Probing direct binding affinity in electrochemical antibody-based sensors

Analytica Chimica Acta 496 (2003) 103–116

Probing direct binding affinity in electrochemicalantibody-based sensors

Bobby Corrya, Janelle Uilkb, Charlene Crawleya,∗a Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA

b Chemical Engineering, Virginia Commonwea1th University, Richmond, VA 23284, USA

Received 29 January 2003; accepted 29 January 2003

Abstract

Antibodies (Ab) are commonly used in the development of biosensors for large molecules. Their effectiveness in the directanalysis of small analyte at low concentrations can be compromised by the mode of Ab immobilization to solid substrates.The association of atrazine with anti-atrazine IgG monoclonal antibodies bound to gold-deposited quartz-crystal (GQC) andindium-doped tin oxide (ITO) electrode surfaces was characterized by electrochemical impedance spectroscopy (EIS) asa function of immobilization chemistry. Relative differences in the capacitance of unmodified and antibody modified goldelectrode surfaces varied inversely with the degree of surface roughness as determined by atomic force microscopy (AFM).In addition, the surface resistance and capacitance elements measured at antibody modified GQC and ITO-electrodes wereshown to reflect antibody–antigen binding.© 2003 Published by Elsevier B.V.

Keywords:Biosensors; Monoclonal antibody; Atrazine; Electrochemical impedance spectroscopy; Atomic force microscopy; Quartz-crystalmicrobalance

1. Introduction

Numerous studies have been conducted over thepast decade in the development and application ofantibody-based methods for large and small analyte[1–4]. Although the bulk of studies have been appliedin the field of clinical analysis, more recent interestsare focused on the analysis of environmental pol-lutants and biowarfare agents[5]. Immunochemicaltechniques based on the interaction of antibodies withantigens, such as the enzyme linked immunosorbentassay (ELISA) continue to gain wide acceptance inthe analytical and regulatory communities. These

∗ Corresponding author. Tel.:+1-804-828-8599.E-mail address:[email protected] (C. Crawley).

techniques are sensitive, selective, inexpensive, andwidely applicable to the simultaneous analysis ofmultiple aqueous samples with minimal pretreatmentusing the microtiter plate format. Among the disad-vantages of immunoassays are the necessity to uselabels such as an enzyme, fluorophore, or radioac-tive isotope that can provide only an indirect probeof antigen- or hapten-antibody-binding and may notdeliver the anticipated response in some matrices. Inaddition, variability in the label response can occurover long incubation times.

Immunosensors couple the advantages of im-munoassay techniques where immobilized antibodyis used to engender molecular recognition coupledwith some mode of direct signal transduction usingtechniques such as the quartz-crystal microbalance

0003-2670/$ – see front matter © 2003 Published by Elsevier B.V.doi:10.1016/j.aca.2003.01.001

104 B. Corry et al. / Analytica Chimica Acta 496 (2003) 103–116

(QCM), surface plasmon resonance, ellipsometry, orelectrochemical methods[6–8]. In addition, the cou-pling of immunosensors with flow injection analysisenables antibody regeneration and automation proce-dures that are difficult to implement with immunoas-say methods. However, barriers to broad implementa-tion of this technology include limited information onstructure–activity relationships, variability in perfor-mance of antibody, and difficulty diagnosing failurein an assay.

Antibodies have been immobilized to solid supportssince the 1960s[9]. Their utility in molecular recog-nition applications stems from two main factors:

(1) The ability to produce antibodies to almost anyspecies based on rapid development of phage dis-play technology in antibody engineering[10].

(2) The relative homogeneity of theFc portion of an-tibodies which allows development of immobi-lization strategies applicable to a large variety ofspecies[11].

When engineering an immunosensor it is criticalthat the immobilization method allows retention ofthe native conformation and binding characteristicsof the antibody. Other important considerations insensor development are regeneration of the sensorsurface through disruption of Ab–Ag binding andrelease of antigen, long-term stability of the sensor,and insensitivity of the sensing surface to samplematrix species. Traditional immunochemical meth-ods require that either the antibody or antigen areimmobilized onto a solid substrate. However, fac-tors such as “mode of attachment” and “anchoringlocation” can alter the binding affinity and dynamiccapacity of immobilized antibody. There are rela-tively few studies reporting a systematic investigationof immobilization conditions. One reason for this isthe lack of available methods capable of ‘real-time’monitoring of proteins on surfaces under physiologicconditions, which are also good transducers of theAb–Ag binding event. A primary goal in immunosen-sor research is the construction of new surfaces thatimmobilize and concentrate active antibodies to envi-ronmental toxins for sensor applications. This reportexamines the following three separate approachesfor the immobilization of a monoclonal anti-atrazineIgG antibody raised against the triazine herbicide,atrazine, on gold-deposited quartz-crystal (GQC) and

indium-doped tin oxide (ITO) electrode surfaces:

(a) adsorption;(b) adsorption with Tween-20 surfactant modifier;(c) covalent linking agent.

The assembly of an antibody layer and the bindingof the antigen to antibody have been monitored byelectrochemical impedance spectroscopy (EIS) andatomic force microscopy (AFM). AFM is used toimage the Ab-modified surfaces prepared under vary-ing conditions. EIS is used as an alternate means ofquantifying the selective responses of immobilizedantibody in the presence of antigen to time vary-ing stimuli of small magnitude. EIS is the ideal toolfor probing antigen–antibody molecular recognitionin that it allows the characterization of interfacialproperties in the absence of redox reactions[10]. Bymeasuring resistive and capacitive changes accompa-nying removal of solution resident substrate and itsassociation with surface bound species, this techniqueoffers a direct “in situ” probe of the physicochemicalchanges associated with selective and non-specificantibody-binding in heterogeneous environments.

2. Experimental

2.1. Chemicals and reagents

Monoclonal anti-rabbit immunoglobulin (∼1 mg/ml) clone RG-16 and monoclonal rabbit IgG (∼1 mg/ml) purchased from Sigma were used as received.Two IgG� subclass anti-atrazine antibodies desig-nated as AM7B2.1 and AM5D1.1 were generatedfrom mouse splenocytes fused with P3X63AG8.653myelomas[12]. Frozen cell cultures were obtained askind gifts from Professor (emeritus) Alexander Karu(UC-Berkeley). The mouse IgG monoclonal antibodyagainst atrazine was generated by splenocytes incu-bated in Iscove’s dulbecco’s Medium (IMDM) with10% fetal bovine serum at the Monoclonal Hybridomafacility at the Virginia Commonwealth UniversityHealth System Campus, Richmond, VA. When anadequate concentration of antibodies is formed the so-lution was frozen at−20◦C until needed. Aliquots ofthis solution (∼25 mg/ml total solids) were thawed toroom temperature and either dialyzed using a Spectra/Por Cellulose Ester membrane (Spectrum, Houston,

B. Corry et al. / Analytica Chimica Acta 496 (2003) 103–116 105

TX), molecular weight cut-off of 100,000, or puri-fied using a commercial Affinity PAK ImmobilizedProtein A column (Pierce) to yield IgG concen-trations of 0.6 mg/ml. Elution of bound IgG wasmonitored by absorbance at 280 nm. Membranes con-taining the antibody/medium solution were dialyzedagainst 150 mM phosphate buffer containing 15 mMsodium chloride, pH 8.4, and refrigerated at 4◦ for24 h, placed in fresh buffer, and stirred for an ad-ditional 24 h. All buffer solutions were prepared indeionized Milli-Q, Millipore (18 M�) water. Sodiumphosphate dibasic and sodium phosphate monobasic,monohydrate (ACS grade) were from EM Science(Germany), and atrazine was purchased from Supelco(Bellefonte, PA).

Dithiobis(succinimidylundecanoate) (DSU) was akind gift from Prof. James Burgess (Case-WesternReserve University), 3-aminopropyltriethoxysilane(98%) was purchased from Sigma and toluene(0.002% water) was from J.T. Baker Inc., NJ.

2.2. Equipment

2.2.1. Electrochemical cell and potentiostatAn 8 ml three-electrode cell was used to con-

duct the impedance measurements. GQC (0.2 cm2)and ITO (1.33 cm2) electrodes served as workingelectrodes. The counter electrode was a large areacoiled platinum wire. A double junction Ag/AgCl(1 M KCl) reference electrode was used where theouter junction contained test solution. The impedancespectra were measured using an IM6e potentiostat(Bioanalytical Systems, BAS) in the frequency range1 Hz–100 kHz. BAS Thales software was used to col-lect and evaluate the impedance responses. The am-plitude of the ac sinusoidal potential applied to boththe gold and ITO-electrodes was 10 mV. All mea-surements were conducted at gold electrodes poisedat 0.3 V and ITO-electrodes were maintained at 0 V.All impedance experiments were performed at roomtemperature.

2.2.2. Quartz-crystal microbalanceThe quartz-crystal microbalance used for this study

was an in-house system assembled at Virginia Com-monwealth University. The electrical design of thissystem is based on that published by Bruckensteinet al. [13].

2.2.3. Atomic force microscopy imagingDry GQC electrodes were scanned by tapping mode

using cantilevers with silicon tips. The data were pro-cessed through the DI Instrument program providedby the system. Coated glass substrates were imagedusing tapping mode atomic force microscopy in air atambient temperatures within 1 week of preparation. ADimension 3100 scanning probe microscope (DigitalInstruments, Santa Barbara, CA) and nanoprobe can-tilevers (125�m, Digital Instruments) were utilized.Imaging was performed at set point amplitude to can-tilever free oscillation amplitude (Asp/Ao) ratios of0.7–0.8, generally regarded as low tapping force[14].Scan rates were 0.5–1 kHz and scan sizes were 1�mand 250 nm square.

2.3. Procedures

2.3.1. Electrode preparationGQC electrodes with a surface area of 0.2 cm2 were

obtained from International Crystal, OK. Prior to eachexperiment the electrodes were sonicated for 5 minin pure ethanol followed by a 5 min sonication rinsewith Milli-Q water, and then dipped for 3 min intopiranha solution (1:3 35%–H2O2:conc. H2SO4). Theelectrodes were again rinsed with purified water, driedunder nitrogen and placed in a UV cleaner for 60 min.

Indium-doped tin oxide electrodes with a surfaceresistivity of approximately 18� cm2 were purchasedfrom PPG Industries. The ITO-electrode pretreatmentprocedure has been previously published by Bowdenand Hawkridge[15] and Hawkridge and co-workers[16]. Electrodes were polished with 0.3�m alumina(Buehler) on a Gamal Polishing Cloth (Fisher), rinsedwith Milli-Q water. They were then cleaned ultra-sonically in the following solutions: 2% solution ofMicro-90 (International Products Corp., NJ), 95%ethanol, and twice in Milli-Q water (5 min each run).

2.3.2. Electrode surface modification with covalentlinkers

2.3.2.1. GQC–DSU linker. Several drops of a0.3 mM DSU solution in ethanol were placed on thecleaned electrode surface. The electrode was exposedto this solution for 24 h in a humidity chamber fol-lowing an ethanol rinse and was then dried undernitrogen.


2.3.2.2. Indium-doped tin oxide (ITO)-aminosilanelinker. The surface modification of ITO-electrodesconsisted of three successive steps:

(1) cleaning the electrodes;(2) increasing the surface coverage of hydroxyl

groups;(3) silanization with aminopropyltriethoxysilane (AP-

TES).

Steps 1 and 2 were performed according to pub-lished procedures[17]. ITO (∼25 mm×25 mm) sizedplates were cleaned by sonication with acetone anddichloromethane for 10 min each following five 2 minsonication rinses with water. After this, the plates wereplaced for 1 h in a glass beaker with a mixture of 5:1:1H2O+H2O2 (30%)+NH3 (25%) at 70◦C, rinsed withwater and dried in an oven at 100◦C for 4 h.

Silanization was carried out as described in[18].The cleaned ITO-electrodes were exposed to 200 mlof toluene, containing 2 ml of APTES at 110◦C for3 h, followed by a 1 h incubation in the same solutionat room temperature. The electrodes were then washedsuccessively by sonication with toluene (5 min),dichloromethane (five times, 5 min each) and absoluteethanol (three times, 5 min each). The silanized ITOplates were stored in a dessicator until use.

2.3.3. Antibody immobilization procedures

2.3.3.1. Anti-atrazine IgG antibody adsorbed on GQCelectrode. Several drops of the dialyzed antibody so-lution were placed onto the cleaned electrode surface.The electrode was then placed into a humidity cham-ber that was refrigerated at 4◦ for 24 h, rinsed withMilli-Q water, and dried under nitrogen prior to use.

2.3.3.2. Anti-atrazine IgG antibody adsorbed withTween-80 on GQC. Several drops of dialyzed anti-body premixed with Tween-80 surfactant were placedonto the cleaned electrode surface. The electrode wasthen placed into a humidity chamber that was refrig-erated at 4◦ for 24 h, rinsed with Milli-Q water, anddried under nitrogen prior to use.

2.3.3.3. Anti-atrazine IgG antibody-DSU linker onGQC electrode. Several drops of dialyzed antibodypremixed with Tween-80 surfactant were placed ontothe DSU-modified electrode surface. The electrode

was then placed into a humidity chamber that wasrefrigerated at 4◦ for 24 h, rinsed with Milli-Q waterand dried under nitrogen prior to use.

2.3.3.4. Anti-rabbit IgG antibody-DSU linker on GQCelectrode. The covalent attachment of anti-rabbitIgG to DSU-modified gold electrodes was carried outby placing several drops of IgG solution (∼1 mg/ml)onto the DSU-derivatized electrode surface. The reac-tive coupling between amino groups of lysine residueson the antibody with the ester group on DSU[19]was allowed to occur for 24 h at 4◦C in the humiditychamber. The electrode was then carefully rinsed withphosphate buffer solution (pH 7).

2.3.3.5. Anti-atrazine IgG antibody-aminosilanelinker on ITO-electrode. Prior to reaction withamino-group of APTES-modified ITO-electrodes, theoligosaccharide moieties of anti-atrazine IgG were ox-idized to aldehydes following the protocol describedin [20]. The site-directed assembly of oxidized IgGto silanized ITO has been previously published[18].The silanized electrodes were incubated in a solutionof oxidized IgG (1 mg/ml) in acetate buffer of pH5.2 containing 5 mM NaBH4 for 12 h. This procedurewas determined to yield stable and homogeneous im-mobilized IgG layers without loss of antigen bindingcapacity[18]. After removal from IgG solution, theelectrodes were thoroughly rinsed in phosphate buffersolution at pH 7.

3. Results and discussion

3.1. Impedance analysis

Electrochemical impedance spectroscopy is basedon the application of a sinusoidal signal excitation toan electrochemical cell and the analysis of the cur-rent produced. The impedance, or cell resistance toan alternating current, is a parameter used to describerelationships between the voltage and the current inthe electrochemical cell. As a result of impedancemeasurements, imaginary and real portions of theimpedance are produced which are indicative of char-acteristic capacitance and resistance elements of theelectrode–electrolyte interface. Since the formation ofantibody–antigen complexes at an electrode surface


will change both the charge distribution in the elec-trical double layer and the resistance of the surfacelayer, one would anticipate that the impedance couldprovide information about the association-dissociationkinetics of biological complexes at antibody modi-fied electrode surfaces, as well. Indeed, several au-thors[21–23] have reported correlations between theimpedance change and affinity complex formation.

In the absence of a redox couple in the electrolytesolution, the two main elements of the electrochem-ical circuit to be considered are the capacitance,C,and the resistance,R. For a series connection betweenthe resistance and the capacitance, the impedance isdescribed by:

Z = R − j

ωC(1)

whereω = 2πf andf is the frequency of the alternat-ing current.

The real and imaginary parts ofZ areRand−1/ωC,respectively. Using a Nyquist diagram of the imagi-nary versus the real impedance components, one ob-tains a graph represented by a vertical line with anabscissa equal toR, which is the typical model for anelectrochemical interface in the absence of a faradaicprocess.

For the parallel combination ofR and C, the fol-lowing relation describes the impedance:

Z = R

1 + (ωRC)2− R2Cω

1 + (ωRC)2j (2)

The graph representing a parallel combination ofthe resistance and capacitance such as would becharacteristic of an antibody immobilized at anelectrode–electrolye interface, is a semicircle whereat low frequencies the impedance is purely resistive.The impedance of a real system characterized by in-tact self-assembled monolayers of an analyte attachedto the surface in the absence of a faradaic processusually presents some combination of the equivalentcircuits described inEqs. (1) and (2). A generalizedequivalent circuit developed for self-assembled mono-layers on a Au (1 1 1)-electrode proposed by Janek andFawcett[24], is presented inScheme 1shown below.

The components W.E., C.E. and R.E. are the work-ing, counter and reference electrodes, respectively.Rsrepresents the solution resistance,Ru uncompensatedsolution resistance,R1 and R2 are resistances of the

Scheme 1. The equivalent circuit model for electrochemical cellin the presence of self-assembled layers on Au(1 1 1)-electrode.

self-assembled monolayer,CDL is the capacitance ofthe electrical double layer andC2 is the additional ca-pacitance due to the presence of the monolayer.

3.2. Characterization of antibody at gold-modifiedsurfaces

The feasibility of using EIS and other surface sensi-tive methods to gauge the impact of immobilization onantibody-binding was studied by comparing the directadsorption and covalent attachment of anti-atrazineantibody (Ab) to a GQC electrode surface using theDSU linker immobilization method. Gold-depositedquartz electrodes commercially sold for use on aquartz-crystal microbalance were used in these stud-ies. As previously described, three procedures foranti-atrazine immobilization onto gold-depositedquartz-crystal electrodes were investigated:

(a) direct adsorption;(b) adsorption with Tween-80 surfactant;(c) DSU linker-Tween-80.

Although adsorption is the simplest method of af-fixing antibody to a surface, a lack in control of theorientation of the antibody coupled with aggregate for-mation can lead to the generation of a non-uniformsurface resulting in partial obstruction of the antigenbinding site. Covalent linkers such as DSU can pro-vide a more uniform coverage of antibody with site-directed orientation; however, these agents can alsoobscure the detection of antigen–antibody-binding, es-pecially for small antigens.

AFM scans of antibody coated surfaces showedfeatures characteristic of the mode of immobilizationas shown inFig. 1(a)–(c). Antibody adsorption with-out surfactant resulted in formation of ‘non-uniformclumps’ of antibody on the surface. This surfaceyielded the largest degree of surface roughness,


Fig. 1. AFM (height image) scans for anti-atrazine antibody modified GQC electrodes prepared by: (a) direct adsorption, (b) adsorptionin Tween-80 surfactant, (c) covalent binding to DSU self-assembled to the surface (with Tween-80 surfactant).

calculated to have a relative mean square (RMS) of13.9 nm, compared to 2.4 nm for the bare-gold sur-face. This uneven coverage of antibody also results in‘open pore’ regions where the bare-gold surface is ac-cessible to buffer, analyte, and impurities in solution.The darker colored regions shown in the AFM heightimages obtained in tapping mode are indicative of theharder gold surface. The latter two procedures pro-duced more smooth and uniform soft surfaces. Thesurfactant additive serves to disperse aggregate for-mation to give more uniform layers, while the DSUlinker generates a more highly oriented monolayer ofantibody on the surface. The respective RMS valuesfor the Tween-80 and DSU-Tween treated electrodes

were 4.2 and 2.3 nm (Bare GQC surfaces contain-ing just Tween-80 were also imaged and are moresmooth than the bare-gold electrode blank; however,the capacitance response does not vary upon addi-tion of antigen). Electrodes formed by the physicaladsorption of Ab in the presence of surfactant werefound to exhibit selective binding to atrazine whenmeasured using a quartz-crystal microbalance system.The QCM responses for a GQC electrode modifiedby adsorption with anti-atrazine antibody and placedin a flow cell are shown inFig. 2. The electrode wasfirst equilibrated in water until a stable signal wasobtained. After a 50 s baseline frequency responsewas obtained, aqueous pesticide analyte was injected


Fig. 2. QCM responses for an anti-atrazine GQC immunosensorelectrode prepared by adsorption with Tween-80 in a flow cellthat is exposed to 35 ppm of atrazine and alachlor pesticide at2 m/min pesticide is added to the cell at Point ‘a’; after 50 s andis replaced with water at Point ‘b’ after about 3 min.

into the cell at Point ‘a’ for about 3 min. The flowwas changed back to water as indicated by the secondset of arrows indicated as Point ‘b’. The data werefiltered used a moving average (10 point base). TheQCM frequency responses shown inFig. 2 for com-parable concentrations of aqueous alachor (35 ppm),(a non-triazine pesticide), and atrazine (35 ppm)demonstrate that the atrazine response of this sensorwas three times larger than that obtained for alachlor.Although the QCM response obtained for a smallantigen at these levels was significant and validatethe feasibility of using the QCM as a transducer ofselective pesticide responses, mass changes from thenon-specific adsorption of possible aqueous analyteimpurities, irreproducible antibody deposition, andthe eventual stripping of adsorbed antibody under flowconditions, lead to highly variable responses at dif-ferent electrodes. The impedance responses obtainedby EIS for electrodes prepared by (i) adsorption and(ii) DSU-covalent attachment of antibody (withoutTween-80) were compared with the bare GQC elec-trode. The cell capacitance evaluated as a functionof Ab immobilization procedure is summarized inTable 1.

A decrease in surface capacitance is expected witha more uniform coverage of the surface with an or-ganic substrate. The capacitance was evaluated usingthe circuit indicated below whereCstray is the instru-ment stray capacitance in picofarads,Rs is the solu-tion resistance, and CPE is aconstant phase element

Table 1Electrode surface capacitance at GQC electrodes as a function ofantibody immobilization procedure (standard error± %R.S.D.)

Electrode description CPE (�F) α

Bare–Au 2.6± 7 0.88± 7Adsorption (Au–Ab) 2.2± 5 0.84± 0.3Linker (Au–DSU–Ab)a 1.3 ± 2 0.87± 0.2

a Tween-80 was not used to prepare GQC–DSU–Ab electrodesso that its impact would not be reflected in the capacitance mea-surements.

in microfarads that behaves as an ideal double layercapacitance whenα is 1.0 (Scheme 2).

Overall, GQC antibody electrodes prepared usingthe DSU linker yielded electrodes with more repeat-able behavior and larger capacitance changes uponaddition of antigen. (This trend was apparent forGQC electrodes prepared using DSU linker both withand without surfactant.) A comparative evaluationof the impedance response for GQC–DSU–Ab elec-trodes prepared using anti-rabbit IgG and anti-atrazineIgG antibodies (both without surfactant) is shownin Fig. 3. Bode plots of the change in the in-phaseimpedance as a function of frequency were exam-ined for anti-atrazine modified electrodes exposedto 20 ppm atrazine and anti-rabbit IgG electrodesexposed to 10 ppm rabbit-IgG antigen between 100and 1000 Hz. The response was shown to vary whenexposed to either the rabbit IgG or atrazine antigens.The impedance responses are indicated inFig. 3 forthe following three cases:

Case I: (1,1′) Ab-modified electrode exposed to justbuffer (0.01 M phosphate).

Case II: (2,2′) Ab-modified electrode exposed tobuffered antigen solution.

Case III: (3,3′) Ab-modified electrode in buffer after1-h exposure to antigen.

Scheme 2. The equivalent circuit model of the electrochemicalcell used to evaluate the capacitance of the bare-gold and antibodymodified GQC electrodes.


Fig. 3. Real impedance vs. frequency responses for DSU-antibody modified GQC electrodes (no Tween) in the presence of (1) 0.01 Mphosphate buffer, pH 7, (2) antigen in 0.01 M phosphate, and (3) 0.01 M phosphate following exposure to the respective antigens. Dashedcurves are for anti-atrazine modified electrodes exposed to ca. 20 ppm atrazine. Solid curves are to anti-rabbit IgG modified GQC electrodesexposed to ca. 10 ppm rabbit IgG antigen.

The dashed curvesshow the response of the anti-atrazine electrode to 20 ppm atrazine for the variouscases. Thesolid curves indicate the responses for10 ppm rabbit-IgG antibody at it’s respective elec-trode. Increasingly large differences in impedancewere observed for the larger antigen. In addition, themagnitude of the impedance differences observed forCases I-II were found to occur in the 1000–10 kHzrange. Martelet and co-workers[22] also observedcapacitance and in-phase impedance changes as afunction of antigen–antibody interactions.

AFM (phase) images showing individual DSU-immobilized anti-rabbit IgG antibodies (without sur-factant) attached to GQC electrodes are shown inFig. 4 at nanometer resolution. Images of surface im-mobilized IgG antibodies have been shown to rangein size from about 30–40 nm by AFM, although ac-tual geometric dimensions range from 9 to 15 nm (or90–150 Å). Differences will depend upon the point

of attachment and the orientation of the antibody tothe electrode surface, and the formation of antibodyaggregates[18]. X-ray diffraction analysis indicaterespective width, height, and thickness dimensions ofan IgG molecule to be 145 Å× 85 Å × 40 Å, wherethe height is measured from theFab to theFc end ofthe molecule, and the width extends across both endsof the Fab (antigen binding site) region[25]. Thediscrepancy between the AFM image dimensions andthe actual molecule dimensions will also vary whenthe radius of curvature of the probe tip is comparableto the size of the molecule being imaged thereby re-sulting in low resolution, or ‘image broadening’. Theaverage tip radius of curvature used in these studieswas 10 nm resulting in the larger image dimensionsobserved. AFM image color in tapping mode willvary with surface hardness. The darker colored spacesin between the antibodies represent possible under-ivatized sites on the hard gold metal electrode that


Fig. 4. AFM phase image for DSU-immobilized monoclonal anti-rabbit antibody (no Tween) on a GQC electrode. The resolution is shownat 50 nm/div.

can allow non-specific adsorption of analyte at theelectrode surface. This can interfere with capacitivebased sensor responses, as well as result in variableresponses from one electrode to another.

The impedance responses obtained on covalentlyimmobilized antibody electrodes reflect the electrodesurface, additives in solution, as well as the degreeof antigen–antibody-binding. Nyquist plots of theimpedance responses obtained at a GQC electrodemodified with DSU-immobilized anti-atrazine anti-body are shown inFig. 5. The responses were obtainedupon application of a 10 mV ac potential to electrodespoised at 300 mV versus Ag/AgCl at frequenciesranging from 0.1 Hz to 10 kHz. The semicircular re-gion mainly reflects the solution resistance and the

potentiostat stray capacitance. The linear portion ofthe response mainly depicts the surface dependentpseudo double layer capacitance which varies withantibody-binding. Upon addition of an aqueous sat-urated solution of atrazine (20 ppm) to the electrodein a 0.01 M solution of phosphate buffer (pH 7), thecapacitance trace shifted from Trace-1 to Trace-2.After the electrode was rinsed, the response did notvary significantly, as observed in Trace-3 (capacitivedifferences are magnified at higher frequencies). Af-ter the electrode was exposed to a high ionic strengthbuffer (0.1 M phosphate buffer-Trace-4) sufficient tocause antigen–antibody dissociation, the capacitanceresponse began to approach initial values observed inTrace-1. (Unless otherwise indicated, the electrode


Fig. 5. Nyquist plot for GQC electrode modified with DSU-anti-atrazine antibody (no Tween) exposed to (1) buffer, (2) atrazine in solution,(3) buffer rinse following atrazine exposure, (4) partial disruption of atrazine binding with high ionic strength buffer, IM phosphate buffer(PB).

was equilibrated for1h in the various solutions priorto EIS analysis.)

3.3. Characterization of antibody at tinoxide-modified surfaces

Antibody immobilization and response at oxide sur-faces such as silica and tin oxide are also of inter-est in chromatographic and optical sensing methods,such as in the use of silica capillaries in the analy-sis of ppb levels of atrazine by capillary electrophore-sis [26]. Linkers that immobilize the antibody to thesurface through theFab region of the molecule arenot ideal for the development of direct sensors forlow molecular weight analyte since the binding site ishindered and unavailable for binding analyte. There-fore ideal linkers for these surfaces have functionalgroups that react with the surface oxides on one endand amine, hydrazine, or other groups that will re-act with either the disulfide groups in the hinge re-gion or carbohydrate groups near theFc region ofthe IgG molecule. AFM images obtained for mono-

clonal anti-atrazine antibodies covalently attached tosilanized surfaces of indium-doped tin oxide semi-conductor electrodes are shown inFig. 6. A proce-dure that targets the carbohydrate region of the an-tibody was used to immobilize the antibody awayfrom the antigen-binding site. Clean tin oxide elec-trodes were simultaneously silanized and function-alized with amine using aminopropyltriethoxysilane.The silanized electrodes were incubated for 20 h withoxidized antibody prior to imaging. A cleaned andsilanized tin oxide electrode surface is compared withthat obtained after covalent attachment of antibody asshown inFig. 6(a) and (b), respectively at nanome-ter resolution. The surface density of antibody us-ing this chemistry is less than that observed for theDSU-Ab-modified electrodes at gold electrodes. How-ever, amine functionalization, which targets the car-bohydrate regions of oxidized antibody, is reported togive a more optimal orientation of antibody on thesurface[18].

A comparative Nyquist plot obtained for anti-atrazine antibody immobilized at an indium-doped


Fig. 6. (a) (Upper trace) AFM (height) image of indium-doped tinoxide electrode silanized with aminopropyltriethoxysilane reagentat 50 nm/div. (b) (Lower trace) AFM (Phase) image of theITO-electrode surface after reaction with 0.6 mg/ml solution ofoxidized anti-atrazine monoclonal antibody at 50 nm/div.

tin oxide electrode via aminopropyltriethoxysilane isshown inFig. 7(a) and (b)when exposed to atrazine(top) and the sulfonylurea surfactant, Londax (bot-tom). In the top graph (a) capacitive changes areobserved when atrazine is added to buffer.

After rinsing, the response indicates some lossof non-specifically adsorbed atrazine as shown inTrace-3. However, the response does not return backto the original Trace-1 due to atrazine bound to the

Scheme 3.

antibody. The experimental data are indicated by thesolid symbols and the two curves indicate the simu-lated responses that fit to the following circuit shownin Scheme 3, whereRs is the solution resistance,R1is related to the semiconductor surface resistance,Cstray is the instrument stray capacitance, and CPE,or constant phase element isthe pseudo double layercapacitance. Each RC parallel element produces asemicircular shaped response in the Nyquist plot.The bottom graph (b) shows the Nyquist plot forthe same electrode in buffer and when rinsed afterexposure to the non-triazine pesticide, Londax. Aswould be expected Trace-1 and Trace-2 are essentiallyidentical since antibody should not bind Londax.(This behavior was also observed at multiple ITO-electrodes as well as for GQC–DSU antibody mod-ified electrodes exposed to Londax.) The electrical

Table 2Electrical circuit elements for a bare ITO-electrode exposed to0.01 M phosphate buffer (PB) electrolyte and 20 ppm atrazine (Atr)(standard error± %R.S.D.)

0.01 M PB PB+ Atr insolution

PB + Atr (rinsed)

Cstray (nF) 3 ± 66 4 ± 94 4 ± 92Rs (�) 417 ± 4.9 420± 6.2 442± 6.1R1 (k�) 20 ± 14 19± 7.2 19± 7.2CPE (�F) 13.7± 2.4 13.7± 3.4 13.6± 3.5α 0.876± 0.18 0.878± 0.25 0.88± 0.23

Table 3Electrical circuit elements of anti-atrazine antibody modifiedITO-electrode exposed to 0.01 M phosphate buffer (PB) electrolyteand 20 ppm atrazine (Atr) (standard error± %R.S.D.)

0.01 M PB PB+ Atr insolution

PB + Atr

Cstrays (pF) 729± 18.3 583± 22.4 691± 20.8Rs (�) 576 ± 1.11 512± 0.50 510± 0.71R1 (k�) 35.5 ± 3.8 42.1± 6.53 39.3± 5.61CPE (�F) 8.52± 1.14 10.06± 0.69 10.14± 0.68α 0.88 ± 22 0.896± 0.09 0.894± 0.09


Fig. 7. (a) Nyquist plot for anti-atrazine modified ITO-electrode upon exposure to (1) 0.01 M phosphate buffer, (2) 20 ppm atrazine inbuffer, and (3) 0.01 M phosphate after exposure to atrazine. (b) Atrazine modified ITO-electrode exposed to similar concentrations of thesulfonylurea pesticide, Londax, in the same sequence indicated in (a).

equivalent circuit elements evaluated for a bare andantibody modified ITO-electrode for Cases I–III aresummarized inTables 2 and 3, respectively. Thecapacitance of the bare electrode decreases from ap-proximately 14–9�F when modified with antibody.

Also changes in the capacitance of the bare ITO-electrode with exposure to atrazine are negligible incomparison with the capacitance changes observedfor the antibody-modified electrode upon exposure toatrazine.


4. Conclusions

Capacitance changes at gold quartz-crystal (GQC)and tin oxide semiconductor electrodes have beenshown to vary as a function of the electrode modi-fication method for electrochemical immunosensorsexposed to ppm levels of the low molecular weightpesticide hapten, atrazine. The results obtained foranti-atrazine IgG antibody immobilized to goldquartz-crystal surfaces indicate that a combination ofsurface roughness, as determined by AFM, and sur-face capacitance changes as assessed by EIS, can beused as a predictor of electrode activity. Electrodesprepared by the covalent attachment of antibody tothiol succinimide linkers (DSU) or aminosilane agentsthat form organized structures on the electrode surfacewere shown to exhibit larger capacitance changes ascompared to adsorbed or unmodified electrodes uponaddition of antigen. In addition, the selectivity of theelectrodes to analyte can be probed via changes in theimpedance responses.

Immunosensors are especially suited to the de-tection of environmental pollutants and biowarfareagents. An understanding of the interaction of the an-tibody with solid substrates is a key factor in effortsto enhance the sensitivity, selectivity, and repeatabil-ity of these electrodes as sensing devices. Althoughthe sensitivity observed for these sensors is not yetat the environmentally significant ranges apparent inimmunoassay methods such as ELISA, the resultsdemonstrate that increases in the capacitance responseat surfaces such as ITO result from an increased sur-face coverage of “active” antibody. These approacheswill be used in future studies to investigate the use ofmore novel covalent linking agents that may enhancethe selectivity and response of these electrodes tosmall molecules.

Acknowledgements

This work was funded by the Jeffress MemorialTrust Fund (Grant Number J-527), the Sloan Founda-tion Pre-Tenure Leave Fellowship and the VCU De-partment of Chemistry. The authors would also like toacknowledge Dr. Olga Baturina for her invaluable ef-forts in the characterization of these surfaces, EmeritusProfessor Alexander Karu for his kind gift of the mon-

oclonal anti-atrazine hybridoma cells, and Teri Russellof the Massey Cancer Center (VCU) for supplying uswith antibody supernatant.

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25.


Charlene D. Crawley is an assistantprofessor of chemistry at Virginia Com-monwealth University (VCU) in Rich-mond, Virginia. Dr. Crawley receivedBS and MS degrees in chemistry fromVCU, where her Master’s research withProf. Fred M. Hawkridge involved theelectrochemical determination of the het-erogeneous electron transfer kinetics ofbiological molecules. She received her

PhD (1986) in analytical chemistry from the University ofDelaware under the direction of Prof. Garry A. Rechnitz, whereshe evaluated the mechanistic responses of polymer-based im-munosensors. Dr. Crawley began a 9-year career in research anddevelopment serving as a senior research chemist in the Analyticaland Special Problems Group and as team leader in Plant Labora-tory Operations with the Hercules Research Center in Wilmington,

DE. She served a 1-year sabbatical from industry as a program of-ficer in the Analytical and Surface Chemistry Program of the Na-tional Science Foundation under the direction of Dr. Henry Blountprior to joining VCU as a visiting assistant professor and collat-eral professor in 1996. She started her current research programat VCU in 1998. Dr. Crawley’s research interests in bioanalyticalchemistry involve the development of sample preparation and as-say methods for trace level detection of analytes in environmentaland biological matrices. Capillary electrophoresis, immunoassay,electrochemical, and atomic force microscopy imaging methodsare some of the tools used to probe interactions between surfacebound antibody and large and small molecule environmental tox-ins. She currently serves as the Publicity Officer for the Ameri-can Chemical Society’s Division of Analytical Chemistry and is amember of the Society of Electroanalytical Chemistry, the Rich-mond Chromatography Discussion Group, and the National Orga-nization of Black Chemists and Chemical Engineers (NOBCChe).

Documents

Probing direct binding affinity in electrochemical antibody-based sensors