Today biotechnology, and nanotechnology result in the hybrid science

Today nanotechnology considers as a capital of many applications and involved in many different research developments. The vision of nanoscience arises in 1959 when the Nobel Laureate Richard Feynman says, “there’s a plenty of room at the bottom” in the annual meeting of American physical society (Feynman, 1960). The term nanotechnology refers to Greek word Nano, that means manipulation and monitoring of matter at atoms and molecular level (Emerich & Thanos, 2003). Nanoparticles refer to a solid colloidal particle in size range from 10 to 1000 nm (McNamara & Tofail, 2017). The merging of two powerful technologies, biotechnology, and nanotechnology result in the hybrid science Nanobiotechnology; which combines the effectiveness of biological substance and the principle of physics, chemistry, and synthetic structures in basic science.

  This integrated science, capable to investigate below the molecular level to generate favorable results  (Ghosal et al., 2017). Nanobiotechnology applications are tremendous includes, diseases detection and diagnosis using biosensor and lab on chip, drug delivery, gene therapy and molecular imaging  (de Morais, Martins, Steffens, Pranke, & da Costa, 2014).1.    BiosensorA biosensor is an analytical device that provide a concentration of an analyte in the sample by converting the biological binding into a measurable signal (Bhalla, Jolly, Formisano, & Estrela, 2016). It consists of three main parts, recognition receptors (antibodies, aptamer, protein. etc), transducer and signal processing system.

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Biosensors can be classified into catalytic and affinity biosensor based on the recognition receptor. Another classification can be applied to the biosensors based on the transducer mechanism, e.g., optical (Endo & Kajita, 2017), electrochemical (Wang, Xiong, Xiao, & Duan, 2017), mass sensitive (Hoß & Bendas, 2017), calorimetric (Kazura, Lubbers, Dawson, Phillips, & Baudenbacher, 2017), magnetic (Petralia et al., 2017) and micromechanical  biosensors (Okan, Sari, & Duman, 2017). Due to the excellent electronic, chemical, physical, and optical properties of nanomaterials, there are numerous increase in the research papers about of highly functional nanomaterial-based biosensors.

A huge domain of biological analytes, like DNA, RNA, glucose, milk-protein, antigens, and enzymes has been successfully approved to be detected by nanomaterial-based biosensor (Shavanova et al., 2016).2.1 Aptasensor An aptasensor is a unique type of biosensor in which the biological recognition receptor is an RNA or DNA aptamer. In an aptasensor, the aptamer identifies the specific target against which it was selected previously in vitro.  The aptamer-target mechanism is not influenced by both the type of transducer used and the class of detection system. Aptasensors are easily multiplexed to discover a diversity of aptamer-target of existing reactions (O’Sullivan, 2002).2.

    Nanomaterials in biosensor; GrapheneGraphene is one of the nanomaterials that attractive to employ in sensing applications. Defined as a single-layer of carbon atoms firmly stuffed into a two-dimensional (2D) honeycomb structure with zero-overlap semiconductor and consider as a basic constructing block for graphitic materials of all other dimensionalities. The attention to graphene materials in recent year due to its extraordinary electronic, thermal, magnetic, mechanical, furthermore optical properties and in addition surface properties. A large specific surface area to volume ratio of graphene and high porosity makes graphene perfect in adsorption of different gasses like hydrogen (H2), carbon dioxide (CO2) and methane (CH4). With these properties, graphene might find applications in optoelectronic devices, catalysis, biosensing, and energy devices. Electronic properties create the greatest investigation aspect of graphene.

Theoretically, graphene has a high electron mobility and a strong electron conductivity (Rao, Sood, Voggu, & Subrahmanyam, 2010). Graphene is an excellent thermal conductor; their thermal conductivity is much higher than other carbon structure such as diamond. The super thermal property of graphene is helpful in the electronic applications and recognized the graphene as an excellent material for thermal management (Balandin et al., 2008). In mechanical properties, the scientist discovered the stiffness of this graphene material and they found that graphene is harder than diamond and steel.

Graphene has elastic properties, very stretchable material and because of these mechanical properties, graphene materials are useful in the application of making a new generation of strong composite materials (Bhimanapati et al., 2015).  Graphene is a very light material still visible by naked eye and has a unique optical property, able to absorbs a high 2.3% light that passes through it and along combined with its mechanical properties’, graphene can make a difference in flexible display applications (Justino et al.

, 2017).3.    AptamersAptamers are recently noticeable as novel recognition receptors for biomedical application as an alternative to antibodies in bio-sensing devices (Eissa & Zourob, 2017) and has a great potential in many other nanobiotechnology applications includes, imaging, targeted therapy, food analysis and environmental applications (Romero-López & Berzal-Herranz, 2017). Aptamers are short single strands of RNA or DNA oligonucleotides that able to fold into specific three-dimensional structures and bind selectively to target molecules like protein, sugar moieties, lipids, low molecular weight metabolites, ions, and also to a whole cell with high specificity and affinity (Gao, Zheng, Jiao, & Wang, 2016). Systematic Evolution of Ligands by Exponential enrichment (SELEX) method, is the technique that used for aptamer invitro synthesis through screening a random of nucleic acids library, that consists of 1012–1015 oligonucleotide sequences (Sun et al., 2014).

Figure 1, shows example of SELEX process for invitro aptamer selection including binding, partitioning and amplification (Shum, Zhou, & Rossi, 2013). The approach of aptamers as biocomponents in biosensors provides a great number of advantages in affinity sensing, such as; kon and koff rates can be designed and modified according to the type of the process and the analysis time of the requested assay. Aptamer can be used to detect a small molecule by using the sandwich-type procedure, removing the needs of competitive type and the associated assay essentials. Aptamers’ selection can be produced in a process like that of real material and environment. During aptamer immobilization or labeling, the modification can be done without affecting the binding affinity (Pfeiffer & Mayer, 2016). Additionally, aptamer has great advantages over antibodies, including (1) lower molecular weight of aptamer with 8–25 kDa versus ~150 kDa of antibodies, facilitate faster penetration in tissue and they reach their target sites in vivo via penetrating tissues barriers more efficient than the larger-sized antibodies.

(2) Aptamers are nonimmunogenic in vivo (no recognized by the immune system) while antibodies are highly immunogenic. (3) Aptamer has better stability than antibodies, even after denaturation step at 95 °C, aptamers can refold into their 3D conformations correctly once cooled to room temperature. Although, antibodies at high temperatures will permanently lose their activity.

(4) Automated chemical synthesis of aptamer causing rapid, large-scale aptamer production and modification amplitude that include various functionalities. And (5) low cost production (Sun et al., 2014). However, using of aptamers as a recognition receptor in biosensor has been widely applied, Eissa & Zourob, developed a simple, low-cost and stable electrochemical array sensing platform for specific DNA aptamers with high affinity selected against HbA1c- and total hemoglobin (tHb). They immobilized a thiol-modified form of the aptamers on gold nanoparticles (AuNPs)-modified array electrodes (Figure 2) and then applied for label-free detection of HbA1c and tHb by square wave voltammetry method. Their apatasensor voltammetry results showed great sensitive detection limits of 0.2 and 0.34 ng/ml for HbA1c and tHb, individually (Eissa & Zourob, 2017).

On the other hand, a study of optical biosensors that based on the idea of aptamer and quenching ability of graphene oxide (GO), the researchers (Qin et al., 2017) developed an FSA by using fluorescein-labeled aptamer that immobilized onto GO surface to detect ?-lactamase contamination in milk (Figure 3). The FSA showed a good detection range with (1-46 U/mL) with a detection limit of 0.5 U/mL. they validate the sensitivity of FSA with commercial method ELISA. Their results of that developed FSA could be a favorable method for monitoring ?-lactamase contamination in milk.   Figure 1. Cell-SELEX to identify aptamers that targets membrane proteins (Shum et al.

, 2013)  Figure 2. Schematic diagram of the aptasensor array platform for HbA1c detection (Eissa & Zourob, 2017). Fluorescein amidite        Aptamer       Beta-Lactamase      Graphene OxideFigure 3. schematic diagram of the mechanism of aptamer/GO fluorescent sensor for ?-lactamase detection (Qin et al., 2017) 4.1 Truncation Aptamer synthesis by SELEX method usually consist approximately of 80-100 nucleotides, a randomized sequences with 30-50 nucleotides and two constant primer sequences ranging between 30 and 40 nucleotides in length at each terminus that facilitate polymerase chain reaction (PCR) amplification  (Radom, Jurek, Mazurek, Otlewski, & Jele?, 2013).

Anywise, not all the nucleotides participate to the binding affinity to the target molecule. Therefore, a study done by Ellington and his group to validate if constant sequences, rather than random sequences could affect aptamer folding using bioinformatics analysis. However, their result approved that fixed region does not involved or affect the binding features of aptamers and they only contribute less in overall structure (Cowperthwaite & Ellington, 2008). Several studies (Alhadrami, Chinnappan, Eissa, Rahamn, & Zourob, 2017; Kaur & Yung, 2012; Macdonald, Houghton, Xiang, Duan, & Shigdar, 2016) have revealed that it is helpful to minimize the length of the aptamers in order to enhance the affinity and induce better structure switching upon target binding which can be further exploited in different detection assays. Moreover, truncating the aptamers will reduce the production cost (Gao et al., 2016)Basically, on each aptamer there are three regions, essential nucleotides region, supporting nucleotides region, and nonessential nucleotides region. Nucleotides on the essential region are immediately contributed on the interaction between the aptamer and the target molecule.

Therefore, a serious loss of binding affinity will happen for any removal or change in the bases of this region (Battig & Wang, 2014) Those essential nucleotides have secondary structures like G-quartet loops, pseudoknots, hairpin loops, or bulges (Zhou, Battig, & Wang, 2010). Supporting nucleotides on the second region plays important role in the secondary structure stability of the aptamer via indirect involvement to the binding affinity. Those stability take place through the stems formation due the intramolecular base pairing of complementary nucleotides. A small reduction in the binding affinity occur in the chance of decreases the length stem or base replacements. Nonessential region is the third region and it contain the nucleotides that able to be excluded or replaced without affecting the binding affinity to the target. It includes the nucleotides that work as PCR primers in the amplification of aptamer during SELEX process and nucleotides that do not involve in the intramolecular binding. The nonessential nucleotides are frequently extracted from the aptamer due to their irrelevant in aptamer binding (Battig & Wang, 2014).

Truncation of aptamer is highly in demand due to several reasons includes, enhancing the sensitivity, and increase binding affinity of shorted aptamer in comparing to full-length oligonucleotides since the nonessential region does not involve in the stability of aptamer structure nor causing conflict with aptamer-target binding. They are easier to synthesize and in low cost. Additionally, in truncation we can minimize the possibility of being phagocytized for in vivo applications and decrease the chance of cross recognition. Furthermore, these shorter aptamers can give more space for building novel nanostructures in small-scale and required dimensions, that is suitable to construct small and sensitive application like bio-sensing devices (Battig & Wang, 2014; Tian, Wang, Sheng, Li, & Li, 2016; Zümrüt et al., 2017).Before aptamer truncation, it is important to determine the information of nucleotides ordered in the aptamer structure and which nucleotide is appropriate to be removed from the structure. There are some algorithms software available to perform sequence alignments and conclude the consensus high-affinity binding design, such as DNAMAN and ClustalW (Nadal, Svobodova, Mairal, & O’Sullivan, 2013). Furthermore, with the information about secondary structure that can be predicted using computer simulation programs such as Mfold and RNAstructure, the researchers can truncate conserved stem-loop regions which is expected to play a role in target binding.

For example, Cheng et al. obtained a different truncated nucleotides sequences to optimize the detection of polychlorinated biphenyls (PCB 77).  M-fold program was used to predict the secondary structure of aptamer specifying the truncation positions to create different DNA sequences.

They investigate the binding mechanism of PCB 77 against the original full-length aptamer and the binding ability of PCB 77 with different truncated nucleotides sequences too. however, they conclude that truncated aptamer with nucleotides 9–40 that was shortened from the 5?-end of the aptamer has the better binding affinity with PCB 77. Moreover, they used this truncated 35 mer ssDNA aptamer as recognition receptor in the developing of their aptasensor application for colorimetric detection of PCB 77 with the detection limit up to 0.05 nM and a linear range from 0.5 nM to 900 nM (Cheng et al., 2018).Additionally, we can acquire more information from other sequences that obtained from SELEX method. The sequences acquired at the end of the selection process, and based on their sequence similarity, can be separated into different families.

According to the relationship between the sequences of the same family and their affinities, the secondary structure of the aptamer can be concluded and that is important for target binding (Gao et al., 2016). Therefore, the sequences in same family must bind to the analyte with the same secondary structures but dissimilar affinity.

Moreover, comparing the sequences of the predicted structure in the same and different family, are helpful in the secondary structure determination for the binding motif of aptamers and aptamer binding (Shangguan, Bing, & Zhang, 2015). Shangguan et al. selected aptamer against leukemia cell line and three truncations are obtained. In the same family of two sequences, a different potential structure was predicted via algorithms and those sequence had the same potential structure with small differences like base mismatches. On the same basis, the core sequences were obtained with higher binding affinities than the original sequences (Shangguan, Tang, Mallikaratchy, Xiao, & Tan, 2007).However, it is not straight forward to define position at which the binding with the target takes place within the full-length aptamer. Therefore, testing the truncated aptamers after eliminating the non-binding region is mandatory and can lead to enhanced conformational change and even stronger complex with the target (Kaur & Yung, 2012; Macdonald et al.

, 2016). In addition, there are several techniques could be used to help in the identification of short high-affinity binding sequences, such as partial fragmentation, microarray-based binding sequence determination, enzymatic foot-printing or Nuclear magnetic resonance (NMR) spectroscopy (Gao et al., 2016).There are many examples in literature that illustrate the Truncation of aptamers in enhance their affinity. For example, a twenty-six aptamers were selected by (Soheili et al., 2016) and divided into seven groups based on similarities in their sequences.

Mfold analyzer used to predicted secondary structure of each aptamer (Figure 4). A colorimetric method that utilizes unmodified gold nanoparticles (AuNPs) were used to measure the binding constant (kd) of three sequences from different groups.  After the evaluation of the screened aptamers of their affinity and specificity, truncation was performed to the most suitable aptamer and located with a 23-base sequence with highest selectivity and affinity (Kd = 132.3 nM).

This high affinity truncated aptamer was utilized for unmodified AuNPbased assay to detect streptomycin residue in milk. Figure 4 The predicted secondary structure of aptamer 8 a and its two derivates: 8–1 b and 8–2 c (Soheili et al., 2016)  4.    Truncated aptamer /GO based biosensor for detection of HbA1Graphene oxide is one of the attractive carbon nanomaterials that is widely employed in sensing applications due to its extraordinary electronic, thermal, magnetic, mechanical and optical properties (Justino et al., 2017). The large specific surface area of graphene oxide makes it an excellent candidate for biomolecules immobilization (Lee, Kim, Kim, & Min, 2016).

Graphene oxide has been extensively involved in the construction of optical biosensors using fluorescence, surface plasmon resonance and colorimetric methods (Mao et al., 2012). Because GO is a good energy acceptor, it is widely exploited in fluorescence-based analytical assays (Chinnappan et al.

, 2018). In this paper, we report the truncation of previously selected aptamers against Hb and HbA1c (Eissa & Zourob, 2017). The shortened aptamers are then integrated in a GO-based fluorescence assay for the detection of HbA1c level. The truncation of the aptamers leads to increased affinity to their targets. This assay is simple to use, low cost and can be applied in 96-well microtiter plates for high-throughput screening of samples.