ORIGINAL PAPER

Surface plasmon resonance immunosensor for cortisoland cortisone determination

Marco Frasconi & Monica Mazzarino &

Francesco Botrè & Franco Mazzei

Received: 23 April 2009 /Revised: 9 June 2009 /Accepted: 12 June 2009 /Published online: 10 July 2009# Springer-Verlag 2009

Abstract In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detectionof cortisol and cortisone levels in urine and saliva samples.The method proposed here is simple, rapid, economic,sensitive, robust, and reproducible thanks also to the specialfeatures of the polycarboxylate-hydrogel-based coatingsused for the antibody immobilization. The sensor surfacedisplays a high level of stability during repeated regener-ation and affinity reaction cycles. The immunosensor showshigh specificity for cortisol and cortisone; furthermore, nosignificant interferences from other steroids with a similarchemical structure have been observed. The suitability ofthe hydrogel coating for the prevention of nonspecificbinding is also investigated. A good correlation is noticedbetween the results obtained by the proposed method andthe reference liquid chromatography/tandem mass spec-trometry method for the analysis of cortisol and cortisone inurine and saliva samples. Standard curves for the detectionof cortisol and cortisone in saliva and urine are character-ized by a detection limit less than 10 μg l−1, sufficientlysensitive for both clinical and forensic use.

Keywords Cortisol . Cortisone . SPR . Immunosensor .

Antidoping

Introduction

Endogenous glucocorticoids are produced in the fascicularzone of the adrenal cortex under the control of thehypothalamo-pituitary-adrenal axis and are transported totheir target receptors in the form of protein complexes withalbumin and with the specific serum protein transcortin. Theirmechanism of action depends on their binding to specificreceptors, which are widely distributed in a great variety ofbiological districts, thus involving many different targettissues [1]. Cortisol is the principal circulating glucocorticoidin man and is secreted in relatively high amounts, 15 mgday−1. The free fraction is biologically active and representsonly 1% of the total cortisol secretion rate [2]. Themeasurement of the blood, urinary, and salivary free cortisollevel is useful in clinical for the adrenal or pituitary glanddisorder diagnosis and in forensic field as first-levelscreening to detect the illicit use of glucocorticoids for thenonphysiological enhancement of sport performance. Unfor-tunately, the free cortisol and cortisone concentration profilesare dependent on many factors, including patient age,gender, sample population, intensive endurance training,severe emotional or physical stress, and medications, suchas for example glucocorticoids, lithium, diuretics, ketocona-zole, estrogens, and tricyclic antidepressants, so no referencerange are still disposable. The control of the cortisol/cortisone concentration is operated by two isoforms of the11 β-hydroxysteroid dehydrogenase (11 β-HSD) that cata-lyze the interconversion of the active cortisol to its inactivemetabolite cortisone [3]. One of its two isomers, the 11 β-HSD type 1 acts predominantly as an 11-oxo-reductase (i.e.,it converts cortisone to cortisol), while 11 β-HSD type 2catalyzes 11 β-dehydrogenation (converting cortisol tocortisone). Cushing’s syndrome of apparent mineralocorti-coid excess is caused by 11 β-HSD deficiency [4, 5].

M. Frasconi : F. Mazzei (*)Dipartimento di Chimica e Tecnologie del Farmaco,Sapienza Università di Roma,Piazzale Aldo Moro 5,00185 Rome, Italye-mail: franco.mazzei@uniroma1.it

M. Mazzarino : F. BotrèLaboratorio Antidoping Federazione Medico Sportiva Italiana,Largo Giulio Onesti 1,00197 Rome, Italy

Anal Bioanal Chem (2009) 394:2151–2159DOI 10.1007/s00216-009-2914-6

In the last years, clinical and endocrinological studiesevidenced the different expressions of the 11 β-HSD in theperipheral tissues. This enzymatic system is sensitive toseveral regulatory factors local and systemic and to a largenumber of drugs including hormones widely employed inthe doping procedures, like growth hormone, insulin, andcorticosteroids [6, 7].

If the reductase activity is predominant (11 β-HSD1), theglucocorticoid activity is increased (i.e., active cortisol isproduced from inactive cortisone). Conversely, if the dehy-drogenasic activity predominates (11 β-HSD2), inactivecortisone is produced. The aim of the doping procedures isto increase the glucocorticoid activity by increasing the 11 β-HSD1 activity towards the 11 β-HSD2 in the organs target ofthe hormonal action (i.e. muscles, cardiovascular apparatus,etc.). This can be achieved by using substances able to: (a)stimulate the 11β-HSD1 activity, (b) suppress the 11β-HSD2activity, and (c) have both this actions [8].

Elevated cortisol levels in plasma, serum, and/or urinehave been also observed during space missions and areconsidered an indication of stress, as well as of motionsickness and acceleration during launch and re-entry [9].Therefore, detection of cortisol levels before, during, andafter space flight is a crucial factor to be taken into accountfor studying human health in space especially in the case oflong-duration mission [10].

Many analytical methods have been developed for thedetermination of corticosteroid concentrations in biologicalsamples such as liquid chromatograph (LC) coupled with aspectrophotometer detector (high performance liquid chro-matography (HPLC)/UV) or gas and liquid chromatographcoupled with a mass-selective detector (GC/mass spectrom-etry (MS) and LC/MS); they are relatively expensive, timeconsuming, and complex [9]. Additionally, the immuno-assays commonly used to measure salivary cortisol levels(i.e., enzyme-linked immunosorbent assays, luminescenceimmunoassays, time-resolved immunoassay) also takeconsiderable time or lack quantifiable results [11, 12].

Besides the clinical importance of the natural glucocorti-coid determination, another aspect to be considered is thedetermination of these substances in athletes before and afterstress for doping control purposes. With the increasedimportance of regular testing for illicit drug content in clinicalor forensic samples, a clear goal is the development of simple,rapid, economic, sensitive, robust, and highly specific analytedetection method. For this purpose, we propose to measurefree cortisol and cortisone with a real-time analysis using thesurface plasmon resonance (SPR) immunosensor. SPR is asurface-sensitive optical technique to probe and characterizephysicochemical changes of thin layers onmetal surfaces [13–15]. Conventional SPR is based on the construction ofexciting a single surface plasma wave on a metal surface,with the minimum intensity of the reflected laser beam

measured either in terms of its incident angle or wavelengthon resonance. Therefore, real-time tracking of the resonanceangle or wavelength of SPR is able to monitor thebiomolecular interactions in real time in terms of thevariation of effective refractive index of the medium [16].Thus, the SPR-based affinity measurement was proposed andwidely used in areas such as pharmaceutical developmentand life science [17–19] because it provides a rapid, safe,high-selectivity, and high-sensitivity method without anyisotopes or fluorescence labels [20, 21].

In this context, the aim of this work is to demonstrate thefeasibility of SPR-based immunosensors for the analysis ofcorticosteroids in urine and saliva samples. Saliva is amatrix very attractive for doping analysis in general and forcortisol and cortisone detection in particular. In fact, in thesalivary glands, the 11 β-hydroxysteroid dehydrogenaseactivity is prevalently ascribed to the 11 β-HSD2 isoform,so the increase of the cortisol/cortisone ratio value insalivary samples is more representative of 11 β-HSDactivity drug-induced alteration than in other biologicalfluids (blood and urine). To the best of our knowledge, wereport the first SPR-based direct detection of cortisol andcortisone, which exploits a reusable strategy of the sensorsurface via the regeneration of the binding between theanalyte-specific antibody immobilized on the sensor sur-face. The suitability of hydrogel-based coatings for theprevention of nonspecific binding is also investigated.

The results obtained have been then compared withthose obtained by the reference methods, i.e., the screeningmethods—accredited under the ISO17025—presently fol-lowed for the analysis of glucocorticoids by the World-Antidoping-Agency-accredited antidoping laboratory ofRome [22, 23].

Experimental

Chemicals and immunoreagents

Purified standards of cortisol, cortisone, testosterone, epites-tosterone, androsterone, and corticosterone were supplied bySigma-Aldrich (Milan, Italy). Standard solutions were pre-pared at 1 mg/ml in water; the working solutions wereprepared monthly and obtained by successive dilutions atconcentrations from 500 to 10 μg ml−1. All solutions werestored at −4 °C in the dark. The antirabbit monoclonalanticortisol and anticortisone antibody (1-mg ml−1 stock)were purchased from Gentaur (Brussels, Belgium).

SPR experiments

The SPR experiments were performed by an Eco ChemieAutolab SPR system (Ecochemie, The Netherlands). It

2152 M. Frasconi et al.

works with a laser diode fixed at a wavelength of 670 nm,using a vibrating mirror to modulate the angle of incidenceof the p-polarized light beam on the SPR substrate. Theinstrument is equipped with a cuvette. The planar gold SPRdisks and the gold disks modified with polycarboxylatehydrogel 80-nm thickness (HC80) were purchased fromXantec Bioanalytics (Germany). The gold sensor disks(25 mm in diameter) were mounted on the hemicylindricallens (with index-matching oil) to form the base of thecuvette. An O-ring (3-mm inner diameter) between thecuvette and the disk prevents leakage. An autosampler (EcoChemie) with controllable aspirating–dispensing–mixingpipette was used to add samples into the cuvette andprovide constant mixture by aspiration and dispensingduring measurements. This experimental arrangementmaintains a homogeneous solution and reproducible hydro-dynamic conditions. The temperature of the cuvette wasmaintained at 25±1 °C. Data were transmitted to a laptopcomputer and analyzed by an SPR software 4.1.2 versionfrom Eco Chemie.

Immobilization of antibody on the sensor disks

The planar gold SPR disks were extensively cleaned in afreshly prepared piranha solution (3:1 H2SO4 98%:H2O2

30%). After 1 h, the disks were thoroughly rinsed withwater, dried in a stream of nitrogen gas and immediatelyincubated overnight in a 1 mM of 11-mercaptoundecanoicacid (MUA) in ethanol. After self-assembled monolayer(SAM) formation, the disks were washed with ethanol andwater and dried with nitrogen gas.

For the detailed experiment, the MUA-modified goldsensor disk was first placed onto the prism of SPR system;a 10-mM acetate buffer solution (pH 5.0) was flowed overthe sensors to generate a baseline and the changes in theresonance angle were monitored from this point. Thesurface was cleaned with an alkaline buffer (pH 10, ionicstrength 2 M) to remove all ionic contaminants. Thecarboxyl functions on the SAM layer were activated witha mixture containing 0.5 mM ethyl(dimethylaminopropyl)carbodiimide (EDC) and 0.1 mM N-hydroxysuccinimide(NHS) in water. After removing the EDC–NHS mixtureand rinsing the disk, acetate buffer containing 1:20 dilutedrabbit monoclonal anticortisol antibodies (or anticortisoneantibodies for the cortisone assay) was flowed over thesensors surface for 15 min (20 min in the case ofanticortisone binding) to achieve a covalent cross-linkingby amino reactive groups of antibodies with the aldehydeterminals. Because of the steric hindrance and the interac-tion forces between antibody molecules, the deactivation ofnonreacted activated groups with ethanolamine is necessaryfor the reduction the nonspecific adsorption (see alsoFig. 1a).

In order to improve the antibody immobilization yieldsand prevent some nonspecific binding, a polycarboxylatehydrogel-coated sensor disk was used. The antibodies werecovalently immobilized onto this sensor chip by aminecoupling chemistry as described for the SAM-modifiedgold disk. A similar procedure was followed for theimmobilization of anticortisone antibodies (Fig. 1b).

Detection of cortisol and cortisone

The kinetic analysis of the reaction of cortisol (or cortisone)with anticortisol-modified coating (or anticortisone-modified coating) include the following steps (see alsoFig. 2): (1) establishment of the baseline—after coating theantibody, the flow cell was washed with the couplingbuffer, 10 mM phosphate-buffered saline (PBS) pH 7.5with 0.1 M NaCl for the anticortisol/cortisol coupling(0.1 M TRIS buffer pH 7.5, 0.1 M NaCl, and dimethyl

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Fig. 1 a Sensorgrams for the binding interaction of anticortisol onHC80-coated (solid line) and MUA-coated (dashed line) sensor.b Anticortisone immobilization on HC80-coated (solid line) andMUA-coated (dashed line) sensor. Subsequent injections of heightionic strength alkaline buffer (1), EDC-NHS (1), anticortisol (3) oranticortisone (3′), acetate buffer pH 5.0 (4), ethanolamine (5), andacetate buffer pH 5.0 (6)

SPR immunosensor for cortisol and cortisone determination 2153

sulfoxide (DMSO) 0.25% for the anticortisone/cortisonecoupling), during 2 min at flow rate of 100 μl/min. Theresonant angle was monitored until the baseline wasstabilized. (2) Association—the sample containing theantigen (30 μl) was injected in the flow cell and incubatedduring 20 min at stopped flow, while the SPR signal wasmonitored. After that, the flow cell was washed with thecoupling buffer during 1 min. (3) Dissociation—thenonspecific adsorptions were removed and the resonantangle was set up for about 10 min. (4) Regeneration—the

interaction between immobilized antibody and antigen wasremoved by injection of a pH 2.5 solution of 10 mMglycine–HCl for anticortisol/cortisol interaction (10 mMglycine–HCl pH 2.5 with NaCl 0.5 M for anticortisone/cortisone interaction); afterwards, the flow cell was washedwith PBS pH 7.5 and 0.1 M NaCl, and the baseline wasrestored (0.1 M TRIS buffer pH 7.5, 0.1 M NaCl, andDMSO 0.25% for the anticortisone/cortisone coupling).Then, the coated sensor disk was ready to run. Cortisol andcortisone solutions of known concentration were used togenerate a standard curve.

Kinetic analysis of the association phase was performedusing the programCLAMPdeveloped byMorton andMyszka[24]. CLAMP fits the data to a specified model by numericalintegration of the rate equations defined by the model.

The specificity of the biosensor was evaluated by testingthe cross-reactivity of several structural analog cortico-steroids (i.e., testosterone, epitestosterone, androsterone,and corticosterone). The stability and the reproducibilitywere also evaluated.

LC/MS conditions

All LC/MS–MS experiments were performed using anAgilent 1100 Series HPLC pump with binary gradientsystem and automatic injector (Agilent Technologies SpA,Milan, Italy). Reversed-phase liquid chromatography wasperformed on Supelco Discovery C18 column (2.1×150 mm, 5 μm). The solvents were: water containing0.1% (v/v) formic acid (eluent A) and acetonitrile contain-ing 0.1% (v/v) formic acid (eluent B). A gradient programwas set up starting at 15% B and increasing to 60% B in7 min and then, after 6 min, to 100% B in 1 min. Thecolumn was flushed for 1 min at 100% B and finally re-equilibrated at 15% B for 4 min. The flow rate was set at250 μl/min. Data were acquired using an Applied Bio-systems API4000 triple–quadrupole analyzer with bothpositive and negative electrospray ionization. The ionsource was operated at 550 °C; the applied capillaryvoltage was 5,500 V and multiple reaction monitoring(MRM) experiments were performed employing collision-induced dissociation (CID) using nitrogen as collision gasat 5.8 mPa, obtained from a dedicated nitrogen generatorsystem (Parker-Balston model 75-A74). For all experi-ments, the collision energy was 30 eV and the transitionsused for the MRM acquisition are: m/z 361/325, 361/343for cortisone; 363/121, 363/327 for cortisol; and 303/97 forthe internal standard 17alpha-methyltestosterone.

Sample preparation

To 3 ml of urine, 1 ml of phosphate buffer (pH 7.4), 50 μlof β-glucuronidase from Escherichia coli, and 50 μl of the

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Fig. 2 a SPR angle–time curves for the detection of the affinityinteraction between cortisol (50 µg l−1) and anticortisol immobilizedonto HC80-coated (solid line) and MUA-coated (dashed line) sensor.Subsequent injection of 10 mM PBS pH 7.5 with 0.1 M NaCl(1, coupling buffer), cortisol (2), 10 mM PBS pH 7.5 with 0.1 M NaCl(3, dissociation buffer), pH 2.5 solution of 10 mM glycine–HCl(4, regeneration buffer). Inset: curve describing the linear relationshipbetween the SPR angle shift and the cortisol concentration. b SPRresponse for the affinity interaction between cortisone (80 µg l−1) andanticortisone immobilized onto HC80-coated (solid line) and MUA-coated (dashed line) sensor. Subsequent injection of 1-M TRIS bufferpH 7.5, 0.1 M NaCl, and DMSO 0.25% (1′, coupling buffer), cortisone(2′), 1-M TRIS buffer pH 7.5, 0.1 M NaCl, and DMSO 0.25%(3′, dissociation buffer), 10 mM glycine–HCl pH 2.5 with NaCl 0.5 M(4′, regeneration buffer). Inset: curve describing the linear relationshipbetween the SPR angle shift and the cortisone concentration

2154 M. Frasconi et al.

internal standard (17α-methyltestosterone: 200 ng ml−1

final concentration) were added and incubated for 1 h at50 °C. After hydrolysis, 1 ml of carbonate/bicarbonatebuffer (pH 9.0) was added in order to alkalinize the sampleand the extraction was carried out with 10 ml of tert-butylmethylether. After centrifugation, the organic layerwas evaporated to dryness. The residue was reconstituted inthe association buffer and the solution was injected in theflow cell of the SPR. For LC/MS measurements, the residuewas reconstituted in 50 µl of mobile phase and an aliquot of10 µl was injected on the instrument system.

Saliva samples were analyzed as such, without anypretreatment.

Results and discussion

Immobilization of the antibody onto the sensor surface

We used SPR spectroscopy to characterize the immobiliza-tion of the antibody onto the modified gold disks. Anexample of anticortisol and anticortisone immobilizationonto the SAM or HC80 surfaces is presented in Fig. 1. Inthese sensorgrams, the injection steps, activation, antibodyattachment, and subsequent blocking of remaining activatedgroups are shown. Finally, the protein solution was replacedwith buffer for 5 min to quantitate the amount of proteinthat was irreversibly immobilized. Anticortisol boundirreversibly to the gold-modified surface as demonstratedby the stability of the resonance angle when the flow wasswitched back to acetate buffer. Treatment of the boundprotein with sodium dodecyl sulfate (SDS) at a concentra-tion of 0.5 mg ml−1 (SDS, a detergent that serves to removenoncovalently immobilized biomolecules from a surface)[25] did not result in removal of the anticortisol from thesurface (data not shown), supporting the result that theimmobilization was covalent. A complete and homoge-neous immobilization of the anticortisol over the coatedgold surface can be evidenced from the fact that anotherinjection of a higher concentration of the antibody(1 mg ml−1) showed only a small increase in the resonanceangle. These results suggest that the gold-modified surfaceswere fully covered by the immobilized antibody, which enablethe prevention of the nonspecific adsorption problem.

The antibody mass loading was calculated from thebaseline changes before and after protein injection. This canbe calculated from the obtained angle shift as 0.120° isequivalent to an amount of attached protein of 1 ng mm−2

[26]. In the case of anticortisone, the immobilization levelobtained was 1.6 and 4.7 ng mm−2 for MUA and HC80coating, respectively; instead for anticortisol, the valueswere 2.5 and 4.9 ng mm−2 for MUA and HC80 coating,respectively. The higher antibody immobilization levels

obtained with the polycarboxylate hydrogel coated (HC80)discloses a higher binding capacity of the 3D carboxyl-modified hydrogel. The reason for the observed loweradsorption of the antibody onto SAM surface is mainly dueto the smaller area of the SAM 2D surface, compared to the3D hydrogel coating, and also caused by the more orderedand closer structure of the monolayer due to the presence oflong alkyl chains [27]. Thus, fabricated anticortisol andanticortisone immobilized surfaces were used as recogni-tion platforms for repetitive analysis of cortisol andcortisone, respectively.

Response to the cortisol and cortisone by SPRimmunosensor: affinity reaction

The SPR response observed for the binding affinitybetween cortisol and the immobilized anticortisol isreported in Fig. 2a. As shown, a rapidly rinsing signalresponse is generated as soon as the cortisol solution(50 μg l−1) begins to flow over the antibody modifiedsurface (MUA or HC80). This rise is due to a combinationof the bulk shift effect, which is proportional to theconcentration of the binding molecule over the surface[28] and the excess concentration of these molecules on thesurface due to the adsorption. The SPR signal thenstabilizes, which indicates that an equilibrated state hasbeen achieved in which the rate of the cortisol adsorptiononto the surface is equal to the rate of desorption off of thesurface. It should be noted here that a slight amount ofinstrument drift also occurs during this time, with theamount of drift being consistent and different for eachcoating and at each concentration. A similar behavior wasobserved for the anticortisone/cortisone binding interaction(Fig. 2b). This effect gives the appearance of a smallcontinual increase in the SPR signal even after theequilibrium is achieved. Although we are uncertain aboutthe exact cause of this phenomenon, we believe that it mayresult from differences in the structure of the solvent withinthe different interfacial environments (MUA and HC80).This can be expected to result in different values of therefractive index local to the surface and differences in thesensitivity of the refractive index to small changes intemperature, which could subsequently be reflected indifferences in the SPR signal drift [29, 30]. Followingequilibration, as the flow was switched back to buffersolution, the resonant angle shift remained almost stabledue to the strong affinity interaction between antigen andantibody. Only a small decrease was observed, which is dueto the dissociation of the excess of nonbounded antigens,implying on false response of the sensor. Moreover, theeffect due to the different refractive indexes of the sampleand buffer solution stated as baseline are compensatedwhen the flow filled with the buffer is monitored; then, the

SPR immunosensor for cortisol and cortisone determination 2155

resonance angle for the calibration curve (Fig. 2a, b, inset)was calculated from the difference between buffer andprevious baseline.

Regeneration of the biosensor surface is an importantaspect for multiple and cost-effective analysis. It is alsohighly desirable if such a surface can be regenerated with asimple reagent that largely preserves the native bindingstructures. Different regeneration solutions at different pHand ionic strength were evaluated for their ability toregenerate the antibody surface. Among them, 10 mMglycine–HCl buffer (pH 2.5) was shown to be highly effectivein anticortisol regeneration, while for anticortisone regenera-tion the best results were obtained with 10 mM glycine–HClpH 2.5 with NaCl 0.5 M. The sudden drop of the baselineupon regeneration buffer injection is due to the difference inthe solution refractive index relative to the buffer. Rinsingwith the buffer quickly established the original level prior toantigen injection indicating the regeneration of the antibodysurface which could be used for the next affinity interactionwith the next concentration of antigen. According to thecalibration curve, the effectiveness of the HC80 antibodycoating towards the analysis of cortisol and cortisone isdemonstrated from the sensitivity and detection limits that aresignificantly higher than those obtained by immobilizing theantibody onto MUA coating. The anticortisol-modified HC80coating exhibits a linear detection range from 5 to 154 μg l−1

(30÷174 μg l−1 for the cortisone detection on to theanticortisone-modified HC80 coating) with a cortisol detec-tion limit of 2 μg l−1 (9 μg l−1 for cortisone).

The higher response obtained with the antibody immo-bilized onto polycarboxylate-hydrogel-coated (almost two-fold), as compared to the monolayer of antibody onto MUAcoating, is due to the higher content of antibody in the 3Dcomposite structure.

The SPR signal upon binding as a function of time containskinetics information, and this allows a full kinetic character-ization of the interaction between cortisol and anticortisol aswell as cortisone and anticortisone. The kinetic analysis of theassociation phase was performed according to a predefinedbinding model using the program CLAMP.With this program,it is possible to fit the association and dissociation phasesimultaneously but since the dissociation proceeds too fast toget reliable analyses with CLAMP, we only analyzed theassociation phase [31]. Taking into account that the diffusionof analyte to the sensor surface is the rate-determining step(mass transport limitation, MTL), a fit with a model includingsuch a diffusion step should give better result [32, 33]:

A bulk ���!kt A sensor

A sensor þ Bkd

ka���! ��� P

In this mass transport model for a bimolecular interac-tion, A is the antigen and B is the immobilized antibody. kt

is the rate constant of diffusion of analyte from bulksolution to the sensor surface, while ka and kd are the on andoff rate constants. In principle, the off rate constant can beobtained from the dissociation phase of the sensorgrams.On the basis of the dissociation constant KD and the offrate, the association rate constant was calculated (ka=kd/KD). However, as reported previously, for MTL-influencedprocesses, the dissociation phase is strongly influenced byrebinding of released antigen [34].

In the fits of cortisol/anticortisol affinity sensorgram, kdis approximately 0.7 s−1 (the dissociation proceeds ex-tremely rapidly) and the maximum binding capacity Rmax atthe value obtained from equilibrium analysis is 0.179° withthe anticortisol HC80 coating and 0.109° with the MUAcoating. This yielded noncorrelated value for ka and kt. Thetransport model deals very well with the linear increase ofthe signal: this initial increase is an indication for MTL andoccurs when dissociation can be neglected. For thepolycarboxylate-hydrogel-coated, when the signalapproaches saturation, the increase is retarded comparedto the model. This might be attributed to effects in theimmobilizing agent, e.g., less accessible sites deeper in thelayer [33]. The value of kt increases with the bindingcapacity, from 1.01(±0.02)×10−5m s−1 for the MUAcoating to 1.45(±0.07)×10−5m s−1 for the HC80 coating.The dissociation binding constant (KD) obtained for thehigh binding surface was 0.52±0.03 µM for cortisol and0.27±0.04 µM for cortisone, while, for the low bindingsurface, KD was 0.33±0.02 and 0.10±0.02 µM, respective-ly. Based on the values of KD and kd, ka is estimated to be1.3(±0.4)×106M−1s−1 for cortisol/anticortisol interaction,and 2.8(±0.6)×106M−1s−1 for cortisone/anticortisone in-teraction. In the approach presented in this study,determination of kd from the dissociation phase iscritically important. The dissociation phase is stronglyinfluenced by rebinding: addition of competing antigenstrongly enhances dissociation. Furthermore, for lowanalyte concentration (≤ KD), the whole association curvecan be fitted very well with the model also in the case ofmass transport limitation.

Detection of cortisol and cortisone in urine and salivasamples: selectivity of the SPR immunosensor

Biological fluids are complex matrices containing highconcentrations of proteins and other compounds such assteroids with a chemical structure similar to cortisol andcortisone. Thus, the sensor response has to be cautiouslyanalyzed to avoid either false-positive or false-negativesignals or a combination of both. An excellent biosensorshould not only posses a good sensitivity but should alsohave the capability to specifically detect the target analytein in-vivo and in-vitro analysis of complex clinical samples

2156 M. Frasconi et al.

to enable error-free measurement. In order to detect theselectivity of the present biosensors, we examined the effectof four compounds, which belong to the steroid family(testosterone, epitestosterone, and corticosterone), and theyare always present in both biological fluids considered inthis study.

From the selectivity test (Table 1), the polycarboxylatehydrogel HC80-coated SPR sensor disk proves to be thebest suited as immobilized matrix for the detection ofcortisol and cortisone in complex matrices. These resultsconfirm that the developed strategy has sufficient speci-ficity and our target analyte can be identified with highselectivity. The design and preparation of surface coatingsthat suppress or even prevent nonspecific adsorption areimportant in biosensor design to ensure specific recogni-tion of the analyte alone. To assess the suitability of thebiosensor surface coating to prevent the nonspecificadsorption, a model mixture containing ovalbumin, bovineserum albumin, and RNase B (1:1:1 molar ratio) spikedwith cortisol or cortisone was tested. In the case of cortisolimmunosensor, stock solutions of 154 μg l−1 of cortisolwere prepared containing a 10-, 100-, and 1,000-foldexcess, respectively, of each component of the proteinmixture. Dilution series were prepared (154–5 μg l−1)from these solutions, and SPR measurements were carriedout onto the anticortisol-modified HC80 coating. Thedetection limit was not influenced by protein concentra-tion up to 100-fold higher than the cortisol concentration.Experiments performed with 1,000-fold excess of eachcomponent of the protein mixture showed a significativedisturbance of the association phase due to a majorcontribution of the protein mixture to the bulk effect. Thissuggests that the presence of interfering compounds at acertain concentration can affect the sensitivity of thebiosensor. For the antibody immobilized onto MUAcoating, the bulk effect already interfered with the cortisolresponse at a tenfold excess. Similar results were obtainedfor cortisone immunosensor. The hydrogel-based coatingswere previously shown to be able to minimize nonspecificadsorption (i.e., protein) depending on the nature of thesecoatings and also by the way it was grafted on the sensorsurface [35, 36]. However, in view of possibly higheramounts of contaminant proteins in some biological

samples (i.e., blood or plasma) that could influence thedetection limit, a pretreatment procedure (partial purifica-tion/concentration) of these samples should be taken in toaccount.

Detection of cortisol and cortisone in real samples

The calibration curves for cortisol and cortisone determi-nation in saliva and urine samples are reported in Fig. 3.The changes in slopes for samples with cortisol andcortisone in the different matrices were compared. Cortisoland cortisone were detected in saliva and urine at concen-trations as low as 4 and 10 µg l−1, respectively (Table 2).The biosensor shows a useful detection range between 9and 132 µg l−1 for cortisol (30 and 143 µg l−1 forcortisone). Results obtained in the cortisol analysis ofsaliva samples are comparable with those reported in anSPR immunosensor-based paper recently published [37],although our method does not need of any samplepretreatment procedures.

Figure 4a, b shows the correlation of quantitative dataobtained by the SPR technique and the reference LC/MS–MS method for the analysis of cortisol and cortisone inurine samples, respectively. As it can be seen, results arehighly correlated, in a range of concentration that issufficiently extended, for both analytes. The same goodcorrelation has been obtained in the analysis of salivamatrices (data not reported). The results obtained evidencedthe feasibility of the developed method in a variety ofphysiologic and pharmacologic assays.

Conclusion

We report in this work a rapid assay for the measurement ofcortisol and cortisone in saliva and urine samples. Levelsof cortisol and cortisone in biological fluid can be measuredand used as an indicator of stress. While measurements ofthese target molecules in saliva or urine are as much as 100-fold lower than in blood, it is thought that salivary cortisoland free cortisol in blood correlate more closely with stressthan does total blood cortisol. Also, collecting blood mayinduce stress and alter the measurement while saliva

Antibody Coating Response (%)

Cortisol Cortisone Testosterone Epitestosterone Corticosterone

Anticortisol MUA 100.0 12.4 4.2 6.1 9.2

HC80 100.0 7.3 3.6 5.1 6.0

Anticortisone MUA 7.5 100.0 7.8 5.9 10.9

HC80 6.1 100.0 5.3 4.5 7.6

Table 1 Selectivity of the SPRbiosensor for detection of corti-sol and cortisone

SPR immunosensor for cortisol and cortisone determination 2157

collection is stress free. The collection of saliva does notrequire trained medical skills and can, therefore, simplifycollection of samples at numerous time points. In particular,for the saliva samples, method requires no chemicalextraction or complex sample pretreatment. The developedimmunosensor chip is highly sensitive with a rapidresponse time of approximately 15–20 min. Reusability of

the sensor chip is highly impressive; the same sensor chipwas stable and responsive for as many as 100 determinationcycles. The SPR-based immunosensor investigated hereenhanced the sensitivity thanks to the disk coating to afforda low detection limit of 3 μg l−1.

The very good correlation of our data with the referencemethod shows that the proposed technique is of potentialutility for the resolution of clinical, pharmacological, andantidoping issues.

Concentration (µg l-1

)

0 20 40 60 80 100 120 140

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Concentration (µg l-1

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Fig. 3 a Standard curve for cortisol detection in urine (filled circles)and saliva (empty circles). b Standard curve for cortisone detection inurine (filled circles) and saliva (empty circles). Error bars representstandard deviation for experiments run in triplicate

Table 2 Analytic performance of cortisol and cortisone determination in real samples

Analyte Sample Linearity range (μgl−1) Slope (m° lμg−1) LOD (μgl−1) RSD (%) BIAS (%)

Cortisol Urine 9÷132 0.54±0.02 3 8.9 7.6

Saliva 13÷125 0.46±0.03 4 9.3 9.1

Cortisone Urine 30÷143 0.46±0.01 9 8.1 8.9

Saliva 35÷134 0.28±0.02 10 8.7 9.0

LC-MS Response Ratio

0.002 0.004 0.006 0.008 0.010 0.012 0.014

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0.0 0.1 0.2 0.3 0.4 0.5

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Fig. 4 a Correlation curve for SPR and LC–MS response for cortisoldetection in urine sample. b Correlation curve for SPR and LC–MSresponse for cortisone detection in urine sample

2158 M. Frasconi et al.

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