ultrasensitive amperometric magnetoimmunosensor for human c-reactive protein quantification in serum

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Sensors and Actuators B 188 (2013) 212– 220

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Sensors and Actuators B: Chemical

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ltrasensitive amperometric magnetoimmunosensor for human-reactive protein quantification in serum

erta Esteban-Fernández de Ávilaa,1, Vanessa Escamilla-Gómeza,1,usana Campuzanoa,1, María Pedreroa,1, J.-Pablo Salvadorb,c,2,.-Pilar Marcob,c,2, José M. Pingarróna,∗

Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid, SpainNanobiotechnoly for Diagnostics (Nb4D), Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, SpainCIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain

r t i c l e i n f o

rticle history:eceived 13 June 2013eceived in revised form 5 July 2013ccepted 9 July 2013vailable online 17 July 2013

eywords:RP

a b s t r a c t

A highly sensitive magnetoimmunosensor for the determination of human C-reactive protein (CRP)is described in this work. A sandwich format involving covalent immobilization of the captureantibody (antiCRP) onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), theantigen–antibody reaction and incubation of the modified MBs with a biotynilated antibody (biotin-antiCRP), is used. Furthermore, an incubation step with a Streptavidin HRP (Strp-HRP) conjugate isemployed to allow monitoring of the affinity reaction. The resulting modified MBs are captured by a mag-

mperometric magnetoimmunosensorsagnetic beadsuman serum

net placed under the surface of a disposable gold screen-printed electrode (Au/SPE) and the amperometricresponses are measured at −0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate. The CRPmagnetoimmunosensor exhibited a wide range of linearity between 0.07 and 1000 ng mL−1 with a lowdetection limit of (0.021 ± 0.005) ng mL−1. The magnetoimmunosensor was successfully applied to the

al sta

. Introduction

C-reactive protein (CRP) is an alpha globulin with a molecularass of ∼110–140 kDa, composed of five identical monomers of

5 kDa each non-covalently assembled as a cyclic pentamer [1].RP synthesis in liver by the hepatocytes is stimulated by certainytokines (IL-1�, IL-1�, TNF-� and �, and indirectly by IL-6) [2,3].his acute-phase protein is normally present as a constituent oferum or plasma at levels lower than 3 mg L−1. CRP is considered

nonspecific biomarker of inflammation and infection that cane used as a predictive risk marker of cardiovascular disease in

∗ Corresponding author. Tel.: +34 913944315; fax: +34 913944329.E-mail addresses: berta.efa@quim.ucm.es (B. Esteban-Fernández de Ávila),

aneeg@quim.ucm.es (V. Escamilla-Gómez), susanacr@quim.ucm.esS. Campuzano), mpedrero@quim.ucm.es (M. Pedrero), jpablo.salvador@iqac.csic.esJ.-P. Salvador), pilar.marco@cid.csic.es (M.-P. Marco), pingarro@quim.ucm.esJ.M. Pingarrón).

1 Tel.: +34 913944315; fax: +34 913944329.2 Tel.: +34 934006100.

data documenting that chronic low-level CRP concentrations canprovide prognostic information about the risk of future coronaryevents in apparently healthy people, and help to predict the risk ofischemic stroke, acute myocardial infarction and peripheral vascu-lar diseases [7]. Indeed, the American Heart Association and theUnited States Center for Disease Control established three cate-gories of CRP concentration for the evaluation of cardiovasculardisease (CVD) and myocardial infarction risk [8–12]: low risk fora CRP concentration below 1.0 mg L−1; intermediate risk for con-centrations between 1.0 and 3.0 mg L−1; high risk for levels above3.0 mg L−1. In combination with other tests, CRP assays can con-tribute to patient care and can be more useful and accurate indiagnosing ongoing problems than almost any other form of patientassessment. Indeed, high sensitive CRP (hs-CRP) assays can deter-mine heart disease risk in people with undetected heart diseaseand risk of complications for people that have already suffered aheart event.

Nowadays, there are available several tests for C-reactiveprotein that are employed in clinical practice based on nephelo-

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oint-of care analysis [16]. Other routine automated methods avail-ble for CRP quantification used in the clinical laboratories typicallyave limits of quantification of 3–8 mg L−1 [14]. A lot of effort haseen made in the last years to develop highly sensitive methodsuitable for predicting future risk of coronary events in apparentlyealthy individuals. Surface Plasmon Resonance (SPR) biosensinglatforms for CRP were developed reaching a detection limit of

nM (1 �g mL−1) of purified CRP [17] and a linear range within6–40 nM (2–5 �g mL−1) [18]. Meyer et al. also developed a methodased on magnetic detection with a linear detection range between5 and 2500 ng mL−1 [19]. An indirect-competitive quartz crystalicrobalance immunosensor for CRP was reported by Kim et al.ith a wide linear concentration range of 0.130–25,016 ng mL−1

nd a LOD of 0.130 ng mL−1 [20]. A chemiluminiscence method-logy has been developed by Lee et al. within an integratedicrofluidic system for fast and automatic detection of CRP pro-

ein (LOD 0.0125 mg L−1) [12]. Buch and Rishpon developed aisposable sensor with screen-printed electrodes modified withulti-walled carbon nanotubes and protein A to detect CRP levels

f 0.5 ng mL−1 [7]. Recently, a number of CRP assays using electro-hemical impedance spectroscopy (EIS) have also been reported.owever, these impedimetric approaches suffer from limited sen-

itivity [1], not demonstrable specificity [21], or do not encompass clinically relevant range [22,23].

The use of magnetic beads (MBs) in the development of electro-hemical immunosensors has demonstrated to be an useful tool tomprove their sensitivity, reduce the time of analysis and minimize

atrix effects [24–31]. Electrochemical magnetoimmunosensorsllow the analysis of complex samples with no need for pre-nrichment or purification steps due to the “extraction” of thearget analyte from the complex matrix and the measurementf the electrochemical signal in a “clean” environment [32]. Anntecedent of an electrochemical immunosensor for the determi-ation of CRP using MBs is the paper reported by Gan et al. [33].hey immobilized HRP-labeled detection antibodies on Fe3O4@Auagnetic nanoparticles and used the decrease in the electrochem-

cal signal of hydrogen peroxide upon antigen binding on carboncreen-printed electrodes (SPCEs) modified with a chitosan mem-rane and Fe (III) phthalocynanine for monitoring the affinityeaction.

In this paper, an amperometric magnetoimmunosensor for theetermination of CRP using a sandwich configuration, Au/SPEs andOOC-modified MBs (HOOC-MBs) is reported for the first time. Theeveloped methodology involved the immobilization of the cap-ure antibody on the HOOC-MBs, and successive incubation stepsf the modified MBs with the analyte, a specific biotin-labeledetection antibody and a Strep-HRP conjugate. The electrochem-

cal detection of the enzyme reaction product is carried out at aisposable Au/SPE using TMB as electron transfer mediator and2O2 as the enzyme substrate. The applicability of the developedisposable magnetoimmunosensor for clinical diagnosis has beenuccessfully demonstrated by the analysis of a commercial serumith a certified CRP content.

. Materials and methods

.1. Apparatus and electrodes

Amperometric measurements were carried out with an ECOhemie Autolab PGSTAT 101 potentiostat using the softwareackage NOVA 1.7. A P-Selecta (Scharlab) ultrasonic bath and

and Actuators B 188 (2013) 212– 220 213

electrode and an Ag pseudo-reference electrode, were used.Homogenization of the solutions was facilitated with a BunsenAGT-9 Vortex. Magnetic separation steps for incubation/washingprocesses were performed using a Dynal MPC-S (product No.120.20, Dynal Biotech ASA, Norway) magnetic particle concentra-tor. A neodymium magnet (AIMAN GZ) was used to control theattraction of the modified-MBs to the Au/SPE surface.

2.2. Reagents and solutions

Carboxylic acid-modified MBs (HOOC-MBs, 2.8 �m,10 mg mL−1, Dynabeads® M-270 Carboxylic Acid) were pur-chased from Dynal Biotech ASA. 2-(N-morpholino)ethanesulfonicacid (MES), NaCl, KCl, Tween®20, sodium di-hydrogen phosphate,di-sodium hydrogen phosphate, and Tris–HCl were purchasedfrom Scharlab. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide(EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and ethanolaminewere purchased from Sigma–Aldrich.

Anti-human C-reactive protein antibody (antiCRP), and humanC-reactive protein (CRP) were kindly provided from Audit Diag-nostics. Biotin-conjugated-antiCRP (biotin-antiCRP) was preparedby using EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce) according tothe protocol described in Section 2.3. Heparin sodium saltfrom porcine intestinal mucosa (H3149) from Sigma–Aldrich,D-dimer (ab35949) from Abcam, bovine serum albumin (BSA-TYPE VH, 1066.0010) from Gerbu, NT-proBNP and cardiactroponin T (cTnT) from Hytest Ltd., a high sensitivity Strep-tavidin HRP (Strep-HRP) conjugate from Roche (Ref: 11 089153 001, 500 U mL−1) and TMB-H2O2 K-Blue reagent solu-tion from Neogen in a ready-to-use reagent format (K-Blueenhanced-activity substrate, also containing H2O2) were alsoused.

It is important to remark that a new CRP solution was prepareddaily from a small concentrated aliquot kept frozen at −40◦ C dueto the short biological half-time life of CRP (less than 24 h) [34].

Moreover, a WHO 1st International Standard for Human C-reactive protein (National Institute for Biological Standards andControl, NIBSC, code: 85/506), containing 100 �g mL−1 CRP wasused as the sample reference material.

The following solutions prepared in deionized water were alsoemployed: 0.1 M phosphate buffer, pH 7.0; 0.1 M phosphate buffer,pH 8.0; phosphate-buffered saline (PBS) consisting of 0.01 M phos-phate buffer solution containing 137 mM NaCl and 2.7 mM KCl, pH7.5; 0.025 M MES buffer, pH 5.0; 0.01 M sodium phosphate buffersolution (pH 7.5, PBST) consisting of PBS with 0.05% Tween®20, and0.1 M Tris–HCl buffer, pH 7.2.

Activation of the HOOC-MBs was carried out with an EDC/sulfo-NHS mixture solution (50 mg mL−1 each in MES buffer, pH 5.0). Theblocking step was accomplished with a 1 M ethanolamine solutionprepared in a 0.1 M phosphate buffer solution of pH 8.0.

All chemicals used were of analytical-reagent grade, and deion-ized water was obtained from a Millipore Milli-Q purificationsystem (18.2 M� cm).

2.3. AntiCRP biotinylation

4 mg of antiCRP antibody were dissolved in 2 mL of borate buffer(0.2 M boric acid/sodium borate, pH 8.7) and 0.34 mg of sulfo-NHS-LC-LC-Biotin dissolved in 0.2 mL of MiliQ water were added.The reaction was allowed proceeding under continuous stirring

aliquots were prepared in 10 mM PBS (pH 7.5) and stored at4 ◦C.

214 B. Esteban-Fernández de Ávila et al. / Sensors and Actuators B 188 (2013) 212– 220

F r. Them

twctsvmoCa(ta1i6bac3PbwbposAe

ig. 1. Schematic display of the developed CRP sandwich magnetoimmunosensoediated reduction of H2O2 with TMB at the Au/SPE are also shown.

.4. Modification of MBs

A 3-�L aliquot of the HOOC-MBs commercial suspension wasransferred into a 1.5 mL eppendorf tube. Then, the MBs wereashed twice with 50 �L MES buffer solution during 10 min under

ontinuous stirring (600 rpm, 25 ◦C). Between each step the par-icles were concentrated using a magnet and, after 4 min, theupernatant was discarded. The MBs carboxylic groups were acti-ated by incubation during 35 min in 25 �L of the EDC/sulfo-NHSixture solution. The activated MBs were washed twice with 50 �L

f MES buffer and re-suspended in 25 �L of a 50 �g mL−1 anti-RP solution (in MES buffer). The antiCRP was captured onto thectivated beads during 60 min at 25 ◦C under continuous stirring600 rpm). Subsequently, the antiCRP-modified MBs were washedwice with 50 �L of MES buffer solution. Thereafter, the unreactedctivated groups on the MBs were blocked by adding 25 �L of the

M ethanolamine solution (in 0.1 M phosphate buffer, pH 8.0) andncubating the suspension under continuous stirring (600 rpm) for0 min at 25 ◦C. After two washing steps with 50 �L of 0.1 M Tris-uffer (pH 7.2) and another one with 50 �L of PBST (pH 7.5), thentiCRP-coated MBs were re-suspended in 25 �L of a variable con-entration of the free antigen (in PBST, pH 7.5) and incubated during0 min. Then the modified MBs were washed twice with 50 �L ofBST (pH 7.5) and immersed in a solution containing 1 �g mL−1

iotin-antiCRP solution (in PBST, pH 7.5) during 30 min. After twoashing steps with the same buffer, the resulting beads were incu-

ated during 30 min in a Strep-HRP (1:1000) solution in PBST,H 7.5. Finally, the modified-MBs were washed twice with 50 �Lf PBST buffer solution (pH 7.5), re-suspended in 45 �L of 0.1 Modium phosphate buffer solution (pH 7.0) and immobilized on theu/SPE surface by placing a neodymium magnet under the workinglectrode surface.

.5. Amperometric measurements

Amperometric measurements were carried out at an appliedotential of −0.10 V vs. the Ag pseudo-reference electrode. Aftertabilization of the blank current, 5 �L of the TMB-H2O2 solutionere deposited on the working electrode surface and the currentas recorded. The analytical signal was the current measured after

00 s.Unless otherwise indicated, the reported data corresponded to

he average of at least three replicates.

.6. Analysis of a certified serum sample

The magnetoimmunosensor was employed for the analysisf an International Standard from pooled normal human serum

enzyme and electrode reactions involved in the amperometric detection of the

containing 100 �g mL−1 of CRP. According to the instructions rec-ommended by the supplier of the serum sample, the total contentof the ampoule (containing 50 �g of CRP from 0.5 mL pooled nor-mal human serum) was reconstituted at room temperature with0.5 mL of PBST, pH 7.5, dissolved totally by gentle swirling to avoidfroth, subdivided in small fractions and frozen rapidly to below−40 ◦C in the dark. CRP determination was performed after a 1000-times dilution with PBST. The antiCRP-modified MBs, prepared asdescribed in Section 2.4, were re-suspended in a 25 �L aliquot ofthe prepared serum samples during 30 min and a similar procedureto that described in Sections 2.4 and 2.5 was followed.

3. Results and discussion

The fundamentals of the immunosensor configuration as wellas of the electrochemical transduction are displayed in Fig. 1.Briefly, the antiCRP was covalently immobilized onto the HOOC-MBs previously activated with an EDC/sulfo-NHS solution. After ablocking step with ethanolamine of the unreacted activated groupsof the commercial MBs, the antiCRP-MBs were incubated in thesample solution and the target protein was sandwiched with thebiotinylated-detector antibody labeled in a final step with a Strep-HRP conjugate.

The MBs bearing the sandwich immunocomplexes were mag-netically captured on the Au/SPE by means of a neodymium magnetplaced under the working electrode surface, and the biorecognitionevent was monitored by amperometric measurement of the reduc-tion current generated after the addition of the TMB-H2O2 substrate[35]. Using this methodology, the Au/SPE acted only as the electro-chemical transducer while all the immunoreactions occurred onthe surface of the MBs.

3.1. Optimization of the working variables

The influence of the capture and detection antibodies concentra-tions in the incubation solutions was tested by checking the amper-ometric response measured at −0.10 V (vs. Ag pseudo-referenceelectrode) with the corresponding magnetoimmunosensors for1 ng mL−1 CRP. The applied detection potential was optimized pre-viously using the mediated reduction of H2O2 with TMB at anAu/SPE [31].

The effect of the amount of capture antibody immobilized on theactivated MBs is shown in Fig. 2a. The measured current increasedwith the antibody loading up to 50 �g mL−1 and decreased for

antiCRP was selected for further work. Moreover, the influence of

B. Esteban-Fernández de Ávila et al. / Sensors and Actuators B 188 (2013) 212– 220 215

0.0 0.5 1.0 1.5 2.00.00

-i, µ

[biotin-antiCRP], µg mL-1

0 20 40 60 80 10 00.00

-i, µ

[antiCRP], µg mL-1

Fig. 2. Effect of the antiCRP concentration immobilized on the activated HOOC-MBs (a) and of the biotin-antiCRP concentration (b) on the amperometric responses measuredw , 0.1 MV tin-ant

FseEas

for a CRP concentration of 1000 ng mL . This concentration valuewas chosen in order to work under conditions where possibleaggregation of protein may occur. As it can be seen, although theimmunosensor response was sufficient to discriminate between 0

Table 1Optimization of the different experimental variables involved in the preparation ofthe electrochemical magnetoimmunosensor for CRP.

Experimental variable Tested ranges Selected values

[antiCRP], �g mL−1 0–100 50[biotin-antiCRP], �g mL−1 0–2 1[Strep-HRP] 1:500–1:2000 1:1000VMBs, �La 2–6 3

ith the sandwich magnetoimmunosensor for 1 ng mL−1 CRP. Supporting electrolyteinc = 25 �L; Eapp = −0.10 V vs. Ag pseudo-reference electrode. Other conditions: [bioriple of the standard deviation (n = 3).

he biotin-antiCRP concentration was tested in the 0–2 �g mL−1

ange. As can be seen in Fig. 2b, the current increased with theiotinylated antibody concentration up to 1 �g mL−1 leveling offbove this value. Consequently, this biotin-antiCRP loading waselected for further studies.

Another relevant variable to be optimized is the Strep-HRP con-entration used to label the secondary antibody. Several dilutionactors were applied to the original reagent and, as it is shown inig. 3, the maximum signal was obtained using a 1:1000 dilutionhich agreed with the value recommended by the reagent supplier.

The time implied in the different incubation steps was optimizedn a similar manner. All the tested ranges for the different vari-bles as well as the selected values for the magnetoimmunosensorunctioning are summarized in Table 1.

Furthermore, we evaluated the effect of reducing the assayime. This was carried out by doing the antigen capture and

1:2000 1:1000 1:5000.00

-i, µ

[Strep-HRP], dilution factor

ig. 3. Effect of the Strep-HRP dilution factor on the amperometric response mea-ured for 1 ng mL−1 CRP with the sandwich magnetoimmunosensor. Supportinglectrolyte, 0.1 M sodium phosphate solution, pH 7.0; VMBs = 3 �L; Vinc = 25 �L;app = −0.10 V vs. Ag pseudo-reference electrode. Other conditions: [biotin-ntiCRP] = 1 �g mL−1; [antiCRP] = 50 �g mL−1. Error bars calculated as triple of thetandard deviation (n = 3).

sodium phosphate solution, pH 7.0; VMBs = 3 �L; Strep-HRP dilution factor = 1:1000;tiCRP] = 1 �g mL−1 in (a) and [antiCRP] = 50 �g mL−1 in (b). Error bars calculated as

containing biotin-antiCPR and Strep-HRP), instead of the threesteps described in Section 2.3. Fig. 4 compares the amperomet-ric responses obtained using the different incubation protocols

tantiCRP, min 15–120 60tCRP, min 15–60 30tbiotin-antiCRP, min 15–60 30tStrep-HRP, min 15–60 30VTMB-H2O2 , �La 1–10 5

aExperimental variables optimized in previous work [36].

321Blank

Number of incubation steps

-i, µ

Fig. 4. Amperometric responses measured at −0.10 V vs. the Ag pseudo-referenceelectrode for solutions containing 1000 ng mL−1 CRP (gray bars) as a function of thenumber of incubation steps used to modify the MBs in the sandwich immunoassayconfiguration. Error bars calculated as triple of the standard deviation (n = 3).

216 B. Esteban-Fernández de Ávila et al. / Sensors and Actuators B 188 (2013) 212– 220

0.0 1.0 1.5 2.0 2.5

-i, µ

log ([C RP], n g mL-1)

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0.8-i, µ

log ([CRP], ng mL-1)

F he samt

aorSciawn

ftwo(oc

ctsstaamplisatth

ig. 5. Calibration curves constructed for CRP standards prepared in PBST (a) or in the standard deviation (n = 3).

nd 1000 ng mL−1 CRP (cut-off value for this biomarker) using onlyne or two incubation steps, a remarkable better signal-to-blankatio was achieved when the three incubation steps described inection 2.3 were accomplished. It is worth to mention that theurrent measured in the absence of CRP (white bar in Fig. 4) isndependent of the number of incubation steps used. Taking intoccount these results, the protocol involving three incubation stepsas selected to evaluate the analytical characteristics of the mag-etoimmunosensor.

.2. Analytical characteristics

Under the optimized experimental conditions, a calibration plotor CRP was constructed in PBST (Fig. 5a). A linear relation betweenhe measured current and the logarithm of CRP concentrationas found over the 0.07–1000 ng mL−1 range, with a slope value

f (2.03 ± 0.07) × 10−7 A and an intercept of (1.2 ± 0.1) × 10−7 Ar = 0.997). The linear calibration plot, covering more than fourrders of magnitude of CRP concentration, leveled off for higheroncentration values.

The limits of detection (LOD) and determination (LQ) werealculated according with the 3 s/m and 10 s criteria, respec-ively, where m is the slope of the linear calibration plot, and

was estimated as the standard deviation of the amperometricignals obtained in the absence of CRP, expressed in concentra-ion units when determining LQ. LOD and LQ values were 0.021nd 0.07 ng mL−1, respectively. It should be emphasized that thechieved LOD is 48,000 times lower than the 1000 ng mL−1 mini-al clinical threshold recommended in serum/plasma samples to

redict a moderate risk of myocardial infarction and stroke. Thisow LOD means a great practical advantage in the case of exist-ng strong matrix effects for real clinical samples, which makes aample dilution necessary before the analysis. The Kd value of thentibody-antigen interaction was estimated by ELISA (see Suppor-ing Information). The low Kd value obtained, 4.75 nM, indicatedhe high affinity of such interaction [37], therefore supporting theigh sensitivity found with the magnetoimmunosensor.

The reproducibility of the amperometric responses obtainedith different magnetoimmunosensors constructed following

he same protocol was evaluated by performing measurementsor 500 ng mL−1 CRP. Results from 9 different immunosen-ors prepared on the same day yielded an RSD value of 6.5%hus demonstrating that the magnetoimmunosensor fabrication

ple reference material diluted 20,000 times (b). Error bars estimated as a triple of

procedure (MBs modification and magnetic capture on the Au/SPEsurface) and the amperometric transduction method were reli-able.

The developed methodology implies that the immunoreactionsare carried out only on the MBs and not on the electrode surface.Therefore, the storage stability of the antiCRP-MBs conjugates, oncethe blocking step with ethanolamine was performed, was checked.AntiCRP-MBs were stored at 4 ◦C in eppendorfs containing 50 �Lof filtered PBST. Each working day two replicates of the preparedconjugates were incubated in a 500 ng mL−1 CRP solution accordingto the procedure described in Section 2.4. A control chart was con-structed by setting the mean value of the current measured with 9different immunosensors on the first day of the study as the cen-tral value, and ±3× the standard deviation of this value as the upperand lower control limits (data not shown). The antiCRP-MBs con-jugates provided amperometric measurements within the controllimits during 6 days indicating an acceptable stability of the cap-ture antibody-modified MBs. This result supports the possibilityof carrying the stored conjugates to the place where samples aretaken, and performing the determination of the target protein insitu.

3.3. Selectivity of the developed magnetoimmunosensor

The selectivity of the magnetoimmunosensor was evaluatedtoward various non-target compounds at their concentrationscommonly found in serum or plasma (5 mg mL−1 BSA, 500 ng mL−1

D-dimer and 4000 �g mL−1 heparin) and two other non-target car-diac proteins at much higher concentrations than their normalvalues in serum: 7.5 ng mL−1 of amino-terminal pro-B-type natri-uretic peptide (NT-proBNP) and 500 ng mL−1 of cardiac troponin T(cTnT). The tests were performed by comparing the current valuesmeasured with the magnetoimmunosensor for 500 ng mL−1 CRP inthe absence and in the presence of the potential interfering com-pounds. Fig. 6 clearly shows that the presence of BSA, D-dimer andheparin at their usual concentrations found in serum and plasmadid not interfere significantly in the CRP determination. Moreover,the target cardiac protein can be accurately determined in thepresence of other two cardiac proteins (cTnT and NT-proBNP) at

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Table 2Analytical characteristics for different CRP immunosensors and immunoassays reported in the literature.

Methodology Technique Sample Concentrationrange

LOD Assay timea Reference

Electro-chemiluminescence immunoassay using [Ru(bpy)3]2+-containingmicrospheres as labels

ECL Humanplasma

0.010–10 �g mL−1 0.01 �g mL−1 4 h [39]

SPR sandwich based immunosensor SPR – 2–5 �g mL−1 1 �g mL−1 — [18]Electro-chemiluminescence immunoassay using Ru(bpy)3

2+-encapsulatedliposomes as labels

ECL – 100–10,000 ng mL−1 100 ng mL−1 3 h 30 min [40]

Electrical immunosensor based on the use of silicon nanowire arrays Electricalresponses

– 1 fM–1 nM – 3 h 35 min [41]

Electrochemical immunosensor based on a heterogeneous sandwichimmunoassay and SPEs modified with MWCNTs and protein A

Amperometry(Eapp = 0.00 V vs.Ag/AgCl)

Serum 0.5–200 ng mL−1 0.5 ng mL−1 7 h [7]

Label-free impedance immunosensor based on the covalent antibodiesimmobilization on a three-dimensional ordered macroporous (3DOM)gold film MPA-modified electrode

EIS Serum 0.1–20 ng mL−1 0.1 ng mL−1 39 h [22]

Label-free electrochemical impedance immunosensor based on covalentantibody immobilization using EDC/NHS on MUA/MPA-SAMs-modifiedgold electrodes

EIS – 45 ng mL−1–5.84 �g mL−1 30 ng mL−1 18 h 40 min [42]

Electrochemical sandwich immunosensor based on a PDMS-Aunanoparticles composite microfluidic chip and ZnSe quantum dotsbioconjugated with the secondary antibody as labels

SWASV Serum 0.5–200 (g L−1) 0.22 (g L−1) 66 h 10 min [43]

Label-free capacitive immunosensor based on the covalent antibodyimmobilization on

Au interdigitated electrodes fabricated on SiO2 surface Capacitance/dielectricmeasurements

Serum 25 pg mL−1–25 ng mL−1 32 pg mL−1 31 h [44]

Impedimetric immunosensor based on the antibodies immobilization onthe surface of nanocrystalline diamond

EIS Serum Cleardiscriminationbetween 1 �M,100 nM, and 10 nM

10 nM 14 h [1]

Signal-amplified piezoelectric sensor for the detection of hs-CRP using HRPdoped magnetic core-shell Fe3O4@SiO2@Au nanostructures as labels

Piezoelectric Serum 0.01–200 ng mL−1 5 pg mL−1 20 h 10 min [38]

Label-free electrochemical impedance immunosensor based on antibodyimmobilization on standard polycrystalline Au electrodes

EIS Bloodserum

60 �g L−1–6.0 mg L−1 19 �g L−1 18 h 15 min [45]

Chip-based point-of-care testing application based on ananogap-embedded field effect transistor (FET)

FET Serum 0.1–100 ng mL−1 0.1 ng mL−1 65 min [46]

Renewable amperometric immunosensor for hs-CRP based onfunctionalized Fe3O4@Au magnetic nanoparticles attracted on a Fe (III)phthlocyanine/chitosan-membrane modified screen-printed carbonelectrode by a magnet

Amperometry(Eapp = −0.3 V)

Serum 1.2–200 ng mL−1 0.5 ng mL−1 22 h 30 min [33]

Sandwich magnetoimmunosensor involving the use of HOOC-MBs andAu/SPEs

Amperometry(Eapp = –0.10 V vs.Agpseudo-referenceelectrode)

Humanserum

0.5–1000 ng mL−1 21 pg mL−1 4 h 25 min This work

Au/SPEs: gold screen-printed carbon electrodes; ECL: electrochemiluminescence; EDC: N-ethyl-N′-(dimethylaminopropyl) carbodiimide; EIS: electrochemical impedance spectroscopy; MPA: 3-mercaptopropionic acid; MUA:11-mercaptoundecanoic acid; MWCNTs: multiwalled carbon nanotubes; NHS: N-hydroxy succinimide; PDMS: poly(dimethylsiloxane); SPR: surface plasmon resonance; SWASV: square-wave anodic stripping voltammetry.

a Includes immunosensors preparation and final measurement.

218 B. Esteban-Fernández de Ávila et al. / Sensors

1 2 3 4 5 6

-i, µ

Tested compound

Fig. 6. Selectivity control for the CRP magnetoimmunosensor. Current values mea-sured for 500 ng mL−1 CRP in the absence (1) and in the presence of 5 mg mL−1

ssiepatlis(ntbiT1sttvcici9

SA (2), 7.5 ng mL−1 NTproBNP (3), 500 ng mL−1 cTnT (4), 500 ng mL−1 D-dimer (5)nd 4000 �g mL−1 heparin (6). Error bars were calculated as triple of the standardeviation (n = 3).

f false negative results thus offering a secure diagnostic to theser.

.4. Application to the analysis of a certified human serum

The validity of the developed methodology for clinical analy-is was checked by analyzing an International Standard for CRPerum containing a certified concentration of 100 �g mL−1 CRPn the reconstituted sample. The possible existence of a matrixffect was tested by constructing a calibration plot from the sam-le diluted with PBST until a final CRP concentration of 5 ng mL−1

dequately spiked with growing amounts of a standard CRP solu-ion up to 1000 ng mL−1 CRP (Fig. 5b). The slope value of theinear calibration plot (r = 0.994) was (2.1± 0.1) × 10−7 A with anntercept value of (1.5 ± 0.2) × 10−7 A. This slope is statisticallyimilar to that obtained with the buffered standard CRP solutions2.03 ± 0.07) × 10−7 A and, therefore, it could be concluded thato significant matrix effect was apparent after the sample dilu-ion. Accordingly, the CRP quantification could be accomplishedy simple interpolation of the measured current for the sample

nto the calibration plot constructed with buffered CRP standards.he analysis of ten diluted serum samples containing nominally00 ng mL−1 CRP yielded a mean content of (101 ± 2) ng mL−1. Atatistical comparison according to the Student’s t test gave acalc = 0.941 lower than ttab = 2.262. Therefore, it could be concludedhat there were no significant differences between the results pro-ided by the magnetoimmunosensor and the certified value at aonfidence level of 95%. It should be remarked that the magneto-mmunosensor can be applied to the clinical determination of thisardiac marker with a minimal sample treatment (just a dilutionn the buffer solution) and in a total assay time of approximately0 min (once the antiCRP-MBs are prepared).

.5. Comparison with other immunosensors and ELISA assayseported in the literature

The performance of the amperometric sandwich magneto-mmunosensor was compared with data available in the literature

he broadest linear range and one of the best LODs. Only the piezo-lectric sensor reported by Gan et al. [38] achieved a lower value

and Actuators B 188 (2013) 212– 220

(5 pg mL−1). In particular, the comparison with the immunosen-sor developed by Gan et al. [33] should be emphasized. Themagnetoimmunosensor developed in this work shows a muchwider linear range as compared to that achieved by Gan et al. whichranged from 1.2 to 200 ng mL−1. Moreover, the achieved LOD ismore than one order of magnitude lower than that reported in Gan’swork (0.5 ng mL−1).

Furthermore, some important advantages of the magneto-immunosensor rely on the simplicity and much easier potentialautomation and miniaturization of this approach compared withthe complex and time consuming substrate modification or bio-conjugates labels preparation protocols. These constitute relevantfactors to be considered for designing point-of-care (POC) diagnosisand prognosis tests.

Moreover, a performance comparison with commercial sand-wich ELISA spectrophotometric kits for CRP is also made inTable 1 in the Supporting Information. As it can be seen, theirLODs range from 0.002 (Abcam) to 100 ng mL−1 (Abnova). Apartfrom the lower sensitivity of most of the ELISA kits comparedto that of the magnetoimmunosensor, these methods require apretreatment of the biological sample (normally a centrifugationstep), and involve complicated, tedious and time-consuming multi-stage processes. Some other tests based on immunonephelometric,immunoturbidimetric and immunoluminometric measurementsare also available from different Companies, with LODs between0.005 and 0.32 mg L−1 and with a linear response within 0.3 and10 mg L−1 [14].

It is also important to remark that the magnetoimmunosen-sor allows the determination of CRP in other biological samplesbesides plasma and serum, where the biomarker concentrationis significantly lower such as feces and urine (with CRP contents<56 and 6 ng mL−1, respectively). The developed biosensors alsooffers a promising alternative for some other recently demandingapplications, such as the diagnosis and prognosis of cancer wherethe concentrations of tumor related proteins are very low at earlystages of the disease. Recent studies have demonstrated the poten-tial clinical use of slightly increased CRP levels to predict risk ofcertain types of cancer and to improve staging and treatment allo-cation in patients diagnosed with cancer [47].

4. Conclusions

In this work, a disposable amperometric magnetoimmunosen-sor for sensitive measuring of CRP in human serum which can beused in predicting future cardiovascular events is reported. Themagnetoimmunosensor possesses an excellent analytical perfor-mance achieving a LOD of 0.021 ng mL−1, value well below theminimum cut-off value (1000 ng mL−1) to qualify the severity ofrisk for cardiovascular disease, and allowing the reliable detectionof CRP across the clinical relevant range in dilute blood serum.The applicability of the developed magnetoimmunosensor wasdemonstrated by the analysis of a standard serum containing a cer-tified CRP content. Moreover, taking into account the cut-off valuefor CRP and the high sensitivity of this approach, the presentedmethodology can be simplified and shortened by performing onlya single incubation step including all the immunoreagents. Thegreat analytical performance exhibited in combination with theuse of disposable mass-produced biosensors constitutes importantadvantages for an easy integration of the presented method-ology into portable and multiplexed formats. We believe thepresented results serve as an important basis for the imple-

yA1ppvÁCDs

B. Esteban-Fernández de Ávila et al. / Se

cknowledgments

The financial support of the Spanish Ministerio de Economía Competitividad Research Projects, CTQ2012-34238, and theVANSENS Program from the Comunidad de Madrid (S2009PPQ-642) are gratefully acknowledged. A part of the work has beenerformed in the project 120215-CAJAL4 EU, in which the Spanishartners are funded by the Spanish Ministry of Science and Inno-ation, and the ENIAC Joint Undertaking. B. Esteban-Fernández devila acknowledges a FPI fellowship from the Spanish Ministerio deiencia e Innovación. The authors would like to acknowledge Auditiagnostics Company for kindly providing the CRP protein and its

pecific capture and detector antibodies.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.snb.2013.07.026.

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2 nsors

chemical arrays for multiplexed detection. He is AssociateEditor of the journal Electroanalysis and Vice-president ofthe Spanish Royal Society of Chemistry. He has authoredover 275 research papers and several books and book

chapters.

20 B. Esteban-Fernández de Ávila et al. / Se

iographies

Berta Esteban-Fernández de Ávila received her degreein “Science and Food Technology” from the UniversidadAutónoma de Madrid (Spain) in June 2009. She is work-ing on her PhD in Analytical Chemistry in the UniversidadComplutense de Madrid (Spain) since September 2009.She belongs to the “Electroanalysis and Electrochemical(Bio)sensors” research group. She worked as a ResearchScholar in the research group of Prof. G. Palleschi at theDepartment of Scienze e Tecnologie Chimiche in Univer-sitá di Roma Tor Vergata (Italy) from February to June2012. Her areas of interest include the development ofimmuno and DNA electrochemical sensors for clinicaldiagnosis.

Vanessa Escamilla-Gómez received her PhD in Analyt-ical Chemistry from the Universidad Complutense deMadrid (Spain) in 2009. Since 2003, she has been workingin the Analytical Chemistry Department of the Chem-istry Faculty of this University, where she is currentlyworking at the European Project “Chip Architectures byJoint Associated Labs for European diagnostics (Cajal 4EU)”. Her areas of interest include the development ofenzymatic, immuno-electrochemical sensors, bioelectro-catalysis, and electrode modification for the ultrasensitivedetection of bacteria and cardiac markers. She is alsoinvolved in the development of electrochemical arrays formultiplexed detection. She is co-author of ten research

apers.

Susana Campuzano received her PhD in analytical chem-istry from the Universidad Complutense de Madrid (Spain)in 2004. Since 2005 she works as Assistant Professor at theAnalytical Chemistry Department of the Chemistry Fac-ulty of the Universidad Complutense de Madrid where shecollaborates in the “Electroanalysis and Electrochemical(Bio)sensors” research group headed by Prof. J.M. Pingar-rón. She worked as a Research Scholar in the researchgroup of Prof. J. Wang at the Department of Nanoengineer-ing in UCSD (USA) from January 2010 to July 2011. Herareas of interest include the development of enzymatic,immuno and DNA electrochemical sensors and advancednanomachines.

María Pedrero received her PhD in Chemical Sciences(Analytical Chemistry) in 1993 from the Universidad Com-plutense de Madrid. In 1994 she stayed at the NewMexico State University for her postdoctoral training withProf. Joseph Wang. Since 1991, she has been workingin the Analytical Chemistry Department of the Chem-istry Faculty of this University, where she is Professorof Analytical Chemistry since 2002. She collaborates in

and Actuators B 188 (2013) 212– 220

J.-Pablo Salvador received his PhD in Chemistry fromUniversity of Barcelona in 2007. Since 2007 he works asResearch Assistant by CIBER-BBN in the Nanobiotechnol-ogy for Diagnostics group (Nb4D) at IQAC-CSIC. His areasof interest are around the production of antibodies for thedetection of small molecules, peptides or proteins andtheir application as analytical tools such as ELISA. Theuse of these antibodies in biosensing has also studied forthe development of fluorescent microarrays or biosensorsbased on plasmonic properties.

M.-Pilar Marco finished her PhD thesis entitled Synthe-sis and Regulatory Aspects of the Insect Molting HormoneSystem in 1990. She worked as a postdoctoral researcher(1990–1993) at the University of California in Davis in Prof.Bruce D. Hammock’s group on Immunochemical Analyti-cal Methods for Environmental and Biological Monitoring.Nowadays, she is the Coordinator of the NanomedicineArea of the Networking Research Center for Bioengineer-ing, Biomaterials and Nanomedicine (CIBER-BBN). Sheworks on new transducing principles to develop bioana-lytical multiplexed platforms for clinical diagnostics. Shehas been principal investigator of a number of researchprojects and is co-author on more than 160.

José M. Pingarrón obtained his PhD in Sciences from Com-plutense University of Madrid. He is Professor of AnalyticalChemistry at Complutense University of Madrid andhead of the group “Electroanalysis and electrochemical(bio)sensors”. Current research includes the developmentof nanostructured electrochemical biosensors, includingenzyme electrodes, immunosensors and genosensors forthe ultrasensitive detection of bacteria, low moleculeweight hormones and cancer markers as well as electro-

ultrasensitive amperometric magnetoimmunosensor for human c-reactive protein quantification in serum

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