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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 exposed on the surface of activated platelets or other cells. Produced


thrombin initiates feedback amplification loop by generating factor Va
Human has the ability to overcome the excessive bleeding upon in- and VIIIa by the limited proteolysis [8]. In intrinsic or contact activation
jury through hemostasis. During injury, the ruptured endothelium trig- pathway, zymogen factor XII is activated to factor XIIa when in contact
gers a response of protein such as tissue factor that initiates changes to with anionic surfaces such as glass, kaolin and celite [9]. Through the
blood platelet and plasma protein fibrinogen, which form a plug at the limited proteolysis, factor XIIa is generated from zymogen activated by
site of injury, therefore occlude vascular lesion [1]. These sequential in- factor XIIa and thrombin [8,9]. Factor IXa is generated by factor XIIa
teractive response events to the formation of plug stopping hemorrhage using a similar mechanism. In common pathway, thrombin acts as an
and repairing of the damaged vessel are called blood coagulation [2]. In enzyme, which cleaves fibrinogen, inactive circulating plasma mem-
order to strengthen the platelet plug, clotting factors in blood plasma re- brane to form fibrin monomers. These fibrin monomers will aggregate
sponse in cascade mechanism form a fibrin [1]. Generation of fibrin is spontaneously forming an insoluble fibrin polymer [2]. Fig. 1 shows
controlled by series of cofactors modulating cascade of the protease at general blood clotting cascade.
injury [3]. Coagulation involves two pathways (intrinsic and extrinsic), Blood coagulation is a good response to the body upon injury; how-
where they are activated separately, but merges into a single pathway ever, there are several thrombotic disorders that need anticoagulant
to convert factor IX to factor IXa, which subsequently form thrombin therapy for the prevention and treatment. One of the examples is hyper-
as end product [4–7] (Fig. 1). coagulability, it is a state of heightened activation of the coagulation sys-
The intrinsic pathway is also known as contact activation pathway tem, mostly by the increased activation of platelet and loss of thrombo-
and extrinsic pathway is called ‘tissue factor pathway’ [1,3]. The cascade resistant properties of vascular endothelium [10]. A great interest had
begins when tissue factor in tissue adventitia is exposed to the circulat- risen in the perioperative monitoring of blood coagulation to predict
ing blood following the injury. The tissue exposed then binds to factor the risk of bleeding and diagnose causes of hemorrhage during surgical
VII or activate serine protease factor VIIa to initiate the extrinsic path- procedures [1]. During the postoperative stage, patients undergo oral
way. The activated serine factor VIIa complex converts factor IX and fac- anticoagulant therapy to prevent thromboembolic disorders [1]. Hepa-
tor X to factor IXa and factor Xa, respectively. The factor Xa generates rin is used because it is cheaper, safer behavior, and easier to monitor.
thrombin from prothrombin (inactive form) in the presence of calcium However, it has limitations, such as long half-life protamine as an anti-
(Ca2+), non-enzymatic cofactor factor Va and anionic phospholipid dote, induce thrombocytopenia and it has non-specific plasma binding.

Fig. 1. Blood clotting cascade scheme. Both intrinsic and extrinsic pathways are involved. These pathways are completed by about 30 proteins.
C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773 767

Heparin in some cases causes the platelet aggregation and dysfunction commonly preferred and in this overview, we gave the importance to
[2]. In order to overcome these limitations, blood coagulation cascade these two probes.
is studied carefully to know the major factors that affect blood clotting.
On the other hand, due to the genetic disorder some patients may 2.1.1. Antibody
have a severe bleeding symptom, whereby the capability for the blood Since 1967, sensor development involves the use of an antibody, was
to clot is low. Hemophilia A and B are two common disorders with severe the first immobilized antibody on solid substrate proposed by Catt and
bleeding. Both are X-linked recessive disorders resulting from mutations Niall [12]. Followed by the discovery of enzyme-linked immunosorbent
in the gene for blood clotting factor VIII and factor IX, respectively [11]. assay (ELISA), several antibody-sensing strategies on the solid surface
About 20% of patient with X-linked recessive congenital hemophilias have been proposed [12–17]. There are various types of antibody
have factor IX deficiency and the remaining 80% is from factor VIII defi- existed for the detection of specific protein, such as anti-factor IX anti-
ciency [4]. Therefore, it is proven that factor IX and factor VIII are the body against the antigen, factor IX. For the efficient binding of factor
main factors of blood clotting disorder. By determining the main proteins IX to a specific antibody, it has to undergo the confirmation changes
involved in blood clotting disorder, it is vital to monitor their amount in [18]. The conformation changes can be induced by a number of di-
the human body. Therefore, a better sensitive sensor is needed to deter- and trivalent metal ions such as Ca2+, Mg2+, Ba2+, and Mn2+. A recent
mine the amount of clotting factors, especially factor IX and thrombin, study showed that the addition of Mg2+ ions at saturated concentration
as they are the major clotting factors in human. The availability of poten- could increase the affinity of factor IX for anti-factor IX antibodies and
tial sensors will enable detection of blood clotting disorders. Previously, to also could enhance the activation of factor IX by factor IXa [18]. The in-
determine the factor IX and (pro) thrombin methods like one-stage teraction between antibody and antigen will provide a measurable
clotting assays with factor IX-deficient plasma or with anti-factor IX change in the system. The antibody has a binding site terminal with
inhibitory monoclonal antibodies, immunoradiometric assays with an amine (NH2) while and carboxyl (COOH) terminal at the stem for im-
monoclonal antibodies, thrombin generation assays and thrombin-anti- mobilization on the surface of the sensor as shown in Fig. 3 [19]. Struc-
thrombin complex measurements have been demonstrated. In the cur- tural analyses of blood clotting proteins (Fig. 4a & b) made the ways to
rent overview, we focused on the precise methods used for the understand the configuration of antigen and antibody interactions.
diagnosis of factor IX and thrombin.
2.1.2. Aptamer
Aptamer also knew as “chemical antibody” [12] is a single-stranded
2. Sensing clotting factors 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-
2.1. Essential biosensing elements tion and specific binding to the corresponding target by its nature with
the ability to fold into a well-defined three-dimensional structure [20].
Biosensors have been fulfilled with the fundamental essentials, Aptamers behave like antibodies and are generated usually by in vitro
which include a selection of probe, substrate fabrication, chemical selection. The smaller sizes of aptamer are stable, cheaper and ease in
functionalization on the substrate, transducer and the electrical output the modification, make aptamer as a favorable probe for past few de-
(Fig. 2). Among these essentials, selection of probe has been considered cades [21]. Aptamer also has a dissociation constant of as low as
as primary one, which decides the genuine interaction of the target to be picomolar-femtomolar [17].
tested. The probe can be antibody, aptamer, DNA, RNA, glycan or recep- Generally, RNA aptamer appears to have more capability to bind to
tor molecules. Among these probes, antibody and aptamer are quite factor IX with a higher affinity and also at the same time discriminate

Fig. 2. Overview on the biosensor. All necessary elements for the biosensing are displayed.
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]. biological component and liquid environment results in an optical phe-
Therefore, the aptamer is suitable to be used as a probe to bind factor nomenon known as SPR [26,28]. Molecular interaction on the interface
IX for the detection. Aptamers are able to synthesize chemically, will cause variation in the signal obtained in concomitant with spectral
which does not have batch-to-batch variation, and also reduces cost changes [26] as shown in Fig. 5a.
and time of production [17,23]. The aptamer is stable, as it able to with- In the previous study, SPR sensor was used for the detection of factor
stand repetitive denaturation and renaturation (temperature set back to IX, with the surface was fabricated as immunosensing chip [29] to cap-
optimum) [24]. Unlike aptamer, antibodies will undergo irreversible ture factor IX. SPR has been preferred for the detection of factor IX, be-
conformation change at room temperature or higher [17]. Size of the cause of it is label-free and shows online dynamic properties [30].
aptamer is smaller compared to antibodies that able to reach previously Biosensor properties from this sensor are constructed by a prism with
blocked or intracellular targets. Immunogenicity of the aptamer is lesser a coupling method of the attenuated total reflection, the propagation
than antibodies [17]. Since aptamers are nucleic acids, they are easy to constants of incident light wave and surface plasmon wave along x-
be labeled and modified, therefore provide simple means of detection axis [30]. Sandwich assays are used to detect factor IX that is aptamer-
[23]. Because of these properties, this study was used aptamer as a protein-antibody or antibody-protein-aptamer. When compare be-
probe for the detection and antibody was set for comparison. Recent tween these two sandwich assays, aptamer-protein-antibody had
aptamer selection using DNA pool became an additional candidate for been proven to show a greater sensitivity [12]. By a combination of
the detection of factor IX [25]. both co-immobilization of polymers (N6-PEG with PEG-b-PAMA)
completely abolished bio-fouling, whereas BSA shows a higher non-
2.2. Detection of factor IX specificity. Using polymer as a non-fouling agent, 800 fM detection
limit was obtained.
As mentioned earlier, the importance of factor IX is reflected in he-
mophilia B (Christmas disease), where the protein concentration in 2.2.2. Evanescent-field-coupled waveguide-mode sensor
plasma reaches abnormal levels from the normal level of around 5 Development of sensor using waveguide-mode is similar to SPR sen-
μgmL−1 (87 nM) [12]. In order to detect it, aptamer or antibody has sor except for the mode of measurement was wave guided instead of
been used as a detection probe. For factor IX detection, there were dif- surfaced [31]. The principle of this sensor is utilizing a visible light to ex-
ferent sensors have been demonstrated, which include surface plasmon cite the waveguide mode and changed the incident light reflectivity
resonance and waveguide-mode sensors. over a narrow angular region. The sensitivity of the waveguide modes
to the dielectric environment near the surfaces of the waveguides and
2.2.1. Surface plasmon resonance (SPR) variety of dielectric environment cause the changes in reflectivity.
SPR-based instruments utilize refractive index measurement near Upon attachment of biomolecules on the surface, the resonance angle
sensor surface using the optical mode [26]. The optical fiber SPR is will change and the attached biomolecules can be determined from
used to know the target molecular concentration and also to analyze the intensity changes of the reflected light [28,32].
the molecular interactions [27]. The principle of this sensor is the utili- In previous studies, an evanescent-field-coupled waveguide-mode
zation of the light to excite the surface plasmons in the case of a planar (EFC-WM) biosensor was used and it utilizes monolithic SiO2 sensing
surface, which is the basis of many standard tools for measuring adsorp- plates [28,32–34]. Quantitative analysis was performed by using
tion of materials on planar metal surfaces. Total internal reflection of streptavidin-conjugated gold nanoparticles [35]. A waveguide-mode
electromagnetic waves or monochromatic and polarized light at the in- sensor is a good choice in analyzing biomolecular interactions; in the
terface between metal-coated surface and prism surrounded by above case authors performed the aptamer-factor IX interaction. This
C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773 769

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
Fig. 4. (a) Crystal structure of calcium-stabilized human factor IX bound to a Nyquist plot can be produced to visualize the frequency response of
conformation-specific anti-factor IX antibody. (b) Un-liganded wild-type human IDE. Recently Cheen et al. [39] had utilized IDE with two kinds of the
thrombin. probe (antibody and aptamer) and electrochemical impedance spec-
troscopy to detect factor IX. As shown in Fig. 6a, IDE was modified
biosensor has several advantages such as its nanopore-fabricated sur- using (3-Aminopropyl)triethoxysilane (APTES) followed by glutaralde-
face, which enables the waveguide-based sensor to have a larger surface hyde before attachment of biomolecule. Using this method researchers
area with ultimate greater sensitivity [34]. A waveguide-mode sensor able to achieve the detection limit of 10 pM of factor IX protein [39].
utilizes electric field enhancement in the sensor chip (similar to SPR Using similar impedance analysis with different sensing patterns,
sensor) and is, therefore, more sensitive than a reflectance absorption Gopinath et al. [40] recently attained the sensitivity of 1 pM using anti-
spectrometer [36]. The EFC-WM used has a multilayer structure of 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 island film was used to make fluorescence stronger and much more sta-
87 nM [40]. ble, therefore increased the sensitivity of the sensor [41]. The linear re-
lation was observed whereby, when the thrombin concentration is
2.3. Detection of thrombin increased, the fluorescence intensity is decreased. Using this sensor,
0.073 nM detection limit was obtained. This concentration is relatively
Based on blood clotting cascade mentioned earlier, thrombin is in- low compared to the concentration of thrombin in real human blood
volved in cleaving fibrinogen to form fibrin, which is vital for blood serum, makes the ideal situation with this sensor.
clotting system. There are different sensors have been demonstrated
for the detection of thrombin as revealed in the case of factor IX. Even 2.3.2. Electrochemical aptasensor
though detection strategies designed for both factor IX and thrombin Electrochemical aptasensors have been used as one option for bio-
are important, the main difference can be obviously found as factor IX sensing method in recent studies [42–45]. Electrochemical aptasensor
participates in the middle stage of the clotting reaction, whereas throm- utilizes AC impedance spectroscopy to detect the changes in charge
bin forms at the later stage. However, thrombin is still considered to be transfer resistance upon attachment of biomolecule [42]. In the previous
vital for the diagnosis of clotting deficiency. Crystal structure studies on study, the method involves the usage of carboxymethyl-PEG-
thrombin further support the development of high-performance sens- carboxymethyl and thrombin-binding aptamer (TBA) and assembled
ing systems (Fig. 4b). 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
2.3.1. Metal-enhanced PicoGreen fluorescence method shows a great potential for detecting thrombin due to lower
Development of fluorescence spectroscopy had been driven by the cost, label-free operation, ease of miniaturization, and the ability for in-
implementation of metal-enhanced fluorescence due to its increase in tegration with multi-array for diagnostics [46,47]. Cyclic voltammetry is
both fluorophore emission intensity and photostability [40]. The princi- used to monitor various stages of aptasensing on the electrodes. TBA is
ple behind this sensor is utilizing the affinity of chromophore on target grafted onto the surface film with a great membrane resistance. This
protein and the ratio between fluorescence intensity of the chromo- stage causes a high peak current indicating an organic layer of TBA
phore when bound to the target compared to the free chromophore in form on the electrode. The modified electrode immersed in thrombin
solution, to determine the concentration of target protein. In the previ- solution, the lower peak is formed due to its high-density thrombin
ous study, the method of detection used PicoGreen as a signal molecule binding, which causes steric hindrance and inhibits electrotransfer.
and the surface-bound metal-enhanced fluorescence substrate as the From this method of sensing, 15.6 fM detection limit was obtained
signal enhancers [41]. For this sensor to detect thrombin to be a throm- [46] compared to the previous research is showing 0.76 pM [48].
bin aptasensor, 15-bp human thrombin-binding aptamer was pro-
duced. Using this method of analysis, the amount of thrombin can be 3. High-performance sensing
quantified by monitoring the fluorescence intensity. The fluorescence
intensity is proportional to the amount of dissociated aptamer. Silver 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. 6. (a) Schematic illustration for detecting factor IX using aptamer and antibody Fig. 7. Diagrammatic representation of the interaction with interdigitated electrodes as
sandwich on IDE; (b) Schematic illustration for detecting factor IX using antibody on IDE. sensor surface for thrombin detection.
C.C. Ong et al. / International Journal of Biological Macromolecules 116 (2018) 765–773 771

or non-metal particles. An effectiveness of sensor can also be improved PEGylation [51]. PEG is used because it is chemically inert and provides
by making sure the immobilized biomolecules are correctly oriented on terminal hydroxyl groups that can be used as anchors for the functional
the sensing surface and by minimizing the non-specific biofouling in groups. It is also nontoxic and dispersible in water [12]. For PEG-deriv-
order to increase the signal to noise ratio (S/N) [35]. These steps are im- atives are immobilizing effectively on a substrate surface, anchoring
portant in making the sensor to be more specific in detection of the de- strategy must be designed based on specific interactions; hydrophobic
sired protein and also to improve the sensitivity of detection at an or electrostatic interactions or covalent conjugation. As in previous
extremely low concentration of protein. studies, multi-anchoring shows the most effective ways of stably
immobilizing PEG derivatives on the surface of the sensor [35].
3.1. Blocking agents
3.1.3. Ethanolamine
Human clotting factors can be detected by applying the blood sam- For COOH-modified surfaces, ethanolamine is commonly used as
ple to the sensor chip surface to immobilize blood clotting factors. The blocking agent [12]. In presence of ethanolamine, it can be observed
specific interaction of probe with target protein (clotting factors) takes that specific binding of factor IX to antibody/aptamer is in a dose-de-
place on the surface. However, it is important to reduce and overcome pendent manner [12]. However, some of the active groups on COOH
the non-specific interactions of the probe with target protein to sup- surface may facilitate the non-specificity with factor IX. This is due to
press background signals [35]. The most common approach is covalent the amine group on the protein easily binds COOH surface. But still eth-
to bind the oligonucleotides on the surface by the formation of a anolamine is considered as a good blocking agent on silicon substrates
polyorganosiloxane layer with organic functionalities [49]. Fig. 8a [31].
shows the schematic representation of blocking agents binding to the
modified sensing surface. 3.2. Improvement by gold nanoparticle (GNP)

3.1.1. Bovine serum albumin (BSA) GNP is one of the fabricated nanomaterials, which has been used for
BSA is widely used blocking agent [50] to wash away non-specific the development of sensors in recent studies [42,52]. GNP has unique
molecules from the surface that would compete with the specifically properties such as compatibility with surface functionalization, non-re-
targeted protein [49]. However, in an experiment conducted using dif- activity towards biological materials, the capability to easily disperse in
ferent preparations of BSA as blocking agent in ELISA give a range of water, and compatibility to be tailored with uniform and different nano-
the varied amount of non-specific binding with reactants [50]. BSA sizes [52–54]. Adsorption of visible light for GNPs is around 520 nm en-
also shows to cause stiction of the Micro-Electro-Mechanical Systems ables these particles to be used in several optical sensors [52]. Besides
resonators. Besides that, BSA does not bind strongly to the underlying that, GNPs have the propensity for hydrophobic absorption, electro-
layer, which causes detachment and leads to weight calculation error static attractions, and covalent bonding, therefore, possess the capabil-
[49]. ity to adsorb small oligonucleotides [52]. GNP can also provide a
spatial arrangement for the molecular immobilization and aid for in-
3.1.2. Poly (ethylene glycol) (PEG) creasing the sensitivities to several folds. Fig. 8b displays schematic il-
PEG is an effective hydrophilic polymer commonly used for the sur- lustration for GNP-mediated high-performance sensing.
face modification [51]. This technique of modification is known as
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
Fig. 8. (a) Schematic illustration of the function of blocking agent in preventing biofouling. possibility of non-invasive strategies for the beneficial to the child and
(b) Schematic illustration for enhancing sensitivity using gold nanoparticle. 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 [27] S. Shi, L. Wang, R. Su, B. Liu, R. Huang, W. Qi, Z. He, A polydopamine-modified optical
fiber SPR biosensor using electroless-plated gold films for immunoassays, Biosens.
Bioelectron. 74 (2015) 454–460, https://doi.org/10.1016/j.bios.2015.06.080.
None declared. [28] S.C.B. Gopinath, K. Awazu, M. Fujimaki, K. Sugimoto, Y. Ohki, T. Komatsubara, J.
Tominaga, K.C. Gupta, P.K.R. Kumar, Influence of nanometric holes on the sensitivity
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