Graphene-based nanopore approaches for DNA sequencing: A literature review

Author’s Accepted Manuscript Graphene-based Nanopore Approaches for DNA Sequencing: A Literature Review Asma Wasfi, Falah Awwad, Ahmad I. Ayesh

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To appear in: Biosensors and Bioelectronic Received date: 25 May 2018 Revised date: 20 July 2018 Accepted date: 30 July 2018 Cite this article as: Asma Wasfi, Falah Awwad and Ahmad I. Ayesh, Graphenebased Nanopore Approaches for DNA Sequencing: A Literature Review, Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2018.07.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphene-based Nanopore Approaches for DNA Sequencing: A Literature Review Asma Wasfi1, Falah Awwad1*, and Ahmad I. Ayesh2 1

Department of Electrical Engineering, United Arab Emirates University, Al Ain, United Arab Emirates 2

Department of Math., Stat. and Physics, Qatar University, Doha, Qatar, P. O. Box 2713 * Corresponding author. Email: [email protected]

Abstract DNA (deoxyribonucleic acid) is the blueprint of life as it encodes all genetic information. In genetic disorder such as gene fusion, copy number variation (CNV) and single nucleotide polymorphism, DNA sequencing is used as the gold standard for successful diagnosis. Researchers have been conducting rigorous studies to achieve genome sequence at low cost while maintaining high accuracy and high throughput, as such sequencer devices have been developed which led to the evolvement of this technology. These devices are categorized into first, second, and third DNA sequencing generations. One successful endeavor for DNA sequencing is nanopore sequencing. This specific method is considered desirable due to its ability to achieve DNA sequencing while maintaining the required standards such as low cost, high accuracy, long read length, and high throughput. On the other hand, non-nanopore sequencing techniques require extensive preparation as well as complex algorithms, and are restricted by high cost, small throughput, and small read lengths. In this review, the concepts, history, advances, challenges, applications, and potentials of nanopore sequencing are discussed including techniques and materials used for nanopore production and DNA translocation speed control. Additionally, in light of the importance of the nanopore material configuration and fabrication, graphene which is a common and effective material will be discussed in the context of nanopore fabrication techniques. Finally, this review will shed light on some nanopore-related investigations in the area of molecular biology.

Keywords: DNA sequencing, nanopore fabrication, DNA translocation, graphene production.

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1.

Introduction

Deoxyribonucleic acid (DNA) is made up of nucleotides. Every DNA nucleotide consists of three groups: sugar, phosphate, and nitrogen base. The four types of nucleobases are adenine (A), thymine (T), guanine (G), and cytosine (C). The chemical assembly of DNA is displayed in Figure 1. The DNA sequence or the genetic code is distinguished by the sequence of the four nucleobases. DNA sequencing is defined as the process of reading off the sequence of bases within a DNA molecule. It includes all techniques or methods used in determining the sequence of the main bases A, G, C, and T in a DNA strand. DNA sequencing has become substantial in biological and medical research, and in various fields such as biotechnology, medical diagnosis, biology of forensic, and medical systems. It provides better understanding for the relationships among inheritance, diseases, and individuality. DNA sequencing is a critical step to improve human health and to achieve personalized medicine where the suitable procedure will be applied to patients. DNA Sequencing applications include environmental monitoring, clarifying the spaceflight effect on organisms’ molecular basis, and disease diagnosis (Castro-Wallace et al. 2017). In addition, it is a promising process, that is why there have been huge efforts to develop DNA sequencing techniques throughout the years. In fact, the advancement toward fast, reliable, and cheap sequencing has witnessed huge improvement since the human genome project in 2001(Lander et al. 2001). One particular promising DNA sequencing technique is nanopore sequencing; this strategy allows for the identification of DNA sequence by passing it through a pore and generating specific and identifiable DNA signals. Nanopore sequencing is of interest to researchers due to its low cost as it only requires the amplification of label-free DNA molecules to produce recognizable DNA strands. However, a number of critical challenges need to be resolved before this approach can be openly utilized. This review discusses the significance, history, progress, challenges, applications, and potentials of nanopore sequencing. Additionally, approaches and materials used for nanopore production and DNA translocation speed control will be discussed. Moreover, graphene nanopores fabrication techniques are illustrated below.

Figure 1: DNA chemical structure. 2

2.

History of DNA Sequencing

Over the last fifty years plenty of DNA sequencing techniques were established. This period of time witnessed a massive improvement in these techniques from sequencing few bases to millions of them.

2.1

First-Generation DNA Sequencing

In 1965 the first whole nucleic acid sequence was produced (Holley et al. 1965), and a related technique was introduced during the same year by Sanger et al. (Sanger et al. 1965). The first thorough protein-coding gene was sequenced in 1972 (Min Jou et al. 1972). Few years later, a complete sequence for nucleotide of bacteriophage MS2 RNA was performed (Fiers et al. 1976). During mid-1970s Sanger et al. developed a new rapid technique ‘chain-termination’ to define the sequence of nucleotide in single strand DNA (Sanger and Coulson 1975). In addition, Maxam et al. used chemical agents to break the DNA molecule to its bases in order to find out the sequence (Maxam and Gilbert 1977). Sanger et al. and Maxam et al. techniques were adopted widely and considered as the birth of first generation DNA sequencing. These first generation techniques provided a very short read length.

2.2

Second-Generation DNA Sequencing

Second generation techniques are different from the previous ones where they did not affect nucleotide identity. These techniques started in 2005 with the report of parallel pyrosequencing from 454 Life Sciences (Margulies et al. 2005). Sequencing machines produced by 454 allowed the parallelization of the sequencing reaction which increased the DNA amount that can be sequenced in one run (Margulies et al. 2005). Various parallel sequencing techniques were established after the success of 454 such as Solexa technique (Voelkerding et al. 2009). Another notable second generation sequencing technique is “DNA nanoballs” which is based on sequence by ligation (Drmanac 2010). In 2011, one more remarkable technique called post light sequencing was developed by Rothberg et al. (Greenleaf and Sidow 2014; Rothberg et al. 2011). Illumina sequencing technique which provides the lowest cost and highest throughput was considered the most successful second generation sequencing technique (Greenleaf and Sidow 2014).

2.3

Third-Generation DNA Sequencing

Third generation techniques are usable for single molecule sequencing without the necessity for amplification. These techniques lead to cost reduction and procedure simplification. One of the most broadly used third generation techniques are the single molecule with real time sequencing techniques (Clarke et al. 2009). Nanopore sequencing is of the greatest promising third generation technique. Nanopore techniques include biological, solid state, and hybrid nanopores. Oxford Nanopore Technologies (ONT) is a leading company in nanopore technology (Branton et al. 2008; Eisenstein 2012). Nanopore sequencing is based on the conductivity of the pore where the ion current changes when the pore is blocked by a nucleobase. Each base blocks the electrical ionic current in a different way (Branton et al. 2008). Nanopore techniques do not need 3

polymerase chain reaction (PCR) amplification or other molecule modification and promise cost reduction, speed increment, error rate reduction, and an increment in the read length. The crucial issue with this technology is slowing the DNA translocation speed down across the nanopore.

3.

Nature of Nanopores

Nanopores are the primary component of nanopore sequencing. It is essential to understand their physical and chemical characteristics of the pores that are utilized in DNA sequencing, since the generated signal is due to the interaction among the DNA strand and the nanopore. The three classes of nanopores are biological nanopores, artificial nanopores, in addition to hybrid nanopores. These types of nanopores are illustrated in more details below.

3.1

Biological Nanopores

Biological pores are normally placed into a membrane such as liposomes, lipid bilayers, and polymer membranes. This type of nanopore is also known as transmembrane protein channels. Biological pores are highly reproducible in specific size and structure and can be changed easily using molecular techniques (Feng et al. 2015). The various types of biological nanopores displayed in Figure 2 will be illustrated in this section. There are three types of the beneficial biological nanopores which are α-hemolysin (Song et al. 1997), mycobacterium smegmatis porin A (MspA) (Faller et al. 2004), and Phi29 (Wendell et al. 2009). The first biological nanopores were made of protein membrane such as α-hemolysin protein (Deamer and Branton 2002; Feng et al. 2015; Kasianowicz et al. 1996; Meller 2003; Olasagasti et al. 2010b; Wanunu 2012). This protein usually forms a cylindrical nanopore. The smallest part of the α-hemolysin protein channel is approximately 1.5 nm which allow single-stranded RNA or DNA to pass through it (Deamer and Branton 2002; Meller 2003; Wanunu 2012). α-hemolysin is the most commonly used nanopore since it has many advantages such as: the channel is stable even with thermal and chemical conditions. It works with a pH that ranges from 2 to 12 and a temperature reaching 100ºC. Moreover, chemical modifications can be made to these pores (Feng et al. 2015; Meller 2003; Olasagasti et al. 2010b; Wanunu 2012). It was experimentally proved that α-hemolysin can discriminate the different nucleobases (Haque et al. 2013; Meller 2003; Wanunu 2012). Using αhemolysin for DNA sequencing has few drawbacks. It was observed that 10 to 15 nucleobases occupy the channel, so the produced signal is for all of them and it is difficult to figure out the signal for each nucleobase (Wanunu 2012). These drawbacks motivated researchers to find better nanopore where a single base signal can be achieved. An alternative protein membrane, MspA, was successfully used (Butler et al. 2008; Wanunu 2012). MspA channel has a funnel shape with a diameter of 1 nm which enables the DNA sequencing process. The DNA sequence resolution is better for MspA than α-hemolysin. Manaro et al. were able to find the DNA sequence for single nucleobases using MspA (Manrao et al. 2011). MspA maintains the channel active even with extreme conditions such as temperature at 100 °C and PH value that varies from 0 to 14 (Feng et al. 2015). Moreover, MspA channel has better stability than α-hemolysin (Feng et al. 2015). αhemolysin and MspA nanopores are small channels that enable single stranded RNA or DNA to go through them (Meller 2003; Wanunu 2012). Phi29 protein was proposed to detect double stranded DNA (Wendell et al. 2009). The channel diameter is approximately 3.6 nm. The phi29 channel has stable specifications when the voltage is between −150 mV to +150 mV, regardless of the various pH range conditions (Feng et al. 2015). Measurements for large molecules 4

including dsDNA, proteins and DNA complexes can be done with phi29 since it has large diameter. This large diameter offers better flexibility for biochemical modifications (Feng et al. 2015). Phi29 has better conductance and resolution than the previously mentioned channels, however successful DNA sequencing was not achieved with it (Feng et al. 2015; Wendell et al. 2009). In general, biological nanopores have numerous merits in DNA sequencing applications such as their sensitivity, flexibility and simplicity (Astier et al. 2006; Venkatesan and Bashir 2011; Wanunu 2012). On the other hand, sensors based on biological pores have many problems such as limited stability and restricted sizes. There are recent developments that promise to achieve successful sequence with biological nanopores.

Figure 2: Biological nanopores: (a) Illustration of α-hemolysin (Deamer and Branton 2002). Reprinted with permission from American Chemical Society. (b) Illustration of MspA (top and side view of MspA from Mycobacterium smegmatis )(Butler et al. 2008). Reprinted with permission from Proceedings of the National Academy of Sciences of the United States of America. (c) Illustration of phi29 connector (side and top view of phi29 displaying the acidic in red, basic in blue, and the other amino acids in white and showing the diameter measurements) (Wendell et al. 2009). Reprinted with permission from Nature Nanotechnology.

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3.2

Solid State Nanopores

Even though biological pores showed interesting experimental results for single stranded sequencing, these nanopores have some limitations. Therefore, researchers developped an alternative solution: artificial pores in solid material (Dekker 2007; Li et al. 2001; Storm et al. 2005). Biological nanopores are more stable mechanically, chemically, and thermally than solid state pores (Dekker 2007; Miles et al. 2013) while solid state nanopores can be produced in various shapes and sizes (Li et al. 2001). In 2001, the first solid state pores were fabricated from a silicon nitride (Si 3N4) film by using argon ions focused beam (Li et al. 2001). Solid state nanopores have attracted researchers’ interest because of the improvement of micro fabrication techniques. Recently, artificial nanopores were applied in different fields and applications such as disease diagnosis, protein detection, and DNA sequencing (Feng et al. 2015). These nanopores work fine under various conditions and can be fabricated by semiconductor fabrication procedures. Moreover, solid state nanopores were also fabricated using SiC, SiO2 and Al2O3 membranes since these films have soft mechanical strength which enabled the holes production process (Dekker 2007; Feng et al. 2015; Miles et al. 2013). Furthermore, these materials could be modified mechanically and chemically (Miles et al. 2013). While slowing the DNA translocation, it was noticed that Al2O3 has lower noise and higher resolution (Feng et al. 2015; Haque et al. 2013). However, DNA sequencing using solid state nanopores still require further experimental testing to ensure feasibility and effectivness (Dekker 2007). The main advantage of solid state nanopores over biological nanopores is their high stability. Silicon nanopores are being widely utilized because of their high chemical resistance and low mechanical strength (Deng et al. 2013; Wu et al. 2005). The nanopore diameter can be changed by the experiment (Li et al. 2001). The traditional thickness of Si3N4 is ~ 30 nm which is greater than other pores (Wu et al. 2009). This thickness can be changed to 5 nm in experiment. Wanunu et al. suggested that some chemical modification to Si3N4 membrane can improve the DNA sequence (Wanunu and Meller 2007). Al2O3 pores are easier to control than Si3N4 and have better signal to noise ratio since the surface is positively charged and can strongly interact with DNA strands (Murali Venkatesan et al. 2011; Venkatesan et al. 2010). Recently, single layer membranes such as boron nitride (BN), molybdenum disulfide (MoS 2) and graphene are used to fabricate synthetic nanopores (Feng et al. 2015; Garaj et al. 2013; Liu et al. 2013; Merchant et al. 2010b; Paulechka et al. 2016). These membranes are used because their thickness is similar to the DNA nucleobase size ~ 0.3 nm which improve the resolution of the sequencing process. However, there are drawbacks for utilizing artificial nanopores for DNA sequencing such as blocking the nanopore because of the strong interaction among the pore and the nucleobase, the strong DNA adsorption to the membrane out of the channel and the large noise rate generated by the narrow pores (Schneider et al. 2013). Chemical modifications and cleaning of the membrane are required (Ivankin et al. 2014; Larkin et al. 2013; Larkin et al. 2014). These limitations affect the interpretation of the signals. Moreover, the translocation velocity is very high to detect the signals that enable clear DNA sequence (Venkatesan and Bashir 2011). Numerous attempts were done to overcome this issue (Keyser 2011). Another major issue is that different nucleobases interact similarly with the channel. The fabrication process of these pores usually produces undesired chemical changes to the membrane outside the 6

pore which might affect the translocation process (Keyser 2011; Wanunu 2012). These limitations indicate that solid-state pores were not able to achieve accurate DNA sequence and various improvements should be done.

3.3

Hybrid Nanopores

A main disadvantage of solid state nanopores is the lack of chemical discrimination from the target DNA of similar size. This can be improved by functionalizing surfaces or adding specific receptors or recognition sequences (Feng et al. 2015). To overcome biological and artificial nanopores limitations, numerous efforts proposed combining the best features of both (Iqbal et al. 2007; Wanunu and Meller 2007). Thus, hybrid nanopores were fabricated to control DNA or protein translocations (Hall et al. 2010; Iqbal et al. 2007; Keyser 2011; Wanunu and Meller 2007). This idea was first suggested by Hall et al. who inserted α-HL proteins in SiN film (Hall et al. 2010). The main concept is to connect biological groups into solid nanopores. This leads to improve the chemical specificity while keeping the chemical and mechanical stability. However, hybrid nanopores should be improved to achieve successful DNA sequencing.

4.

Graphene for DNA Sequencing

Graphene based DNA sequencing has grown tremendously because of graphene unique structure and properties. There are various graphene based DNA sequencing approaches.

4.1

Graphene

Graphene or Graphene Oxide (GO) is a unique material that provides novel opportunities and approaches for DNA sequencing. Graphene is a mono-layer of carbon atoms that are bonded in a repeating hexagon pattern. It is an extremely thin material that is considered two dimensional. Graphene has a number of superb properties where it is (i) extremely thin (single layer of carbon atoms), (ii) robust material because of the strong bonds between the carbon atoms, (iii) flexible, (iv) transparent, (v) conductive due to its narrow bandgap, and (vi) can be produced cheaply (Sheka 2014). Graphene has tremendous potential for use in electronic field effect transistors (FETs) due to its remarkable electronic properties, good stability, and reduced dimensionality(Ayesh and Awwad 2012). GO can be used as an inexpensive alternative to graphene (Sheka 2014). Biosensors based on GO films sensitivity can be improved by decorating the films with metal nanoparticles such as silver, platinum, or gold (Sheka 2014).

4.2

Graphene Based DNA Sequencing Approaches

Graphene thickness, stability and electrical conductivity encouraged the researchers to investigate and study the potential use of graphene to detect and analyze the DNA sequence. Below, few graphene based approaches are explained.

4.2.1 Graphene Nanopore for DNA Sequencing

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The first approach is displayed in Figure 3 which includes ionic current signal of a graphene nanopore where DNA molecule bases block the ionic current passing within nanopore in a graphene sheet differently. The concept of graphene nanopore is simple, where a single nanopore is fabricated in a graphene film using an electrolyte solution. Nanopores are fabricated in graphene membrane using a transition electron microscope (TEM). An ionic current is introduced through the pore after applying a voltage to the film and ions are driven across the pore. DNA bases pass within the pore and each base affect the ionic current differently. The period of current blockage and change in its magnitude provide an indicator to determine the sequence of the DNA bases. As mentioned in section 3, nanopores are categorized into three types: (i) biological nanopores based on proteins, (ii) solid state nanopore fabricated using solid substrates such as graphene, and (iii) hybrid which is a combination of both. The channel constituting the solid state nanopores is considered to be very long compared to the DNA bases. It is around 100 times the separation between two DNA bases which is a major limitation (Schneider et al. 2010). Graphene provides an ideal solution for this issue since graphene thickness is ∼0.3 nm which is only a single atomic layer (Novoselov et al. 2004). This indicates that graphene thickness is the same as the distance between two bases. DNA sequencing through graphene nanopores attracted researchers’ attention since it is amplification free, label free and high throughput approach. Nanopore sequencing can be achieved with biological and solid state nanopores. Solid-state nanopores exhibit many features which are their high stability, high sensitivity, and well suited for massive upscaling. Furthermore, it has high noise levels and lack of atomic control. The simulations revealed some challenges with this approach, as they showed that sequencing errors occur since the bases move stochastically through the pore and conformational fluctuations of the bases occur (Heerema and Dekker 2016).

Figure 3: Schematic display for DNA sequencing through graphene nanopore (Heerema and Dekker 2016). Reprinted with permission from Nature Nanotechnology.

4.2.2 Tunneling Through Graphene Nanopores Figure 4 displays tunneling through a graphene nanopores approach where each base within a nanogap leads to a different tunneling current through the gap since different bases have different electronic level structure. The idea is to measure the conductance through two graphene based 8

electrodes and to control the current change when DNA bases go through the nanopore. When different DNA bases fall within the voltage range of the electrodes, a special and different current is noticed (Heerema and Dekker 2016). The transverse conductance of the DNA passing through the graphene nanopore result in a nonlinear current voltage characteristic were that current change by 5 orders of magnitude. This high value of current helps in finding out the nucleobase type independently without being affected by the width of the nanopore. The likely sequencing error is based on the nanopore width. The nanopore sequencing can be done with a very small gap with a size ranging from 1-2 nm enable (Prasongkit et al. 2011). Graphene layers thickness is not a major issue when using this approach since the tunneling current is highly affected by the spacing between the nucleobase and the electrode. The graphene layer in this approach is considered as the membrane and the electrodes for the fabricated tunneling device (Arjmandi-Tash et al. 2016; Bayley 2010; Heerema and Dekker 2016). This feature is advantageous since the film is the electrode which resolves the issue of fabricating nanoelectrodes aligned with the nanopore. Graphene nanopores can be fabricated using various methods such as nanolithography with a scanning tunneling microscopy (STM) (Tapasztó et al. 2008), electromigration (Venema et al. 1997), local anodic-oxidation (Weng et al. 2008), TEM fabrication (Fischbein and Drndić 2008), and catalytic nano-cutting (Ci et al. 2008; Datta et al. 2008). The optimal nanopore width is 1-1.5 nm to achieve single stranded DNA (ssDNA) sequencing. There are few theoretical promising proposals for DNA detection using graphene nanopores, but till now there is no complete experimental work on DNA sequencing using tunneling across graphene nanopores due to its experimental challenges. The experimental challenges for DNA detection using graphene nanopore are as follows: (i) small tunneling current, (ii) large fluctuations, (iii) high DNA translocation speed, and (iii) high noise rate (Heerema and Dekker 2016).

Figure 4: Schematic display of DNA sequencing using tunneling across graphene nanopore (Heerema and Dekker 2016). Reprinted with permission from Nature Nanotechnology.

4.2.3 In-plane Transport within Graphene Nanoribbon Containing a Nanopore 9

Figure 5 shows the third approach which is in-plane detection for graphene nanoribbon where DNA bases modulate the ionic current passing through graphene nanoribbon differently. This approach has an advantage over the previous one since the current in the nanoribbons is larger. It is predicted that graphene nanoribbons can provide better base recognition results (Heerema and Dekker 2016). The interactions among the nucleobases and graphene nanopore modulate the nanoribbon current. The different coupling strength of the nucleobases within the graphene nanoribbon enabled the researchers to identify the base type and find the DNA sequence (Heerema and Dekker 2016). Zigzag and armchair nanoribbons provide promising DNA sequencing platform. This approach has been studied theoretically (Nelson et al. 2010) and experimentally (Vicarelli et al. 2015). In 2010, the first density functional theory (DFT) study of graphene nanoribbon with a nanopore was conducted where a graphene nanopore device was fabricated to detect the nucleobases sequence of single stranded DNA (ssDNA or/and RNA). The nucleobases pass through the pore in graphene nanoribbon. The conductance patterns and the charge densities were examined for each nucleobase in the graphene nanopore. The fabricated device has enough sensitivity to distinguish between the different nucleobases. This technique is helpful for developing fast, low cost DNA sequencing (Nelson et al. 2010). In 2013, the first experimental study for DNA sequencing using DNA translocation within graphene nanoribbons with nanopores was described (Traversi et al. 2013). This study showed that graphene nanoribbon transistor can be integrated with solid state nanopore to fabricate a DNA biosensor. Using this sensor DNA sequence is detected by evaluating the ionic current drops and the changes in the transistor local voltage. The fabricated device measures the ionic current in real time to accomplish real time sequencing (Traversi et al. 2013). Various methods are used to produce graphene nanoribbons using scanning transmission electron microscopy (STEM), STM lithography, and electron beam lithography (Heerema and Dekker 2016).

Figure 5: Schematic display of in-plane detection using graphene nanoribbon with a nanopore (Heerema and Dekker 2016). Reprinted with permission from Nature Nanotechnology.

4.2.4 DNA Physisorption on Graphene Nanostructures 10

The interactions among the DNA nucleobases and graphene allowed researchers to use these interactions for DNA sequencing applications. This approach is displayed in Figure 6 which is based on the graphene current modulation due to DNA physisorption, the different measurements based on the variations in the electrochemical activity, or the adsorption and desorption of DNA strand (Dontschuk et al. 2015; Vicarelli et al. 2015). Due to graphene-DNA complex binding nature, various mechanisms have been studied such as electrostatic, Van der Waals, π–π stacking, and hydrophobic interactions (Oliveira-Brett and Paquim 2003). Studies showed that single-stranded DNA has a higher binding affinity to graphene than does double-stranded DNA sequence. The strength of the interaction varies based on the DNA bases polarizability (Lee et al. 2013; Sh et al. 2007). It was reported that G base has stronger binding to graphene compared to A, T, and C bases which have similar or lower interaction strength (Antony and Grimme 2008; Le et al. 2012; Sh et al. 2007). DNA adsorption on graphene nanostructure surfaces (for example, nanoribbons) can help vastly in identifying the DNA bases. The DNA adsorption studies showed that the base fluctuations are minimized using this approach which lead to lower noise rate (Heerema and Dekker 2016).

Figure 6: Schematic display of DNA sequencing using DNA physisorption on graphene (Heerema and Dekker 2016). Reprinted with permission from Nature Nanotechnology.

4.2.5 Comparative Analysis Graphene-based electronic DNA sensors are attractive as they are label-free and offer real-time detection that directly reflects the binding of target DNA. In particular, nanoporous graphene represents a suitable system for single-stranded DNA and double-stranded DNA sequencing due to its speed of processing, cost-effectiveness, and scalable production (Reiner et al. 2012). Molecular dynamics MD simulations were carried out to evaluate the DNA movement across graphene nanopores (Liang et al. 2013) and it was identified that poly(GC) and poly(AT) can be differentiated at 1 V bias voltage (Sathe et al. 2011). Two main issues related to graphene-based DNA sensors are noise and current blockage. Noise levels can be reduced by decreasing the freestanding graphene area or utilizing a multilayered graphene (Garaj et al. 2013). DNA adsorption technique, which calls for minimizing base fluctuations in angle and position, is also effective in decreasing noise levels inside the sensors (Heerema and Dekker 2016). High current blockage occurs due to the graphene membrane thinness; tunneling current through a nanogap would help resolve this issue as electrodes would be aligned with the nanogap hence allowing passing of high currents (Postma 2010). The nanoribbon current approach is another way to pass high currents through graphene-based sensors; this approach may be superior to tunneling as 11

insulation of nanoelectrodes is not required and signals are detected at lower frequency (He et al. 2011a). Additionally, it is predicted that the nanoribbon approach caries out measurements at higher bandwidths, allowing DNA sequencing information to be measured at faster speed (Heerema and Dekker 2016). Finally, the currents passing across graphene nanoribbons are larger than the currents going through graphene nanogaps and ionic currents in graphene nanopore.

5.

Graphene Nanopores

The principal of DNA sequencing using graphene nanopore is straightforward which is done using approaches such as: resistive pulse and coulter counter technique. A nanopore is created using an electrochemical device with suitable electrolytes. An ionic electrical current is induced by an applied voltage leading the ions to move within the nanopore. DNA passing through the nanopore affects the ion flow. The nanopore diameter is extremely small so DNA can go through in a linear stand. Each nucleobase will be affect the ionic current differently, so the current blockage and the magnitude variation will be a good indicator to achieve DNA sequencing as shown in Figure 7. Graphene provide an ideal membrane for electronic DNA sequencing since the single layer thickness is almost identical to the distance among two nucleobases in single stranded DNA (ssDNA). Numerous experimental and theoretical studies on this topic were done in order to improve DNA sequencing techniques using graphene nanopores.

Figure 7: Scheme of graphene nanopore device for DNA sequencing (Wells et al. 2012). Reprinted with permission from American Chemical Society.

5.1

Experimental Studies of Graphene Nanopores for DNA Sequencing

The special properties of graphene present it as a suitable material for DNA sequencing. Graphene thickness enables the graphene nanopore to interact with only one base at a single time (Merchant et al. 2010a). In 2008, Fischbein et al. were able to use TEM to create graphene nanopore in suspended multilayer graphene sheets using focused electron beam (Fischbein and Drndic 2008). In 2010, Merchant (Merchant et al. 2010a), Garaj (Garaj et al. 2010), and Schneider (Schneider et al. 2010) were able to detect individual double stranded DNA (dsDNA) 12

with graphene nanopores. They used a graphene sizes ranging between 2 and 25 nm and graphene thickness from 1 to 5 layers. Their experiment results were slightly different, but they all showed excellent results. Venkatesan et al. fabricated multilayered graphene-Al2O3 nanopore membranes for DNA and DNA-Protein detection. The developed nanopore has low electrical noise which is less than the noise levels in pure graphene. Moreover, it is sensitive to electrolyte pH at low concentrations of KCl (Murali Venkatesan et al. 2011). Garaj et al. found that the monolayer graphene nanopore conductance is related to the nanopore aperture (Garaj et al. 2010). However, Schneider et al. demonstrated that the electrical conductivity is varies with the square of the graphene nanopore diameter and that the conductivity is not affected by the graphene nanopore thickness (Schneider et al. 2010). Nanopore size is a major factor to achieve the desired sensitivity and selectivity. The nanopore size specifies the translocated molecule size and the ionic current magnitude. Numerous methods are used to fabricate graphene nanopore such as focused electron beam (FEB), helium ion beam (HIB), focused ion beam (FIB), reactive ion etching (RIE), and dielectric breakdown (Chen et al. 2017). Moreover, STM lithography was used for highly accurate sculpting of graphene nanostructure (Tapasztó et al. 2008). During the early studies, the velocity of the translocation reached 3000 nucleobase/ms in solid state nanopores (Li et al. 2003). DNA translocation speed is very high which makes quick sequencing promising. However, this high speed makes it impossible to measure the small current blockades. This means that the error rate increases dramatically. Issues facing DNA translocation are the DNA translocation speed of and the control of the dynamics of DNA. These issues should to be studied and resolved. Fologea et al. found that the DNA translocation speed could be decreased using solvents with higher viscosity or by decreasing the temperature (Fologea et al. 2005). Also, Kowalczyk et al. were able to reduce the DNA translocation speed by changing the ion type (Kowalczyk et al. 2012). Processive DNA enzymes were used to reduce the DNA translocation speed by unzipping the DNA oligomers into the biological pore (Deamer 2010). Experiments of processive DNA enzymes in DNA sequencing using graphene with nanopores revealed many problems such as it is difficult to reach the anzymes stability on the graphene nanopore membrane. Moreover, modification to the graphene nanopore membrane leads to different DNA and pore interactions and to different strength of interactions. Optoelectronic control technology can be used to change the DNA translocation dynamics in solid state pores (Di Fiori et al. 2013). It is a promising technique since it will raise the time of translocation to ~1000 folds (Di Fiori et al. 2013). The time of translocation for DNA can be increased highly by applying hydrophilic substance on the graphene pores which will raise the non-covalent interaction (Schneider et al. 2013). Garaj et al. produced a graphene nanopore that successfully captured the DNA molecule with 0.6 nm resolution (Garaj et al. 2013). The DNA translocation velocity was highly reduced by applying the previous methods.

5.2

Theoretical Studies of Graphene Nanopore for DNA Sequencing

In recent years, many theoretical studies were performed to improve the DNA sequencing resolution with graphene nanopores (Heerema and Dekker 2016; Novoselov et al. 2004). Moreover, many theoretical investigations and calculations for the translocation time of DNA sequencing through the nanopores have been carried out. Molecular dynamics (MD) simulations showed that the speed of DNA translocation was heavily reduced by using Na+ ions instead of K+ ions since the Na+ ions have stronger interactions than K+ (Luo et al. 2008). This means that 13

the translocation time increase exponentially with the interaction strength between DNA and the nanopores. There are huge efforts to develop the nucleotide detection resolution. Molecular dynamics (MD) simulations showed that the base pairs A-T, G-C can be easily identified using graphene nanopores (Qiu and Guo 2012). Qiu et al. showed that using MD simulations that force peaks with 2 nm graphene nanopore enable the direct read off the DNA sequence (Qiu and Guo 2012). Reducing the configuration vibration of a nucleobase will make it possible to detect the sequence repeatedly which will improve the detection resolution. Nelson et al. utilized calculations based on density functional theory to prove that the DNA nucleobases can be identified using the electric conductivity of DNA translocation across a graphene pore (Nelson et al. 2010).

6.

Production of Graphene and Graphene Oxide

One of the most common difficulties is to produce adequate quantity of high quality graphene. Various techniques were developed to produce graphene such as epitaxial growth (Tetlow et al. 2014), mechanical cleavage (Jayasena and Subbiah 2011), chemical method (Park and Ruoff 2010), chemical vapor deposition (CVD) (Juang et al. 2010), and total organic synthesis (Koehler and Stark 2012). The earlier effort to make graphene was in 1960 when FernandezMoran was searching for a uniform support film that is electron beam transparent (FernándezMorán 1960). Micromechanical exfoliation was used to produce very thin graphene films ~ 5 nm which includes around 15 layers of graphene (Fernández-Morán 1960). Boehm et al. reported multilayers graphite oxide using electron microscopy (Boehm et al. 1962). After that, exfoliation and chemical intercalation of oxidized graphite were extensively studied (Soldano et al. 2010). After the discovery of nanotubes and fullerenes, huge efforts were done to study all types of carbon materials such as graphene (Soldano et al. 2010). Rubbing fabricated graphite pillars enabled the development of sub nanometer graphite (Lu et al. 1999a) and provided a chance to develop single layer graphene using this technology (Lu et al. 1999b). Monolayer graphene membranes were successfully developed in 2004 by Novoselov et al. (Novoselov et al. 2004). CVD and epitaxial growth techniques allowed generating high quality graphene. It is necessary to find large scale production techniques for graphene to provide large amounts of graphene for industry applications (Ruoff 2008).

6.1

Mechanical Cleavage

Mechanical cleavage technique uses scotch tape to separate graphene by removing it off from graphite (Novoselov et al. 2004; Soldano et al. 2010). With interaction energy of ~ 2 eV/nm, Van der Waals force is very weak in graphite (Fernández-Morán 1960) which allows peeling graphene using scotch tape (Zhang et al. 2005). Optical microscope, atomic force microscopy (AFM), and Raman techniques can be used to check graphene thickness after identification and to transfer them to clean membrane (Novoselov et al. 2004; Novoselov et al. 2005). This technique includes looking for single layers of graphene films manually among multilayer flakes. Results of this technique are not efficient enough due to the manual part. It is extremely important to choose optimal substrates to deposit graphene such as SiO2/Si (Blake et al. 2007; Jung et al. 2007). This graphene production technique is un-scalable and incapable of mass production. However, graphene produced using this technique can be used for primary studies such as characterizing graphene properties. 14

6.2

Epitaxial Growth

In epitaxial growth, graphene monolayer is grown on silicon carbide crystal (SiC) using vacuum based graphitization. The number of graphene layers can be controlled using time and temperature, and the uniformity can be amended by vapor phase annealing (Emtsev et al. 2009; Tedesco et al. 2010). The main benefit of this technique is that the produced graphene can be patterned by applying standard lithography techniques. The physical properties of graphene exfoliated mechanically are different from those grown epitaxially. Similar techniques were performed to other metallic membranes as substrates, for example, ruthenium (Ru) was used as a substrate to fabricate graphene films (Sutter et al. 2008; Vázquez de Parga et al. 2008). However, it is difficult to monitor the graphene thickness in the normal production process. Moreover, unexpected graphene piling was noticed in monolayer graphene because of the various epitaxial production patterns on various SiC polar crystal faces which had an influence on the electronic and physical specification of graphene (Sprinkle et al. 2009). Furthermore, the structure and electronic specifications of the above interface among graphene and the substrate should be clarified. These growth techniques require detailed investigations.

6.3

Chemical Vapor Deposition (CVD)

In this technique, a transition metal substrate is used to grow graphene through introduction of carbon due to hydrocarbon gas exposure at elevated temperature (Bae et al. 2010; Reina et al. 2009; Won Suk et al. 2011). Cu and Ni membranes are usually utilized as the substrate using methane as a precursor gas. The solubility of carbon is reduced on the substrate when the substrate is cooled to develop monolayer graphene films. The compatibility with the matching metal oxide semiconductor is one of the main advantages for using CVD growth and epitaxial growth. A typical drawback is monitoring the thickness of the graphene film, which is challenging (Allen et al. 2009). Another major drawback is the cost of membranes that are needed for growing graphene which limits the application of this technique for scalable production. However, CVD technique has evolved as an important technique for graphene mass production with few electronic and structural disorders.

6.4

Total Organic Synthesis

One more approach to produce graphene is using polyacrylic hydrocarbons (PAHs). PAHs have a structure that is intermediate between molecular and micromolecular phases; it can be replaced by aliphatic chains in order to change its solubility. Yang et al. were able to synthesis nanoribbon-like PAHs (Wissler 2006). These electrical properties of the developed nanoribbonlike are unknown, but they might have similar properties to graphene (Allen et al. 2009). PAHs might be usable to produce a new way for graphene fabrication if their size can be extended. However, it is hard to totally remove the defects from molecule boundaries.

6.5

Other Chemical Methods 15

Flake graphite is the most popular source for graphite utilized in oxidation, which might be generated by eliminating heteroatomic defects from natural graphite (Bae et al. 2010; Tapasztó et al. 2008). Because of the spaces among the layers in graphite, the intercalating agents can reside among the graphene layers due to chemical reactions, resulting in graphite intercalation compound (GIC) (Chung 2002). After the successful production of the first intercalation compound by utilizing potassium (Allen et al. 2009), extensive work have been done to investigate the insertion of chemical compounds among the basal planes. The space between the layers of GIC can be increased based on the intercalant which reduce the Van der Waals between neighboring layers (Chung 2002). The weak Van der Waals force make it easy to produce single graphene layer or graphene oxide (GO). The spacing between the layers in GICs can be extended using thermal shock to get expanded graphite (EG). The expression ‘sheets’ refers to a number of layers while the word ‘platelets’ is used to represent thicker monolayer GO sheets. Brodie was the first one to prepare graphite oxide 150 years ago by treating graphite frequently by nitric acid and potassium chlorate (Eda and Chhowalla 2010). More efficient way for GO preparation was done by Hummers and Offeman (Dreyer et al. 2010), who used a mixture of potassium permanganate, sodium nitrate, as well as concentrated sulfuric acid (Eda and Chhowalla 2010).

7.

Fabrication of Graphene Nanopores

Nanopore size is a major important factor to accomplish the required selectivity and sensitivity. It can define the magnitude of ionic current and the size of the translocated molecule (Bacri et al. 2011; Cai et al. 2006). Different techniques are used for the fabrication process using FEB, FIB, HIB, RIE, and dielectric breakdown. These techniques are used to establish different shapes and sizes of the nanopores (Deng et al. 2015; Rollings et al. 2016). FIB resolution of fabrication is limited by the physical characteristics of the membrane, that’s why it is rarely used (Gierak et al. 2007).

7.1

Focused Electron Beam (FEB) Drilling

Focused electron beam drilling in TEM is the most popular technique for fabricating nanopores because of its imaging capability, atomic precision and orientation control (Miles et al. 2013; Storm et al. 2003). CVD or mechanical exfoliations are used to obtain graphene membranes for nanopores (Park and Ruoff 2010). Using mechanical exfoliated graphene has advantages such as controlling the number of layers easily and having fewer defects. On the other hand, CVD provides large-scale graphene that can be easily manipulated (Merchant et al. 2010a; Schneider et al. 2010). Fischbein et al. (Fischbein and Drndić 2008) demonstrated that increasing TEM magnification to 800000× make it possible to achieve very high resolution modification of the graphene sheets ranging from 1-20 layers. Various graphene nanostructures such as nanogaps, nanobridges, and nanopores can be established and maintain their stability. In order to fabricate nanopores in graphene, a number of TEM parameters should be adjusted such as beam current density, acceleration voltage, drill time, and temperature (Fischbein and Drndić 2008; Schneider et al. 2010; Song et al. 2011). Freedman et al. (Freedman et al. 2013) analyzed the effect of using various beam intensities while developing reproducible nanopore sizes, establishing various structures around the nanopore edge and reducing membrane damage. Applying low electron beam intensities leads to slower pore growth rate which allow accomplishing accurate nanopore size without damaging 16

the surrounding membrane. Using high beam intensity was extremely undesirable for fabricating nanopores (Freedman et al. 2013; Girit et al. 2009).

7.2

Dielectric Breakdown

Another effective pore fabrication technique is dielectric breakdown which was established by Kwok et al. (Kwok et al. 2014; Tahvildari et al. 2015; Ying et al. 2016). This technique can be applied in electrolyte solution which makes it more convenient and cost effective than FIB and FEB based techniques. It is a simple and cost effective method, i.e. current stimulus dielectric breakdown (CSDB), was developed by Wing et al. to sculpture the nanopore shape in a thin SiN membrane (Ying et al. 2016; Zhang et al. 2016). Kuan et al. (Kuan et al. 2015) discovered that pores can be consistently fabricated in graphene membrane using dielectric breakdown technique.

7.3

Helium Ion Beam (HIB) Drilling

One more recently growing technique is using field ionization as an accurate lithography tool to control the different nanostructures (Bell et al. 2009). HIB gives direct and rapid patterning (Yang et al. 2011). Yang et al. group was able to use HIB drilling to fabricate a nanopore in SiN membrane for DNA sequencing (Yang et al. 2011; Zahid et al. 2016). HIB technique was used to develop several graphene based devices (Archanjo et al. 2014; Kalhor et al. 2014).

7.4

Focused Ion Beam (FIB) Drilling

FIB was used to produce solid state pores to control the translocation of single molecule (Li et al. 2003; Li et al. 2001). FIB does not have enough resolution to fabricate nanopores of sub-10 nm, but some researches are pushing the limits to use FIB drilling on graphene. Wang et al. (Wang et al. 2016) used both FIB drilling and electron beam to accurately tune the size of graphene pores.

8.

Challenges of DNA sequencing

Despite the promising developments and the huge efforts, various problems in DNA nanopore sequencing are not completely resolved. There are two main issues for the nanopore sequencing which are the high speed of translocation and the low sensitivity (Branton et al. 2008; Feng et al. 2015; Liang et al. 2017; Venkatesan and Bashir 2011). Recent experiments showed that the DNA translocation speed strand is 1 base/μs which is high for accurate sequencing. This high speed leads to low frequency of ions in the pore which reduces the resolution. Due to the low signal to noise rate, the discrimination among the four nucleobases is difficult. Short time with thermal fluctuations reduce the reliability of the measurements (Zwolak and Di Ventra 2008). Velocity of the ssDNA should be reduced to 1 nucleobase per millisecond which enable the identification process of the DNA nucleobases. One way to slow down the translocation is by adding bulky groups through chemical modifications (Mitchell and Howorka 2008), or by optical traps method (Keyser 2011). Experimental investigations showed that slowing down the translocation speed can be accomplished via biological nanopores (Kawano et al. 2009). One more issue, is the scaling gap between experimental and theoretical work (Liang et al. 2017). Another major issue is improving the chemical selectivity and sensitivity for all nanpores types. It was improved in α17

hemolysin nanopores by incorporating DNA enzymes in the pore (Olasagasti et al. 2010a), labeling the nucleobases chemically (Borsenberger et al. 2009), and attaching aminocyclodextrin adapter (Venkatesan and Bashir 2011). In the bulk solution, the enzyme attaches to the DNA and reduces the motion speed (Lieberman et al. 2010). For artificial nanopores the sensitivity can be improved by choosing the best conditions (pH, temperature, viscosity)(Olasagasti et al. 2010a), chemical functionalization (Kim et al. 2007), changing the membrane thickness (Chen et al. 2010; Wanunu et al. 2010), and using small pore diameter (Wanunu et al. 2010). Despite all of these enhanced conditions, the resulting resolution is not high enough to achieve single base sequencing (Branton et al. 2008; Feng et al. 2015; Hong et al. 2008; Yang et al. 2013). During the last decades, the DNA sequencing using graphene nanopore has been massively studied in order to achieve single molecule real time detection to provide new gene detection devices. Nevertheless, there are still various issues that need to be resolved such as mass production, repeatable high quality graphene nanopores with low cost, improving the electrical detection methods to achieve high resolution detection, controlling the speed of DNA translocation within the nanopores, reducing the signal overlap among the different nucleobases, and suppressing the stochastic nucleobase motions. Moreover, pure graphene has a high noise ratio, thus, to overcome this issue, a multilayered graphene-Al2O3 can be produced to achieve low electrical noise (Murali Venkatesan et al. 2011). In general, our understanding to the dynamics of DNA translocation within graphene pores is inadequate. In addition, the nanopores of multiple layer graphene have not been thoroughly studied which provides a great opportunity for researchers to investigate and overcome the previous issues.

9. Control Methods of DNA Translocation Behavior Through a Solid-State Nanopore One of the most serious problems facing DNA sequencing using solid state nanopores is the high DNA speed going within a nanopore. Different approaches and theories were used to address the translocation process of the DNA. These approaches include controlling the voltage bias, temperature, and solution viscosity (Fologea et al. 2005; Wong and Muthukumar 2007). Changing these factors might lead to some issues such as weakening the readout signals and affecting the optimal conditions for the sequencing process (Branton et al. 2008; Nakane et al. 2004). Effective approaches to control the behavior of DNA translocation were developed such as modifying nanopores with biological, physical, and chemical methods, the tweezers methods, and decoration the DNA molecule.

9.1

Solid State Nanopore Size Reduction

Wanunu et al. noticed an order of magnitude decrease in DNA translocation speed when the diameter in SiN pore decreases (Wanunu et al. 2008). Reducing the nanopore size leads to better interaction among the DNA molecule and the nanopore. These interactions are affected by several factors such as temperature, DNA length, and nanopores size (Wanunu et al. 2008). Keyser et al. found similar approach by applying a small modification in the nanopore diameter to affect the translocation time because of the hydrodynamic coupling between nanopores and molecules (Keyser et al. 2010). It was found that a nanometre-sized bead structure fabricated around a nanopore can decrease the speed of ssDNA by setting the bead diameter and the chemical group of the beads’ surface (Goto et al. 2015). Fabricating nanopores with controllable 18

diameter is a major problem. However, DNA strands going through smaller nanopores leads to more collision and reduces the signal to noise ratio.

9.2

Solid State Nanopores Surface Charge Condition Modulation

The density conditions of the surface charge of a pore affect the translocation performance of the DNA molecules since they are self-charged in electrolyte solutions. Nanopores that are positively or negatively charged might be utilized to modulate the translocation process of DNA (Behrens and Grier 2001; Bruno Schoch et al. 2008; Venkatesan et al. 2010). He et al. suggested modulating the surface charge density by monitoring the gate voltage instead of keeping a fixed pore surface charge conditions (He et al. 2011b; Novoselov et al. 2005). Asymmetry channel has rectification property (Siwy and Howorka 2010) which offers a highly effective method to modulate DNA velocity through the pore (E Gracheva et al. 2007; Siwy et al. 2007). Changing the applied gate voltage is used to manipulate the surface charge conditions. Karnik et al. used these properties to improve and stop the protein in the transistor-reservoir-transistor circuit (Karnik et al. 2006). Asymmetry pores appear to be preferable platform for the sequencing process.

9.3

Hybrid of Synthetic and Biological Nanopores

Biological pores can result in improvement of DNA translocation speed and performance (Majd et al. 2010; Venkatesan and Bashir 2011). Hall’s group was able to insert 𝛼-HL into SiN pores (Hall et al. 2010). Instead of including bio pore into solid state pore, DNA origami can be utilized to adjust the solid state pores (Bell et al. 2011; Hernandez-Ainsa et al. 2013). The two modes that were used to improve the control of DNA translocation speed are the physical mode which is based on self-tuning and the chemical mode which relies on the interaction between the pore and the bases. These interactions among the pore and the DNA lead to reduced DNA translocation speed (Douglas et al. 2009; Hernandez-Ainsa et al. 2013). Experiments were performed using hybrid pores in “chemical” mode and it was noticed that the translocation time increased (Hernandez-Ainsa et al. 2013).

9.4

DNA Molecule Modifications

One more approach to reduce the translocation speed through the pore is modifying the DNA strands. These modifications can be done by combining enzymes (Benner et al. 2007; Hornblower et al. 2007) or oligonucleotides to the DNA strands. A biological nanopore was used by Banner et al. to differentiate the enzymes that bind to the DNA strand (Benner et al. 2007). A huge reduction in the translocation speed resulted from enzyme bound DNA. Many experimental and theoretical studies were performed in utilizing the unzipping approach to reduce the translocation speed (Bockelmann and Viasnoff 2008; McNally et al. 2008).

9.5

Magnetic and Optical Tweezers

The earlier mentioned methods have one common problem where it is hard to expect the actual force exerted and position on the DNA molecule which can only be done using MD simulations. Optical tweezers technique offers a direct way to reduce the DNA speed. It was first done by 19

Keyser et al. (Keyser et al. 2006b). DNA translocation speed can be adjusted to any required value by controlling the velocity and direction of the optical tweezers (Keyser 2011; Keyser et al. 2006a). Trepagnier et al. was able to use this method to decrease the DNA translocation velocity to 150 bp/ms and monitor the DNA going back and forth inside a pore (Trepagnier et al. 2007). This approach can fulfill three dimensional spatial monitoring of the DNA molecule and achieve parallel detections. The magnetic tweezers uses neighboring magnet to produce a magnetic field gradient which produces a force to monitor the DNA molecule. Peng et al. used this technique to achieve a DNA translocation speed of 0.0096 bases/𝜇s (Peng and Ling 2009). However, magnetic tweezers is not usable to control the whole translocation behavior of DNA (Keyser 2011).

10.

DNA Sequencing Applications

DNA sequencing ultimate goal is to achieve cheap, fast, and accurate sequencing. Below are some of the DNA sequencing applications:

10.1 Developing Personalized Medicine Three decades ago, one drop of human blood was used to identify to which one of the four blood groups the human blood belongs. Nowadays, this drop is used to identify the genetic information of human and provide interesting opportunities in biomedical treatment (Schuster 2008). Nanopore sequencing technologies enabled better thoughtful of the basis of genetic diseases. Recent developments have explained the clinical applications of sequencing techniques in describing the genetic mechanisms of tumor development pathways, inherited diseases, and specific medication response (Jones et al. 2009). Although the difficulties facing the genome analysis and the needed studies to make sure that these technologies will be applied within clinics in an ethically and medically responsible way, latest genome findings and the developed genome sequencing potential are showing a promising future of personalized treatment and individualized medicine (Robinson et al. 2011). Researchers are still arguing the need for healthy people should sequence their genome. Other than finding a new way for drugs generation, diseases prevention, and treatment methods, DNA sequencing techniques can be used to achieve better knowledge of genotype-phenotype connections, offering precious details regarding susceptibility to diseases, defining family pedigrees as well as predicting individual’s adaptability and vulnerability to an environment. Moreover, various efforts are done to use the DNA sequence to investigate the associated genes with skin aging and develop personalized skin care products.

10.2

Better Perception of Ourselves

Genome sequencing can be used to obtain better insight of species which is hard to be cultivated inside the lab such as archaea in marine sediments (Lloyd et al. 2013) or to evaluate the diversity of genes encoded from microbial communities (Hugenholtz and Tyson 2008).

10.3

Safe Food

20

The entire genome sequencing for meat animals, food plants, and bread wheat is highly important for their genetic improvement and evolution (Ling et al. 2013)‎. The quick analysis of genome for foodborne pathogens will enable us to have a better understanding of outbreaks and develop better diagnostic (Chin et al. 2011).

10.4

Data Storage

DNA is considered as a stable material to store data which encodes the entire required information for the function and development of living organisms. Thus, data can be stored in the DNA base sequenced. Nature journal reported that approximately 5 mega bits of data information were encoded completely by Agilent Technologies’ OLS (oligo library synthesis) and restored with accuracy of 100% (Goldman et al. 2013). This process was highly expensive. However, the reduction in the cost of DNA sequencing and synthesis is showing a promising future for finding a practical way for DNA based data storage.

11.

Conclusion

Various efforts have been done to accomplish low cost DNA sequencing. Nanopore sequencing techniques are evolving in an unexpected high speed while the other techniques are paving. It can be anticipated that the essential objective of sequencing for clinical applications will be accomplished in the future due to the advancement in sequencing techniques. Single molecule detection using nanopores has evolved as one of the main robust tools for DNA sequencing. Nanaopore has significant importance over other techniques since it is label free and amplification-free, it has low material requirement, long read length and high throughput. These advantages simplify the experimental procedure and enable it to be used in other applications. This technique has potential in fields including analysis of RNA, DNA, proteins, polymers, peptides, drugs, and ions. Moreover, the advances in the fabrication process of nanopores will enhance the nanopore devices performance. The need for fast, cheap and reliable DNA sequencing technologies is increasing, that is why a variety of nanotechnology-based methodologies such as graphene-based DNA sequencing have been established and studied. Graphene-based DNA sequencing has grown tremendously because of graphene unique structure and properties. Graphene is a unique material that provides novel opportunities and approaches for DNA sequencing, and expected to witness new advances in the coming years. DNA sequencing is of significant importance in biological research. Over the last (few years, researchers from around the world worked and invested in improving DNA sequencing techniques. Sequencing innovations improved the sequencing capabilities, reduced the cost, and enabled long read lengths. Understanding the rich history of DNA sequencing techniques provides new insights for the development of novel strategies for DNA sequencing. Nanopore DNA sequencing are still facing challenges that should be resolved to achieve accurate, fast, and reliable DNA detection. One of the major challenges is the DNA high speed which can be resolved using various approaches and theories to control the temperature, the solution viscosity, and the voltage bias. MD simulations provide a powerful tool to investigate effects of various factors, such as shape and size of nanopores, voltage, and ion concentration on DNA sequencing techniques. The MD simulation results provide a good indication for the experimental work. Graphene can be produced using various techniques such as mechanical cleavage, epitaxial growth, chemical vapor deposition (CVD), chemical method, and total organic synthesis.

21

Moreover, various techniques are utilized to fabricate graphene nanopores using FEB, FIB, HIB, RIE, and dielectric breakdown.

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Highlights: 

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The concepts, history, advances, challenges, applications, and potentials of nanopore sequencing will be discussed including techniques and materials used for nanopore production and DNA translocation speed control. In light of the importance of the nanopore material configuration and fabrication, graphene which is a common and effective material will be discussed in the context of nanopore fabrication techniques. This review will also shed light on some nanopore-related investigations in the area of molecular biology.

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