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Full Paper

Detection of C Reactive Protein (CRP) in Serum by an

Electrochemical Aptamer-Based Sandwich Assay
Sonia Centi,a* Laura Bonel Sanmartin,a Sara Tombelli,a Ilaria Palchetti,a Marco Mascinia, b
a
Dipartimento di Chimica, Università degli Studi di Firenze, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino,
Firenze, Italy
b
Istituto Nazionale di Biostrutture e Biosistemi, Viale delle medaglie doro 305, 00136, Roma, Italy
*e-mail: sonia.centi@unifi.it

Received: November 11, 2008

Accepted: December 8, 2008

Abstract
A disposable electrochemical assay involving magnetic particles and carbon-based screen-printed electrodes (SPCEs)
was developed for the detection of C Reactive Protein (CRP). CRP is a plasma protein and is among the most
expressed proteins in acute phase inflammation cases, being a known biomarker for inflammatory states. The assay
was based on a sandwich format in which a RNA aptamer was coupled to a monoclonal antibody and alkaline
phosphatase (AP) was used as enzymatic label. After the sandwich assay, the modified magnetic beads were captured
by a magnet on the surface of a graphite working electrode and the electrochemical detection was thus achieved
through the addition of the AP substrate (a-naphthyl-phosphate) and a-naphthol produced during the enzymatic
reaction was detected using differential pulse voltammetry (DPV). The parameters influencing the different steps of
the assay were optimized in order to reach the best sensitivity and specificity. With the optimized conditions, the assay
was applied to the analysis of CRP free serum and serum samples.

Keywords: Aptamers, Biosensors, Magnetic beads, C reactive proteins, Electrochemical transduction, Serum

DOI: 10.1002/elan.200804560

1. Introduction detect CRP levels in the range 5 – 10 mg/L [11]. The hsCRP
assays detect CRP concentrations equal to 0.2 mg/L. These
Detection and quantification of C-reactive protein (CRP) in assays have the aim to predict the risk of a possible disease
an easy, cheap, and fast way can improve clinical diagnostics related with CRP levels. The cCRP assays are designed to be
in order to prevent serious inflammatory states. CRP is a an independent auxiliary prognostic test in populations with
protein present in plasma and is one of the most expressed stable coronary diseases or possible future development of
proteins in acute phase inflammation cases, being a known cardiovascular accidents. The levels of detection are the
biomarker for inflammatory states [1]. CRP is organized in a same for hsCRP concentrations [11].
two-chain form, but it can adapt a circular form constituted The techniques normally used to detect CRP are radial
by five equal subunits [2]. Each subunit binds to two calcium immunodiffusion (RID), radioimmunoassay (RIA), immu-
ions, interacts by intermolecular noncovalent salt bridges [3] nonephelometry (IN), immunoturbidimetry (IT), immuno-
and comprises 224 amino acids with a molecular weight of fluorescence, immunochemiluminescence and standard en-
approximated 25 kDa [4, 5]. zyme immunoassay (as ELISA). The limit of detection of
The CRP reference concentration of healthy subjects is these assays ranges between 0.1 and 0.2 mg/L.
< 5 mg/L in serum [6] and the clinical range of interest is 1 – In the last years several biosensors for CRP detection
500 mg/L. High levels of CRP are related with a large have been published. Many of them employ antibodies as
clinical state of diseases [7] such as cardiovascular diseases biorecognition elements [12 – 18], whereas few papers are
[8, 9] and bacterial infections [7, 10]. based on the use of aptamers [19, 20]. Aptamers are single-
The detection of CRP levels is of extreme importance stranded DNA or RNA oligonucleotides generated by an in
because of the clinical role of CRP. There are several assays vitro selection process called SELEX (systematic evolution
to detect CRP levels in human fluids. Their denomination is of ligands by exponential enrichment). It is possible by the
connected with the CRP detection range. According to the SELEX process to identify RNA/DNA molecules from a
Food and Drug Administration, the assays might be very large population of random sequence oligomers (DNA
qualified as Conventional C reactive protein (CRP), High or RNA libraries), which bind to the target molecule with
sensitivity CRP (hsCRP) and Cardiac C reactive protein very high affinity and specificity. Aptamers have been
(cCRP) [11]. The assays with the aims to detect the level of selected against a wide number of diagnostically relevant
inflammatory damage are denominated conventional and marker molecules, among these cancer-associated proteins

 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2009, 21, No. 11, 1309 – 1315
1310 S. Centi et al.

[21], thrombin [22, 23] and HIV-1 Tat protein [24]. In Moreover, the detection method is different, because in
comparison to antibodies, aptamers have a number of this case an electrochemical detection is employed using
advantages that make them very promising in analytical and differential pulse voltammetry (DPV) as electrochemical
diagnostic applications: they are chemically stable, show technique and disposable screen-printed electrodes as
high detection sensitivity and selectivity and can be selected transducers.
in vitro for any given target. Finally, this approach was evaluated using CRP free
In this paper an electrochemical aptamer-based sandwich serum samples and also testing some serum samples of
assay performed on magnetic particles for detection of CRP healthy subjects.
in serum samples is reported. The interesting approach of this
work is related to the coupling of an aptamer with a
monoclonal antibody as biorecognition elements for the
2. Experimental
development of a sandwich assay, to the use of magnetic
particles as solid support and of the electrochemical detection
2.1. Materials and Equipment
by using screen-printed electrodes. The coupling of the
aptamer and the antibody as ligands for the sandwich assay Planar three electrode strips, composed of a carbon working
results from the fact that only one aptamer has been selected electrode, a carbon counter electrode and a silver pseudo-
for CRP, therefore the only way to perform a sandwich assay reference electrode [23] were used as electrochemical cells.
is to combine it with a CRP specific antibody. Anyway, this The electrodes were screen-printed in-house using a DEK
work is the first step in the realization of an assay for CRP 248 screen-printing machine (DEK, Weymouth, UK). Silver
based on the use of the specific aptamer exploiting the known based (Electrodag PF-410) and graphite-based (Electrodag
advantages of these biomimetic receptors. 423 SS) polymeric inks were obtained from Acheson (Milan,
In the literature only two other papers report the Italy); the insulating ink (Vinylfast 36 – 100) was purchased
development of a biosensor for CRP detection using the from Argon (Lodi, Italy). A polyester flexible film (Autostat
specific aptamer. Bini and co-workers [19] used such CT5), obtained from Autotype (Milan, Italy) was used as
receptor for the development of a direct assay with an printing substrate.
optical transduction; they studied the immobilization pro- Electrochemical measurements were performed using a
cedure of the aptamer on a solid phase evidencing the mAutolab type II PGSTAT with a GPES 4.9 software
importance of modifying the aptamer with a suitable tail for package (Metrohm, Italy). All measurements were carried
a better recognition with the protein. Moreover, they out at room temperature by using Differential Pulse
studied the binding protocol, e.g. the best conditions for Voltammetry (DPV) with the following parameters: range
the formation of the complex aptamer-CRP and the potential 0/þ 600 mV, step potential 7 mV, modulation
developed assay was very sensitive in buffer exhibiting a amplitude 70 mV, standby potential 200 mV, interval time
detection limit of 0.005 mg/L and a coefficient of variation 0.1 s.
equal to 11%. However, when the optical biosensor was The sample mixer with 12-tube mixing wheel and the
applied to the analysis of serum samples, a very high matrix magnet were purchased from Dynal Biotech (Milan, Italy).
effect was observed and it was not even removed performing
a serum pre-treatment with magnetic particles. The other
work [20] concerns the development of a chip based on a
2.2. Chemicals
sandwich assay with a fluorescent detection. The assay was
performed combining the CRP aptamer with a monoclonal Biotin, human immunoglobulin (hIgG), streptavidin-alka-
antibody labeled with a fluorophor and its performance was line phosphatase conjugated, a-naphthyl phosphate, and
compared with the respective antibody-antibody chip. human serum albumin (HSA) were provided by Sigma
Moreover, the performances of the chip were evaluated (Milan, Italy).
spiking known CRP concentration in CRP free serum. The CRP aptamer used in this study is a 44-mer RNA
Compared to such reported chip, the proposed approach aptamer with the following sequence:
uses the same CRP aptamer even if modified in order to 5’-GCCUGUAAGGUGGUCGGUGUGGCGAGU-
enhance its stability. The aptamer was otherwise modified at GUGUUAGGAGAGAUUGC-3’
the 5’ end because of the different immobilization chemistry The RNA aptamer biotinylated in 5’ was purchased by
used, but, being a RNA based, it was very sensitive to the IBA (Germany) with a spacer arm, a TEG (triethylene
action of RNAse and for this reason it was very important to glycol) tail. The TEG tail (C6H14O4) is a spacer based on a
stabilize it. Because the nucleases that are most abundant in triethylene glycol, containing 4 oxygen atoms. Moreover, to
biological fluids, such as serum and plasma, are the enhance its stability, the aptamer was modified with F-
pyrimidine-specific nucleases, introduction of specific modi- pyrimidines.
fications at the 2’-position of pyrimidine nucleotides pro- Purified Human C Reactive Protein (CRP) and Biotin
tects an RNA oligonucleotide from degradation, increasing conjugated monoclonal antibodies against CRP (clones 5, 6
the half-life up to 15 hours [25]. With this modification, it is and 7) were purchased by Exbio (Praha, Czech Republic).
not necessary to work in an RNAse-free environment as Streptavidin magnetic beads (1.05 mm) were from Dynal
reported by Pultar and co-workers [20]. Biotech (Milan, Italy). CRP free serum was purchased from

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Detection of C Reactive Protein 1311

HyTest (Turku, Finland): this serum is prepared by immu- washing solution for 2 minutes, followed by the separation
noaffinity chromatography from pooled normal human with the magnet holding block to remove the supernatant.
serum and contains less than 0.02 ng/mL of CRP. The beads were resuspended in 500 mL of biotin solution
The composition of the buffers used for the experiments is 800 mg/L in order to saturate the sites of streptavidin which
reported below: have not bound to the biotinylated aptamer. The beads were
Buffer for beads washing: 5 mM Tris-HCl, 0.5 mM EDTA, then washed with buffer B and resuspended in 500 mL of
1 M NaCl and 0.1% of Tween 20 pH 7.5 (Buffer A). buffer B.
Buffer for the immobilization of the aptamer onto the Addition of the analyte: 50 mL of suspension containing
beads: 5 mM Tris-HCl, 0.5 mM EDTA and 1 M NaCl pH 7.5 aptamer-coated beads were mixed with 450 mL of solution
(Buffer B). containing CRP at different concentrations in the range
Binding buffer: 10 mM HEPES pH 7.4 containing 2 mM 0 – 100 mg/L. After 15 minutes of incubation time, the beads
CaCl2 (Buffer C). were magnetically separated to remove the supernatant and
Detection buffer: 0.1 M diethanolamine buffer containing then washed twice using buffer C.
1 mM MgCl2, 100 mM KCl pH 9.6 (Buffer D). Addition of the secondary ligand: the beads were re-
suspended in 500 mL of a solution of monoclonal antibody
anti-CRP (clone 6) 1 mg/L in buffer C for 15 minutes and
then separated and washed twice using buffer C.
2.3. Electrochemical Assay Procedure
Addition of the Enzyme-conjugate: The beads carrying the
The assay developed for CRP detection was based on a affinity complex (aptamer-analyte-antibody) were re-sus-
sandwich format, following the scheme reported in Figure 1. pended in 500 mL of streptavidin-alkaline phosphatase
All steps of the assay were carried out onto magnetic conjugated (0.2 U/mL) in buffer D containing 0.5% (w/v)
particles; only the electrochemical detection was performed of casein for 10 minutes. After separation and washing, the
transferring the functionalized beads onto the working beads were re-suspended in 50 mL of buffer D.
electrode of a screen-printed electrode with the aid of a Electrochemical detection: 10 mL of the beads suspension
magnet holding block. were transferred onto the surface of the working electrode.
Beads washing: Magnetic beads were treated with buffer To better localize the beads onto the electrode, the magnetic
A before the development of the assay in order to remove block was placed on the bottom of the electrode. Then 60 mL
the NaN3 preservative. of a solution containing the enzymatic substrate (a-naphthyl
Aptamer immobilization on magnetic beads: The immo- phosphate) 1 mg/mL in buffer D were deposited on the
bilization of the biotinylated aptamer was based on strepta- screen-printed strip. After 5 minutes, the enzymatic product
vidin-biotin interaction using streptavidin-coated magnetic was determined by DPV.
beads. For this purpose, a suspension of 50 mL of magnetic
beads was introduced in a tube containing 250 mL of
aptamer solution 0.1 mM prepared in buffer B. After
2.4. CRP Free Serum and Analysis of Serum Samples
15 minutes of incubation time, the tube was positioned on
a magnet holding block to allow the magnetic separation of Standard solutions of CRP were added to CRP free serum
the beads; the supernatant was then removed and the beads diluted 1 : 10 to test the performance of the electrochemical
were washed twice with 500 mL of washing solution. Each aptamer-based assay.
washing step consisted of a re-suspension of the beads in the

Fig. 1. Scheme of the electrochemical assay based on the use of magnetic beads and disposable electrochemical sensors.

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1312 S. Centi et al.

Fig. 2. Optimization of the CRP aptamer concentration: several concentrations of aptamer have been tested in the range 0.05 – 1 mM.
The aptamer concentration has been chosen comparing the relative signals measured in presence and in absence of CRP.

Two serum samples (sample A and B) were tested. They 3.1.1. Optimization of the Aptamer Concentration
were collected and characterized by the Medical University
of Graz, Department of Internal Medicine, Division of Different concentrations of aptamer, in the range 0.05 –
Diabetes and Metabolism, Auenbruggerplatz (Graz). The 1 mM, were used to modify the beads. The assay was
CRP concentration in the two samples evaluated by the performed incubating the modified beads first with a
reference laboratory was < 1.0 and 2.9 mg/L respectively. solution containing 0 or 50 mg/L of CRP and then with a
For the analysis, aptamer-modified magnetic beads were solution 1 mg/L of monoclonal IgG anti-CRP (clone 6).
incubated with samples diluted 1 : 10, then the assay was Before the electrochemical detection as described in para-
carried out by adding the secondary ligand and the graph 2.3, beads were incubated with a solution of strepta-
conjugate as previously reported. vidin-alkaline phosphatase 0.2 U/mL. The results are re-
ported in Figure 2, which shows the differences in the
binding increasing the aptamer concentrations. The signal
measured in absence of CRP was the same recorded for all
3. Results and Discussion
aptamer concentrations while in presence of CRP (50 mg/L)
the signal measured using the aptamer concentration equal
3.1. Sandwich Assay Development
to 0.05 mM was lower than those obtained using the other
Some parameters such as the concentration of the biotin- concentrations.
ylated aptamer to immobilize on magnetic beads, the 0.1 mM was chosen as aptamer concentration for the
concentration of the biotinylated antibody anti-CRP and, immobilization onto the beads, since it was the lower
at the end, that of the enzymatic conjugate, need to be concentration showing the best ratio between the signal in
optimized for the development of an electrochemical presence and in absence of CRP.
sandwich assay. Another important parameter to take into
consideration is the aptamer-analyte binding conditions. In
3.1.2. Choice of the Secondary Ligand and Optimization of
a previous article [19] HEPES buffer (10 mM pH 6.5 con-
Its Concentration
taining 2 mM CaCl2) was chosen as binding buffer, since in
this buffer the best ratio between the specific signal (in the Some pairs of monoclonal antibodies anti-CRP, able to
presence of CRP) and the unspecific signal (in the presence recognize different sites of the target analyte, are commer-
of HSA) was found. Therefore, such buffer was used as cially available for the development of a sandwich assay for
binding buffer even if the pH was adjusted to 7.4 in order to the detection of CRP; for example the clone 5 is used as
have the optimum pH also for dilution/analysis of serum capture antibody and coupled to clone 7 as detection
samples. antibody [26, 18] or clone 2 is matched with clone 6 [13]. In
The starting conditions were: biotinylated 44-mer RNA the case that aptamer against CRP is used as capture ligand,
aptamer with a TEG tail as capture ligand; biotinylated it is important to identify the best clone to couple to it as
monoclonal antibody anti-CRP as detection ligand (con- detection antibody for the development of a sandwich assay.
centration 1 mg/L, incubation time 15 min); binding buffer, For this purpose, some clones of monoclonal antibody anti-
HEPES 10 mM at pH 7.4 containing 2 mM CaCl2 and CRP (clones 5,6 and 7) at the concentration of 1 mg/L were
0.005% (v/v) of Tween 20. tested and the performances of the corresponding assay
were compared. The signals recorded for the solution
without CRP was the same using all the three clones, while
those measured for the solution containing 50 mg/L of CRP

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Detection of C Reactive Protein 1313

Table 1. Comparison between the signals obtained using two different concentrations of streptavidin-alkaline phosphatase.
Concentration of streptavidin-alkaline phosphatase 0 mg/L CRP 0.1 mg/L CRP 10 mg/L CRP
2 U/mL 4.0  0.4 mA 7.5  0.1 mA 13.5  0.8 mA
0.2 U/mL 0.2  0.06 mA 0.5  0.09 mA 3.2  0.3 mA

was much lower in the case of clone 5 (1.5  0.4 mA, CV ¼ parameters of the corresponding dose-response curve are
29%) than in the case of clone 6 (3.6  0.4 mA, CV ¼ 10%) shown in Table 2.
and 7 (3.3  0.4 mA, CV ¼ 13%). The difference between the The dose response fitting curve is the following:
DPV signals obtained with the three clones could be due to
the different binding properties between the protein and Y ¼ 0.13 þ 3.76 [1 þ 10log(1.49X)0.82]1 (1)
these ligands. Experimentally it was found that in the case of
clone 5 the DPV signals were lower than those measured for The assay was repeated in order to evaluate the reprodu-
the others two clones. Among clone 6 and 7, the clone 6 was cibility; for this purpose, five repetitions of each standard
chosen as detection ligand for the development of the solution in the concentration range 0 – 1000 mg/L were
sandwich assay according to higher signal values and better carried out. The average coefficient of variation (ACV) was
reproducibility. 13%, calculated as mean of all the concentrations consid-
After the choice of the clone to couple with the aptamer, ered.
the concentration of the biotinylated antibody (clone 6) to The limit of detection (LOD) of the assay was evaluated
bind the aptamer-analyte complex was studied. To this as minimum detectable concentration, which is the lowest
purpose, some concentrations of the antibody were tested concentration of analyte which can be distinguished at a
(0.5, 1 and 5 mg/L) with CRP bound to the aptamer and the stated level of probability from a sample not containing the
best performances in terms of sensitivity and reproducibility analyte. Moreover, the limit of quantification (LOQ) is
were obtained with the 1 mg/L antibody solution with an considered as the level above which quantitative results may
incubation time of 15 min (data not shown). be obtained with a specified degree of confidence. The LOD
value was calculated by the evaluation of the average
response of the blank plus three times the standard
3.1.3. Optimization of Streptavidin-Alkaline Phosphatase
deviation, whereas the LOQ considering the average
Conjugate Concentration
response of the blank plus 10 times the standard deviation.
Some experiments were carried out in order to find the best In this case the recorded blank signal was 0.2  0.06 mA,
concentration of streptavidin-alkaline phosphatase conju- leading to a LOD of 5.4  102 mg/L and a LOQ of 2.3 
gate to use for the sandwich assay. At this purpose, the assay 101 mg/L.
was performed incubating the beads modified with the The clinically relevant borderline for CRP is 8 mg/L [18]
biotinylated aptamer with a solution of CRP in the concen- and this value is within the dynamic range of the sandwich
tration range 0 – 10 mg/L and then with a solution 1 mg/L of assay, giving the possibility to diagnose pathology with a
monoclonal IgG anti-CRP (clone 6). Beads were incubated high accuracy.
with streptavidin-alkaline phosphatase conjugate solution To test the specificity of the sandwich assay, a solution of
(2 or 0.2 U/mL) and then the electrochemical measure- hIgG at physiological concentration (10 g/L) was used and
ments was performed as usual. The results are reported in the recorded response was compared with that measured for
Table 1, where the signals obtained using the two different CRP in the same conditions. This experiment demonstrated
concentrations of enzymatic conjugate are compared. the good specificity of the assay since a high signal was
Using 0.2 U/mL of enzymatic conjugate the solution obtained only when the specific protein (CRP) was tested,
without CRP gave a lower background signal (0.2 mA) than whereas the signal recorded in presence of hIgG resulted as
that measured using 2 U/mL (4.0 mA). The signal measured 3% of the specific signal (data not shown). Moreover, the
for such solution is correlated with the nonspecific binding, low signals obtained with nonspecific IgG showed that the
therefore it is advisable to work with the lowest background. modification of the aptamer with 2’-F pyrimidines to
Moreover, a better discrimination between the several enhance its stability did not affect its specificity.
concentrations of CRP is observed using 0.2 U/mL of
streptavidin-alkaline phosphatase. For these reasons, the
concentration 0.2 U/mL was chosen for the following Table 2. Analytical characteristics of the electrochemical sand-
experiments. wich assay performed using the CRP aptamer and the monoclonal
antibody ( C6) as capture and detection biorecognition elements.
Measuring range 0 – 1000 mg/L
Dynamic range 0.1 – 50 mg/L
3.2. Dose Response Curve of CRP Limit of detection ( LOD ) 5.4  102 mg/L
Limit of quantification (LOQ ) 2.3  101 mg/L
The sandwich assay was performed using different concen-
Average CV% 13%
trations of CRP standard solutions and the main analytical

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1314 S. Centi et al.

Fig. 3. Dose-response curve for CRP carried out in CRP free serum diluted 1 : 10. The points correspond to the current  SD calculated
for n ¼ 4 repetitions and the line that joins the points is referred to the theoretical curve calculated by sigmoidal data interpolation.

3.3. Dose Response Curve of CRP in CRP Free Serum figure they are compared with the voltammogram measured
in presence of CRP free serum.
The next experiments were focused on evaluating the ability Both samples gave a higher peak than that measured in
of the electrochemical aptamer-based assay to detect CRP CRP free serum (0.070  0.009 mA). The current signal
in serum. CRP free serum, diluted 1 : 10, was tested alone or measured for sample A (0. 093  0.007 mA) containing the
spiked with CRP (in the concentration range 0 – 1000 mg/ lowest CRP concentration (< 1 mg/L) was lower than that
L), and the corresponding dose response curve, shown in measured for sample B (0.12  0.01 mA) containing 2.9 mg/
Figure 3, covered the whole dynamic range from the region L of CRP.
of signal noise to a saturation value. The signal increased Using the logistic equation reported before and consid-
with the increased concentration of the analyte and a matrix ering the dilution factor, the concentrations found for
effect was observed considering the lower currents mea- sample A and B correspond to 1.1  0.1 mg/L and 5.6 
sured in serum with respect to buffer. Probably this decrease 2.3 mg/L respectively.
is due to a reduced concentration of CRP available for the The difference in the concentration values obtained using
binding caused by the interaction with some matrix compo- the electrochemical assay and the reference method is not
nents. However, an interference on the binding properties of relevant considering that the measured response is related
the aptamer-modified beads can not be excluded. The to the logarithm of the concentration as reported in Figure 3.
calculated LOD and LOQ were 0.2 and 6 mg/L respectively The proposed method is able to detect even low levels of
and the average coefficient of variation (ACV) was 8%. The
matrix effect increased the value of the detection limit even
if such value is comparable with that reported by hsELISA
(0.2 mg/L) [27]. Moreover, it is interesting to consider that
the LOD of the assay is twenty-five times lower than the
biological CRP concentration (< 5 mg/L) and that both the
LOD and LOQ calculated in serum, even if higher than
those measured in buffer, are lower than the clinically useful
borderline value (8 mg/L) [18].
The dose response fitting curve is the following:

Y ¼ 0.066 þ 0.25 [1 þ 10log(13.44X)0.44]1 (2)

3.4. Analysis of Serum Samples

The performance of the developed electrochemical assay
was tested on two serum samples (A and B). The recorded Fig. 4. Comparison between the current peaks obtained for CRP
voltammograms are shown in Figure 4 and in the same free serum ( – - – ), sample A ( – & – ) and sample B ( – ~ – ).

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Detection of C Reactive Protein 1315

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