Ultra-sensitive and selective electrochemical biosensor with aptamer recognition surface based on polymer quantum dots and C60/MWCNTs- polyethylenimine nanocomposites for analysis of thrombin protein
Graphical abstract
Introduction
At the molecular level, all biomechanisms in cells are carried out through proteins, nucleic acids and interactions between them. The molecular basis of most diseases is a disorder or interference in normal activities of proteins active in cellular biomechanisms [1], [2], [3]. If proteins’ activities diverge from their normal values, it can lead to various diseases including cancer [4]. Cancer is one of the largest predicted medical challenges and estimations have shown that by year 2030, more than 24 million people worldwide will be suffering from cancer [5]. Measuring and analysis of proteins in biological samples plays an important role in medical treatments, bioengineering, and nutritional safety and health applications [6]. One of the preferred methods for dealing with various conditions is their fast diagnosis through biomarkers and tumor-markers which plays an important role in proper treatment of these conditions [7]. Although various advancements have been reported in fast identification of proteins in biological samples, investigations are still ongoing for creating methods for fast and reliable detection of various proteins [8], [9], [10].
Thrombin or Factor II is one of the important proteins and biomarkers in humans which plays a central role in blood clot dissolution and tissue regeneration [11], [2]. This protein is used as a protein model in clinical studies and diagnoses and has a chain of 258 amino acids. Thrombin molecule has a length of 84 Å and a width of 34 Å, with a molecular weight of 28,400 Da [6]. This protein is a Serine protease enzyme that turns soluble Fibrinogen to insoluble Fibrin [12]. Thrombin can also act as a hormone for Platelet aggregation, activation of endothelial cells and other important biological responses. Furthermore, in a healthy brain, thrombin is the regulator for duplication, differentiation and development of neurons [13], [14]. Therefore, the development of thrombin sensors with high sensitivity and selectivity can play an important role in clinical diagnoses [15].
Today, characteristics and capabilities of aptamers (short single-stranded DNA/RNA molecules) have resulted in increased attention toward these molecules for biosensor manufacturing [16]. Along with high selectivity of aptamers for binding of an analyte, their unique characteristics have resulted in various advantages compared to other identifying biomolecules such as antibodies [17], [2]. These characteristics include small size compared to antibodies, high stability, structural stability during physical changes such as changes in pressure, temperature, pH, metal ions, etc. [18], [19], their ability for binding with a diverse set of targets from ions such as K+ to cancer cells and microorganisms such as bacteria and viruses, the ability for various modifications with different chemical groups, easy immobilization on various surfaces, stability for long-term storage and easy transportation at ambient temperature, higher affinity binding and low dissociation rate as well as specific and selective binding [20]. On the other hand, the use of antibodies as biosensors comes with problems such as low stability, difficult manufacturing, limited storage time and irreversible temperature denaturing [21].
To this day, various aptamer-based biosensors have been reported for analysis of thrombin using methods including optical methods [22], quartz crystal microbalance (QCM) [23], field-effect transistors [24], fluorescence [25], surface-enhanced Raman scattering (SERS) [26], quartz microbalance crystal [27], surface plasmon resonance [28], colorimetric assay [29] and electrochemical methods [30], [31], [32], [33], [34], [35]. However, the advantages of electrochemical aptasensors such as cheap manufacturing, simple structure, portability, the possibility for miniaturization, and high selectivity, have attracted various researchers to this type of aptasensors [36], [37], [38], [39].
One of the most important steps in the manufacturing of an aptasensor is the covalent immobilization of aptamer on the transducer which has a direct effect on sensor stability and sensitivity [6], [40]. As a result, various nanomaterials are often used for development of aptasensors and better covalent immobilization of aptamers [41], [42]. To this day, studies have reported the manufacturing of electrochemical aptasensors using various polyelectronic polymers, carbon nanomaterials such as multiwalled carbon nanotubes, graphene, carbon quantum dots as well as metal nanoparticles such as gold and silver nanoparticles [43], [44], [45], [46].
One of the nanomaterials with significant use in manufacturing of electrochemical aptasensors is multi-walled carbon nanotubes (MWCNTs). Multi-walled carbon nanotubes offer characteristics such as suitable mechanical stability, high surface to volume ratio, inexpensive price, electrocatalytic characteristics, and biocompatibility, making them attractive for manufacturing of biosensors [46], [47]. One of the other important carbon-based nanomaterials is fullerene (C60) which has received a great deal of attention. Fullerene is one of the synthetic carbon morphologies which is created by heating graphite. Fullerene offers characteristics such as high chemical and mechanical stability, high surface to volume ratio, lack of metallic impurities, electrocatalytic properties, high electronic conductivity, good biocompatibility and inert behavior [48], [49].
Surface modification using conductive or non-conductive polymers is used to improve the adsorption of biological species on carbon-based materials [42], [46]. Carbon electrodes modified with polymers show significant improvements in selectivity, stability, and repeatability of electrode response for various analytics [9], [46]. PEI is one of the polymers which can be used as a stabilizing film on the surface of carbon-based materials. PEI offers advantages such as formation of thin films, biocompatibility, inexpensive price, low toxicity, ease of separation and recycling and odorless nature while presence of a large number of amine groups on its structure helps in better immobilization of biological compounds on electrochemical aptasensors [50], [51], [52]. polymer quantum dots are formed through agglomeration of polymer chains attached to a carbon core and are known as non-conjugated polymer quantum dots. Along with the advantages of carbon quantum dots, polymer quantum dots offer other advantages such as inert behavior and easy synthesis at room temperature [53], [54], [55], [56].
In this study, for the first time, the characteristic of polymer quantum dots was used in the manufacturing of electrochemical aptasensors. To develop a sensitive thrombin aptasensor, C60/MWCNTs-PEI/PQdot composite was used for the development of a biorecognition surface. Better aptamer covalent immobilization, increased sensitivity and higher stability due to the synergy between various nanomaterials in this nanocomposite resulted in a very sensitive and selective electrochemical aptasensor with high stability and repeatability. The proposed nanocomposite offers advantages such as high surface to volume ratio, high electrical conductivity, and presence of a large number of surface amine groups for better covalent immobilization of aptamer and high mechanical and chemical stability. In order to investigate the performance of the proposed aptasensor, a recovery test was carried out in real human blood serum samples. The results indicated that the proposed aptasensor can be used as a suitable tool for biological and clinical studies for samples with complex matrices.
Section snippets
Chemicals and equipment
The amino-linked aptamer sequence used in the current study for analysis of thrombin protein was 5′–NH2- AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3″ [57] which was acquired from Bioneer Co., South Korea. 1-ethyl-3-(3-di-methylaminopropyl) carbodiimide hydrochloride (EDC), glutaraldehyde (GLA), N-hydroxysuccinimide (NHS), diethylenetriamine (DETA, 99%), nitric acid (65%), MWCNTs (with a length of 5–9 μm bundles and a diameter of 50 – 100 nm), polyethyleneimine (PEI), L-ascorbic acid (99%) and
Characterization of polymer quantum dots
In this section, DLS, XRD, TEM, UV–vis-NIR and fluorescence spectroscopy techniques, FT-IR and zeta potential were used to determine successful synthesis of polymer quantum dots from L-Ascorbic acid and diethylenetriamine. The detailed results of these studies are presented in section 4 of the supplementary materials (Fig. S-3).
Surface morphology of nanocomposites
FE-SEM and TEM techniques were used to investigate the nanoscale morphology of electrodes surface after modification with different nanoparticles and nanocomposites.
Conclusion
In this study, a novel SPCE/C60/MWCNTs-PEI/PQdot/GLA/APT aptasensor was developed for the analysis of thrombin protein. The C60/MWCNTs-PEI/PQdot nanocomposites were used to modify the surface of SPCE electrode in order to manufacture a sensitive electrochemical aptasensor. These C60/MWCNTs-PEI/PQdot nanocomposites offer unique characteristics such as high electrical conductivity, fast electron transfer kinetics, high surface to volume ratio, suitable stability and a high number of surface amine
Ethical approval
All procedures performed in studies involving human samples were in accordance with the ethical standards of the institutional and committee Isfahan University of Technology and with its later amendments or comparable ethical standards.
Permissions
The management of the Health Center of Isfahan University of Technology has authorized the use of blood samples in this study, and the publication of the results of the study of the mentioned samples has no conflict of interest.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors would like to thank the Research Council of Isfahan University of Technology, (IUT), the Iranian Nanotechnology Initiative Council and Center of Excellence in Sensor and Green Chemistry for their support and cooperation in this study.
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