Aptamer-antibody dual probes on single-walled carbon nanotube bridged dielectrode: Comparative analysis on human blood clotting factor
Introduction
Haemophilia is the bleeding defect of the blood clotting factors, leads incomplete clotting pathways with ultimate interference in the platelet formation to stop the bleeding at the injury site [1]. People with haemophilia produce a lower amount of factor IX or VIII, this cause difficulty in clotting process within the time frame and leads to bleed for a longer period after an injury [2], [3], [4]. In this conjunction, it is mandatory to quantify the level of clotting factor IX (FIX), which makes the way to take the proper treatment for the Haemophilia people [5], [6], [7]. In this research, fabricated the single-walled carbon nanotube (SWCN)-modified interdigitated electrode (IDE) sensor is to quantify the level of FIX using the dual probe system with aptamer or antibody.
Aptamer, an ‘artificial antibody’ is either DNA or RNA, generated from the randomized library of molecules by three simple processes namely binding, separation and amplification [8], [9], [10]. The selected aptamers have higher binding affinity to the target molecule. Compared with antibody, aptamers have more advantages, such as high sensitivity with target molecular detection, stable, easy to synthesize and non-immunogenicity [11]. The application of aptamers are widely spread in all the fields, which includes medical diagnosis, environmental, and drug delivery [12], [13], [14]. Among others, aptamer application in the field of diagnosis is most welcome due to its high selective and sensitive detection of the target. Various aptamers have been selected for diagnosing purposes and used to detect the diseases, predominantly with cancer, viral infection and bacterial infection [15], [16], [17]. Moreover, aptamers can bind to their target in the small region, and they can discriminate the closely related molecules [18]. Previously, Lakshmipriya et al. have selected the aptamers against Influenza virus B, which could clearly discriminate the closely related influenza viruses [14]. Since aptamers and antibodies having their unique characteristics in the application of medical diagnosis, this work compared the detection of FIX by its aptamer and antibody on the SWCNT-modified IDE sensing surface.
Moreover, the sandwich pattern with aptamer and antibody is also carried out to improve the detection of FIX. In any sandwich assay, capturing probe molecule plays a major role to improve the detection of target molecule. If more target molecules are captured, it is more possible to interact the higher number of targets. In general, polyclonal antibody is used as the capture molecule to attract a greater number of target molecule in the sandwich assay, and then monoclonal antibody used as the detection molecule for the higher specificity [19]. The current assay tried both aptamer and antibody as the capture molecule interpedently for comparison in detection of FIX. Further, aptamer-FIX-antibody and antibody-FIX-aptamer sandwich patterns were tested on the SWCNT-IDE sensing surfaces against the target and compared.
Graphene is preferred in this research, as this material has unique chemical, physical and electrical properties, which make it to be suitable for biosensor applications [20], [21]. Graphene materials have been used to immobilize the biomolecules such as DNA, RNA, protein, antibody and enzyme efficiently on the sensing surfaces to bring out the selective and sensitive detection [22]. Carbon nanotube is one of the efficient nanomaterial was utilized in biosensors to detect range of diseases [17], [18], [19]. SWCNT act as the transducers, which involved in converting the interactions of target and analyte molecule into the measurable units. The conjugated graphene structure anchors the electron to transfer between the transducer and bioreceptor, eventually it generates a high signal for biosensing [26]. In the present study, SWCNT-modified IDE sensing surface was immobilized with aptamer or antibody as the probe and quantified the level of clotting protein FIX.
Section snippets
Reagents and biomolecule
Streptavidin, 3-Aminopropyl)triethoxysilane (APTES), Single-walled carbon nanotube (SWCNT) and HEPES were purchased from Sigma-Aldrich (USA), FIX FIX-antibody were bought from Abcam (Malaysia), Ethanolamine was received from Fisher Scientific (UK). Factors XI and FVII were American Diagnostica Inc., USA and Haematologic Technologies Inc., USA, respectively. Aptamer with poly-A (A24) at the 3′ end was prepared enzymatically using transcription kit (Epicenter Technologies, USA). Biotin-dT20 was
Results and discussion
Identification of disease at an earlier stage is necessary to treat and cure for the healthier life. Biosensor is the sensing technology to diagnose the diseases by converting the biological interaction on the sensing surfaces into the readable measurements [30]. Sensitivity and selectivity are the main two key features identified to improve the biosensing. Biomolecular interaction and surface functionalization are mainly involved in enhancing the selective detection of target [3], [31], [32].
Conclusion
Factor IX (FIX) is the one of the clotting proteins, plays a major role during the process of blood clotting. Insufficient level of FIX cause the problem in clotting, so that it is mandatory to quantify the level of FIX for getting the proper warning. In this research FIX was detected on the single-walled carbon nanotube modified interdigitated electrode surfaces by its specific aptamer and antibody as independent dual probes. With aptamer, the limit of detection was found to be 100 fM, while
Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
Disclosure
The funder had no role in the design, analysis, or writing of this article.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
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