International Journal of Biological Macromolecules 116 (2018) 765–773

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Diagnosing human blood clotting deficiency

Chong Cheen Ong a,b, Subash C.B. Gopinath c,d,⁎, Leong Wei Xian Rebecca a,b,
Veeradasan Perumal b,e, Thangavel Lakshmipriya b, Mohamed Shuaib Mohamed Saheed a,b
a
Department of Fundamental & Applied Science, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak Darul Ridzuan, Malaysia.
b
Centre of Innovative Nanostructure & Nanodevices (COINN), Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak Darul Ridzuan, Malaysia
c
School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia
d
Institute of Nano Electronic Engineering, University Malaysia Perlis, 01000 Kangar, Perlis, Malaysia
e
Department of Mechanical Engineering, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak Darul Ridzuan, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: There are different clotting factors present in blood, carries the clotting cascade and excessive bleeding may cause
Received 10 April 2018 a deficiency in the clotting Diagnosis of this deficiency in clotting drastically reduces the potential fatality. For en-
Received in revised form 13 May 2018 abling a sensor to detect the clotting factors, suitable probes such as antibody and aptamer have been used to cap-
Accepted 14 May 2018
ture these targets on the sensing surface. Two major clotting factors were widely studied for the diagnosis of
Available online 15 May 2018
clotting deficiency, which includes factor IX and thrombin. In addition, factor IX is considered as the substitute
Keywords:
for heparin and the prothrombotic associated with the increased thrombin generation are taking into account
Medical diagnosis their prevalence. The biosensors, surface plasmon resonance, evanescent-field-coupled waveguide-mode sensor,
Factor IX metal-enhanced PicoGreen fluorescence and electrochemical aptasensor were well-documented and improve-
Thrombin ments have been made for high-performance sensing. We overviewed detecting factor IX and thrombin using
Bleeding these biosensors, for the potential application in medical diagnosis.
Blood clotting © 2018 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
2. Sensing clotting factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
2.1. Essential biosensing elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
2.1.1. Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
2.1.2. Aptamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
2.2. Detection of factor IX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
2.2.1. Surface plasmon resonance (SPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
2.2.2. Evanescent-field-coupled waveguide-mode sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
2.2.3. Electrochemical impedance spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
2.3. Detection of thrombin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
2.3.1. Metal-enhanced PicoGreen fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
2.3.2. Electrochemical aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
3. High-performance sensing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
3.1. Blocking agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
3.1.1. Bovine serum albumin (BSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
3.1.2. Poly (ethylene glycol) (PEG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
3.1.3. Ethanolamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
3.2. Improvement by gold nanoparticle (GNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
4. Current trends and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771
Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772

⁎ Corresponding author at: School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia.
E-mail address: subash@unimap.edu.my (S.C.B. Gopinath).

https://doi.org/10.1016/j.ijbiomac.2018.05.084
0141-8130/© 2018 Elsevier B.V. All rights reserved.
766 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
1. Introduction
Human has the ability to overcome the excessive bleeding upon in-
jury through hemostasis. During injury, the ruptured endothelium trig-
gers a response of protein such as tissue factor that initiates changes to
blood platelet and plasma protein fibrinogen, which form a plug at the
site of injury, therefore occlude vascular lesion [1]. These sequential in-
teractive response events to the formation of plug stopping hemorrhage
and repairing of the damaged vessel are called blood coagulation [2]. In
order to strengthen the platelet plug, clotting factors in blood plasma re-
sponse in cascade mechanism form a fibrin [1]. Generation of fibrin is
controlled by series of cofactors modulating cascade of the protease at
injury [3]. Coagulation involves two pathways (intrinsic and extrinsic),
where they are activated separately, but merges into a single pathway
to convert factor IX to factor IXa, which subsequently form thrombin
as end product [4–7] (Fig. 1).
The intrinsic pathway is also known as contact activation pathway
and extrinsic pathway is called ‘tissue factor pathway’ [1,3]. The cascade
begins when tissue factor in tissue adventitia is exposed to the circulat-
ing blood following the injury. The tissue exposed then binds to factor
VII or activate serine protease factor VIIa to initiate the extrinsic path-
way. The activated serine factor VIIa complex converts factor IX and fac-
tor X to factor IXa and factor Xa, respectively. The factor Xa generates
thrombin from prothrombin (inactive form) in the presence of calcium
(Ca2+), non-enzymatic cofactor factor Va and anionic phospholipid
Fig. 1. Blood clotting cascade scheme. Both intrinsic and extrinsic pathways are involved. These pathways are completed by about 30 proteins.
exposed on the surface of activated platelets or other cells. Produced
thrombin initiates feedback amplification loop by generating factor Va
and VIIIa by the limited proteolysis [8]. In intrinsic or contact activation
pathway, zymogen factor XII is activated to factor XIIa when in contact
with anionic surfaces such as glass, kaolin and celite [9]. Through the
limited proteolysis, factor XIIa is generated from zymogen activated by
factor XIIa and thrombin [8,9]. Factor IXa is generated by factor XIIa
using a similar mechanism. In common pathway, thrombin acts as an
enzyme, which cleaves fibrinogen, inactive circulating plasma mem-
brane to form fibrin monomers. These fibrin monomers will aggregate
spontaneously forming an insoluble fibrin polymer [2]. Fig. 1 shows
general blood clotting cascade.
Blood coagulation is a good response to the body upon injury; how-
ever, there are several thrombotic disorders that need anticoagulant
therapy for the prevention and treatment. One of the examples is hyper-
coagulability, it is a state of heightened activation of the coagulation sys-
tem, mostly by the increased activation of platelet and loss of thrombo-
resistant properties of vascular endothelium [10]. A great interest had
risen in the perioperative monitoring of blood coagulation to predict
the risk of bleeding and diagnose causes of hemorrhage during surgical
procedures [1]. During the postoperative stage, patients undergo oral
anticoagulant therapy to prevent thromboembolic disorders [1]. Hepa-
rin is used because it is cheaper, safer behavior, and easier to monitor.
However, it has limitations, such as long half-life protamine as an anti-
dote, induce thrombocytopenia and it has non-specific plasma binding.
 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
Heparin in some cases causes the platelet aggregation and dysfunction
[2]. In order to overcome these limitations, blood coagulation cascade
is studied carefully to know the major factors that affect blood clotting.
On the other hand, due to the genetic disorder some patients may
have a severe bleeding symptom, whereby the capability for the blood
to clot is low. Hemophilia A and B are two common disorders with severe
bleeding. Both are X-linked recessive disorders resulting from mutations
in the gene for blood clotting factor VIII and factor IX, respectively [11].
About 20% of patient with X-linked recessive congenital hemophilias
have factor IX deficiency and the remaining 80% is from factor VIII defi-
ciency [4]. Therefore, it is proven that factor IX and factor VIII are the
main factors of blood clotting disorder. By determining the main proteins
involved in blood clotting disorder, it is vital to monitor their amount in
the human body. Therefore, a better sensitive sensor is needed to deter-
mine the amount of clotting factors, especially factor IX and thrombin,
as they are the major clotting factors in human. The availability of poten-
tial sensors will enable detection of blood clotting disorders. Previously, to
determine the factor IX and (pro) thrombin methods like one-stage
clotting assays with factor IX-deficient plasma or with anti-factor IX
inhibitory monoclonal antibodies, immunoradiometric assays with
monoclonal antibodies, thrombin generation assays and thrombin-anti-
thrombin complex measurements have been demonstrated. In the cur-
rent overview, we focused on the precise methods used for the
diagnosis of factor IX and thrombin.
2. Sensing clotting factors
2.1. Essential biosensing elements
Biosensors have been fulfilled with the fundamental essentials,
which include a selection of probe, substrate fabrication, chemical
functionalization on the substrate, transducer and the electrical output
(Fig. 2). Among these essentials, selection of probe has been considered
as primary one, which decides the genuine interaction of the target to be
tested. The probe can be antibody, aptamer, DNA, RNA, glycan or recep-
tor molecules. Among these probes, antibody and aptamer are quite
Fig. 2. Overview on the biosensor. All necessary elements for the biosensing are displayed.
767
commonly preferred and in this overview, we gave the importance to
these two probes.
2.1.1. Antibody
Since 1967, sensor development involves the use of an antibody, was
the first immobilized antibody on solid substrate proposed by Catt and
Niall [12]. Followed by the discovery of enzyme-linked immunosorbent
assay (ELISA), several antibody-sensing strategies on the solid surface
have been proposed [12–17]. There are various types of antibody
existed for the detection of specific protein, such as anti-factor IX anti-
body against the antigen, factor IX. For the efficient binding of factor
IX to a specific antibody, it has to undergo the confirmation changes
[18]. The conformation changes can be induced by a number of di-
and trivalent metal ions such as Ca2+, Mg2+, Ba2+, and Mn2+. A recent
study showed that the addition of Mg2+ ions at saturated concentration
could increase the affinity of factor IX for anti-factor IX antibodies and
also could enhance the activation of factor IX by factor IXa [18]. The in-
teraction between antibody and antigen will provide a measurable
change in the system. The antibody has a binding site terminal with
an amine (NH2) while and carboxyl (COOH) terminal at the stem for im-
mobilization on the surface of the sensor as shown in Fig. 3 [19]. Struc-
tural analyses of blood clotting proteins (Fig. 4a & b) made the ways to
understand the configuration of antigen and antibody interactions.
2.1.2. Aptamer
Aptamer also knew as “chemical antibody” [12] is a single-stranded
DNA or RNA that binds to a wide range of molecules with higher speci-
ficity and affinity [17]. Aptamers have the ability for molecular recogni-
tion and specific binding to the corresponding target by its nature with
the ability to fold into a well-defined three-dimensional structure [20].
Aptamers behave like antibodies and are generated usually by in vitro
selection. The smaller sizes of aptamer are stable, cheaper and ease in
the modification, make aptamer as a favorable probe for past few de-
cades [21]. Aptamer also has a dissociation constant of as low as
picomolar-femtomolar [17].
Generally, RNA aptamer appears to have more capability to bind to
factor IX with a higher affinity and also at the same time discriminate
768 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
Fig. 3. Diagrammatic representation of different regions of the antibody. Antigen binding regions other important regions are indicated.
other structurally similar coagulation factors by over 5000-fold [22].
Therefore, the aptamer is suitable to be used as a probe to bind factor
IX for the detection. Aptamers are able to synthesize chemically,
which does not have batch-to-batch variation, and also reduces cost
and time of production [17,23]. The aptamer is stable, as it able to with-
stand repetitive denaturation and renaturation (temperature set back to
optimum) [24]. Unlike aptamer, antibodies will undergo irreversible
conformation change at room temperature or higher [17]. Size of the
aptamer is smaller compared to antibodies that able to reach previously
blocked or intracellular targets. Immunogenicity of the aptamer is lesser
than antibodies [17]. Since aptamers are nucleic acids, they are easy to
be labeled and modified, therefore provide simple means of detection
[23]. Because of these properties, this study was used aptamer as a
probe for the detection and antibody was set for comparison. Recent
aptamer selection using DNA pool became an additional candidate for
the detection of factor IX [25].
2.2. Detection of factor IX
As mentioned earlier, the importance of factor IX is reflected in he-
mophilia B (Christmas disease), where the protein concentration in
plasma reaches abnormal levels from the normal level of around 5
μgmL−1 (87 nM) [12]. In order to detect it, aptamer or antibody has
been used as a detection probe. For factor IX detection, there were dif-
ferent sensors have been demonstrated, which include surface plasmon
resonance and waveguide-mode sensors.
2.2.1. Surface plasmon resonance (SPR)
SPR-based instruments utilize refractive index measurement near
sensor surface using the optical mode [26]. The optical fiber SPR is
used to know the target molecular concentration and also to analyze
the molecular interactions [27]. The principle of this sensor is the utili-
zation of the light to excite the surface plasmons in the case of a planar
surface, which is the basis of many standard tools for measuring adsorp-
tion of materials on planar metal surfaces. Total internal reflection of
electromagnetic waves or monochromatic and polarized light at the in-
terface between metal-coated surface and prism surrounded by
biological component and liquid environment results in an optical phe-
nomenon known as SPR [26,28]. Molecular interaction on the interface
will cause variation in the signal obtained in concomitant with spectral
changes [26] as shown in Fig. 5a.
In the previous study, SPR sensor was used for the detection of factor
IX, with the surface was fabricated as immunosensing chip [29] to cap-
ture factor IX. SPR has been preferred for the detection of factor IX, be-
cause of it is label-free and shows online dynamic properties [30].
Biosensor properties from this sensor are constructed by a prism with
a coupling method of the attenuated total reflection, the propagation
constants of incident light wave and surface plasmon wave along x-
axis [30]. Sandwich assays are used to detect factor IX that is aptamer-
protein-antibody or antibody-protein-aptamer. When compare be-
tween these two sandwich assays, aptamer-protein-antibody had
been proven to show a greater sensitivity [12]. By a combination of
both co-immobilization of polymers (N6-PEG with PEG-b-PAMA)
completely abolished bio-fouling, whereas BSA shows a higher non-
specificity. Using polymer as a non-fouling agent, 800 fM detection
limit was obtained.
2.2.2. Evanescent-field-coupled waveguide-mode sensor
Development of sensor using waveguide-mode is similar to SPR sen-
sor except for the mode of measurement was wave guided instead of
surfaced [31]. The principle of this sensor is utilizing a visible light to ex-
cite the waveguide mode and changed the incident light reflectivity
over a narrow angular region. The sensitivity of the waveguide modes
to the dielectric environment near the surfaces of the waveguides and
variety of dielectric environment cause the changes in reflectivity.
Upon attachment of biomolecules on the surface, the resonance angle
will change and the attached biomolecules can be determined from
the intensity changes of the reflected light [28,32].
In previous studies, an evanescent-field-coupled waveguide-mode
(EFC-WM) biosensor was used and it utilizes monolithic SiO2 sensing
plates [28,32–34]. Quantitative analysis was performed by using
streptavidin-conjugated gold nanoparticles [35]. A waveguide-mode
sensor is a good choice in analyzing biomolecular interactions; in the
above case authors performed the aptamer-factor IX interaction. This
 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
Fig. 4. (a) Crystal structure of calcium-stabilized human factor IX bound to a
conformation-specific anti-factor IX antibody. (b) Un-liganded wild-type human
thrombin.
biosensor has several advantages such as its nanopore-fabricated sur-
face, which enables the waveguide-based sensor to have a larger surface
area with ultimate greater sensitivity [34]. A waveguide-mode sensor
utilizes electric field enhancement in the sensor chip (similar to SPR
sensor) and is, therefore, more sensitive than a reflectance absorption
spectrometer [36]. The EFC-WM used has a multilayer structure of
769
Fig. 5. (a) Schematics representation of surface plasmon resonance principle. (b)
Schematics representation of waveguide-mode sensor. Both sensing systems behave in a
similar way, however, in SPR has surface mode whereas waveguide has a guiding mode.
SiO2 waveguide, a thin single crystalline Si layer, and a SiO2 glass sub-
strate [34,35]. Structure of EFC-WM is as shown in Fig. 5b. From the di-
agram, the light source is from xenon lamp and light is detected using a
spectrophotometer. Factor IX protein bound to aptamer is attached to
the top of the SiO2 layer. Since PEG-b-PAAc was used as a blocking
agent to prevent non-specific binding on the surface sensor, the factor
IX detection limit of 0.1 pM was obtained using this biosensor.
2.2.3. Electrochemical impedance spectroscopy
Interdigitated electrode is a part coming from electrochemical sen-
sors, which use impedance spectroscopy to transduce signals [37]. Com-
binations both electrochemical impedance spectroscopy and
interdigitated electrode (IDE) have shown interest in biological sensing
[37,38]. The array produced can be used for low cost point-of-care clin-
ical test systems, such as cancer diagnosis and heart diseases [38]. The
low cost is due to ease of miniaturization, batch fabrication and automa-
tion. Detection of factor IX can also be done using surface modified in-
terdigitated electrode and use electrochemical impedance
spectroscopy as means of measurement [39].
The sample (factor IX) binds to the probe (aptamer or antibody). The
binding will cause changes in electric properties in the vicinity of elec-
trode [38]. The change in electric properties will cause the change in
the impedance; therefore direct electric signal can be measured. The
Nyquist plot can be produced to visualize the frequency response of
IDE. Recently Cheen et al. [39] had utilized IDE with two kinds of the
probe (antibody and aptamer) and electrochemical impedance spec-
troscopy to detect factor IX. As shown in Fig. 6a, IDE was modified
using (3-Aminopropyl)triethoxysilane (APTES) followed by glutaralde-
hyde before attachment of biomolecule. Using this method researchers
able to achieve the detection limit of 10 pM of factor IX protein [39].
Using similar impedance analysis with different sensing patterns,
Gopinath et al. [40] recently attained the sensitivity of 1 pM using anti-
body and gold nanoparticle conjugate (Fig. 6b). These sensitivities are
770 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
quite fair as the real concentration of factor IX in human blood serum is
87 nM [40].
2.3. Detection of thrombin
Based on blood clotting cascade mentioned earlier, thrombin is in-
volved in cleaving fibrinogen to form fibrin, which is vital for blood
clotting system. There are different sensors have been demonstrated
for the detection of thrombin as revealed in the case of factor IX. Even
though detection strategies designed for both factor IX and thrombin
are important, the main difference can be obviously found as factor IX
participates in the middle stage of the clotting reaction, whereas throm-
bin forms at the later stage. However, thrombin is still considered to be
vital for the diagnosis of clotting deficiency. Crystal structure studies on
thrombin further support the development of high-performance sens-
ing systems (Fig. 4b).
2.3.1. Metal-enhanced PicoGreen fluorescence
Development of fluorescence spectroscopy had been driven by the
implementation of metal-enhanced fluorescence due to its increase in
both fluorophore emission intensity and photostability [40]. The princi-
ple behind this sensor is utilizing the affinity of chromophore on target
protein and the ratio between fluorescence intensity of the chromo-
phore when bound to the target compared to the free chromophore in
solution, to determine the concentration of target protein. In the previ-
ous study, the method of detection used PicoGreen as a signal molecule
and the surface-bound metal-enhanced fluorescence substrate as the
signal enhancers [41]. For this sensor to detect thrombin to be a throm-
bin aptasensor, 15-bp human thrombin-binding aptamer was pro-
duced. Using this method of analysis, the amount of thrombin can be
quantified by monitoring the fluorescence intensity. The fluorescence
intensity is proportional to the amount of dissociated aptamer. Silver
Fig. 6. (a) Schematic illustration for detecting factor IX using aptamer and antibody
sandwich on IDE; (b) Schematic illustration for detecting factor IX using antibody on IDE.
island film was used to make fluorescence stronger and much more sta-
ble, therefore increased the sensitivity of the sensor [41]. The linear re-
lation was observed whereby, when the thrombin concentration is
increased, the fluorescence intensity is decreased. Using this sensor,
0.073 nM detection limit was obtained. This concentration is relatively
low compared to the concentration of thrombin in real human blood
serum, makes the ideal situation with this sensor.
2.3.2. Electrochemical aptasensor
Electrochemical aptasensors have been used as one option for bio-
sensing method in recent studies [42–45]. Electrochemical aptasensor
utilizes AC impedance spectroscopy to detect the changes in charge
transfer resistance upon attachment of biomolecule [42]. In the previous
study, the method involves the usage of carboxymethyl-PEG-
carboxymethyl and thrombin-binding aptamer (TBA) and assembled
on the electrode (Fig. 7). Thrombin will catch on the electrode via TBA
and generate the inhibition in electro-transfer by a barrier [46]. This
method shows a great potential for detecting thrombin due to lower
cost, label-free operation, ease of miniaturization, and the ability for in-
tegration with multi-array for diagnostics [46,47]. Cyclic voltammetry is
used to monitor various stages of aptasensing on the electrodes. TBA is
grafted onto the surface film with a great membrane resistance. This
stage causes a high peak current indicating an organic layer of TBA
form on the electrode. The modified electrode immersed in thrombin
solution, the lower peak is formed due to its high-density thrombin
binding, which causes steric hindrance and inhibits electrotransfer.
From this method of sensing, 15.6 fM detection limit was obtained
[46] compared to the previous research is showing 0.76 pM [48].
3. High-performance sensing
With the aforementioned sensors even though there were satisfied
levels of sensitivities attained, developing high-performance sensors
need a fine-tuning. The fine-tuning to improve the performance of sen-
sors can be fulfilled by different ways. The common strategies followed
are co-immobilization of block-polymers or other potential blocking
agents along with the probe and usage of nanoparticles, such as metal
Fig. 7. Diagrammatic representation of the interaction with interdigitated electrodes as
sensor surface for thrombin detection.
 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
or non-metal particles. An effectiveness of sensor can also be improved
by making sure the immobilized biomolecules are correctly oriented on
the sensing surface and by minimizing the non-specific biofouling in
order to increase the signal to noise ratio (S/N) [35]. These steps are im-
portant in making the sensor to be more specific in detection of the de-
sired protein and also to improve the sensitivity of detection at an
extremely low concentration of protein.
3.1. Blocking agents
Human clotting factors can be detected by applying the blood sam-
ple to the sensor chip surface to immobilize blood clotting factors. The
specific interaction of probe with target protein (clotting factors) takes
place on the surface. However, it is important to reduce and overcome
the non-specific interactions of the probe with target protein to sup-
press background signals [35]. The most common approach is covalent
to bind the oligonucleotides on the surface by the formation of a
polyorganosiloxane layer with organic functionalities [49]. Fig. 8a
shows the schematic representation of blocking agents binding to the
modified sensing surface.
3.1.1. Bovine serum albumin (BSA)
BSA is widely used blocking agent [50] to wash away non-specific
molecules from the surface that would compete with the specifically
targeted protein [49]. However, in an experiment conducted using dif-
ferent preparations of BSA as blocking agent in ELISA give a range of
the varied amount of non-specific binding with reactants [50]. BSA
also shows to cause stiction of the Micro-Electro-Mechanical Systems
resonators. Besides that, BSA does not bind strongly to the underlying
layer, which causes detachment and leads to weight calculation error
[49].
3.1.2. Poly (ethylene glycol) (PEG)
PEG is an effective hydrophilic polymer commonly used for the sur-
face modification [51]. This technique of modification is known as
Fig. 8. (a) Schematic illustration of the function of blocking agent in preventing biofouling.
(b) Schematic illustration for enhancing sensitivity using gold nanoparticle.
771
PEGylation [51]. PEG is used because it is chemically inert and provides
terminal hydroxyl groups that can be used as anchors for the functional
groups. It is also nontoxic and dispersible in water [12]. For PEG-deriv-
atives are immobilizing effectively on a substrate surface, anchoring
strategy must be designed based on specific interactions; hydrophobic
or electrostatic interactions or covalent conjugation. As in previous
studies, multi-anchoring shows the most effective ways of stably
immobilizing PEG derivatives on the surface of the sensor [35].
3.1.3. Ethanolamine
For COOH-modified surfaces, ethanolamine is commonly used as
blocking agent [12]. In presence of ethanolamine, it can be observed
that specific binding of factor IX to antibody/aptamer is in a dose-de-
pendent manner [12]. However, some of the active groups on COOH
surface may facilitate the non-specificity with factor IX. This is due to
the amine group on the protein easily binds COOH surface. But still eth-
anolamine is considered as a good blocking agent on silicon substrates
[31].
3.2. Improvement by gold nanoparticle (GNP)
GNP is one of the fabricated nanomaterials, which has been used for
the development of sensors in recent studies [42,52]. GNP has unique
properties such as compatibility with surface functionalization, non-re-
activity towards biological materials, the capability to easily disperse in
water, and compatibility to be tailored with uniform and different nano-
sizes [52–54]. Adsorption of visible light for GNPs is around 520 nm en-
ables these particles to be used in several optical sensors [52]. Besides
that, GNPs have the propensity for hydrophobic absorption, electro-
static attractions, and covalent bonding, therefore, possess the capabil-
ity to adsorb small oligonucleotides [52]. GNP can also provide a
spatial arrangement for the molecular immobilization and aid for in-
creasing the sensitivities to several folds. Fig. 8b displays schematic il-
lustration for GNP-mediated high-performance sensing.
4. Current trends and future developments
Biosensors have been in a great development for detecting blood
clotting factors and enhance the potential clinical diagnosis. This is im-
portant to prevent loss of excessive blood that may cause fatality upon
injury. Current routine clinical methods for factor IX are measuring
the activity before and after incubation through a partial thromboplas-
tin time assay. To move forward, research has been done enable samples
to be analyzed by the direct quantitation within an hour. Factor IX pro-
tein can be quantified up to pico-scale using the advancement of nano-
technology. On the other hand, fluorescent-based quantifications are
yielding a further strength. It utilizes 7-amino-4-methyl coumarin
fluorogenic peptide by releasing fluorophore when cleaved by throm-
bin. Over the decade, studies had been conducted to increase the sensi-
tivity of the biosensor and to be more selective on the targeted clotting
factors, factor IX and prothrombotic associated thrombin generation.
The sensitivity limit of current clinical technologies is easily attaining
1 ng. The future development of nanotechnology is giving an expecta-
tion to reduce the analytical time and increase the sensitivity further
down. Technology advancement is also the co-current development of
biosensors; more portable devices may be developed in future to detect
several clotting factors instead of complicated and lengthier procedures.
Nanotechnology has shown us a portable handheld device with the pos-
sibility of determining any desired analyte. In parallel to the diagnosis,
progress with the clinical phase trials has been done with factor IX
and thrombin. Both clotting factors are under different phase trials
and progressing well. Further, cancer and gene therapy researches con-
nected with hemophilia are documented [55–57] and need to be pro-
moted further to get a deep insight. It is also wise to generate the
possibility of non-invasive strategies for the beneficial to the child and
older patients by replacing the current invasive methods.
772 C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773
Conflict of interests
None declared.
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