugation has beenhighlighted by various groups [17,18].

It is also important that the hapten-protein conjugateis designed so as to preserve and enhance the epitopicregions on the hapten for getting an Ab specific against atarget molecule. The site of linkage of hapten and proteinshould usually occur away from any suspected Agdeterminants. Using a spacer arm (bridging groups) be-tween hapten and protein structure also helps in theproduction of hapten-specific Abs. The spacer arm can beenvisioned as a pedestal for the hapten to accommodaterecognition and binding by the large Ab molecule. Ingeneral, it is necessary to increase the length or thehydrophobicity of the spacer arm to help generate Abs ofhigher specificity and good titer. The same haptenemploying a shorter spacer arm may not elicit the Abresponse.

Design of a suitable hapten, and selection of a spacerarm and a suitable carrier protein are therefore critical toensure successful generation of Abs having the desiredspecificity [16].

3.2. Antibody generation and characterizationA general protocol of immunization, for the generationof either pAbs or mAbs, involves injecting the immu-nogen mixed with the adjuvant (first using completeFreund�s adjuvant followed by boosters with Freund�sincomplete adjuvant) at multiple sites. The dose ratevaries with animal type (50–1000 lg generally coversall needs). Sera are then collected for the isolation ofpAbs. For mAb generation, spleen cells of an immunizedanimal are fused with an appropriate myeloma cell line,and extensive screening is carried out to select a clonesecreting the mAb desired. In our earlier work [10], weextensively described production and characterization ofAbs against low molecular-weight pesticides (e.g., atra-zine and 2,4-dichlorophenoxyacetic acid) with majoremphasis on immunization protocols.

Cross-reactivity of an Ab to unexpected metabolites isa major problem in any immunodetection technique forpesticide applications. Depending upon the conjugateused for immunization and the class of chemicals underinvestigation, cross-reactivity of the Abs with metabo-lites similar to the analyte of interest are frequentlyobserved [18]. It is therefore useful to check whichcompounds cross-react to what extent with the Ab usedby comparing the standard curves of the analyte underinvestigation with similar haptens, using analyteconcentrations at 50% of the inhibition curve as the


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reference. Cross-reactivity with a particular analyte isnot the same over the whole range of standard curve.Higher cross-reactivity is usually observed at lowconcentrations of the cross-reacting analyte [19].Cross-reactivity, in general, could be minimized by usingcompound-specific (monospecific) pAbs or mAbs, be-cause there are then fewer binding epitope-recognizinganalogues and metabolites other than the target com-pound.

4. Transducer aspects of immunosensors

Immunosensors are designed so that the formation of theimmune complex on the transducer surface is directlydetermined by measuring the physical changes (electri-cal or optical) induced by the binding reaction. Either theAb or the Ag is immobilized on the transducer carriersurface to form a sensing device and allowed to reactwith the complementary Ag or Ab to form an immunecomplex. This formation alters the physical properties ofthe surface. Depending upon the transducer technologyemployed, immunosensors are broadly classified intooptical, piezoelectric (PZ), electrochemical and micro-mechanical (Fig. 1).

4.1. Optical immunosensorsOptical immunosensors are based on the measurementof a change in the optical characteristics of the trans-ducer surface by the formation of Ab–Ag complexes [20].

Optical sensors can be further classified as direct orindirect.

Signal generation in direct immunosensors dependson the formation of Ab–Ag complex on the transducersurface which in turn changes the optical properties ofthe surface.

An indirect optical immunosensor may be configuredto include labels (e.g., fluorophores or chromophores) todetect binding. Indirect immunosensing methods pro-duce better signal-to-noise discrimination, and aretherefore quite sensitive for most applications.

Widely explored optical transducers include evanescentwave (EW), surface-plasmon resonance (SPR), fluores-cence and chemilumenescence devices.

4.1.1. Evanescent wave. An optical immunosensorbased on EW combines the potential of molecular rec-ognition by the Ab with the good signal-transductioncapability of a waveguide or fiber-optic probes. Signalgeneration can be obtained directly or enhanced bycoupling a reactant with a label.

When light is propagated through a waveguide (g1)by multiple total internal reflections in the x direction,an electromagnetic (EM) wave (called an EW) is gener-ated in the optically less dense external medium (g2)with g1 > g2. For visible light, the evanescent fieldpenetrates 50–500 nm in the y direction, so only sur-face-bound molecules interact with the incident light.This allows the absorption of energy by the moleculeslocated in the evanescent field, leading to attenuation ofthe light reflected in the waveguide. For immunosensingapplications, planar-waveguide systems exploit the sameoptical phenomenon as fiber optics with some minorvariations [20]. The waveguide can be mass produced at

Membrane potential changes

Electrode potential changes

Piezoelectric properties change

Micromechanical approach

Antibody

Solid Matrix Optical properties change

Figure 1. Principle of non-labeled immunosensor. Antigen binding to antibody-coated surface changes the physical properties of the transducerthat can be converted into detectable electrical signal.




very low cost by injection molding. A suitable detectormonitors the formation of Ab–Ag complex. A majoradvantage of this system is minimal interference fromthe bulk media.

There are two possible methods for measuring EW:attenuated total reflection (ATR) and total internalreflection fluorescence (TIRF).

In ATR mode, the energy absorbed by the surface-bound molecules is monitored as an attenuation of theinternally reflected light. Since the absorbed light isusually a small percentage of the total incident light, thesensitivity of the assay is very poor.

In TIRF mode, the evanescent photons captured bysurface-bound molecules are re-emitted at a longerwavelength as fluorescence. Fig. 2A shows the basicprinciple of an EW biosensor where light is directedinto the waveguide and generates an EM field,allowing excitation of fluorophores used as labels withAbs or Ags.

EW biosensors based on measurement of fluorescenceexcitation or emission via the evanescent field of thewaveguide have been developed for water analysis, andhave allowed real-time monitoring of labeled Abs. Thelimits of detection (LODs) of the assay for 2,4-D andsimazine were 0.035 ng/ml and 0.026 ng/ml, respec-tively [21].

4.1.2. Surface-plasmon resonance. In a different evan-escent format, using SPR, the excitation is provided bythe evanescent field, which is produced at the site of totalinternal reflection from a glass-metal interface. In thisformat, laser light first enters the optical substrate atcertain angle and then strikes the glass-metal boundary

by first passing through the layer coated with biomole-cules on the metal film before contacting the glass. Theintensity of the reflected light is a function of therefractive index at the glass-metal interface. Any changein the refractive index, caused by the formation of theimmune complex on the metal surface, signifies immu-nological recognition (Fig. 2B).

The SPR phenomenon was first utilized in an immu-nosensing application by Liedberg and his team [22].Immunosensor systems based on SPR detection havebeen developed for various pesticides in aqueous solu-tions [23,24]. An inhibition immunoassay using theBIAcore system achieved an LOD of 0.005 ppb for atra-zine in water with the analysis time of approximately15 min.

A competitive immunoassay based on SPR for detec-tion of pesticide 2,4-D was reported recently [25]. Theassay was based on regeneration of the chip surface bythe reversible interaction between monosaccharide (D-glucose) and lectin (concanavalin A). The interactionbetween anti-2,4-D Ab and the surface-bound conca-navalin A-2,4-D conjugate was monitored by SPR andthe response was used to quantify 2,4-D. The dynamicrange of the curve was 3–100 ng/ml.

SPR-based immunoassays for small molecules havebeen reviewed by Mullett and his colleagues [26].

4.1.3. Fluorescence polarization. On absorbing light ofparticular wavelength, some substances are excited to ahigher energy level and then emit light of a greaterwavelength. This phenomenon is known as fluorescence.If these substances are excited by polarized light, themolecules change their orientation and the emitted light

QQ

n1n2

EW

EW

n2n1

Figure 2. Evanescent wave (EW)-based optical immunosensor. (A) Optical waveguide, and (B) surface-plasmon resonance (SPR) configuration.The monochromatic laser light is directed into the prism/waveguide, which generates an electromagnetic (EM) field allowing excitation offluorophores used as tracers in optical waveguide and resonance shift in SPR, respectively.




is also depolarized. Fluorescence polarization (FP) isdetermined by exciting these compounds with verticallypolarized light and measuring the intensity of both thevertically (Iv) and horizontally (Ih) polarized componentsof the fluorescence light emitted. The polarization (P)value is determined as the ratio of the difference and thesum of Iv and Ih components:

P ¼ ðIv� IhÞ=ðIvþ IhÞTheoretically, the value of P is 0 or 0.5 for full depolar-ization or fixed molecules without movement, respec-tively.

The use of FP-based immunoassay (FPIA) for thedetection of pesticides has been described extensively[27]. FPIA measures the increase in FP when a tracer(fluorophore-labeled Ag) is bound to a specific Ab; the FPsignal is decreased when free analyte competes with thetracer for binding. FPIA for small pesticide molecules is acompetitive method based on detection of the differenceof FP between a small fluorescent-labeled Ag and itsimmune complex with a specific Ab. When there is noanalyte present in the sample, the tracer will be com-pletely bound to the specific Ab and the FP value willincrease. However, if the analyte concentration insample is greater than the concentration of the tracer,the Ab-binding sites will be occupied by the analyte sothere will be free tracer. This will decrease the FP valueof the reaction mixture.

In a different optical approach, a simple, sensitivemethod based on an optical immunosensor was devel-oped to determine different types of organic pollutants inwater samples [28]. The system, called river analyzer(RIANA), is based on a rapid, solid-phase, indirect,inhibition immunoassay that takes place at an optical-transducer chip chemically modified with an analytederivative. Fluorescence produced by labeled Abs boundto the transducer is detected by photodiodes and can becorrelated with analyte concentration. The sensor sur-face can be regenerated, thus, allowing the performanceof several measurements (around 300) with the sametransducer. Each test cycle, including one regenerationstep, is accomplished in 15 min. The LOD achieved in thedirect determination of bisphenol A in water with thissystem was 0.014 lg/l.

Similarly, a parallel affinity-sensor array (PASA)system, based on chemiluminescence labels (peroxidase/luminol) and charge-coupled device (CCD) detection wasdeveloped for monitoring pesticide contaminants in water[29]. An LOD of this system of 20 ng/l was achieved forterbutylazine. The assay could be regenerated more than100 times.

4.2. Piezoelectric crystal immunosensorThe PZ crystal immunosensor works on the principle ofmeasuring a small change in resonant frequency of an

Metal electrode(s)

z Quartz wafer

x x

Receptor

Crystal Substrate

Gold Electrode

Antigen

Blocking Agent

Figure 3. (A) Piezoelectric crystal construction, (B) crystal vibrating in thickness shear mode, (C) piezoelectric crystal bound with receptor mol-ecules for antigen detection, and (D) quartz-crystal set-up that our group designed and developed for pesticide analysis. The quartz-crystal micro-balance (QCM) unit is coupled to a flow-through system for on-line monitoring applications.




oscillating PZ crystal due to a change in mass on thesensor surface, as a result of the formation of the Ab–Agcomplex. The resonant frequency of a PZ crystal is massdependent, giving LODs for analyte concentration at thesub-ng level [30].

Fig. 3 (A–C) shows the PZ crystal vibrating in thick-ness shear mode and the formation of immunocomplex(Ab–Ag) on electrodes. Fig. 3D shows a prototype of aquartz-crystal microbalance (QCM) system that ourgroup developed with an auto-flow attachment (patentfiled) for pesticides monitoring.

The basis of this mass-frequency dependency [i.e. thesurface mass change (Dm) and resonant frequency shift(DF) of a PZ crystal vibrating at fundamental frequency(F)] is attributed to the Sauerbrey equation [31]:

DF ¼ �k F2 Dm=A�1 ¼ �2:26� 10�6F2 Dm=A�1

where DF is the change of frequency (Hz) due to coating,k is the proportional constant depending upon densityand shear modulus of quartz crystal (for AT-cut quartz,the density is 2.648 g/cm3 and shear modulus is2.947 · 1011 dynes/cm2), F is the fundamental fre-quency (MHz) of the quartz crystal, Dm is mass (g) ofcoating deposited, and A is the coated area of the crystal(cm2). The oscillating frequency of the quartz crystalimmersed in a aqueous medium is also influenced by thedensity (q) and the viscosity (g) of the medium, asdescribed by the following equation [32]:

DF ¼ �2:26� 10�6F3=2ðq � gÞ1=2

This equation gives a shift in fundamental frequency ofthe order of 7 kHz for a 10-MHz quartz crystal with onlyone face in contact with aqueous medium.

Various groups have reported using PZ-crystal-basedimmunosensor to demonstrate how the Ab–Ag complexcan be used to estimate environmental pollutants (e.g.,pesticides). PZ-crystal-based immunosensors for detec-tion of atrazine in aqueous samples have been reported[33]. Characterization of mAbs to 2, 4-D using a PZcrystal microbalance in solution has also been reported[34].

Because of their low cost and high quality factors (Q),these miniaturized sensors have fast response time andhigh sensitivity, and they can be mass produced usingstandard fabrication techniques. Arrays of such sensorscould be fabricated to cover ranges of a particularsensing property and could be seamlessly integrated withCMOS-based electronics. The future use of this techniqueseems to be very promising for environmental applica-tions.

4.3. Electrochemical immunosensorsFormation of Ab–Ag complex in electrochemical trans-ducers alters the change in ion concentration or electrondensity on the electrode surface, which, in turn, ismeasured by electrodes. Electrochemical transducers,

classified as amperometric, potentiometric, conducti-metric and capacitative, measure changes in current,potential (voltage), conductance and capacitance,respectively.

Applications of electrochemical transducers for detec-tion of different pesticide molecules have been reported.

A rapid assay based on an immunoenzyme electrodeand peroxide conjugates was developed for 2,4-D and2,4,5-trichlorophenoxy acetic acid (2,4,5-T) [35]. Theassay monitors the competitive binding of free pesticideand pesticide-peroxide conjugate with anti-pesticide Abimmobilized on a graphite electrode by measuring per-oxide activity in the immune complex on the electrodesurface. LODs for 2,4-D and 2,4,5-T were about50 ng/ml showing no interference with serum proteinpresent in solution.

A simple electrochemical immunosensor-based assayfor the field-based quantification of 2,4-D in soil extractshas been demonstrated [36]. The sensor utilized acompetitive immunoassay format, incorporating animmobilized Ag complex at the surface of a disposable,screen-printed, working electrode element. The extent ofglucose oxidase-labeled Ab binding to the Ag electrodewas determined amperometrically and was related to thesample-analyte concentration.

A disposable immunosensor with liposome enhance-ment and amperometric detection technique for moni-toring herbicide triazine in real samples has also beenpresented [37]. Hapten-tagged liposome-entrappingascorbic acid was chosen as a marker molecule forgeneration and amplification of the signal. Responsetime of the developed assay was 1–3 min., with mea-surement sensitivity in tap water below 1 ng/ml ofatrazine.

In a different approach, an acetyl choline (Ach)receptor-based electrochemical biosensor was developed[38]. The Ach receptor was fixed to the gate of an ion-selective field-effect transistor (ISFET) and binding of Achwith the receptor resulted in a potential change that wasdetected by the ISFET.

4.4. Micromechanical immunosensorsThe merging of silicon-microfabrication techniques withsurface-functionalization chemistry offers an excitingnew opportunity in developing ultra-sensitive micro-scopic immunoanalysis devices. Micromechanicaltransducers make use of cantilevers as force sensors inan atomic force microscope (AFM), which can transducea signal caused by the formation of immune complexonto the cantilever surface into a mechanical motionwith high sensitivity [39].

The origin of this nanomechanical bending of such ahybrid structure has been shown to be by a change in thesurface stress due to ligand–receptor binding [40], sodetection of nanomechanical bending offers a quantita-tive measure of both the occurrence and the kinetics of




specific molecular binding events on the microcantileverbeam. The selectivity of molecular interactions isdictated by the molecular probe (receptor) layer on themicrocantilever surface, while the sensitivity is governedby the extent of microcantilever deflection resulting fromthe molecular binding event. Because of the thermo-dynamic origin and the amenability to conventionalmicrofabrication protocols, microcantilever-based sensorspromise a label-free technique for detecting diversespecific biomolecular and chemical binding events (e.g.,DNA hybridization and protein–protein interactions).Several deflection methods have been proposed to mea-sure microcantilever deflection with a sub-A resolution(e.g., optical lever and piezoresistive [41,42]).

The potential of microcantilever-based immunosen-sors in label-free monitoring of the interaction ofpesticide with a mAb has been studied [43]. Binding of 2,4-D to its mAb was monitored on a thin gold layer(30 nm) deposited on a cantilever. Binding characteris-tics of anti-2, 4-D Ab to the protein-2, 4-D conjugatewere analyzed by measuring the bend of cantilever dueto Ab–Ag interaction on its surface.

Recently, we demonstrated label-free detection ofhighly-specific atrazine Ab–Ag interactions at the nmscale on microcantilevers up to ppt sensitivity [15]. In

this study, we reported a scheme for site-directed Abimmobilization on one side of a gold-coated cantileversurface (450 lm long and 0.8 lm thick) for ultra-sen-sitive detection of atrazine. Cantilever response as afunction of different atrazine concentrations was ob-served linear in the range 4.65 pM–46.5 lM.

Fig. 4 shows the preparation of the thiolated anti-atrazine Ab and the corresponding site-directed immo-bilization scheme on the gold-coated cantilever. Theabsolute deflection of the cantilever, Dz, was measuredusing an optical detection system in liquid with constantflow of Ag (Sentric System, Veeco Instruments). Thedeflection signal resulted in a differential surface stress,Dr, so, using Stoney�s equation:

Dr ¼ 1=4ðt=LÞ2E=ð1� mÞDz;

where L is the effective length of the cantilever, t is thethickness, and E/(1�m) is the ratio between the Young�smodulus, E (130 GPa), and Poisson ratio m of Si (0.28).Because the cantilever deflection also strongly dependedon the geometry, all the cantilevers used in these mea-surements were exactly similar in their geometry. As aresult, the surface-stress change due to chemical inter-actions was directly related to quantitative measure-ments for the bindings.

Figure 4. Nanomechanical detection of atrazine antibody–antigen interactions on a cantilever. Atrazine in solution binds with the antibody,causing the cantilever to bend downward due to compressive surface stress. Antibody molecules are already immobilized on the cantileverin a site-directed orientation pattern to expose antigen-binding sites free for binding.




5. Immunosensor-based assay

Since pesticide molecules are organic compounds ofmolecular mass less than 1 kD, formation of immuno-complex (Ab–Ag) on a transducer surface with thesemolecules in direct immunoassay format does not usu-ally produce significant shifts in physical parameters oftransducer devices, so the sensitivity of this type of assayfor low-molecular mass pesticide is limited.

These molecules are, therefore, coupled with somemacromolecules (e.g., proteins or enzyme tracers) andthen used in a competitive approach based on binding-inhibition tests. This assay format monitors the com-petition between labeled and unlabeled Ag for limitedavailable binding sites of Abs on the transducer surface.By using a fixed amount of immobilized Ab and labeledAg, an inhibition curve is drawn by introducing dif-ferent amounts of unlabeled Ag. The concentration ofunlabeled Ag in a test sample can then be determinedby comparison with the inhibition curve plotted.Competitive immunosensor formats rely on the use ofan Ag-tracer conjugate, which competes with theanalyte for a limited number of binding sites of the Abimmobilized on the transducer surface. Free bindingsites of the Ab exposed to the Ag are then monitored bybinding to the immobilized Ab [44]. This can also beaccomplished by immobilizing Ag on the carriersurface. Fig. 5 shows an immunosensor using Abs incompetitive inhibition for Ab (A) or conjugate (B)immunoassay.

In another approach, based on a flow-immunodetec-tion system using fluorescent detector, atrazine wasscreened with high-throughput-sample capacity [45].The immunochemical-detection principle was based onchromatographic separation of the immunocomplexformed (AbAg or AbAg*) and the free Ag (Ag) by a re-stricted access (RA) column, utilizing size-exclusion andreversed-phase mechanisms. A fluorescein-labeled ana-lyte (Ag*) was used in the competitive assay format withfluorescence detection. The speed and the simplicity ofthe assay were its greatest advantages, allowingmeasurement of the analyte to be carried out in less than1 min. The LODs were 1.4 nM (300 pg/ml) for atrazine.

Recently, we reported a lateral-flow-based dipstickimmunoassay using a novel hapten-protein-gold conju-gate for the determination of atrazine in water samples[46]. The dipstick system was quite simple, rapid, andsensitive enough for pesticide detection. This dipstickformat for the rapid screening of pesticides is increas-ingly important because of the low cost and rapidscreening capabilities [47].

The immunoassay is based on competitive inhibition,in which a newly developed hapten-protein-gold conju-gate competes with the free Ag present in the sample, forthe limited Ab-binding sites available in the test zone onthe dipstick membrane, housed in a plastics cartridge(Fig. 6). The tracer used as the detection reagent wasprepared by first conjugating hapten to a carrier protein(BSA) via its surface-lysine residues and then linkingcolloidal gold nanoparticles to the hapten-protein con-

Substrate Product

Free analyte

Enzyme-labeled hapten

Substrate Product

Enzyme-labeled secondary antibody

Antibody analyte complexFree analyte

Hapten protein conjugate

Anti-pesticideantibody

A B

Figure 5. Principle of labeled immunosensor. Concentration of analyte to be monitored is correlated with the amount of labeled antigen (A) orlabeled antibody (B) to the corresponding ligand coated on the transducer surface.




jugate via cysteine residues of the carrier protein (patentapplied). The color developed due to the conjugate, basedon competitive inhibition approach, was correlated withthe concentration of atrazine sample. The sensitivity ofthe dipstick was enhanced by gold nanoparticles, as anamplification tag, attaining an LOD of atrazine in stan-dard water samples down to 1 ppb level.

The kit could serve as a rapid screening methodologyfor visually screening atrazine contamination of watersamples with an analysis time of 5 min, and, whencoupled with a portable colorimeter, it could provide aninexpensive semi-quantitative assay. The method re-ported could be useful in the field for screening a largenumber of pesticide samples in a very short time.

6. Conclusions

Immunosensors have great potential to become cost-effective, sensitive devices for on-site monitoring of pes-ticide pollutants. They are becoming an attractive optionbecause Abs can be produced against low-molecular-mass pesticides.

Since specificity and sensitivity of immunoassay arebasically determined by the biological recognition system(e.g., Abs), a major challenge is development of differenttypes of Ab that could be used for class-specific andcompound-specific monitoring of pesticides. The crucialstep is to synthesize a suitable hapten molecule, which,after conjugation with a carrier protein, can be used asan Ag to generate appropriate Abs with the desiredspecificity. The hapten needs to be synthesized and

conjugated with the carrier protein so that it mimics thestructure of small pesticide molecules in order to gener-ate the type of Ab suitable for a particular immunoassayfor the target molecule.

Another challenge is development of a multi-analyte-detection system for simultaneous screening of target-pesticide molecules and their analogues or metabolites.Our group is actively involved with some internationalcollaborators in developing a really useful immunobio-sensor system based on micromechanical transductionor a rapid screening immunochromatographic dipstickas effective alternatives to the physico-chemical tech-niques used at present.

AcknowledgementsThis work was supported by the ISCB (Indo-Swiss col-laboration in biotechnology) and NSF (USA)-DST (India)research collaborative program. The authors arethankful to Dr. Neeraj Khatri, In-charge, Animal House,IMTECH, Chandigarh, India, for providing all necessarysupports for production of specific Abs against pesticidemolecules.

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Immunoanalytical techniques for analyzing pesticides in the environment

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