Biomarkers, 2008; 13(78): 637657

Biosensors for biomarkers in medical diagnostics

M. MASCINI1,2, & S. TOMBELLI1

1
Università degli Studi di Firenze, Dipartimento di Chimica, Via della Lastruccia 3, 50019
Firenze, Italy and 2Istituto Nazionale Biostrutture e Biosistemi (INBB), Unit of Florence,
Viale delle Medaglie d’Oro 305, 00136, Roma, Italy
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Abstract
At present, most biomarker testing is taking place at centralised dedicated laboratories using
large, automated analysers, increasing waiting time and costs. Smaller, faster and cheaper
devices are highly desired for replacing these time-consuming laboratory analyses and for
making analytical results available at the patient’s bedside (point-of-care diagnostics).
Innovative biosensor-based strategies could allow biomarkers to be tested reliably in a
decentralised setting, although several challenges and limitations remain, which need to be
improved, in the design and application of biosensors for the appropriate interpretation of the
identified and quantified biomarkers. The development of biosensors is probably one of the
most promising ways to solve some of the problems concerning the increasing need to develop
highly sensitive, fast and economic methods of analysis in medical diagnostics. In this review,
some consideration will be given to biosensors and their application in medical diagnostics,
For personal use only.

taking into account several crucial features.

Keywords: aptamers, biosensors, cancer, cardiovascular disease, hormones, point-of-care

(Received 1 July 2008; accepted 4 November 2008)

Introduction
Since stricter requirements regarding human health have led to a rising number of
clinical tests, there is an increasing need to develop highly sensitive, fast and economic
methods of analysis. The elaboration of biosensors is probably one of the most
promising ways to solve some of the problems concerning sensitive, fast and cheap
measurements.
The definition of a biosensor has recently been selected by IUPAC (Thevenot et al.
2001), but a more ‘modern-time appropriate’ definition has been chosen by Newman
et al. (2004). They referred to a biosensor as: ‘a compact analytical device
incorporating a biological or biologically-derived sensing element either integrated
within or intimately associated with a physicochemical transducer’.
The earliest biosensors were catalytic systems that integrated especially enzymes
with transducers that convert the biological response into an electronic signal. The
next generation of biosensors, affinity biosensors, took advantage of different
biological elements, such as antibodies, receptors (natural or synthetic), or nucleic

Correspondence: M. Mascini, Università degli Studi di Firenze, Dipartimento di Chimica, Via della
Lastruccia 3, 50019 Firenze, Italy. Tel:
39 055 4573283. Fax:
39 055 4573384. E-mail:
marco.mascini@unifi.it

ISSN 1354-750X print/ISSN 1366-5804 online # 2008 Informa UK Ltd.

DOI: 10.1080/13547500802645905
 638 M. Mascini & S. Tombelli

acids. In all of these interactions, the binding between the target analyte and
the immobilised biomolecule on the transduction element is governed by an affinity
interaction, such as the antigenantibody (AgAb), the DNADNA or the protein
nucleic acid binding. The specificity of the biosensor system is given by the
immobilised molecule. The transducers used in biosensors are electrochemical,
optical, thermometric, piezoelectric and magnetic. A general classification of
biosensors is given in Figure 1, where the possible biorecognition elements and
transducers are listed.
The research in the field of biosensors was initiated by Clark, whose study on the
oxygen electrode was published in 1956 (Clark 1956). Since then, a huge number of
biosensors have appeared in the literature and a great number with an application in
medical diagnostics. Actually,
80% of the commercial devices based on biosensors
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are used in this domain (Dzyadevych et al. 2008), starting with the first commercial
apparatus for glucose determination produced by Yellow Spring Instruments (YSI
Incorporated 1975).
Apart from the huge space occupied in the market and in the literature by the
glucose biosensor (Table I, (Newman et al. 2004)), recently reviewed by Wang (2008),
and other enzyme-based catalytic biosensors (Singh et al. 2008, Arya et al. 2008),
many examples related to the analysis of clinical relevant analytes by immunosensors
have been reported in the last 20 years, when this approach first started (Lin & Ju
2005). More recently, in the last decade, DNA-based sensing has appeared for real
applications in clinical diagnostics to detect the presence of pathogenic species
responsible of infections, to identify genetic polymorphisms and to detect point
For personal use only.

mutations (Dell’Atti et al. 2006).

This paper reviews the recent (200608) literature on biosensors for medical
diagnostics: the review is structured by focusing on biomarkers for different problems

Figure 1. Schematic representation of a biosensor. The different biorecognition elements and

transducers are depicted in the figure.
 Biosensors for biomarkers in medical diagnostics 639
Table I. Market-leading biosensor companies and target/sector involved.

Company Target/sector

Abbott (Molecular Diagnostics and Glucose

Diabetes Care)
Affymetrix Pharmaceutical
and general bioscience research
Applied Biosystems and HTS Affinity chip
Biosystems
ARKRAY, Inc. Glucose
Bayer Diagnostics/Kyoto Daiichi Glucose
/Menarini
Becton Dickinson Glucose
Biacore Affinity sensors for pharmaceutical
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and general bioscience research

Biosite Medical
Eppendorf Glucose and lactate
LifeScan Glucose
Molecular Devices Diagnostics, pharmaceutical and defence
Nanogen Diagnostics
Nova Biomedical Glucose, urea, lactate and creatinine
Roche Diagnostics Glucose
YSI Glucose and lactate

(cancer, cardiac diseases, hormones). A separate section is centred on DNA biosensors,

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which can represent a valid alternative to the well-accepted immunosensors.

Despite the impressive number of publications on biosensors in the diagnostics
field, the commercialisation of this technology is feasible only minimally in the near
future, besides the blood glucose and lactate biosensors and a few others (Table II)
(http://www.biotechblog.org/entry/srinakharinwirot-universitys-technology-detects-
tb-within-60-seconds). This can partially be due to the need to prove the stability and
reliability of the biosensors: validation by well-established procedures must be
conducted with the analysis of real samples to prove the effectiveness of biosensors
for the technology to be transferred to the laboratory diagnostics market (Loung et al.
2008).
For this reason and to make a selection among the huge number of works published
in the last 3 years, the authors centre their attention on those papers that report a real
application of the developed biosensor with the analysis of real samples and/or a
comparison with a reference method.

Review of the literature related to biosensors for biomarkers

Biosensors for cancer biomarkers
Cancer is the second most common cause of mortality and morbidity in western
countries and most other countries, with
1.4 million cases in the US this year, with a
similar incidence across the EU, with almost 1.5 million cases. The diagnosis and
treatment of cancer represents a major area of unmet need across Europe and all other
areas of the world (www.cancerworld.org). There is, however, a strong connection
between early detection and positive patient outcome: early detection is the best hope
 640 M. Mascini & S. Tombelli
Table II. New biosensors that have appeared in the literature for several biomarkers.

Biorecognition
Target/biomarker Disease element Transduction Ref.

a-fetoprotein (AFP) Cancer Antibody Electrochemical Wilson & Nie 2006

Competitive assay (array)
Antibody Electrochemical Wu et al. 2007a
Sandwich assay
BRCA1 gene Breast cancer DNA Electrochemical Castaneda et al.
2007
Cancer antigen Ovarian cancer Antibody Electrochemical Wilson & Nie 2006
125 (CA 125) Competitive assay (array)
Cancer antigen Breast cancer Antibody Electrochemical Wilson & Nie 2006
15-3 (CA 15-3) Competitive assay (array)
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Cancer antigen Gastrointestinal Antibody Electrochemical Wilson & Nie 2006

19-9 (CA 19-9) tract carcinoma Competitive assay (array)
Carcinoembryonic Cancer Antibody Electrochemical Wilson & Nie 2006
antigen (CEA) Competitive assay (array)
Antibody Electrochemical Wu et al. 2006
Direct assay
Antibody Electrochemical He et al. 2008
Competitive assay
Cardiac troponin Acute myocardial Antibody Direct assay Optical (SPR) Fireman et al. 2007
T (cTnT) infarction
Cardiac troponin Acute myocardial Antibody Electrochemical Ko et al. 2007
I (cTnI) infarction Sandwich assay
Antibody Optical (FRET) Cody Stringer et al.
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Direct assay 2007

Cortisol General physical Antibody Optical (SPR) Stevens et al. 2008
stress Competitive assay
C-reactive protein Inflammation Antibody Magnetic Meyer et al. 2007
(CRP) Cardiovascular Sandwich assay
diseases
Epidermal growth Breast cancer Antibody Direct assay Optical (SPR) Martin et al. 2008
factor receptor
2 (HER-2)
Ferritin Anaemia Cancer Antibody Electrochemical Wilson & Nie 2006
Competitive assay (array)
Hepatitis B virus Hepatitis DNA Electrochemical Ding et al. 2008
PNA Acoustic (QCM) Yao et al. 2008
Human chorionic Cancer Antibody Electrochemical Wilson & Nie 2006
gonadotrophin Competitive assay (array)
(hCG)
Antibody Electrochemical Chai et al. 2008,
Direct assay Tan et al. 2007
IL-6 Various (Cancer) Antibody Acoustic (SAW) Krishnamoorthy
Direct assay et al. 2008
IL-8 Oral cancer Antibody Optical Tan et al. 2008
Sandwich assay (Fluorescence)
BCR/ABL gene Chronic DNA Electrochemical Chen et al. 2008
myelogenous
leukaemia
L1 viral region Human papilloma DNA Magnetoresistive Xu et al. 2008
virus
DNA Acoustic (QCM) Dell’Atti et al. 2007
 Biosensors for biomarkers in medical diagnostics 641
Table II (Continued)
Biorecognition
Target/biomarker Disease element Transduction Ref.

Platelet-derived Atherosclerosis, Aptamer Electrochemical Lai et al. 2007

growth factor fibrosis, malignant
(PDGF) diseases
Progesterone Antibody Optical (SPR) Yuan et al. 2007
Competitive assay
Prostate-specific Prostate cancer Antibody Optical (SPR) Cao et al. 2006
antigen (PSA) Sandwich assay
Antibody Electrochemical Yu et al. 2006
Sandwich assay
Thrombin Cardiovascular Aptamer Electrochemical Centi et al. 2007,
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diseases Sandwich assay Zheng et al. 2007

in all cancers to improve patient survival and disease prognosis, and may lead to
cancer prevention.
At present, the most important cancer diagnostic indicators are morphological and
histological characteristics of tumours or single biomarkers, such as prostate-specific
antigen (PSA). Several examples of biosensors for the detection of cancer biomarkers
can be found in the literature and further attention will be given to the detection of
PSA in the next subsection, as this antigen is a biomarker strictly associated with one
kind of cancer, prostate cancer. Among other cancer biomarkers that have been
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considered as possible targets for a single-analyte biosensor, a-fetoprotein (AFP)

(Maeng et al. 2008), carcinoembryonic antigen (CEA) (Wu et al. 2006, He et al.
2008), epidermal growth factor receptor-2 (HER-2) (Martin et al. 2006) and
interleukin-8 (IL-8) and interleukin-6 (IL-6) (Krishnamoorthy et al. 2008, Tan
et al. 2008) have recently been taken into account.
No single oncogene or tumour suppressor, however, has been discovered to be
universally altered in all adult cancers and, besides genome-related changes, other
complex molecular alterations, such as protein over/underexpression, can result
during the course of tumorigenesis (Soper et al. 2006). Considering these statements,
a plethora of molecular biomarkers can be analysed for tumour classification, for
diagnosis and for monitoring treatment and disease recurrence.
In this regard, two works are described here that report the development of
biosensors with an array format for the simultaneous detection of different tumour
markers (Wilson & Nie 2006, Wu et al. 2007a). These can be considered of particular
significance because most markers are not specific to a particular tumour and the use
of panels of tumour markers can increase their diagnostic value (Wu et al. 2007b).
Both biosensors are immunosensors with an electrochemical detection. The first one
(Wilson & Nie 2006) was constructed for the detection of seven tumour markers
(AFP, ferritin, b-human chorionic gonadotropin (b-hCG), CEA, cancer antigen
125 (CA 125), cancer antigen 15-3 (CA 15-3) and cancer antigen 19-9 (CA 19-9)) by
a competitive immunoassay conducted on an array of IrOx electrodes. The detection
was accomplished by using in the competitive assay a secondary anti-IgG antibody
labelled with alkaline phosphatase (AP): the enzyme substrate hydroquinone dipho-
sphate (HQDP) was added at the end of the assay and the oxidation current was
registered simultaneously for all the electrodes after the application of a potential of
320 mV. The sensor had good precision and accuracy and was comparable with the
 642 M. Mascini & S. Tombelli

corresponding single analyte ELISAs with a detection limit of B2 ng/ml for all the
markers. Serum control samples were used to validate the sensor, which demonstrated
a good correlation with clinical analysers (within 8% agreement).
The second multi-analyte biosensor (Wu et al. 2007a) was created for the
simultaneous detection of AFP, b-hCG, CEA and CA 125 by using a screen-printed
carbon electrode to capture the specific horseradish peroxidase-labelled antibodies
in a competitive assay format. The detection was achieved by monitoring the
mediator-catalysed enzymatic response to hydrogen peroxide. Clinical serum samples
were analysed with good inter-assay and intra-assay precision, and good correlation
coefficients with a commercial electrochemiluminescence analyser (Elecsys 010,
Roche).
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Biosensors for the detection of PSA. Prostate cancer has become an important health
issue because it is, on a global scale, the third most common cancer in men (Jemal
et al. 2006). Prostate-specific antigen has been identified as a biomarker to screen
prostate cancer patients and it has been shown that PSA is the most reliable tumour
marker to detect prostate cancer at the early stage and to monitor the recurrence of the
disease after treatment (Stephan et al. 2006). The PSA is found in serum, either free
or in a complex with various protease inhibitors, and a total PSA level of 10 ng/ml or
higher is a highly probable indicator for prostate cancer (Benson et al. 1992). Anyway,
a PSA measurement above cutoff value of 4 ng/ml is generally regarded as positive and
might indicate the need for a biopsy (Stenman et al. 1999).
At present, most PSA testing takes place at centralised dedicated laboratories using
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large, automated analysers, increasing waiting time and costs (Healy et al. 2007, Lin
et al. 2008). Faster and cheaper devices are highly desired to replace these time-
consuming laboratory analyses.
Innovative biosensor strategies could represent alternative strategies for reliable
cancer testing: several biosensors for PSA detection have been presented in the last
few years based on different transduction techniques, from electrochemical (Meyerh-
off 1999, Fernandez-sanchez 2004, Sarkar 2002) to piezoelectric (Wu et al. 2001, Wee
et al. 2005) and optical (Besselink et al. 2005, Huang et al. 2005) methods.
More recently, surface plasmon resonance (SPR) based detection of different PSA
isoforms down to 1 ng/ml has been reported (Cao et al. 2006). The detection of a
complex of PSA with a1-antichymotrypsin (PSA-AC) (Stenman et al. 1991) in both
buffer and serum was demonstrated by using an alkanethiolated-modified surface for
the attachment of specific antibodies. The detection limit was improved further with a
sandwich format down to 10.2 and 18.1 ng/ml in buffer and in serum, respectively.
Lower detection limits have recently been reached by several published electro-
chemical biosensors using carbon nanotubes (Yu et al. 2006, Okuno et al. 2006) and
nanoparticles (Lin et al. 2008, Choi et al. 2008) for signal amplification. In particular,
an immunochromatographic electrochemical biosensor coupled to nanoparticles (Lin
et al. 2008), with a detection limit of 0.02 ng/ml, was tested also in serum samples.
This biosensor was validated with a human serum sample and a commercial ELISA
PSA kit: the results of the biosensor were consistent with those of ELISA, with
recoveries for spiked samples of 105  111%.
Particular attention should be given to an electrochemical immunosensor based on
carbon nanotubes (Yu et al. 2006), which reached a detection limit for PSA of 4 pg/ml
and was also tested in several human serum and tissue samples. The application of
 Biosensors for biomarkers in medical diagnostics 643

biosensors to the analysis of tissue samples is particularly challenging because very low
volumes are available and sensitivity should be very high. In this case, PSA was
detected with high sensitivity by using only 10 ml of serum or tissue lysates
containing 1000 cells.
The biosensor was composed of 2030-nm-long terminally carboxylated single-
wallet carbon nanotubes (SWNTs) self-assembled (Chattopadhyay et al. 2001) on
Nafion-iron oxide decorated conductive surfaces. Primary antibodies for PSA were
attached to the SWNT forest and used to capture the target protein. Secondary
antibodies labelled with horseradish peroxidase (HRP) were then used for the
electrochemical detection (Figure 2). The high sensitivity was achieved by using
these secondary antibodies linked to multiwallet carbon nanotubes (CNT) at high
HRP/antibody ratio. PSA in human serum was measured with a 95% accuracy
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compared with a referee ELISA method. Moreover, prostate tissue lysates were tested
and the results demonstrated that differences in PSA concentration could be detected
among different samples that cannot be distinguished by the immunohistochemical
staining reference method (Gannot et al. 2005).

Biosensors for detection of hormones

Sex steroids are thought to participate in the regulation of immune response and may
play a part in the modulation of some inflammatory and autoimmune disorders.
Among these hormones, the measurement of progesterone is important in women.
Progesterone, a C21 steroid secreted by the corpus luteum, promotes the develop-
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ment of the endometrial lining. Reference concentration ranges of progesterone in

plasma are from 0.1 to 28 ng/ml in women.
Immunoassays are commonly used for the determination of progesterone in serum
or saliva. In particular, analysis laboratories use commercially available radioimmu-
noassay (RIA) kits, which are able to detect progesterone with sufficient precision in
the clinically relevant concentration range (Reinsberg et al. 1997). Given the inherent
problems, different non-radioactive methods have been developed for measuring
progesterone (Kakabakos & Khosravi 1992, Choi et al. 1997, Hong & Choi 2002).
Among the high number of immunoassay techniques, ELISA combined with a
colorimetric measurement is the most widely used for measuring hormone concen-
trations (Basu et al. 2006). The use of immunosensors is another interesting
alternative approach. Both electrochemical and optical biosensors have been reported,
using screen-printing technology coupled to cyclic voltammetry (Xu et al. 2005) and
chronoamperometry (Hart et al. 1997). For optical sensing, SPR has been used (Gillis
et al. 2002, Yuan et al. 2007).
Screen-printed electrodes have been used in the immunosensor development for
progesterone detection as solid phase for a competitive immunoassay (Minunni et al.
2007). The EC50 value (the analyte concentration necessary to displace 50% of the
enzyme label) was calculated as 2 ng/ml, whereas the limit of detection (LOD),
calculated by evaluation of the mean of the blank solution (containing the tracer only)
response minus two times the standard deviation, was estimated as 32 pg/ml.
Among other hormones, human chorionic gonadotropin (hCG) has also been
considered as a target for biosensors. Human chorionic gonadotropin is an important
diagnostic marker of pregnancy and one of the most important carbohydrate tumour
markers. Several immunoassay kits or strategies have been presented, including some
 644 M. Mascini & S. Tombelli
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Figure 2. Illustration of detection principles of single-wallet carbon nanotube (SWNT) immunosensors.

Bottom left: a tapping mode atomic force microscope image of a SWNT forest. Above this on the left is a
cartoon of a SWNT immunosensor that has been equilibrated with an antigen, along with the biomaterials
used for fabrication (horseradish peroxidase (HRP) is the enzyme label). (A) The immunosensor after
treating with a conventional HRP-secondary antibody providing one label per binding event. (B) The
immunosensor after treating with HRP-CNT (carbon nanotube) secondary antibody to obtain amplification
by providing numerous enzyme labels per binding event.Adapted with permission from Fernandez-sanchez
et al. 2004.AFM, a-fetoprotein; PSA, Prostate-specific antigen.

electrochemical immunosensors (Lim & Matsunaga 2001). More recently, other

electrochemical immunosensors have been presented based on the use of gold
nanoparticles (Chai et al. 2008) and of ormosil sol-gel membranes (Tan et al. 2007).
The assays, for their analytical performances, fabrication reproducibility and opera-
tional stability, have been presented as promising tool for clinical diagnostics.
A very recent paper has appeared that is focused on the determination of another
hormone, cortisol, by an SPR immunosensor (Stevens et al. 2008). Cortisol is a
steroid hormone required for metabolic activities and cardiovascular function. It is
considered to be an indicator of stress or disease state of a patient with normal
serum levels between 20 and 140 ng/ml. Cortisol can also be found in saliva in a
concentration range 18 ng/ml for healthy subjects: the most important advantage of
detecting cortisol in saliva and not in blood/serum is the good correlation between
salivary cortisol and levels of ‘free’ cortisol in serum, which is more biologically active
than cortisol bound to transport proteins or albumin. The system that has been
presented recently is based on a competition immunoassay with a six-channel portable
SPR biosensor using cortisol-specific monoclonal antibody. In addition, an in-line
 Biosensors for biomarkers in medical diagnostics 645

filter composed of a hollow fibre membrane (20,000 molecular mass cutoff) served to
separate small molecules from large molecular mass saliva components such as
mucins. The detection limit of the biosensor was of 0.36 ng/ml in buffer and 1 ng/ml
in saliva.

Biosensors for cardiovascular diseases

Laboratory tests are an important part in diagnosing heart infarction (World
Health Organization 1979), and fast and cost-effective diagnostics is needed. At
present cardiac troponin I or T (cTnI/T), myoglobin and natriuretic peptide (ANP),
particularly of the B type (BNP), are determined by different immunoassay methods,
such as ELISA (Katus et al. 1989), radioimmunoassay (Cummins et al. 1987) and
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immunochromatographic tests (Penttila et al. 1999). The first two are the most used
but they are time-consuming because they require several steps; the last one is a
qualitative test. As during the heart infarction the troponin T (TnT) is immediately
released to the bloodstream, a biosensor able to monitor this biomarker in a short time
(B 10 min) could improve patient care by allowing a definite diagnosis of myocardial
infarction in real time. In this regard, several biosensors for the detection of TnT and
troponin I (TnI) have been published in the last few years (Fireman Dutra et al. 2007,
Ko et al. 2007, Cody Stringer et al. 2008) based on electrochemical and optical
transduction.
A very good sensitivity and specificity were reached by an optical biosensor
coupled to quantum dots (Cody Stringer et al. 2008). The biosensor was based on
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fluorescence resonance energy transfer (FRET), a signal transduction method that

occurs between two fluorescent molecules, a donor and an acceptor. In this regard,
protein A was modified with carboxy-functionalised quantum dots, used here as
donors, whereas an anti-TnI antibody was modified with a fluorescent dye, the
acceptor. The two modified molecules were then incubated and, because protein A
bound to the antibody, the donor and the acceptor were brought within an energy
transfer distance. As the antigen TnI bound to the antibody, a conformational change
occurred in the antibody molecule, altering the distance between the donor and the
acceptor, resulting in a change of the energy transfer that could be detected by the
fluorescence detector. The biosensor achieved a detection limit of 32 nM of TnI in
buffer and of 55 nM TnI in human plasma. The response time of the biosensor was
determined to be B1 min, which was much lower then the analysis time required by
other TnI diagnostic assays.
Moreover, an SPR immunosensor has recently been presented (Fireman Dutra
et al. 2007) for the detection of TnT based on the use of immobilised monoclonal
antibodies specific for TnT. The biosensor presented a linear response range for TnT
between 0.05 and 4.5 ng/ml with a good reproducibility (CV  4.4%). The amount
of TnT was measured in human serum samples and the results were compared
with a reference method (Roche Elecsys 2010 immunoassay analyser based on
electrochemiluminescence immunoassay (ECLIA)). The measurements with the SPR
biosensor showed good agreement with the ECLIA method at 95% confidence level.
Among other biomarkers, recently, the importance of inflammatory markers in the
early detection of cardiovascular diseases has been shown (Rackley 2004). In
particular, C-reactive protein (CRP), which is used for conventional inflammation
diagnosis, can also serve as a diagnostic marker for low-grade inflammation for risk
 646 M. Mascini & S. Tombelli

estimation of cardiovascular events (Ridker et al. 1998). Normal blood serum

concentrations of humans range from 1 to 5 mg/l and protein levels
5 mg/l are an
indication of inflammatory processes (Black et al. 2004). In routine clinical analysis
CRP levels are determined by ELISA (Dominici et al. 2004) with detection limits
down to 0.2 mg/l.
New approaches in medical CRP diagnosis for cardiovascular disease require rapid
quantification in native matrices, such as saliva and urine (Christodoulides et al.
2005), which are not yet accessible for a CRP determination. A new magnetic
biosensor was presented for the determination of CRP in human serum, saliva and
urine (Meyer et al. 2007). Two CRP antibodies (clones C2 and C6) were used: C2
was used as a capturing antibody and it was immobilised onto polyethylene-sintered
filters in ABICAP† plastic columns. Clone C6 served as a secondary antibody and
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it was biotinylated and attached to streptavidin-coated magnetic beads. This

antibodymagnetic complex interacted with the captured CRP on the primary
antibody, and it could be quantified by a magnetic reader. A very low detection limit
(0.025 mg/l) was achieved with this system, which was also tested in CRP-spiked
samples of serum, saliva and urine. Calibration curves were constructed for CRP in
these matrices with good results.

Nucleic acid-based biosensors

A nucleic acid biosensor is defined as an analytical device incorporating an
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oligonucleotide, even a modified one, with a known sequence of bases, or a complex

structure of nucleic acid (such as DNA from calf thymus) either integrated within or
intimately associated with a signal transducer (Palchetti & Mascini 2008). Nucleic
acid biosensors can be used to detect DNA/RNA fragments or either biological or
chemical species. Most nucleic acid biosensors are based on the highly specific
hybridisation of complementary strands of DNA or RNA molecules; this kind of
biosensor is also called a genosensor. The probe, immobilised onto the transducer
surface, acts as the biorecognition molecule and recognises the target DNA, whereas
the transducer is the component that converts the biorecognition event into a
measurable signal. Assembly of numerous (up to a few thousand) DNA biosensors
onto the same detection platform results in DNA microarrays (or DNA chips), devices
that are increasingly used for large-scale transcriptional profiling and single-nucleotide
polymorphism (SNP) discovery.
In nucleic acid biosensors, the detection of the hybridisation event has been carried
out through different detection technologies, from label-free methods, such as
piezoelectric and SPR transduction, to other methods often requiring labels, such
as electrochemical techniques. Several reviews have recently appeared in the literature
(Lucarelli et al. 2008, Wang 2006) that elucidate all the critical aspects related to the
transduction step. A possible scheme of a genosensor is shown in Figure 3 for the case
of an SPR transduction.
As the specificity of the hybridisation reaction is essentially dependent on the
biorecognition properties of the capture oligonucleotide, design of the capture probe is
undoubtedly the most important preanalytical step. The probes can be linear
oligonucleotides or structured (hairpin) oligonucleotides, which are being used with
increasing frequency.
 Biosensors for biomarkers in medical diagnostics 647

The design of linear probes takes advantage of much commercially available

software that can design capture oligonucleotides within the hypervariable or highly
conserved regions of different genomes after their assembly and alignment. Candidate
sequences, usually 18  25 nucleotides in length, are finally tested for theoretical
melting temperature (Tm), hairpins and dimer formation and for homologies using a
Basic Local Alignment Search Tool (BLAST) search (Lucarelli et al. 2008).
The experimental variables affecting the hybridisation event at the transducer
solution interface are referred to as stringency and they generally include hybridisation
and posthybridisation-washing buffer composition and reaction temperature. When
dealing with complex sets of probes the basic requirement for a functional system is
the ability of all the different probes to hybridise their target sequences with high
affinity and specificity under the same stringency conditions.
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In addition, some probes, variable for chemical composition and conformational

arrangement, have been used to assemble DNA biosensors. Peptide nucleic acids
(PNAs) are DNA mimics in which the nucleobases are attached to a neutral
N-(2-aminoethyl)-glycine pseudopeptide backbone. If compared with the conven-
tional oligonucleotide probes, PNAs have appeared particularly interesting for the
development of electrochemical genosensing, the main reason being the drastically
different electrical characteristics of their molecular backbone.
Limiting the discussion to applications of this kind of biosensor to the field objects
of this review, several DNA-based biosensors have been developed recently for the
detection of virus-related sequences, such as hepatitis B virus (Ding et al. 2008, Yao
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et al. 2008) or papilloma virus (Xu et al. 2008, Dell’Atti et al. 2007), or for the
recognition of disease-related sequences, such as leukaemia (Chen et al. 2008) or
breast cancer (Castaneda et al. 2007). Several other papers could be listed related to
this argument, but the opinion of the authors is that weight should be given to those

Figure 3. Schematic representation of a surface plasmon resonance (SPR) sensorgram recorded during a
hybridisation cycle. The sensorgram reports the SPR signal in resonance units (RU) versus time. A baseline
can be recorded with only the probe immobilised onto the SPR chip (1). The analyte (the complementary
oligonucleotide) is then added and the signal is recorded. After the interaction the chip is washed with buffer
and the signal is again recorded (2). The analytical datum usually considered for quantitative analysis is the
signal difference between step (2) and step (1) when the sensor is in contact with the same running buffer. A
regeneration step can then be performed to dissociate the hybrid and again have the immobilised probe
available for a new hybridisation cycle.
 648 M. Mascini & S. Tombelli

works in which real samples have actually been tested (Arora et al. 2007), not to
papers where only a ‘model’ sequence has been considered using more simple
synthetic oligonucleotides.

New frontiers in nucleic acid biosensors: aptamer-based biosensors for diagnostics

The need for analytical systems capable of rapid multi-analyte measurements of
complex samples is experienced in the medical field where multi-parameter diagnostic
systems are increasingly required in order to detect all the well known and the more
recently known biomarkers for different diseases. When the detection system requires
a biomolecular recognition event, antibody-based detection methodologies are still
considered to be the standard assays in clinical analysis. These assays are well
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established and they have been demonstrated to reach the desired sensitivity and
selectivity. However, the use of antibodies in multi-analyte detection methods and in
the analysis of very complex samples could encounter some limitations, mainly
deriving from the nature and synthesis of these protein receptors. To circumvent some
of these drawbacks, other recognition molecules are being explored as alternatives.
The awareness that nucleic acids can assume stable secondary structures and that
they can be easily synthesised and functionalised has led to the idea of selecting new
nucleic acid ligands called aptamers. Aptamers are artificial single-stranded DNA or
RNA ligands that can be generated against amino acids, drugs, proteins and
other molecules (Tombelli et al. 2005). Their name derives form the Latin word
‘aptus’, which means ‘to fit’. They are generated, exploiting combinatorial chemistry
For personal use only.

technology, from very large random sequence oligonucleotide libraries, through an

iterative process of absorption, recovery and reamplification called SELEX (systema-
tic evolution of ligands by exponential enrichment) (Tuerk & Gold 1990, Ellington
& Szostak 1990).
Several reviews on aptamers have appeared in the literature, even very recently
(Famulok et al. 2007, Mairal et al. 2007), and the appealing features of their use in
bioanalysis have been thoroughly reported (Ng et al. 2006). With respect to their
application, aptamers were selected in the past mainly for their use as therapeutic
agents; for the first time, an aptamer has recently been approved by the US Food and
Drug Administration for the clinical treatment of age-related ocular vascular disease
(Cole et al. 2007). In addition to the therapeutic field, aptamers were then used in
several analytical methodologies, such as mass spectrometry (Cole et al. 2007) or
biosensors (Tombelli et al. 2005). These aptamers-based methods have been used
mainly in the clinical area for the development of diagnostic assays, despite the fact
that the analytical application of aptamers in this field is still under investigation and
the scientific community still needs further research to demonstrate the advancements
brought by this new kind of ligand.
The important characteristics for the success of analytical and diagnostic assays
based on aptamers are the affinity and the specificity of the aptamer that provides
molecular recognition. The selected aptamers can bind to their targets with affinity
ranging from the micromolar to the nanomolar level and they can discriminate
between closely related targets. This is due to the adaptive recognition: aptamers,
unstructured in solution, fold on associating with their molecular targets into
molecular architectures in which the ligand becomes an intrinsic part of the nucleic
acid structure (Hermann & Patel 2000, Aptamers in Bioanalysis 2008).
 Biosensors for biomarkers in medical diagnostics 649

This feature represents an almost unique mechanism, which has been exploited in
the design of new electrochemical sensors (Willner & Zayats 2007). In this approach
the interaction of a labelled aptamer with its target can modulate the distance of the
electroactive labels from the sensor electrode altering the redox current. In this regard,
a growth factor aptamer-based detection method has appeared in the literature
centred on the use of a platelet-derived growth factor (PDGF) DNA aptamer (Lai
et al. 2007). This approach has been presented as being well suited for point-of-care
diagnostics, owing to the high sensitivity and selectivity. The assay was based on the
use of the PDGF aptamer modified with methylthioninium chloride (MB) immobi-
lised onto a gold electrode and exploits the capability of the aptamer to fold in its
characteristic structure when in contact with the target molecule (Figure 4).
In the unfolded structure, hence in the absence of PDGF, the aptamer has only one
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of the three characteristic stems and MB, fixed at the aptamer end, is far from the
electrode surface. In the presence of PDGF the aptamer adopts the three stems
structure and the distance between MB and the electrode decreases, improving the
electron-transfer activity, with an increase of current. Exceptional sensitivity was
presented for this method with a detection limit of 50 pM for PDGF, with the
sensitivity examined in buffer and 50% diluted serum.
Most of the reported aptamer-based biosensors, sensors or assays make
use of the thrombin-binding aptamer. This DNA aptamer (15-mer, 5?-GGTTG
GTGTGGTTGG-3?) was the first one selected in vitro specific for a protein without
nucleic acid-binding properties (Bock et al. 1992). It has been investigated extensively
For personal use only.

and its G-quartet structure has been established (Smirnov & Shafer 2004). Moreover,
Tasset et al., in 1997, reported about the selection of a new thrombin-binding
aptamer, a 29-nucleotide single-stranded DNA with a Kd of 0.5 nM for the protein
(Tasset et al. 1997). This new aptamer binds to the heparin-binding exosite of
thrombin, whereas the previously selected 15-mer aptamer was known to bind the
fibrinogen-recognition exosite.
Thrombin (factor IIa) is the last enzyme protease involved in the coagulation
cascade and it converts fibrinogen to insoluble fibrin, which forms the fibrin gel either
in physiological conditions or of a pathological thrombus (Holland et al. 2000).

Figure 4. An approach based on the interaction of a labelled aptamer with its target modulating the distance
of the electroactive labels from the sensor electrode and altering the redox current. The platelet-derived
growth factor (PDGF) aptamer is immobilised onto the biosensor transducer (electrode). In the absence of
target (PDGF) the aptamer is partially unfolded keeping the electroactive label (methylthioninium chloride;
MB) far from the electrode. Upon target binding the interaction of the label is more efficient because the
aptamer forms a stable structure holding the label close to the electrode surface, leading to an increased
recorded current.
 650 M. Mascini & S. Tombelli

Therefore, thrombin plays a central role in several cardiovascular diseases and it is

thought to regulate many processes in inflammation and tissue repair at the vessel
wall.
Many assays, mainly biosensors, based on the thrombin-binding aptamer for the
detection of thrombin have been developed in the last few years (Radi et al. 2006,
Zhang et al. 2006), but only a few of them have really been used as an analytical
method for the detection of thrombin in real samples.
A sandwich assay was developed by using two selected aptamers binding thrombin
in two different sites (Centi et al. 2007). The protein captured by the first aptamer was
detected after the addition of the second biotinylated aptamer and of streptavidin
labelled with alkaline phosphatase, and the detection of the product generated by the
enzymatic reaction was achieved by differential pulse voltammetry (DPV). Good
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sensitivity and selectivity were demonstrated by the sensor with a detection limit of
0.45 nM in buffer and negligible signal generated by negative control proteins. The
system was also demonstrated to recognise the target analyte in protein-rich media
such as thrombin-spiked serum and plasma samples. Moreover, mimicking the
physiological clogging event, thrombin was generated in situ by the conversion of its
precursor prothrombin present in plasma and its concentration was measured at
different incubation times. The results correlated well with thrombogram-mimicking
software.
The best sensitivity for the detection of thrombin with a detection limit of 7.82 aM
was reached by an electrochemical assay coupled to a new amplification stra-
tegy (Zheng et al. 2007). This ultrasensitive aptamer-based bioanalytical method is
For personal use only.

based on a sandwich format with the target protein captured by the first aptamer-
functionalised magnetic nanoparticles. A second aptamer was attached to gold
nanoparticles, which served for the electrochemical transduction (Figure 5). The
innovative amplification strategy was achieved by forming network-like thiocyanuric
acid/gold nanoparticles whose aggregation greatly enhanced the transduction of the
aptamer-protein recognition event.

Discussion
Over the last decade we have witnessed a tremendous amount of activity in the area of
biosensors. Biosensors for monitoring blood glucose at home have achieved
prominence in the world diagnostics market and are now being joined by a diverse
array of biosensors for detecting other analytes of clinical importance.
Now the problem has shifted to the miniaturisation of the devices, which means
making a quantitative detection with B1ml of whole blood, then with minor pain and
the possible use of multiple sites for taking the sample, such as legs, forearms, and so
on.
Some consideration regarding biosensors and their application in medical diag-
nostics can be done taking into account several crucial features.

Point-of-care testing
Point-of-care systems are viewed as integrated systems that can process clinical
samples for several different types of biomarker in a variety of settings, such as clinical
laboratories, ambulances, doctors’ offices and the patient’s home.
 Biosensors for biomarkers in medical diagnostics 651
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Figure 5. Schematic representation of a thrombin aptasensor. Electrochemical assay coupled to a new

amplification strategy based on a sandwich format with the target protein captured by the first aptamer-
functionalised magnetic nanoparticles. A second aptamer is attached to gold nanoparticles, which served for
For personal use only.

the electrochemical transduction. Signal amplification is achieved by forming network-like thiocyanuric

acid/gold nanoparticles.

Diagnostics at hospitals is based on either large-scale automated equipment or

ELISA techniques based on bioassays, which are not suitable for bedside and
emergency medicine. In the case of general practitioners (i.e., family doctors),
diagnostics is limited to either non-quantitative sticks or sending samples to a central
lab. At present, in the case of home care of patients, patients with diabetes do self-
monitoring in a simple and cost-effective way.
Therefore, validated, intelligent, next-generation diagnostic devices and systems
based on biosensor technology with new, radically enhanced detection capabilities and
integrated sample-handling to address the most common diagnostic problems are
greatly needed. These systems must reduce sample volumes and improve limit of
detection, and make the point-of-care diagnostics fast, easy to handle and cost-
effective. Interestingly, such devices could be assembled for specific biomarkers such
as thrombin, C-reactive protein, troponin, and so on, limiting the hospital stay; this
could bring an important cut in hospital expenses and would be very welcome in many
industrial countries.

Disposable systems?
Some considerations are important in this regard: when developing a biosensor for
clinical diagnostics, a relevant aspect is the simplification of operation, and test strips
are at present still superior. Their mass production allowed the development of very
cheap devices that are user friendly; the well-known glucose electrode is a typical
example. Nowadays it is also possible to print many different working electrodes by
 652 M. Mascini & S. Tombelli

using screen-printing technology, allowing multi-analyte detection in one measure-

ment shot. However, the dilemma of disposable or multi-use sensing is still open:
savings provided by reusable sensors should not be exceeded by the expenses of
necessary maintenance. The choice of the device to be used depends on the final user:
test strips are mainly used in home monitoring, in the doctor’s consulting room and in
small clinical laboratories, whereas reusable devices are better used in clinical
laboratories (as bench instruments) or in bedside instrumentation in hospitals.
The choice between disposable or multi-use systems could also be driven by the
specific assay format: in general, for affinity biosensors in which a regeneration step of
the affinity interaction is often necessary, a disposable device could be more useful in
order to decrease the number of steps for the measurements and the time required for
analysing different samples. Anyway, in this specific field, a good compromise should
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be found between the reduction of the analysis time and the cost of the measurement
because the chips/transducers for affinity biosensors are often very expensive and
multiple use of the same chip could be more convenient.

Real samples
Applications dealing with ‘real’ clinical samples are still rare. Many papers describing
the use of biosensors in this field have only exemplary character. Detailed data as well
as validation with established methods for particular parameters are missing in most
cases. The analytical potential of biosensors in the medical diagnostics field still has to
be strengthened by the demonstration of their applicability to real matrices testing.
For personal use only.

The main problem connected with the lack of experimental data on the real
samples is the difficult ‘communication’ between technologists and hospitals. A better
connection between the researcher in the biosensor field and physicians in hospitals
could assure a real understanding of physicians’ needs, the choice of correct
applications and the availability of real samples for biosensor optimisation and
validation.

New receptors: aptamers

The commercial success of biosensors different from the glucose one has been
partially hampered by the lack of suitable biological recognition molecules that are
inexpensive to produce and stable enough to withstand storage. This problem
becomes particularly acute when designing high-density analytical arrays to support
future needs in medical diagnostics, functional genomics and proteomics. DNA
technology has furnished one powerful way to increase natural diversity and libraries
of new receptors for integration into sensors. These new biomimetic receptors, such as
aptamers, may provide a viable alternative to solving these problems.
The aptamer science in its complexity is still evolving. The therapeutic use of
aptamers is now well established, as the first aptamer was approved for clinical use in
2004. On the contrary, the different fields of analytical chemistry, when dealing with
systems based on biomolecular interactions, are still under the supremacy of
immunoassays but deep analytical studies are now demonstrating that some of the
limitations of these conventional assays can be circumvented by alternative recogni-
tion reagents such as aptamers.
Aptamer receptors, owing to their production by chemical synthesis, have several
advantages that make them very promising in analytical applications such as the
 Biosensors for biomarkers in medical diagnostics 653

development of assays for diagnostics. Several important aspects have to be examined

in detail when using aptamers as immobilised ligand, such as the immobilisation
process and the assay protocol. Moreover, several findings demonstrated that
aptamers could have differing susceptibility to assay protocols and that optimal
operating conditions could be different from one aptamer to another.
If all these important aspects are taken into consideration, the application of
aptamers as biocomponents in diagnostic assays offers a multitude of advantages, such
as the possibility of easily regenerating the function of immobilised aptamers and the
possibility of using different detection methods owing to easy labelling.
Despite the application of nucleic acids in the ‘genomics’ being well established, the
possibility of selecting aptamers for a wide range of targets has opened the possibility
of also using these nucleic acid molecules for proteomics with very low detection limits
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reaching the attomolar limit. This is important because amplification is becoming an

easy task and large dilution of the sample is then allowed.

Declaration of interest: The authors report no conflicts of interest. The authors

alone are responsible for the content and writing of the paper.

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