A biosensing expedition of nanopore: A review

Sensors & Actuators: B. Chemical 284 (2019) 595–622

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A biosensing expedition of nanopore: A review Anuj Nehra

a,b,1

, Sweeti Ahlawat

a,1

, Krishna Pal Singh

a,c,⁎

T

a

Bio-Nanotechnology Research Laboratory, Biophysics Unit, College of Basic Sciences & Humanities, G.B. Pant University of Agriculture & Technology, Pantnagar, U.S. Nagar, 263145, Uttarakhand, India b Centre for Bio-Nanotechnology, College of Basic Science & Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, Haryana, India c Department of Molecular Biology, Biotechnology and Bioinformatics, College of Basic Sciences & Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, Haryana, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanopore Sequencing Biosensors Nanoporous Graphene Graphene oxide

The fascinating domain of nanoscience presents vast opportunities and challenges for developing myriad nanometer-scale biosensing systems. The advent of the nanopore and nanopore-incorporated devices and their wide utilization as next-generation diagnostics have recently had a remarkable impact on the sensing of biologically relevant species over the past few decades. Due to the continuous progress in the field of nanotechnology, the emergence of nanopore-based, powerful detection devices has become the subject of intense research. Devices bearing pores in the nanometer range have been overwhelmingly used in a variety of potential applications. The excellent properties and attributes of nanopores make them suitable candidates for the development of biosensors and for the detection, analysis, and sensing of single molecules, proteins, peptides, drugs, polymers, ions, nucleotides, and a variety of other macromolecules. This review encompasses the hegemony of graphene-assisted, nanopore-based detection over different types of porous matrices, including biological, solid-state, twodimensional metal-organic framework, and hybrid matrices along with their detailed descriptions, keeping present and future perspectives in mind.

1. Introduction The marked reduction in the costs of biomolecule sensing has facilitated the recent scientific investigation and promoted rapid advances in research areas. The development of a versatile, widely applicable biosensor technology would be perfect for those efforts [1,2]. Ideally, a common biosensor should be capable to monitor all biomolecules and measure their concentration at the same instant/moment, without changes in the chemical environment or need for additional instrumentation. With respect to nanosensors for label-free single biomolecule detection, one candidate that has drawn considerable attention is resistive-pulse-biosensing using nanopore [3–6]. A nanopore is a nanoscale pore with diameters ranging in nanometers and is made on material that is of biological origin or based on polymer, graphene, graphene oxide (GO, a derivative of graphene), and hybrid (biological with synthetic matrix). In the present era, nanopores have shown for potential application in the field of sensing, energy conversion, filtration, and nanofluidic and physiological devices [7–16]. These small apertures are generally found in the membranes that are either biological or synthetic in nature. The nanopore-based

detection is an emerging label-free, amplification-free, electrochemical gradient driven and membrane matrix assisted technique which monitors ion trafficking through the nanopore during their passage. This process allows for the detection of charged polymers, including singlestranded deoxyribonucleic acid (ssDNA), double-stranded DNA (dsDNA), and ribonucleic acid (RNA), with sub-nanometer resolution, thereby precluding the need for labels or signal amplification. Advances in nanopore-based sequencing technologies have led to nanopore-based sensing devices becoming more competent than other third-generation sequencing technologies. Over the few years, research is rapidly progressing toward developing biological and solid-state nanopores (SSNs) for direct label-free sequencing of nucleic acid molecules in real time. It is highly plausible that in the near future, nanopore-based sensors/biosensors may progress toward making available other third-generation sequencing technologies for affordable and personalized DNA sequencing [17,18]. These ionic channels, commonly appearing in biological nanopores, namely, α-hemolysin (α-HL) and Mycobacterium smegmatis porin A (MspA), embedded in a lipid bilayer nanopore membrane were the first type of the nanopores analytically used, with evidence that sequence fact can be acquired [19]. However,

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Corresponding author at: Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, Haryana, India. E-mail addresses: [email protected], [email protected] (K.P. Singh). 1 Equally contributed to this work. https://doi.org/10.1016/j.snb.2018.12.143 Received 21 March 2018; Received in revised form 25 December 2018; Accepted 27 December 2018 Available online 28 December 2018 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. A general depiction of translocation of ions using the unique nature of nanopore.

instantaneous passage of ions in the or through the channel/pore allows the analyte of interest. These biosensing devices take advantage of the phenomena that increase at the nano level, such as ion current rectification, surface conductance, and insight properties of targeted analytes. These biosensing devices are used to detect a variety of analytes such as ions, small molecules, proteins, nucleic acids, and particles [31].Biosensors use two types of detection techniques, namely, as label-free detection and label-based detection. In label-based detection, the detection of an analyte of interest relies on the use of specific properties of labels. In contrast, the label-free detection approach detects analytes that are not labeled as well as to screen such analytes where tagging is not an easy task. With the considerable advances made in the fields of both nanotechnology and biotechnology, a wide range of label-free biosensors have been developed [32,33]. This review encompasses the trends of evolution of nanopore as a sensing and sequencing device, called the third-generation sequencing techniques, with specific consideration of various kinds of nanoporous structures used for biosensing matrices/platform.

SSNs are highly stationary alternatives. The tunability of their physical and chemical merits and biocompatibility for mass production are considerable advantages [20]. SSNs offers a tunable pore size; increased strength over a large range of operating conditions (such as pH, voltage, and temperature); mechanical strength; and a natural inclination for unification with semiconductor and micro- and nanofluidic technologies. However, plausible fabrication of nanopores in thin SSN membranes, which are little enough to verify single-file transport of dsDNA and ssDNA, has been a major task [21]. While approaches for creating nanopores in the sub-5-nm range consist, such as either based on a transmission electron microscope (TEM) [22,23] or ion-beam sculpting instrument [24], these approaches are time-consuming, labour profound, and have minimum yield. Usually, collimated beams of greatenergy particles cannot be focused firmly enough to dependably achieve 2-nm nanopores. Thus, larger pores must become smaller through localized melting of the nanoporous membrane in order to acquire the desired pore size [22], though at the cost of topically improving the membrane nanomaterial composition [21]. In addition, for the case of the broadly employed TEM-based drilling, the nanopores creation is checked by eye, recording flickering on a fluorescent screen slit, confounding automation. In spite of these problems, such nanopores are not able of confirming and detecting single nucleotides composing a DNA strand or various biomolecules. The integration of two-dimensional nanomaterials, mainly graphene and GO in nanopore devices, has drawn a lot of interest in the last two decades [25,26]. Graphene and GO nanomaterials have been extensively used as bio-nanomaterials due to their multi-functional nature [27]. Graphene and GO-based nanopores are biologically inspired biosensors. These are used for label-free detection of single-biomolecules that show great promise for a large variety of applications. These applications can be the investigation of proteins, DNA − protein interactions, protein − receptor binding, and DNA sequencing [28,29]. The simple working principle of nanopore biosensing is as follow: the passage of biomolecules through a nanopore sensor modulates the nanopore ionic conductance [30]. This serves as a means for detection and investigation of the target analyte. Yet, graphene and GO nanopores face challenges. For example, control over the translocation speed of the biomolecule is crucial for base pair (bp) recognition on a DNA polymer, as already demonstrated using biological nanopores. Furthermore, it would be advantageous to expand the nanopore approach with new measuring modalities (for example, optical detection) beyond mere electrical probing. Improvement in nanoscale sensing has resulted in the advancement of fabrication techniques, which has, in turn, led to the development of devices bearing channels and pores with reproducible dimensions in a variety of materials. Variation in conductance during accumulation or

2. Nanopore-based sensing devices The basic principle of all nanopore-based devices is simple: analytes pass and interact with the nanopore causing a detectable change in an ionic current generated inside the pore under the applied bias [34], shown schematically in Fig. 1. In the device, the current is transmuted into a voltage signal by using a transimpedance amplifier (TIA) for an additional signal form, without noise or recovery. The sampling rate of analog-to-digital converter (ADC) is twice more than the ionic current signal bandwidth to confirm the blockade incident with minimum dwell time being noticed in accordance with the Nyquist-Shannon sampling theorem. The biased potential is controlled by the digital-to-analog converter (DAC). This digital circuit in the apparatus is employed as a joint bridge between the ADC, DAC and computer for whole experiment control, data process and information collection [35]. Nanopores are currently gaining popularity as robust single-molecule sensing devices for the detection and analysis of various ions, DNA, RNA, proteins, polymers, peptides, drugs, and a variety of macromolecules [20,36–38]. The nanopore-based device allows differentiation of individual molecules or ions based on the fact that a different value of ionic current is obtained for each of these molecules while passing through a nanoscale pore [39–43]. Typically, in a particular nanopore experiment, a specific molecule is forced inside a nanopore under the influence of a biased voltage in the electrolyte solution. When the molecule passes across the nanopore, it generates a characteristic, namely blocking the ionic current. By statistical analysis of the currents and durations of the blockade events, the properties of an individual molecule can be elucidated [44,45]. 596

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last few decades. These bionanopores display the excellent property of selectivity and multi-detection. Pores bearing channels allow the selective transport of small molecules and ssDNA and RNA [69]. The functionality of bare nanopores has been considerably enhanced by the fruitful efforts of engineering sciences revolutionized by biological systems in nature. Generally, it begins with a chemical alteration and covering of bare nanopores that allows modulation of their chemical properties [70]. Electroless deposition is a chemical method that is frequently used for polymeric membranes where the membrane is coated with a gold nano-layer, followed by functionalization of gold via thiol chemistry [71]. However, this methodology is also applied to silica or alumina membranes; polymeric membranes can also be functionalized by direct reaction with functionalized silanes [72], thereby reducing the needed for the two or more chemical treatments to a single one. An artificial system has also been established with major components similar to the nuclear pore complex (NPC) for receptor-mediated transport [73]. In this case, nanoporous-based membrane permeates are immobilized with polyisopropylacrylamide (pNIPAM), allowing rapid movement of a ssDNA-pNIPAM complex collated with the small ssDNA in isolation. It is very similar to the mediated transport occurring through the NPC, in which a transporter transports the biomolecule through the pore. Several other approaches do not mimic biological pores; instead, they rely on the use of biomolecules to confer selectivity. In an early study, it has been shown that the inner walls of silica nanochannel can be functionalized by antibodies to select for enantiomers [74]. Recent research on nanopore functionalization has also shown that the selective recognition for enantiomers is possible with the use of single β-cyclodextrin-modified nanochannel [75]. Similarly, the binding of apoenzymes to porous membrane results in the transport of its substrate molecule [76]. By attaching FG-nucleoporins to commercial nanoporous polycarbonate membrane filters, it is possible to achieve similar transport selectivity similar to the NPC in an artificial environment [77]. Here, the procedure similar to NPC transport conducted by track-etched polycarbonate membranes involves coating it with a gold thin film by sputtering onto one side of the membrane. Then, SAM-modified yeast nucleoporins were attached to the gold nanolayer. The Nsp1 immobilized membranes placed between two fluid chambers was used to measure the intensity of flux of fluorescently labeled karyopherin proteins using confocal microscopy through the pores. Recently, Wang et al. demonstrated a newer nanopore based-biosensing tool for the rapid and sensitive detection of anthrax lethal factor (aLF). The nanopore-based sensing device was used for recording the real-time hybridization interplay between the cDNA probe and the target DNA using a single-stranded aLF gene segment at a nanomolar concentration in approximately a minute [17]. Furthermore, Bell et al. presented a good review on nanopore generated by DNA origami and DNA origami with SSN (hybrid). This paper described the first exciting avenues towards improving nanopore biosensing by using DNA nanostructures, which can be applied to various analytes. In this review study, some minor problems were addressed solved easily such as better fabrication and pore size. They have focused extensively on how simple DNA origami and different nanopores compatible with DNA origami used in nanopore-based biosensors are easily addressed; however, their explanation, application, and major tasks were not compared due to the limitations in the detection, their measurement time, and signal to noise ratio (SNR) [78]. A variety of biological nanopores composed of channel proteins are being used for myriad applications. These are classified as α-HL, MspA, Cytolysin A (ClyA), Phi29, aerolysin, Outer Membrane Porin F (OmpF) and MinION nanopore. A brief discussion about these proteins is presented in the following subsections.

Nanopore-based sensors are completely electrical in nature and can detect an analyte in concentrations/volumes similar to those in a blood or saliva sample [36,37]. Investigations concerning nanopore technology date back to the year 1958, when the Coulter Counter was invented, the first time, for the counting of blood cells. The following approach did not attract much attention from the scientific community [46,47]. One apparent reason for this attention is the directness of the approach and nanoporous membrane/nanopores-based biosensing device have been depicted in Fig.1. Initially, the use of nanopore-based biosensing devices to DNA sequencing was reported by Church et al., in 1995, in a patent application that was awarded in 1998 [48]. Since then, considerable research has been conducted using protein nanopores [19,49]. The versatile approach introducing the concept of nanopore utilizing the biological α-HL pore was the first breakthrough in the origin of nanopore technology. Over the past 15 years, nanoporebased biosensors have successfully been employed in numerous applications such as investigating biomolecular sensing, folding, and unfolding [50,51]; studying metal-biomolecular interactions [52–56]; studying covalent and non-covalent bonding interactions [57]; and probing enzyme activity and kinetics and more [58]. Furthermore, nanopore data could be evaluated and visualized using a modular toolbox for analyzing data from single molecule experiments using modular single molecule analysis interface (MOSAIC) website. In addition, this process permits us to precisely analyze the transient events before they asymptotically attain a steady state. In nanopore applications, this analysis method has resulted in a 20-fold enhancement in the number of states notified per unit time. Recently, Forstater et al. reported a MOSAIC method for decoding of multi-state nanopore data through two key algorithms, namely adaptive time-series analysis (ADEPT) and cumulative sum analysis (CUSUM+). The ADEPT algorithm was used as a physical model to analyze short-lived events of the nanopore that are not reached their steady-state current, as well as CUSUM + algorithm which was optimized for longer events of the nanopore that do. Forstater et al. presented that ADEPT algorithm was detected previously unnoticed conductance states that are occurred as dsDNA translocates via SSN (2.4 nm), and demonstrated newer interactions between ssDNA and vestibule of a biological nanopore. These findings were shown the value of MOSAIC and the ADEPT algorithm and proposed a newer tool that could improve the characterization of nanopore-based data [59]. A variety of nanopores, in different biological and synthetic matrices and their potential biosensing tracks, routinely usable and commonly available are discussed in the following sections. 3. Biological protein nanopores Engineered protein nanopores are widely used for the stochastic detection of myriad biological molecules [39,60] and as an ultra-fast, cost-effective alternative for single-molecule sequencing [60–63]. The biological nanopores were recognized as the first nanopore devices to be used for the selective translocation of biomolecules. These types of pore constitute two major ingredients, namely, channel proteins and biological cell membranes present in all eukaryotic cells. The biological nanopores were first used for protein conformation analysis, and the report was first published around the year 2004 [64]. In their study, Jeremy S. Lee and co-workers synthesized a collagen-like sequence peptide containing different repeats (Gly-Pro-Pro)n which are terminated in ferrocene. Peptides having more repeats cause large and prolonged current blockade. Their conformations can be analyzed individually as single, double, and triple helices, based on the characteristics contour plots [64]. These protein nanopores are well suited for peptide and protein pore interactions at a single molecule level, since they show versatility in shape, size, and function and can capture the peptides under applied bias with their variable conformations [38,65–68]. The biological pores to study the DNA translocation events and other biomolecules have been successfully employed in the 597

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Fig. 2. Schematic diagram of biological nanopores composed of channel proteins used in biosensing device (a) α-HL (b) MspA (c) ClyA (d) Phi 29.

3.1. α-HL nanopore

applications with noise (DBSCAN) clustering algorithm, with the hidden Markov model (HMM) for intelligent and self-activating decoding of multi-level responses for cancer sensors. They calculated the primary state distribution vector (π), the primary state transition probability distribution (A) and the primary statement probability distribution matrix (B) by using the limitation re-approximation formulae whose results were based on the data clustering. The results of this work recorded that the probable range for pre-setting the primary values of the limitations can be large extensive. The following method was insensible to the setting of primary limitations. The primary limitation is not very good with the various nanopore data, as the current level density is generally free of the event pattern. Furthermore, they applied it to the microRNA21 experimental data, probe21 (poly(dT)20) and microRNA21∙probe21 complex utilizing α-HL pores. A duplex strand with microRNA21 and microRNA21∙probe21 was passed through unzipping method after the prominent sequence poly(dT)20 examines for the admission of β-barrel of the nanopore. All translocation process of the duplex strand is led to a usual four-level blockade. In addition, investigators enforced this protocol to the sensing of microRNA21 in serum samples from the colorectal cancer patients. This method gained a correct evaluation of the time duration and amplitude of specific multi-level blockades [82]. In this regard, aptamer-based nanopores have displayed well-defined arrays for the parallel detection of multiple analytes. Furthermore, the α-HL protein has been used to study the translocation dynamics of an unfolded protein forced through the nanopore, which revealed different steps of the unfolding process [83,84]. Stefureac et al. noticed thousands of negatively charged α-helical peptide events. In this method, each peptide was synthesized using Fmos chemistry through solid-phase synthesis on poly(ethylene glycol)polystyrene resin. After that, each peptide has blocked the resin, purified and characterized by high-performance liquid chromatography using preparative reverse-phase and time-of-flight mass spectrometry, respectively. All tested peptides were Fmoc-D2A10K2, Fmoc-D2A14K2, Fmoc-D2A18K2, Fmoc-D2A22K2, Fmoc-D3A14K2, Fmoc-DA14K, and D2A10K2. Inside pore lumen, the short and long peptides were showed a blocking percentage of 62% and 78%, respectively and it was interpreted as an average measurement of the fraction of the pore volume in which ion was excluded during the translocation of the peptide via pore. The dipole moment showed the major influence on the transport

The most commonly and widely used naturally occurring channel protein is α-HL (as shown in Fig. 2a), and it was the first protein nanopore to be used as a sensor for DNA sequencing. This protein behaves as a nanopore by embedding itself spontaneously in a lipid bilayer that is present inside the cell membrane and the α-HL toxin is obtained from staphylococcus aureus. The α-HL is a heptameric protein composed of two domains called cap domain and β-barrel domain (5 nm) separated by an inner constriction of diameter 1.4 nm. The long β-barrel of α-HL has three recognition sites: R1, R2, and R3 [79,80]. Both single- and double-stranded DNA has been studied using this biological pore. Biological pores or channels can be functionalized by various biomolecules and can be used as sensitive biosensors. Recently, Gu et al. presented a precise and strong data process method that recognize the blockades of current and the dwell time evaluation through a unique second-orderdifferential-based calibration (DBC) technique as well as current amplitude through an integration method. They applied the data process method to examine generated current blockades and test data of poly (dA)60. These results showed the exponential decay of the conventional method (CM) that produce a dwell time of 0.33 ms, which was higher than the dwell time of the DBC technique (i.e. 0.13 ms). This important difference in dwell time is attributed mainly to blockade over analysis resulting from the procedure of the CM and the current histogram of poly(dA)60, and split into two populations namely population first (PI) and population second (PII). These distributions were attributed using their integration method resulted in PI and PII at 33.4 and 77.5 pA, respectively. In the meantime, the CM was led to the strength of character as the peak of two populations are shown at 23.7 pA (PI) and 76.7 pA (PII). This method showed that the blockade falls into the PI population (31.5%), while for PII population it was 18.5% for single poly(dA)60 translocated via α-HL. In addition, this method was used to examine the nanopore data of β-amyloid 42. The relative maximum error of the DBC technique is 71% less than CM with blockades having a dwell time (i.e., 0.05 ms). To achieve better the blockades of current amplitudes, they have made a unique integration method that improved the cut off frequency from 2.78 to 5.74 kHz for current amplitudes where dwell times was less than 2Tr [81]. Zhang et al. suggested a changed density-based spatial clustering of 598

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molecules. Modern nanopore technologies are based on material transport to the opposite side of the cell membrane fully aided by channel proteins [19,90]. Wang et al. suggested that a label-free nanopore-based microRNAs (miR-155, and let-7 microRNAs with one or two different nucleotides from lung cancer patients) biosensor can be simply developed by α-HL protein. An α-HL protein was inserted into the lipid bilayer membrane of 1,2-diphytanoyl-sn-glycerophosphatidylcoline with the centre of Teflon film hole (100–150 nm) for easy biosensing the microRNAs. Further, the α-HL protein-based nanopore was fitted in the chamber and filled by the buffer solutions under different conditions i.e., buffers of different pH in cis and trans chamber to calculate the mean translocation time and relative residual conductance (RRD) and then ordered to keep a mean translocation time of 270 ± 30 μs and RRD of 0.08. Therewithal, the advantage of sensing device was that it could be helpful for detecting the microRNAs in the blood serum and, mainly quantified sub-pM levels of microRNAs (cancer-associated) [91]. Kawano et al. showed the rapid detection of cocaine using a microfluidic device consisting of eight chambers. In addition, this microfluidic device can detect cocaine at a minimum concentration of 3 μg mL−1 at 25 s with high selectivity. In this fabricated device, cocaine at a minimum concentration correspondent to that of the drug analysis deadline limit was recognized lower than 1 min with great selectivity [92]. Wang et al. fabricated an aLF biosensor using the mutant α-HL protein grown on the bilayer of 1,2-diphytanoylphosphatidylcholine in Teflon septum (150 μm), which was small aperture i.e., 1.5 nm. In this device, the α-HL protein was fabricated onto the bilayer at 180 mV, which was used the concentration range of 0.2–2.0 ng mL−1. The aLF was hybridized to the complementary ssDNA probe in the trans compartment and, importantly, current significantly declined from lower to a higher concentration at room temperature. Therefore, it had a very good sensitive and quick for sub-nM concentrations of aLF-DNA within 1 min. Furthermore, the pros of this biosensor is that it could be quickly unzipped via α-HL protein, whose diameter is greater than the pore construction, short dsDNA molecules [17]. Rosen et al. examined the distinct states of thioredoxin (Trx) protein phosphorylation by monitoring the detectable changes in the ionic current amplitude and noise as the protein unfolds and moves through the α-HL pore [60]. The same model protein employed in their previous research work and was tagged with Oligo (dc)30 at its C-terminal cysteine residue. They reported that by applying voltage; the protein unfolds due to an exerted force at one end by the DNA leader sequence. Following the same concept further, they studied a set of PKA phosphorylated Trx mutants (TrxS112−P and TrxS 112+P) and concluded that ionic currents are voltage dependent on the basis of differences in mean residual time (IRES %) and noise (In) under an applied potential of + 140 mV. Thus, they utilized the unique attributes of protein nanopores in detecting site-specific phosphorylation. Ding et al. presented a model to understand the unzipping kinetics of the hairpin DNA in a lipid bilayer embedded α-HL nanopore. They studied the unzipping mechanism of internal and fishhook hairpins leading to electrophoretically driven translocation from cis to trans side of α-HL. According to their findings, both types of hairpins showed different unzipping characteristics. It was determined that the duration of unzipping was affected by the unzipping process and that the level of current blockade recorded during the denaturation step was different for the two types of hairpins [93]. Wang et al. proposed an α-HL protein nanopore-based sensor that can quantitatively detect unlabelled cancer-associated microRNAs in plasma samples with enhanced sensitivity and selectivity. The sensor incorporates an oligonucleotide probe that generates analyte-specific signature signals. Similar studies have been conducted on the same biological nanopores for the analysis of metal ions. A stochastic nanopore sensor was proposed where the polyhistidine probe molecule was used to chelate the metal ions for the detection of trace amounts of the metals. The sensor identified copper (Cu2+) ions with a significant limit of detection of 40 nM, along with Co2+, Ni2+, and Zn2+ ions [94]. The

phenomenon of the polymer, so, the bumping phenomenon was only showed for Fmoc-DA14K, which was the minimum dipole moment but when no electric field applied in the chambers (i.e., cis and trans) of the pore. As dipole moment increased, it is possible for a peptide to bump condition into the admission to the pore rather than translocate straight through. Finally, the results showed that the nanopore sensing technology provided valuable structural information [85]. Ying et al. examined that a newer stimuli-responsive α-HL nanopore was presented as a basic tool to study the individual motions of supramolecular tools which are based on photoresponsive host-guest system. Ying et al. employed para-sulfonato-calix [4] arene (SC4)-based host-guest supramolecular tool to evolve synthetic gating mechanisms aiming at regulating wild-type α-HL commanded by both ligand and low weight stimuli. They analyzed the host-guest interactions between SC4 and 4, 4′-dipyridinium-azobenzene (V2+-Az) using the gating merits of α-HL at the single-molecule level. Subsequently, this method extended the application of this gating system to the real-time analyses of light-induced molecular shuttle based on SC4 and V2+-Az at the single-molecule level. Due to the light-induced molecular machine by an α-HL:SC4 system, inhibition number per unit time showed a linear growth with slops of 1.21 (SC4), 0.32 (SC4:V2+-trans-Az after UV irradiation) and 0.05 s−1 (SC4:V2+-trans-Az). The inhibitions frequency after UV irradiation is close to 6.4 times greater than that for the SC4:V2+-trans-Az without irradiation [86]. Meng et al. reported a self-assembly method for single molecule sensing by using host-guest interactions in the α-HL pore. Here, further, the self-assembly method was generated by separate monomer through the α-HL pore. In this method, para-sulfonatocalix [6] arenes and methyl viologen (MV2+) are used as host and guest respectively. The 1:1 and 1:2 complexes self-assembled by SC6 and MV2+ were distinguished based on their specific blockade distributions in the [MV2+]/[SC6] mixtures. In addition, this nanopore-based singlemolecule reading was capable to display the method for 1:1 complex self-assembly into the 1:2 complex [87]. Li et al. suggested a novel strategy for nanopore biosensing-based on aptamer via host-guest interactions inside α-HL. Aptamers are single-stranded oligonucleic acids that were linked to specific analytes (such as vascular endothelial growth factor, thrombin, and cocaine) with large affinities. Authors hybridized an aptamer with a DNA probe which is entirely corresponding to the aptamer or contained a limited deliberate mismatch. After that, the DNA probe was altered with a complex of host-guest in the centre of the strand. When passed via α-HL nanopore, it was speculated to produce characteristic current events. However, the existence of analytes may create the aptamer-probe duplex to unzip if the affinity of the analytes–aptamer is greater than that of the duplex. This technique ultimately frees the DNA probe that generated characteristic ionic current phenomenon when translocated via α-HL under the transmembrane potential. Furthermore, magnetic beads were used which reduced the detection limit by nearly two to three orders of magnitude. However, the aptamer has been shown strong binding affinities with an extensive variety of analytes. In addition, the aptamerprobe duplexes were emulated with the DNA probes for occupation in α-HL, which extremely decreased the efficacy of the generation of signature events. This condition deteriorated when the concentration of analyte was comparatively low while the aptamer-probe duplexes were in great excess. Therefore, removal of nosiness of the aptamer-probe duplexes might significantly increase detection limits of this approach [88]. Rotem and colleagues used nanopores equipped with a single 15mer DNA oligonucleotide aptamer, which when attached to α-HL pore forms a cation-stabilized quadruplex and shows reversible binding with a thrombin protein; this protein could be effective for the real-time sensing on the basis of aptamer-thrombin interaction, which alters the current passing through the nanopore. This chemically modified approach could be found suitable for the fabrication of nanopore sensor arrays [89]. Further, Kubitschek and Kasianowicz et al. employed biological nanopore α-HL to detect the translocation mechanism of DNA 599

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Recently, Rauf et al. showed a newer manuscript which is based on nanopore assay for real-time detection of the action and kinetics of Escherichia coli DNA adenine methyltransferase using nanopore method attached with enzyme linkage reactions. This biosensor had shown the detection limit of 0.03 U/ml for DNA adenine methyltransferase in a low assay time of 150 min. The biosensing device is easy to use, simple fabrication and circumvents the employ of radioactive substances, resulting in good detection of the action of DNA adenine methyltransferase, even in complex matrixes as human serum samples [103]. Pederson et al. suggested a single biological nanopore sensor based on the proximal capture dynamics using same and different electrolytes in the chambers, with timescales of μs to ms. In this fabricated sensor, a soft-walled electrostatic block model develops for the α-HL pore that generated a vector map of drift-generating forces on the applied particles diffusing near the entrance of pore surface. This sensor showed access to intensely large simulation volumes (i.e., ˜ 1010 nm3) and ˜ 10−3 s long simulation times. Therewithal, this study allows for the assessment of the interaction between the electrophoretic, electroosmotic, as well as, the thermal driving forces in terms of applied potential. The obtained results demonstrate the competition between the various drift-producing forces and diffusion force at the nanopore. The results also highlight the spatial and temporal limitations associated with nanopore detection and provide a theoretical framework that can serve as a basis for the placement and kinetics of reaction sites located at, or adjacent to, the nanopore cap [104].

signature of events produced by peptides and proteins are very dissimilar in the case of metal absence and presence. These events, generated due to the biomolecules, underwent conformational changes caused by the metal biomolecular interaction [55,94–96]. Similar metal-biomolecular investigations are also reported with the frequent use of nanopores. Related work was extended to lead (Pb2+) and barium (Ba2+) ions. DNA probe, containing specific DNA sequences such as G-quadruplex when subjected to translocation through the α-HL pore, illustrates the longer translocation events revealing the quantification of these analytes at concentrations as low as 0.8 nM [97]. Liu et al. fabricated a membrane–channel biomimetic tool (MCBT) using the α-HL and the phospholipid membrane to examine the oxidative signal to free radicals. Real-time sensing of the oxidation method of membrane–channel biomimetic tool to free radicals at concentrations ranges from 0.01 μM to 10 mM was acquired. The oxidative rates of free radicals to MCBT exhibited a good relationship of free radicals with such concentration range and could be split into three levels, which was deduced by the buffering capacity of MCBT. It has the potential to be used for quick sensing of contaminants in water, toxicity in drugs etc [98]. Wang and co-workers found that the native unfolded α-syn monomer could be translocated under the applied potential of + 100 mV. At a potential higher than + 100 mV, blocking current increases and captures the partially folded intermediate in the vestibule of α-HL. Further, current decreases at a potential + 70 mV to produce the desired intermediate. At + 40 mV, this intermediate is exited from the vestibule. This phenomenon predicts that the applied potential can have a strong influence on the structural conformation of α-syn, producing intermediate species [99]. Dvir Rotem and co-workers employed, engineered an α-HL pore modified with 15-mer DNA aptamer that detects thrombin. Quadruplex, comprising of thrombin and aptamer stabilized by a cation, was detected and analyzed. The concentrations of thrombin were measured by the data generated as a current blockade and a dwell time in the interior of the nanopore, thereby confirming the interactions between thrombin and aptamer. The authors recommend the development of aptamer with variable characteristics so that they can be used for protein detection through the aptamer-modified nanopores [89]. Payet and coworkers developed a protocol for single-molecule detection, where the folding and unfolding behavior of a protein was discriminated on the basis of the thermal parameter. They suggested a higher temperature of around 45 °C at which the native protein unfolds itself and moves through the nanopore, causing an increase in the ionic current blockades. The unfolding I–V curve gives a sigmoid shape that fits well in the normalized event frequency since it is independent of the structure and charge residing on nanopore [100]. Yi-Tao Long and coworkers demonstrated the mechanism of peptide-oligonucleotide conjugates translocating across α-HL nanopores and derived two important conclusions that as the length of the peptide-oligonucleotide conjugate increases, the duration of translocation events also increased. Their second conclusion was that the protein nanopore discriminates the structure of the conjugate at the single-molecule level [101]. Further, Ying Yilun and co-workers analyzed the conformational changes resulting from the weak interactions between P53 and DNA by α-HL nanopore. Here, it was demonstrated that the complex between p53-P and B40 was formed by weak interactions between the two changes the conformation of B40 when it binds to p53-P; this, in turn, enhances the interaction between the analyte and the pore. Thus, α-HL also favors the identification of weak interactions between any two biomolecules at the single-molecule level [102]. By applying the finite element method, we developed a soft-walled electrostatic block (SWEB) model for use in the α-HL channel. This channel generates a vector map of drift-producing forces on the particles that diffuse adjacent to the pore entrance. Subsequently, maps are generated that are then combined with a single-particle diffusion simulation in order to probe the capture statistics and to determine to track the trajectories of the individual particles on the microsecond to millisecond timescales.

3.2. MspA and phi 29 nanopore Mycobacterium smegmatis porin A, designated as MspA (as shown in Fig. 2b), is derived from Mycobacterium smegmatis. This channel protein is considered to be an ideal platform for constructing nanopore sequencing devices since it has a short and narrow (˜1.2 nm wide and ˜0.6 nm long) channel constriction. Recently, Derrington et al. proposed a simple method for the single-nucleotide resolution with engineered M1-NNN-MspA, making use of a series of double-stranded sections, which alters the movement of the polymer by temporarily holding the nucleotides in the pore constriction [105]. Following the same approach, Manrao et al. [106] demonstrated the feasibility of the mutant M1-MspA protein to resolve the homopolymers of the four nitrogen bases with fairly large current signatures when compared to α-HL. In addition to this, they also established that it could be possible to identify single nucleotide substitutions within a homopolymer chain while threaded through the MspA constriction. Further, the same group employed the mutant form of MspA with phi29 (as shown in Fig. 2d) DNA polymerase to determine the changes in current levels while transversing the single-stranded DNA molecules threaded through the small and narrow constriction of MspA. They suggested that MspA, in combination with phi29 polymerase, yields distinct current levels for the discrimination of individual nucleotide sequences since MspA exhibits high nucleotide sensitivity. Their studies open up newer avenues toward eliminating the major long-standing hurdles faced by nanopore sensors with regard to single-nucleotide resolution and translocation speed of biomolecules [63]. Bhattacharya et al. the results of total-atom MDSs that illuminate the physical event of ionic current blockades in the biological nanopore through MspA. They reported that the quantity of water removed from the MspA nanopore by the DNA strand reflected the nanopore ionic current and that the steric and base-stacking merits of the DNA nucleotides determined the amount of water removed. Surprisingly, an effective force on DNA in MspA undergoes maximum fluctuations, which may generate insertion errors in the DNA sequence readout [107]. 3.3. ClyA nanopore ClyA from Salmonella typhi is another new biological nanopore that is investigated for the detection of small and medium-sized proteins (as 600

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polymer. So, the ratio of bumping/translocation phenomenon was decreased as the dipole moment is increased. Finally, the results showed that the nanopore sensing technology is provided valuable structural information [85]. Cao et al. presented a newer aerolysin nanopore with a lipid membrane for the detection of single-oligonucleotide. They accomplished total internal reflection fluorescence (TIRF) computations to approve dA2 translocation via the aerolysin pore after creating a fluorescently labeled dA2 strand (dA2-FAM). Further, the adding of dA2FAM to the cis chamber of the solution, the ionic current was continually collected, and the trans chamber was recorded at one-hour interval for further experiments based on TIRF. Both the cumulative TIRF intensity and blockade phenomenon were increased with the collecting time. This result offered the direct proof that dA2 translocated via the aerolysin pore from the cis chamber to the trans chamber and it exhibited a maximum ionic current and temporal resolution for the short oligonucleotides detection [111]. Wang et al. showed a newer aerolysin protein nanopore for good performance genetic biosensing using a pH taxis-mimicking technique. The fabricated nanopore device was a simple and easy-to-handle approach to non-covalently transform aerolysin into a highly nucleic acids-sensitive nanopore. They plainly lower the pH on trans side of the nanopore, then aerolysin is directly “activated” and allowed to arrest the target DNA/RNA efficiently from the opposite side of the cis nanopore through a remote pH-modulation technique. This technique as well decelerated DNA translocation, the desired merit for sequencing and gene sensing, permitting temporal separation of DNAs in various lengths. In addition, this fabricated nanopore presented a capture rate of 10 folds (i.e., 0.21 ± 0.31 to 2.8 ± 0.4 μM−1s−1) in the voltage range + 20 mV to + 80 mV, which was highly attractive to the α-HL nanopore capture rate of 1 μM−1s−1 at + 100 mV and close to near zero at + 80 mV [119]. After that, Xi et al. presented a new nanopore-based scheme for good sensitive detection of Ramos cells, which is based on the coupling the enzymatic signal amplification with an aerolysin nanopore biosensor. Furthermore, the fabricated nanopore presented a 100 nM of output DNA with no symmetrical buffer solutions (0.5 M in cis/3 M in trans) allowed approximately 70 events in one minutes, while 100 nM of the DNA with symmetrical buffer solutions (1 M in cis/trans) was generated approximately 4 events in one minute. This technique ultimately produced a maximum number of outputs DNA, which could quantitatively generate characteristic current phenomenon when passed through aerolysin nanopore [120]. Similar studies were conducted using the ionic current for the unfolded proteins translocation through a conducting channel with varying stability. In this device, Pastoriza-Gallego et al. verified that the long ionic current blockade was a translocation time using a wild-type maltose-binding protein (MalEwt-MalEwt), a double-size protein. Furthermore, the translocation time of MalEwt-MalEwt protein was 2fold greater than that was acquired for the MalEwt single protein at the same concentration. The event frequency in such cases varies as a function of applied voltage and protein concentration and unfolded proteins were transported more gradually via aerolysin nanopore compared to α-HL nanopore [121]. Cao et al. suggested that polynucleotide (ssDNA) could be easily detected by wild-type aerolysin nanopore. Through the fabricated nanopore, a very short polynucleotide (four nucleotides in length) could be transported via aerolysin nanopore and was found to acquire approximately 50% amplitude of the open nanopore ionic current. These results of total internal reflection fluorescence calculations provided a direct indication for driven translocation of single polynucleotide via aerolysin nanopore [122]. Recently, Aguilella-Arzo et al. demonstrated a new biological nanopore in which the bacterial channel OmpF under properties identical to those under in vivo conditions were studied; in that study, acidic resistance events are understood to generate oscillations in the electric potential applied to the cell membrane in cis- and trans- chambers. They used a three-dimensional feature, based on a theoretical approach, to incorporate the possibility of calculating fluctuation-driven transport and

shown in Fig. 2c). This protein pore with 90% sequence similarity with the Escherichia.coli ClyA is preferred for the label-free detection of the large proteins analytes that pass through the pore lumen. Recently, Soskine et al. used covalently linked aptamer bearing ClyA nanopores for detecting the target proteins in the folded state, namely human and bovine thrombin. These proteins, despite showing greater sequence identity, can be identified by their characteristic current blockades generated at the time of entering the lumen of a ClyA pore. Under applied conditions, between +60 and −90 mV, ClyA pores were not gated notably and exhibited currents with SNR, approximately 10-fold greater than that of other biological pores [108]. Further, Meervelt and co-workers isolated two types of ClyA nanopores and named them to type I and type II. Both these types showed different run nature on blue native PAGE and showed distinct current blockades by protein analytes. The blockade events originate from TBA binding to thrombin exists in two isomeric orientations. Their findings suggest that ClyA nanopores can be used as a nano-trap channel, which allows the detection of the isomeric conformations of the protein: DNAaptamer complex-binding interactions trapped inside the ClyA nanopores [109]. A model was devised based on rotaxane the protein system, where the translocation behavior of proteins could be adjusted by applying a biphasic voltage across a ClyA nanopore. It was observed that the residence time of molecules inside a nanopore decreases with an increase in the applied voltage, which could be an indication of the translocation of protein molecules [110]. 3.4. Aerolysin and OmpF nanopore In the present era, aerolysin has some exclusive nanopore merits, including none-vestibule and longer pore of a similar radius or diameter of the α-HL pore (˜1.0–1.4 nm). Howard et al. suggested the aerolysin nucleotide sequence, showing its hydrophilicity. The water-soluble monomers can naturally oligomerize to a heptameric shape and subsequently transform into a transmembrane mushroom-shaped nanopore. Furthermore, other characteristic merits of aerolysin are its maximum number of charged residues positioned near the lumen pore. Seven of these residues remain positively unmatched, which expressively improved the electrostatic interaction between the nucleic acid and the nanopore, and led to a decrease in the DNA translocation velocity as the ‘molecule brakes’. Reduction of the translocation speed was led to a prolonged period, which may result in more detailed and correct insights into single molecules [111,112]. Researchers and coworkers from an extensive range of disciplines, including water purification, and biomedical applications have chosen this nanopore. It was formed from E. coli cells having the cloned aerolysin gene and developed generally by the elimination of the signal sequence, however, it is often not released from the cell. This protein was performed to be translocated across the E. coli inner membrane using a signal sequence which is further eliminated and the processed protein can be discharged by osmotic shock [113]. This is a passive protein channel governing the phenomena of translocation as well as shown in Fig. 3a. It can be used as a potential alternative tool for protein folding studies. In a recent study, an auto-transporter virulence protein, pertactin, was used as a model protein to evaluate the dynamics of transport through the aerolysin channel in an unfolded state. Comparison among the mono and dimer forms depicts the exponential variation in the frequency of current blockades as a function of applied voltage and a simultaneous decrease in the duration of individual independent events [114]. Furthermore, it has surged as a newer biosensing technique for biological relevant polymers [115], peptides [85,116], proteins [117] and polysaccharides [118]. Stefureac et al. also noticed the blocking percentage to the tune of 63% and 76%, respectively in the case of α-HL and it was interpreted as an average measurement of the fraction of the pore volume in which ion is excluded as the peptide is translocated. The dipole moment was shown the major influence on the transport phenomenon of the 601

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Fig. 3. Schematic diagram of (a) Aerolysin nanopore with monomer and Heptamer (side and top view). (b) OmpF with side view and top view.

substantial gaps) by comparing with annotated genomes from related parasite comparatives. These mitochondrial genomes have also been described with an initial electrical consensus sequence, using raw signal results created from a MinION R9 nanopore flow cell. The analysis results proved that the competently MinION-created mitochondrial genome of N. brasiliensis is of large sufficient superiority for phylogenetic use [126]. Recently, Wei et al. demonstrated a quick and practical method for developing and sequencing a one dimensional (1D) complex genomic DNA short-read sequencing library using a MinION sequencer. The recent MinION MIN106 platform has been provided advanced sequencing excellence for 1D reads than a prior MinION sequencer. The read length of the majority of reads was felt between 500–1000 bp, and the excellent score of each bp ranged from 4 to 16. The mean excellence score was approximately 9-10. This platform was created approximately 70 K raw reads per hour. The protocol has some limitations such as analysis can be presented in 30 min with certain appropriate computational resources in parallel. A newer nanopore sequence aligner (i.e., minimap 2), would perhaps a robust alternate for downstream data analysis within 10 min grabbing less computational sources with a minimum cost of 5–6% loss in UA reads [127].

showed in Fig. 3b. All calculations showed that excessively high voltages would be essential to calculate the main transport of ions against their concentration ascent [123].

3.5. MinION nanopore Oxford nanopore technologies have been enlisted to various hundreds of research laboratories to beta-test i.e 100-gram MinION sequencing tool since 2014 [124]. It is the portable real-time nanopore tool for sequencing of DNA and RNA. Each consumable flow cell can generate 10–20 Gb of DNA sequence data. The sequences of single DNA strand can be recorded while they are driven via biological nanopores by an applied electric field in the cis and trans chamber. In this device, the rate of each DNA strand passes via nanopore, and is governed by a processive enzyme bound to the DNA molecules at the nanopore orifice. The bases of DNA are revealed using cloud-based software (Metrichor) offered by oxford nanopore technology that uses hidden Markov models to suppose sequences from these current alterations [125]. Recently, Jain et al. calculated and optimized the accomplishment of the MinION nanopore sequencer using M13 genomic DNA molecule and employed expectation enlargement to find robust maximum-likelihood estimate error for insertion, deletion and substitution rates such as 4.9%, 7.8% and 5.1%, respectively. Furthermore, they solved the copy number for a cancer-testis gene family (CT47) within an unsolved region of human chromosome Xq24 by pairing their great-confidence alignment approach with high MinION counts. They have sequenced complete replicative-shape M13 phage dsDNA using three MinION flow cells that enclosed 337–473 functional nanopores channels calculated by online methods. Each 48-h copy process is created between 184 and 450 million bases from 63%, 24% and 13% template, complement and 2D read, respectively. Using pairing highconfidence alignment strategy with long MinION reads, the copy number for a cancer-testis gene family (CT47) within an unresolved region of human chromosome Xq24 was resolved [125]. Chandler et al. fabricated a one contig mitochondrial genome from N. brasiliensis by the data of MinION R9 nanopore. The fabricated assembly was error-accurated using existing Illumina HiSeq reads and decoded in full information (namely gene boundary definitions without

4. Solid-state nanopores Synthetic nanopores have risen as a convenient way of highthroughput single-molecule characterization and analysis. The exciting features of SSNs (as shown in Fig. 4) for a single-molecule detection

Fig. 4. Schematic diagram of SSN. 602

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were first employed for the translocation of dsDNA through the nanopore [24]. The main disadvantage with the SSNs is the scarcity of chemical discrimination of the nearly same size analytes, which can be overcome by functionalizing surfaces and attaching specific recognition sequences and receptors to the nanopores [18,128]. The SSNs are modified at the nanopore surfaces with a myriad of recognition molecules. The SiN-based SSNs covalently modified with nucleoporin 98 kDa and nucleoporin 153 kDa exhibit selective translocation of bovine serum albumin and importin beta (Imp β) [129]. In addition, the human telomere repeat sequence T8 was employed as a probe to ascertain the electrochemical confined consequence in a nanopore [130]. After modification, there is no change in ion current blockade although differences are evident in the electric current duration. Surface modification of the same pore is achieved using gold and a self-assembled monolayer of thiol immobilization on the gold surface. The nitrilotriacetic acid (NTA) was immobilized over the monolayer of gold-thiol bonds as a receptor and was used to detect histidine-tag, which participated with the Ni atoms present in NTA for intermolecular interactions [131]. A single-molecule measurement system was discovered earlier to detect ssDNA using group functionalized SSN channels with DNA hairpin-loop as a probe. The results of the study showed that under the influence of an applied electric field, nanopores selectively transport shorter target molecules complementary to the probe with a shorter translocation time. These promising functionalized nanopores are stable and robust enough for use in an array format [128]. Anderson et al. developed a simple analytical model based on amine-functionalized SSN to trace the DNA translocation time by changing the pH of electrolyte solution under in situ conditions. They observed that by modifying certain properties of SSNs, they can be tuned independently. Any small change in surface charge chemistry can lead to a large shift in the residing time of the analyte. This significant property of these nanopores can be exploited to set the translocation time irrespective of the other experimental parameters [132]. Kececi et al. developed conical shaped nanopore fabricated with a membrane-based resistive-pulse sensor for the detection of short 50-bp and 100-bp DNAs. The nanopores were chemically etched into membranes having anionic carboxylate sites on the nanopore walls. Further, ethanolamine functional groups were linked to these sites using well-known EDC chemistry. The resistive-pulse method utilizes the properties of ethanolamine functionalized pores and can easily detect 50 and 100 bp DNAs and distinguish between both types of DNAs [133]. Subsequently, Gyurcsányi presented a review that on the chemically modified nanopores for biosensing. This review discusses many types of SSNs such as multichannel nanopores, chemically modified single-nanopore, and single nanopore. However, this review indicated promising result on less explored phases of nanopore biosensing such as the use of nanopore arrays, the unification of nanopores in compact biosensing tools, and the improvement of their execution by using the hyphenated apparatus, integration in micro- and nano-fluidic tools, different interrogation methods, and several alternatives techniques for functionalizing nanopores. However, this study could not determine the minimum detection limit and better nanopore for future utilization [134]. In one study, SSNs have been utilized to compare the differential blockade signatures of dsRNA and RNA homopolymers by applying high voltages across the nanopore. It was observed that conductance blockade versus applied voltage was nonlinear for the two categories of molecules as dsRNA blocks more when compared to single-stranded homopolymers. Furthermore, the pros of this biosensor are that the interactions of DNA with the membrane was increased at high voltage, thus easily explaining the enhanced conductance of blockade [135]. Wanunu et al. suggested a new single-molecule technique for rapid binding of small-molecule to particular DNA molecules. DNA-binding molecules have been detected in ultrathin silicon-membrane-fabricated nanopores with bulk fluorescence measurements by measuring the shift in the residual ionic currents. The level of residual current might help

quantify the number of intercalated dye molecules bound to DNA. The dye affinities for two different regions demonstrated the capability of the nanopore method to discriminate the interaction among native and dye-bound regions. Furthermore, this fabricated nanopore shows a well current signal within the time resolution approximately 12 μs, which was the more tremendous average speed of approximately 0.3 μs/bp for short DNA fragment (i.e., 100–1000 bp) translocated via a 3.5 nanopore. Further, the technique could be applied to those protocols where no chemical labelling is required, thereby allowing the interaction DNA and hinders their translocation as well as it offers fast analysis with single-molecule sensitivity [136]. Further, Mulero et al. published a review on nanopore devices for bio-analytical sensing. This review article discusses various advancements in nanotechnology and nanopore-based tools from the earliest application of α-HL nanopores to the development of SSNs to the improvement in the organic-inorganic hybrid nanopores operated with a view the maximum results of DNA sequencing at minimum cost. Furthermore, they also described another well-discussed nanopore that identifies other kinds of biomolecules, namely, protein. This review also explains the application of SSN in various fields such as DNA sequencing, protein analysis, and ultrafine molecular sieving. Each of the applications discussed is not addressing technologies but it drives excellent performance towards exciting newer and great impact bioanalytical sensing methods [137]. Even though much progress has been made since the path-breaking work by Kasianowicz et al. the supreme goal of fast DNA sequencing is yet to materialize. The SSNs were also seen in experiments on the translocation of ssDNA and ssDNA-dsDNA constructs, where ssDNA yields larger current blockade events as a function of voltage as compared to hybrid construct [138]. Recently, Hout et al. presented a new tool for the measurement of forces on the dsRNA, dsDNA molecules in the SSN using optical tweezers such as fdsRNA = 0.11 + 0.02 pN/mV and fdsDNA = 0.14 + 0.03 pN/mV. In that study, one or more molecules with an optically trapped bead connected to the nanopore surface was placed in the cis chamber of the nanopore. Then, they were checked to the change in position signal of the bead. The position related to as a displacement of approximately 64 + 2.3 nN, corresponding to the force 8.6 + 2.3 pN presented the usual stiffness of 135 + 25 pN/μm. Kowalczyk et al. showed the translocation of bare and fully RecA-coated DNA using SSN, which is generated by the DNA with discontinuous patches of the DNArestoration protein RecA connected along its length [139]. Similarly, Wanunu et al. demonstrated the electrostatic focusing on the dsDNA using a salt gradient in the SSN. This fabricated device encompasses two different steps; first - dsDNA coil was impenetrated the nanopore from amount to a distance greater than the size of the coil. According to this phenomenon, its motion was transited from accurately diffusive to biased motion generated due to the applied electric field inside the chambers. The ionic current event was kept apart from the nanopore, that generated a potential characteristic V(r) near the nanopore mouth. It exerted the dsDNA coil from some distance (r) having orders of magnitude larger than the length of Debye screening scales i.e., 0.1–1 nm. Second - one time a dsDNA or DNA was within one coil length/size (rg) of the nanopore, then threads of DNA end into the nanopore, and that mechanism involved passing a free energy barrier. Furthermore, the detection of picomolar dsDNA concentration in 20fold salt gradient solution at large throughput was determined [140]. In 2011, Venkatesan et al. published a review on nucleic acid analysis using nanopore sensors. They reviewed nanopore sensing technologies in medical devices, genetics, and DNA sequencing. These nanopores are analyzed with the charged polymer (including ssDNA, dsDNA, and RNA) with subnanometer resolution and without the necessity for amplification and labels. The authors supported the notion that nanopores-based biosensors would be useful in combination with another third-generation DNA sequencer and may be capable of rapid and credible sequencing of the human genome for under $1000 [18]. Afterwards, Xie et al. proposed a novel nanowire nanopore sensor that 603

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combines SSNs with silicon nanowire field-effect transistors (FETs) on a Si3N4membrane, where the detection signals are achieved by highly localized self-alignment at the nanopore. SSN fabricated in Si3N4 nitride membrane has diverse applications [9]. Mo et al. reviewed the application of synthetic nanoporous membranes in sensing. In this review article, synthetic nanoporous membranes are classified as a nanoporous polymeric membrane, alumina nanoporous anodized, nanopores fabricated using nanolithography method, and CNTs. The review addressed the detection methods, detection limit, and membrane types for various biosensing applications. The review highlighted the lack in the consensus regarding the most superior methods for biosensing in future [5]. Prior to this, biological nanopores were utilized to study the conformational dynamics of nucleic acids. Apart from RNA conformational studies, they have also been used for studying the protein conformations. In a previous study, a protein biomarker for the HIV-1 virus, nucleocapsid protein 7, could be successfully detected upon binding with the RNA aptamer of stem-loop 3 (SL3). The investigators incorporated two categories of pores, with small and large diameters, which can detect the protein-aptamer complex more precisely owing to the smaller diameter pores and allows measurement of accurate dissociation and association constants of the two interacting analytes [141]. Furthermore, Makra et al. published a mini review on electrochemical biosensing using nanopores. This mini-review presented analytical information of resistive pulse sensing (RPS) and potentiometric sensing. The RPS method calculates the pulse height, pulse frequency, and pulse duration which depend on the indicative of the volume of the target is proportional to the target concentration and depends on the mean translocation velocity and relative lengths of the pore and the target species, respectively. The potentiometric sensing method calculates the alteration in the flux of ions due to the chemicophysical merits of the surface, due to the small diameter of the nanopore. These methods are important for the analyses of the electrical resistance, pulse amplitude, SNR, selective detection, and detection limit. However, this review did not identify the best nanopore for biosensing [142]. The first use of synthetic SSN of precise geometry was to study the difference in translocation dynamics of viral RNA motif sequence; the study revealed that the nanopore can be utilized as a viable label-free detector for conformational change in RNA and for detecting the structures of various RNA motifs. Notwithstanding the minimum size of the HCV IRES domain IIa, this is almost same to a ∼ 25 bp fragment in length and nanopore diameter (3 nm), simply discriminate straight from bent RNA conformations. Structural changes in the RNA upon binding with small drugs or even the small quantity of material present can be effectively detected by this approach in the future [143]. Marshall et al. reported the detection of DNA depurination using SSN at the single-molecule scale. In this experiment, they selected four separate SSN devices ranging from 5 to 6 nm in diameter for translocation of 61 bp DNA in 1 M KCl solution (high-ionic-strength) over the range of pH (2 to 10). This nanopore was ascribed to an ameliorative loss of the ds-helix, which escalates confinement affected due to exposed regions of ss-structure where unpaired nucleotides can be attached directly with the SSN. This ability of the nanopore displayed powerful interactions between threading biomolecules and the nanopore and inhibiting translocation speeds [144]. Subsequently, Plesa et al. detected the velocity of linear dsDNA molecules translocating through the SSN. This method is used to observe the mean velocity of all segments of 7560-bp synthetic DNA molecules as well as record the significant fluctuations in translocation velocity at both the intra- and intermolecular levels between different nanopores of the same diameter [145]. Subsequently, Deng et al. demonstrated fabrication technologies for the development of SSNs, which are used in biosensing. This review paper describes and compares the currently available typical fabrication methods of the SSN. The review highlighted the benefits and specific application of each type of nanopore. By serving as nanobiosensing

surfaces, lithography nanostencils, light modulators, and ionic rectification tools, several SSNs showed potential for application in various other fields such as energy conversion, water purification, desalination, and organic pollutant degradation. However, this review did not elucidate the detection limit, response time, SNR, and sensitivity based on fabrication biosensing [146]. Furthermore, Feng et al. demonstrated a fourth-generation DNA sequencing technology based on nanopores and the same nanopores were discussed by other such as Haque et al. [147]. The latter showed that the fourth-generation DNA sequencing technologies (as nanopores based sequencer) have the potential to rapidly and credibly sequence the entire human genome for less than $1000 or less than $100. Additionally, other benefits were also listed, including least sample preparation, erasure of necessity for modification or amplification (namely nucleotides, polymerases or ligases), as well as calculate the length of the long molecules such as those with 10,000–50,000 bases. In this review, the detection limit, sensitivity, efficacy, and response time were not addressed in term of future planning [148]. Pla-Roca et al. developed a new technique to selectively (bio)-functionalize the nanoscale features by using the same material in combination with nanosphere lithography (molecular assembly photolithographic lift-off) to generate SSNs. The unique pros of nanosphere lithography are that the nanopore surface to be designed in the next step was completely protected during the functionalization of the first step and that the total surface has similar surface chemistry. To achieve this, poly(ethylene glycol)-brushes, along with lipid membranes and functional proteins were used over large areas. The method is applicable to achieve both tomographic and plane surface modifications preclude the need for specialized devices and are low in cost [149]. Rodríguez-Manzo et al. developed a new silicon nanopore device that monitors changes in the ionic conductance (ΔG) when certain biomolecules pass through it. The generation of large SNRs for calculating the molecular structure in various applications, namely DNA sequencing, requires low noise and great ionic conductance. This is achieved by decreasing the nanopore size and nanoporous thickness. While the lowest diameter of the nanopore is limited by the biomolecule size, the thickness of the nanopore membrane depends on the type of material used. They have been used in molecular dynamics simulations (MDSs) to calculate the theoretical width of amorphous Si nanopore membranes to be approximately 1 nm, and they fabricated an electron-irradiation-based thinning technique to achieve this and drill nanopores in the thinned areas. dsDNA translocations via these nanopores (up to 1.4 nm in thickness and 2.5 nm in diameter) reach the minimum intrinsic ionic conductance detection limit of biomolecules in Si-based nanopore membrane. These results are contrasted with recent publication describing DNA translocations via nanopores drilled in membranes of various materials with thicknesses less than 10 nm [150]. Pud et al. developed a more up-to-date cost-effective system for the fabrication of self-adjusted plasmonic nanopores by optically controlled dielectric breakdown. Excitation of a plasmonic necktie nanoantenna on a dielectric film confines the high-voltage-driven breakdown of the layer to the hotspot of the upgraded optical field, thereby making a nanopore that is consequently self-adjusted to the plasmonic hotspot of the tie. They showed that their approach allows for exact control over the nanopore estimate, thereby making them suitable as single particle DNA sensors with functions similar to TEM-penetrated nanopores. The standard of optically controlled breakdown can also be employed to develop nonplasmonic nanopores at a controlled position. Their technique ensures arrangement of the nanopore with the optical hotspot of the nanoantenna which allows the interaction of the pore-translocating biomolecules with the concentrated optical field that can be applied in the recognition and control of analytes [151]. Recently, Bell et al. presented a translocation frequency of dsDNA via SSN. They showed a comprehensive technique for the evaluation of the effect of length, salt, and voltage dependence on the frequency of dsDNA translocations via conical quartz nanopores, with an average 604

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the evaluation of the required electrical factors [10]. Wen et al. demonstrated the low-frequency noise characteristics of SSNs of 7–200 nm in diameter drilled via a 20 nm thick SiNX nanopores membrane using a focused ion beam. In this work, bulk and surface ionic currents in the pore are received to allow for the shimmer noise, with their respective effects calculated in term of the concentration of salt and electrolytes pH as well as in the bias condition. With the increase in the concentration of salt with a constant pH value and voltage, the bias pioneers increase the ionic current in the bulk and noise. Furthermore, changing pH with a constant concentration of salt and current biases outcomes in the variation of surface charge density, resulting in alterations in the surface ionic current and noise. The improved understanding has led to the development of a generalized device encompassing greatest noise mechanisms particularly in the minimum frequency range [154]. Recently, Wang et al. presented a high-sensitive cocaine biosensor which is based on a single nanopore linked with DNA aptamers. This fabricated biosensor was developed by immobilization of DNA aptamers (C-aptamers) onto the inside wall of the nanopore. The C-aptamers were attached to selectively bind cocaine and DNA aptamer biomolecules (T-aptamers) that acted as target onto the pore wall, resulting in limited or whole occlusion to shield surface charge of the pore, thereby decreasing the transmembrane nanopore ionic current. As, a result, this biosensor showed the good linear relationship between the output ionic current and target cocaine concentrations (1 nM to 10 μM) and had shown the minimum detection limit as 1 nM [155]. Lastly, Pérez-Mitta et al. showed newer perspectives on the conical SSN. This perspective was shown to fetch together the current fabrications by employing nanopores as “iontronic” transducing parts. The transduction of these flickers into a pre-defined “iontronic” signal can be magnified by availing nanoconfinement and the physic-chemical impact namely changes in the ionic concentration, steric constraints, charge distribution or the equilibrium displacement. This work promoted a cascade of innovative efforts in nanopore-based biosensing devices which facilitated only the rational fabrication method and design of the gateable nano-fluidic device but did not provide much clarity on detection limit, nanopore size, and materials useful for nanopore device [156].

opening radius (diameter) of 7.5 nm (15 nm). Subsequently, they analyzed an entropic barrier-finite, length-dependent and -independent translocation frequency of salt concentration at 4 M (M) LiCl, and 1 M KCl, respectively, as well as a drift-dominated. At 4 M LiCl electrolyte concentration, they calculated an enhancing translocation frequency where the DNA length works as a function in the range of 0.5–10 kbp. This was consistent with the nature of entropic barrier-finite movement in a one-dimensional convection-diffusion equation. At a minimum concentration of 1 M potassium chloride (KCl), where the DNA charge is greater, they calculated the characteristic transport merits of a driftgoverned regime. This study provided extensive insights into the understanding and forecasting of polymer transport via nanopores across a wide range of features [152]. Subsequently, Tsang et al. suggested a luminescence method of an assay involving of BaGdF5:Yb/Er upconversion nanoparticles, which corresponds with oligonucleotide probe and gold nanoparticles (AuNPs) attached with target Ebola virus oligonucleotide. They developed a homogeneous assay platform with a detection limit at a picomolar level. Furthermore, the luminescence resonance energy transfer was attributed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. In addition, they anchored the upconversion nanoparticles and gold nanoparticles to obtain a heterogeneous assay platform on a nanoporous alumina membrane. Importantly, the detection limit was greatly increased, exhibiting a considerable value at the femtomolar range; this improvement is attributed to the light-matter interaction throughout the walls of a nanopore of the nanoporous alumina membrane. This specificity test provided that the nanoprobe was specific to Ebola virus oligonucleotides. The strategy of combining up conversion nanoparticles, gold nanoparticles, and nanoporous alumina membrane provided the advantages of low-cost, quick, and ultrasensitive detection of several diseases. Further, they showed the feasibility of a clinical device using the inactivated Ebola virus sample and found the good potential for the practical application of their heterogeneous assay platform [153]. Recently, Roy and Hall demonstrated a modified SSN-sensing method for the detection of alcohol-soluble proteins. In their work, they showed short duration signals with exponential distributions. Furthermore, their study showed a majority of the observed phenomena was less than the 33 μs in duration, below which distortions would be accepted. They also used the biosensing device to calculate α-zein, a model protein with solubility only in maximum concentration alcohol samples. This work was about an order of magnitude greater than previously published manuscripts of protein translocation in conventional liquids and may be suggested that either (i) a minimum driving force is acted on α-zein under conditions; (ii) transfer of proteins to the nanopore entrance is detracted by changed diffusive kinetics in ethanol azeotropes samples; and/or (iii) the large amount of translocations are unnoticeable, as has been noticed for hydrophilic proteins in aqueous conditions. Analysis of electrical translocations showed that only slowmoving translocations could be solved, identical to the results obtained the traditional solvents [11]. Later, Yusko et al. developed new methods to characterize proteins that generally undergo physical or chemical changes or cannot be used to investigate individual biomolecules in the solution. They showed that the zeptolitre biosensing volume of bilayerfabricated SSNs can be used to determine the approximate size, volume, charge, shape, rotational diffusion coefficient, as well as the dipole movement of individual proteins. To achieve this, they developed a new theory for the quantitative measurement of gradients in ionic current based on the rotational dynamics of single proteins during transportation by the applying a voltage inside the nanopore. This measurement technique allowed for the assessment of five parameters simultaneously. Their method allowed for the identification, characterization, and quantification of proteins, which makes it suitable for various fields such as structural biology, proteomics, biomarker detection, and routine protein analysis. However, this method is limited by the application of about 10% of resistive pulses, lasting at least 400 μs to allow for

5. Nanocapillaries as nanopores These types of special nanopores have recently been investigated by many research groups, and have shown potential for application in DNA sensing, scanning conductance microscopy, and ionic current rectification. The laser-pulled glass nanocapillaries of conical shape with a small hole at their tip recently works as an inexpensive alternative to silicon nitride (SiN3)-fabricated SSNs. They have the ability to sense the folded state of translocating ds-DNA at the single-molecule level. The nanocapillaries, a new and exciting class of nanopores, have opened up the bright possibility of avoiding the conductive coating over the glass. Steinbock et al. examined the effects of scanning electron microscopy (SEM)-induced shrinking of glass nanocapillaries on the conductance change caused by translocating dsDNA. They explored and evaluated the effect of smaller diameter on decreasing current by shrinking the nanocapillaries to a diameter of 100 nm to 10 nm. They compared the SNR of shrunken glass nanopores with that of the samesized nanopores in the SiN3 membrane. They reported higher SNR of around 25 for 14 nm pores for shrunken nanopores and lower SNR of around 15 for 3 nm pores in the silicon nitride membrane. From their findings, they have concluded that SNR is more useful for glass nanocapillaries with a diameter below 30 nm than standard nanopores in the SiN3 membrane [157]. Another excellent use of the glass nanocapillary was shown by Chen and coworkers who developed a nanocapillary-based regenerable sensing platform with the aid of polyglutamic acid (PGA) non-immobilized probe for the rapid and selective detection of cupric ions. Polyglutamic 605

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Fig. 5. Schematic diagram of (a) nanoporous graphene and (b) nanoporous GO layers.

an approach where the graphene-embedded fluidic nanochannel device functions as an ultrasensitive platform to control the movement of the bases of the DNA via π- π interaction coupled with the conductance characteristics of the nanoribbon, thereby allowing for the differentiation of different bases one-by-one in real time, by using data mining technique and a 2D autocorrelation analysis [161]. Several groups have recently realized and demonstrated experimentally the use of graphene sheets to fabricate nanopores and their use as a sensing device for detecting the electric-field-driven translocation [25,29,162]. Sathe et al. utilized SMD as a computational investigatory method for the detailed atomic study of the translocation kinetics of a nucleic acid along with the magnitude of the ionic current blockades associated within graphene nanopores. Since graphene is a subnanometer thick, the designing of graphene nanopore-based device can be directed to understand the influence of certain factors associated with the blockade of ionic current signals rooting from translocation phenomena. They suggested that the graphene nanopore could be required averting DNA adherence to the graphene layers in order to have DNA stretched in the nanopore for DNA sequencing purposes. For example, such type rectification can be achieved by using optical tweezers to keep the DNA in the stretched form [163]. Venkatesan et al. demonstrated a newer multilayered graphene-Al2O3 nanopore device for the sensitive biosensing of DNA and complexes of DNA protein at the single biomolecule level. This device evaluated voltage-dependent DNA transporters in terms of parameters such as a decrease in the translocation time (tD) with an increase in the voltage. Thus, this nanopore is greatly sensitive at not only biosensing of the existence of single molecules but also a molecule’s secondary structure, that is, folded or unfolded. Furthermore, this experiment also demonstrated the detection of an 8-kbp-long recombination protein A (RecA)-coated dsDNA biomolecule via a 23 nm diameter the graphene-Al2O3 pore with 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8 electrolyte solutions at an applied voltage of 500 mV. In that study, single-biomolecule transport experiments using graphene-Al2O3 pore completely resolved the λ-DNA translocation (folded and unfolded) in addition to the transport of RecA-coated DNA; the protein-DNA complex reveals deeper ionic currents blockades relative to native dsDNA [164]. Puster et al. also reported the use of sensitive graphene nanoribbon (GNR)-based nanopore devices for the detection of DNA by preventing the TEM-induced electron-beam damages [165]. Avdoshenko et al. performed theoretical attempts for DNA translocation by modifying the dynamic and electronic transport analysis properties of the graphene nanopore-based sensing device. Their findings reveal that although it is not possible yet to distinguish the four nucleobases, it may be possible to have full control over the translocation dynamics if four-fold layers that are deposited onto the graphene platform-bearing pore [166]. Liu et al. suggested a new atomic thin molybdenum disulfide (MoS2) nanopore for use in the highly sensitive detection of DNA biomolecule. The application of this nanopore was checked in several types of dsDNA with changed length and conformations, demonstrating greater sensitivity (SNR greater than 10) as compared to the conventional SiNx pores with a thickness of 10 nm. As with nanopores of graphene, no

acid is highly selective for cupric ions as compared to other metal ions. The detection phenomena chiefly depend on the detection of a decrease in ionic current across the lumen of nanopore while reversal ion current rectification signals are regenerated as the cupric ions present on the probe surface gets chelated [158]. Lan et al. utilized conical pores present inside glass membranes to differentiate the polystyrene nanoparticle sizes of 80 and 160 nm on the basis of pulse height and translocation time using the Coulter counter-principle. The authors have drawn a satisfactory conclusion that these nanopores show better resolution for the particles greater than 40 nm. These can also be applied to the smaller diameter particles if the instrumentation and electronics can be further improved [159]. 6. Graphene and GO-based nanopore Graphene, a two-dimensional thin, flexible carbon lattice has been recently discovered and has been found to be attracted to an excellent potential platform for sensing a variety of gases and molecules. Due to its unique electronic and mechanical properties, the nanopores can be ideally fabricated. Graphene nanopores are drilled via a focused electron beam inside the TEM as shown in the schematic diagram, Fig. 5a. Molecules present in the electrolyte solution are driven through these nanopores. Suitable use of graphene nanomaterials as a membrane open up avenues for a new class of nanopore devices (as shown in Fig.5a) that enables both electronic sensing and control to be performed directly at the pore interface. After the translocation of the molecules, the flow of ions is blocked, this could be detected as a drop in the current measured. Recently, Merchant et al. demonstrated the possibility of graphenebased nanopore devices by utilizing the nanopores created inside graphene membranes for the purpose of DNA translocation. They visualized larger current blockades as compared to traditional SSNs due to the thin nature of the graphene layer [29]. Postma suggested a newer method for studying the bp sequence of a single DNA biomolecule using a nanogap of graphene to read the transverse conductance of DNA biomolecule. In the current fabricated device, the nonlinear characteristic of current-voltage was employed to calculate the base type independent of nanogaps-width differences that reason the current to alter by five orders of magnitude. The residual ionic current between the unpassivated carbon atoms at the edges of the nanogap of the graphene will be caused an additional contribution to the ionic current signal as well as its first derivate of dI/dV. This offset was rectified before and after the DNA has been translocated via nanogap which reduced to recompensate the effect [160]. Furthermore, Nelson et al. explored a graphene nanochannel nanopore device that simultaneously included two essential parameters for device performance in a single proposed device. The device based on GNP excludes the independency of the conductance to nucleobase orientation while passing through the nanopore, as in the case of tunneling currents. Secondly, for discrimination of individual bases, precise and sufficient data is provided by the device that presents the strong proof for their sensitive behavior over ionic and tunneling currents [49]. Min et al. theoretically reported 606

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MoS2 thin membrane [173], but not graphene and GO nanomaterials. Since graphene is also a two-dimensional, layered nanomaterial, it shows the same merits as MoS2 [33,174]. In this fabricated device, they have explored a single-sheet MoS2 membrane with 10 nm pore size and a 30% porosity by exploiting parallelization. The estimated power density could be reached 106 Wm−2 with a salt gradient. These results were increased-by two to three orders of magnitude the values recorded with boron nitride nanotubes, and are million times larger than the power density noticed by reverse electrodialysis with classical interchange membranes [173]. After that, Shim et al. demonstrated a new MoS2 nanopore-based assay to detect the dsDNA. In this fabricated method, the current blockades and the translocation duration of the molecules were noticed during dsDNA transport via the MoS2 nanopore. According to this, the current blockades and transport durations of 10 kb dsDNA via a MoS2 6.5 nm nanopore were 1.27 ± 0.24 nA, 1.7 ± 0.41 nA, 2.35 ± 0.43 nA, and 188 ± 17 μs, 118 ± 8 μs, 83 ± 6 μs at 500 mV, 700 mV, 1000 mV, respectively. The current blockades amplitudes were enhanced at larger biased voltages, in accordance with the trends of dsDNA analyzed in other related nanopores. To analyze the transport of dsDNA transport in another nanopore, it has been demonstrated that with the application of higher voltage normally a higher current blockade is achieved with shorter duration times. These findings with dsDNA molecule transports via MoS2 nanopore were in compliance with experiments in previously published studies [175]. Subsequently, Shim and et al. demonstrated a new MoS2 nanopore-based assay to detect the naked and methylated dsDNA fragments. They used a similar approach employing MoS2 and methylated form of DNA to present the capability of MoS2 pore to well distinguish naked DNA from hypermethylated DNA (hyMethDNA). Target DNA molecule was showed 90 bp sequences with 30 CpG sites. Ten (10) methylcytosine domains were attached to the naked DNA along with hypermethylated DNA to create homogeneously distributed methylation sites. In this fabricated method, the current blockades and the translocation duration of the molecules were noticed during 90 bp naked dsDNA transport via the MoS2 nanopore. According to this, the current blockades and transport durations of 9 bp dsDNA via a MoS2 of 7.2 nm nanopore were 2.82 ± 22 pA, 312 ± 13 nA, 376 ± 16 pA, and 53 ± 3 μs, 36 ± 4 μs, 32 ± 2 μs at 50 mV, 80 mV, 100 mV, respectively. Moreover, the complex of hyMethDNA bound with MBD1x was generated detectable events through the MoS2 nanopore. Unpredictably, the complex showed a signature current blockade at 200 mV [175]. After that, Shim et al. demonstrated a new MoS2 nanopore-based assay to detect the single CpG site in dsDNA fragment. In this study, the single MBP is in the centre of the biomolecule and the electrical peak representing the methylation site is large. In addition, they reported the methylation summary of dsDNA via a MoS2 pore with a slow translocation speed due to the difference in salt gradient. The current blockades and the translocation duration were noticed during naked and complex dsDNA transport via the MoS2 nanopore. According to this, the current blockades and transport durations of naked and complex dsDNA via a MoS2 of 9 nm nanopore were −277 ± 20 pA, −600 ± 66 pA, and 1.23 ± 0.21 ms at 200 mV, respectively. The complex translocated via MoS2 pore at 1.39 ± 0.23 ms was shown nearly 160 μs slower translocation compared to the naked DNA. While they had formerly found the approximately 20-fold difference between locally methylated DNA and naked DNA via SiNx pores, the similar range of translocation duration between endMethDNA and naked DNA was unpredicted [175]. Furthermore, Farimani et al. demonstrated that single-layer nanoporous graphene is highly capable of biosensing and distinguishing between dissimilar subclasses of IgG antibodies, notwithstanding their minor and subtle changes in the atomic structure. This study showed the translocation of IgG2 (91 ns) and IgG3 (111 ns) via the nanoporous graphene. This work showed distinguishable properties of nanoporous graphene, such as the dwell time with respect to the hinge region length, during the translocation of IgG2 and IgG3 via the 10 nm

noteworthy slowing down could be acquired with small-diameter nanopores of ∼2 nm. The DNA translocation velocity is approximately 20 ns per bp, still away from the 1 MHz ionic current amplification bandwidth for translocation tests. Even although the improvement of current signal amplitude is affected in MoS2 pore, the absence of temporal resolution is the foremost obstacle that would be controlled for multiple applications. Vital pros of MoS2 over the graphene and boron nitride nanopore is that the MoS2 intrinsic bandgap nature would render the implementation of sequence-specific transistors extra promising [167]. You et al. used the graphene layer with its reduced counterpart rGO layered membrane to achieve 100% desalination and suggested that the membrane layer formed from graphene elegantly achieved the same result since the fabrication of rGO and its precise pore size control seems to be very convenient and economical from the practical aspect in several industrial-based filtration processes in future [168]. Crick et al. presented the in-situ, controlled opening of graphene nanopores through electro-etching. In this article, the few-layered graphene membrane is presented to be applicable to receive nanopores of any size between that of fully-closure and fully-opened nanopores. Thus, it is possible to achieve in-situ nanopore opening, thereby allowing a change as translocation methods are carried out. This nanopipette tool showed a distinction in DNA translocation, with small nanopores exhibiting a maximum change in dwell time. These functions are made possible by nanomaterial coating using graphene nano-flakes (GNFs) to immobilize the nanopipette tools with a 25 nm diameter pores. The thickness of the nanofilm of GNFs was improved to achieve consistent coating and effortlessness of opening. This was achieved by using GNFs solution (1.5 mg.mL−1) using dip-coating, which endowed a 3–4 nm thick nanofilm. The pore size reduction could increase the energy barrier for the translocation of DNA. An additional effect of the small sized-pore is that the DNA must be opened in order to translocate, this conformation essentially has a higher translocation time of DNA compared to a more constricted. In addition, the DNA may be relating expressively with the graphene layers coating as it is moved through the pore. The electrochemical method used to open the nanopore may cause hydrophilic groups to interact at the graphene surface. These groups attracted the DNA biomolecules toward the nanomaterial coated over the membrane – this effect was utmost at smaller nanopore sizes [169]. Recently, Rollings et al. demonstrated a graphene nanopore for ion selectivities such as potassium (K+) and chloride (Cl−). This nanomaterial nanopore was widely used in electrodialysis desalination due to its inherent properties such as atomic thickness associated with its mechanical strength, whereby selective ions are displaced under the applied voltage through ion-selective pores. Rolling et al. showed that a single layer nanoporous graphene preferably allows the passage of potassium cations over chloride anions with selectivity ratios of over hundred and behaves as mono-valent cations up to five times greater than divalent cations [170]. Furthermore, Tash et al. presented a new review on single molecules biosensing with graphene and other 2D nanomaterials. Currently, these nanomaterials are fabricated into nanoscale chips that may sequence genomes in the future but did not present the efficacy, sensitivity, and detection limit clearly [171]. Feng et al. explored a viscosity gradient device based on room-temperature (RT) ionic liquids to handle the DNA translocation dynamics via a nanometer-sized pore invented in a simple thin molybdenum disulfide (MoS2) membrane. DNAs (nucleotides) are noticed according to the ionic current response collected during their transient residence in the fine nanopore orifice of simply thin MoS2 nanopore. They demonstrated the velocity of single nucleotide translocation that is an optimum velocity (i.e., 1–50 nt/ms) for DNA sequencing, whereas keeping the SNR greater than 10 [172]. Feng et al. reported that single nanosheets MoS2based nanopores are sensitive to generate power called as “blue energy”. This fabricated sensor obtained a wide, osmotically generated current created from a salt gradient with a calculated power density of up to 106 watts/m2. A current can be ascribed notably to atomically 607

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comparison to the challenging synthesis of monolayer graphene membranes, which necessitates the preparation of large-sized graphene sheets and punching of nanopores with a narrow size distribution [179]. Once dry, GO membranes prepared by vacuum filtration are so closely packed (void spacing, ˜0.3 nm between nanosheets) that only water vapor that is aligned in a monolayer can pass through [180]. Joshi et al. reported that when this GO membrane is immersed in an ionic solution, the GO spacing increases to ˜0.9 nm [30], allowing the entry of any ion or molecule with a hydrated radius of 0.45 nm or less, but blocking larger species. This sharp cut-off by the GO membrane has a variety of applications. By modifying the GO spacing by the placement of spacers between the nanosheets, a wide range of GO membranes can be established precise isolate target ions and molecules of a specific size from a bulk solution. This certainly offers more advantages in sieving as compared to the commonly used polymeric membranes with wide pores. Hydration of GO in aqueous solution is a major challenge to restrict GO spacing within a sub-nanometer range. For example, desalination requires to GO spacing to remain less than 0.7 nm to sieve hydrated Na+ (with a hydrated radius of 0.36 nm) from the water. This can be achieved by partially reducing the GO such that the size of hydrated functional groups is reduced or by causing covalent bonding of the GO nanosheets with small molecules to counter hydration forces. On the contrary, increased GO spacing (1 to 2 nm) can be easily achieved by introducing large, rigid chemical groups [178] or soft polymer chains (for example, polyelectrolytes) between GO nanolayer/nanosheets, resulting in GO-based nanosieve membranes that are suitable for application in water purification, wastewater reuse, and pharmaceutical and fuel separation. By using larger nanoparticles or nanofibers are used, GO-based nanosieve membranes with a spacing greater than 2-nm can be developed for use in the biomedical applications (i.e., artificial kidneys and dialysis) involving the separation of large bio-molecules and small waste molecules [179]. Recently, Hu et al. proposed a new method to fabricate a novel type of water separation membrane by using the GO nanolayer such that these molecules can flow via the nanopores between GO nanosheets, while the undesired solutes are removed using the size exclusion and charge effects. The GO nanosieve membrane was fabricated through LBL deposition of GO nanolayer, which was bound through 1,3,5-benzenetricarbonyl trichloride, on polydopamine-coated polysulfone support. The fabricated platform was showed GO membrane flux of 80–276 LMH/MPa, approximately 4–10 times greater than that of most common nano-filtration membranes. Even though, this platform was exhibited minimum rejection of mono- and divalent salts (approximately 6–46%), medium rejection of methylene blue, and high rejection of Rhodamine-WT (approximately 46–66% and 93–95%, respectively) [178]. Kim et al. used the few ultrathin layers of GO membranes for CO2 gas separation profiles. A group such as a hydroxyl and a carboxyl investigated the transit of gases by means of even thick layers of GO membranes at elevated transmembrane pressures. Further, gas permeability can be modulated by changing the average GO sheet size. On the basis of findings, gas permeability via the ultrathin GO membranes is believed to be strongly proportional to the enforced transmembrane pressure for a particular average size of GO sheet [181]. Current existing commercial technologies for providing fresh water for human consumption are limited by their excessive energy consumption and high costs. Desalination with the aid of nanopore-based materials can surely overcome these problems [182]. Lin et al. explored the use of reduced GO thin-film membranes for alienation and CO2 removal in natural gas purification based on the direct relationship between the rGO synthesis parameters and the defect size. They investigated that if appropriate synthesis condition is selected for the rGO membrane synthesis; it is possible to achieve excellent separation and permeate fluxes as compared to the available membranes [183]. Abraham et al. have shown that the GO membranes

nanoporous graphene. Extensive simulations of molecular dynamics, meticulous statistical analysis with all assemble simulation responses of 2.7 μs, classification tools, and supervised machine learning are used to differentiate IgG2 from IgG3. This nanoporous graphene membrane was employed for the biosensing and discriminating the subclasses of the antibody with better accuracy compared to the Si3N4 (same diameter as graphene nanopore) [176]. Li et al. demonstrated the fundamental properties of DNA translocation via nanoporous graphene. In their work, MDSs of ssDNA translocation via nanoporous graphene were employed to confirm the nucleobase trajectories and to test the impact of graphene layers with the applied electric field on the ssDNA translocation. They reported that the velocity of ssDNA translocation increased with the application of high voltage, and two- and five-layered nanoporous graphene with 1.0 nm diameter could discern several DNA strands by the translocation time. In this fabricated device, when the ssDNA has entered the nanopore, the density of Cl− ion nearby the pore is considerably detracted, while the Na+ current was changed a little due to the two compensating influences of electric attraction and steric exclusion. According to this, the steric and ion density influenced the blockade current, thus the blockade current is not an easy function of the size of the DNA bases, and it is feasible to improve the main role of the steric effect on the blockade current by increasing the nanopore length [28]. Patel et al. demonstrated how dsDNA translocates via graphene nanogaps. These nanogaps were developed with a newer capillary-force generated graphene nanogaps formation method. These results of DNA translocation for the gaps are qualitatively dissimilar from those achieved with simple ring nanopores. In this sensing method, the translocation time and conductance values of DNA vary by approximately 100%, due to the width variations caused by local nanogap. One promising mechanism was observed with the graphene upon interaction with DNA. However, the DNA translocation events do not require an entire unfolding of the molecule. These translocation events itself may be greatly smaller than events that need unfolding of the biomolecule. Therefore, they established a maximum limit to the unfolded translocation time of approximately 15 μs, the progressive resolution of this experiment [13]. A derivative form of graphene popularly known as GO (as shown in Fig. 5b), is expected to be proficient as a probable candidate due to numerous characteristics. In particular, the GO nanolayer (oxygenated graphene layers) bearing functional groups such as carboxyl, hydroxyl, and epoxide, have shown remarkable potential in the fabrication of functional nanocomposite surfaces that are chemically stable, have substantial hydrophilicity, and exhibit good antifouling properties that are chemically stable, have substantial hydrophilicity, and exhibit good antifouling properties [177]; these properties render them particularly suitable for water, gas, and the biomolecules treatment processes. Nanoporous GO membranes are synthesis either by vacuum filtration assembly or by layer-by-layer (LbL) method. Both these procedures are performed in an aqueous solution without the need for the addition of any organic solvent, making them environmentally friendly. The GObased nanosieve membranes are fabricated by vacuum filtration assembly of a pure GO aqueous solution or a mixture of GO and spacers; therefore, the membranes prepared in this manner might be lacking in plenty bonding between the GO nanolayers/nanosheets. Because of the highly hydrophilic nature of GO, the membranes may disperse in water, especially under cross-flow conditions that are typically observed in membrane operations. However, the LbL method is perfect for providing an interlayer stabilizing force by means of covalent bonding [178], electrostatic interaction, or both during layer deposition. The thickness of the membrane can be regulated by modifying the number of LbL deposition cycles. Theoretically, two stacked GO layers may be required to generate sieving channel. However, additional GO layers are necessary to cancel out the harmful effects of the defects and non-uniform deposition of GO nanosheets that may be present on the membrane. LbL synthesis of GO membranes is a highly scalable and cost-effective method, in 608

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transported across such membranes [187]. After that, Lv et al. explored a MOF of Zn4O(1,4-benzenedicarboxylate)3 (MOF-5) with copious hierarchical nanopores for the detection of minimum concentration aniline. Here, a typical micro-gravimetric transducer with resonant micro cantilever was used, and the absorption of the aniline induced mass-addition of MOF-5 was transformed to the sensing signal detectable as a function of frequency shift. The detection limit was better than 1.4 part per million, and the selectivity is acceptable to nine types of mutual interfering gases. The three-cycle repeatability was also examined with an RSD of 4%. After 20 days, this sensor only showed an insignificant response degradation which presents an improved reproducibility. The results of materials studio simulation revealed that MOF-5 crystals with 1,4-benzenedicarboxylic ligand are the superior site for aniline molecules adsorbing [188].

possess the excellent ability for molecular permeation, showing great potential for widespread application. This quality is believed to be related to the minimum opposition to the entrance of water molecules and enlargement of the slip lengths within the graphene nanocapillaries. Thus, they presented a simple, scalable technique to prepare graphene-based membranes with restricted swelling, by achieving a 97% rejection of NaCl [26]. Currently, Nehra et al. demonstrated a newer electrochemical biosensor comprised of a GO nanosieve platform for the rapid and sensitive monitoring of the Human Immunodeficiency Virus Type 1 envelope glycoprotein ectodomain. The GO nanosievebased biosensor had sensitivity (0.87 mA mM−1 cm−1), minimum detection limit (8.3 fM) and considerably fair response times (12 s) [184]. The GO membrane is a represent the next generation of ultrathin, high-flux membranes that have high energy efficient membranes. This membrane has the potential for application in numerous important fields by enabling accurate ionic and molecular sieving in aqueous solutions. Further investigations are necessary to precisely understand the mechanism of transport of water and solutes through the GO membrane. Future research is also warranted to identify the solutions for important concerns regarding the application of these membranes in various applications such as desalination, hydro-fracking water treatment, and energy production, in addition to their application in the biomedical and pharmaceutical fields. Moreover, some other properties of GO membranes, such as their antifouling, adsorptive, antimicrobial, and photocatalytic properties, are yet to be sufficiently explored [179].

8. Hybrid nanopores Nanotechnology has enabled the creation of customized nanostructures containing a nanometer-sized pore, that in combination with SSNs or the lipid bilayer shows remarkable characteristics as hybrid nanopores [157–159,189–192]. Hall et al. developed came up with an experimental platform to produce the functional hybrid nanoporous structures by combining the single α-HL pore with a solid-state fabricated nanopore. The advantage of the developed hybrid potential format is that crucial properties required in terms of precise structure and engineering of the biological protein pore can be merged along with the potency needed during the fabrication of an integrated system [193]. Currently, DNA origami nanoporous structures have attracted much attention for detecting efficiently the translocation behavior of biomolecules. DNA origami is a special nanostructure formed by direct folding of DNA as well as shown in Fig. 6a. Hernandez-Ainsa et al. present the first experimental evidence to investigate the voltage-dependent properties of hybrid DNA origami nanopores. Upon applied voltage, these origami nanopores tend to change their conformation, and due to their voltage sensitive behavior, two different conformational states are observed, which is attributed to the mechanical distortion of the DNA origami nanopores [189]. Their results proved that these artificial structures can be utilized as smart nanopores to control the rate of biomolecular translocations. Recent studies have reported the formation of hybrid nanostructures assembled by constituting DNA origami and SiN3 nanopores (as shown in Fig. 6b) that can detect proteins and DNA [8,131,192]. Bell et al. recently showed the possibility of reversibly forming hybrid DNA origami nanopores by combining DNA origami structures with glass nanocapillaries. According to the findings of the authors, these DNA origami nanopores display two mechanisms that allow them to have control over DNA translocation [191]. Furthermore, Bell et al. demonstrated the establishment of hybrid nanopores related to the 3D DNA origami systems mounted onto an SSN for λ-DNA detection. For this hybrid pore, they noticed the peak location and can be easily resolved by fitting with a various Gaussian

7. Metal-organic frameworks nanopores Among the classes of highly recommended nanoporous nanomaterials, metal-organic framework (MOF) having crystalline structure and almost equated to zeolite is unmatched in its degree of structural diversity and tenability as well as its range of physical and chemical merits. Accordingly, MOFs and zeolites have been employed for numerous of the similar applications as gas storage and heterogeneous catalysis as well as separation. Herein, Yamamoto et al. demonstrated a newer nanopore which has created host-guest charge transfer event in the MOF nanopore. In this nanopore, single crystals of the target complexes are recorded through conversion of crystal-to-crystal. All the crystals are appropriate for structural study using a mild impregnation method [185]. Recently, numerous research have also been commenced searching the MOF potential as nanopore biosensors. Even though it has yet to be thoroughly exploited in terms of excellent tenability and its vital advantage over other types candidate of nanopore-sensory nanomaterials [186]. Recently, Zhang et al. explored a MOF membrane containing zeolitic imidazolate framework (ZIF)-8 and UiO-66 membranes with subnanometer pores involving nanometer-sized cavities and angstromsized windows for an ultra-high selective pass of alkali metal ions. In this membrane, the windows were employed as ion selectivity sieves for selection of alkali metal ions, whereas these cavities were worked as ion conductive pores for ultra-high ions passes. The ZIF-8 and UiO-66 membranes presented a ˜4.6 and ˜1.8 lithium chloride/ rubidium chloride (LiCl/RbCl) selectivity, respectively, which was much higher than the 0.6 to 0.8 LiCl/RbCl selectivity calculated in present nanoporous membranes. MDSs are suggested ultra-high and selective ion passes in ZIF-8 membrane which was modulated with limited dehydration effects. The ZIF-8/GO/AAO fabricated membrane can rapidly and selectively transport Li+ ion over other alkali metal ions based on unhydrated size exclusion, displaying the subsequent order of the ion transport rate such as Li+ (4.6) < Na+ (3.4) > K+ (2.1) > Rb+ (1.0). The ultrafast, selective ion transport in ZIF-8 associated with partial dehydration effects was suggested by MDSs. The theoretical mobility rate of Li+ ions in ZIF-8 was found to be much faster than its transport rate in water. In contrary, artificial nanoporous membranes have exactly similar ion transport rate in order to that measured in water (Li+ < Na+ < K+ < Rb+), indicating the state of hydration of ions

Fig. 6. Schematic diagram of the hybrid nanopore. 609

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peak function in the histogram. A clear peak location at roughly −60 pA was noticed, a number of folded events even though with lower frequency can also be calculated. The conductance changes for the nanopore depends on the length of sensing which was nearly doubled for the hybrid pore with the length of 51 nm when compared to the simple pore with length of 30 nm as recorded by the thickness of the SiN membrane [8]. One is the physical control required for tuning the pore size by which the folding of the dsDNA molecule can be controlled. The other one is chemical control whereby attachment of chemical residues inside the inner walls of the nanopore makes them suitable for employing as a sensor capable of differentiating between ssDNA sequences [131,189,192]. The possibility of precisely tailoring the pore geometry and surface in the DNA origami structures is a novel approach to control the translocation of various biomolecules. Bell and Keyser's groups recently demonstrated voltage-dependent conductivity changes in nanopores modified by DNA origami [194]. Furthermore, Balme et al. developed a hybrid biological/solid-state polymer nanoporous membrane for blocking the proton and handling potassium selectivity using passive diffusion. However, separation of cations and protons is generally achieved by the use of electro-membrane, which needs the application of electric energy. This fabricated membrane is very simple to develop and based on the hydrophobic nature of the polymeric nanopore walls and the confined gramicidin, a biomolecules is detectable [195]. In a recent study, Souza et al. reported the fabrication and development of a novel tool composed of a nanopore shaped within a hybrid sheet, which in turn was made with a graphene nanorod embedded within a hexagonal boron nitride sheet (hBN). This tool is used for the biosensing of DNA nucleotides (such as deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxycytidine monophosphate (dCMP), and deoxythymidine (dTMP)) that translocate via the graphene nanopore, which is based on the current simulation generated in the carbon chain. The sensitive part of this platform was included of an electrically conducting carbon chain creating on the edge of the pore and presented the sensitivity at applied gate voltage: - 0.04 V and + 0.02 V. In both voltages, the all nucleotides were differentiating from the open nanopore by sensitivity values greater than at least 5%. For example, at a gate voltage of − 0.04 V, the SMD demonstrates 12.6%, 40.9%, 23.3%, and 17.9% sensitivity towards dAMP, dGMP, dCMP, and dTMP, respectively. However, for gate voltage of + 0.02 V, the sensitivity is restricted over a small range, i.e., 5.3% (for dGMP) to 17.7% (for dTMP), with the values for dAMP and dCMP being 10.5% and 14.3% respectively. They have quantitatively reported the effective biosensing capability through determining the sensitivity, which can be shown as an alteration in zero-bias conductance relative to an empty pore [12].

mechanical durability and strength of these pores are very low, which restricts their practical application. Further, the geometry of pore displays a variable non-uniform pore size, which cannot be utilized for several types of biomolecules. Currently, well-known applications of nanopore techniques include DNA sequencing, [37,199] conformationbased analysis of biomolecules, [98,200,201], and detection of small molecules, [42,98], heavy metal ion and nanoparticles [202–204]. The present review thoroughly examines various examples of natural and man-made nanopore-based sensing systems, ideally moving from the biological to the biomimetic systems. On the other hand, the SSNs offer wide usability because of their finite geometry bearing flexible shapes and sizes [138] and can be fabricated according to the analyte to be sensed. However, these nanopores lack molecular recognition ability, and it is difficult to distinguish between different types of molecules present in the same volume since such pores only detect ionic current changes arising due to the displaced molecular volumes rather than the molecule type [205]. Thus, they do not undergo selective translocations; as a result, they only perform multi-detection with a low degree of selectivity. Some recent studies have focused on this issue and functionalization of stable SSN surfaces with recognition molecules that allow them to recognize a specific molecular entity [37]. Therefore, it is evident that SSN is mechanically more durable and stronger than biological nanopores. Recently, hybrid platforms have been created by combining biological and SSNs; these platforms are advantageous in that they combine the potential attributes of both types of nanopores [3]. Among the various nanopore sensing nanopore materials, including the biological, solid-state, and hybrid nanopores, the solid-state graphene-based nanogap using an electron beam, graphene, and GO-based nanopore is reported to be one of the best sensing tools along with DNA sequencing via the transverse conductance [49,160]. It appears that this nanopore may be feasible to detect the DNA sequence by exploiting the electron tunneling arising with along the DNA axis. As shown in Table 1, graphene nanogap displays highly interesting attributes in terms of reduced pore size and excellent translocation time in comparison with other available nanopores [3,206]. Single-nanopore drilled graphene membranes and GO-based nanopores are an ideal substrate for very high-resolution, high-throughput nanopore-based single-molecule detectors and biosensors [162]. Graphene or GO-based inter-laminate nanocapillaries have shown great potential in nanosieving [30] and are expected to be increasingly popular in the preparation of modern biosensing or sequencing devices. Of course, it is needless to say that the field of nanotechnology, particularly, nanopores, is being utilized as sensors is much broader in its sense.

9. Present and future perspectives

As shown in Tables 1 and 2, various nanopores for biosensing and sequencing of biomolecules have been analyzed with respect to the physical attributes of nanopore size, mean translocation time, and current blockades. For electrochemical biomolecules separation/DNA biosensing (i.e., cis and trans chamber), graphene-and GO-based nanopores have been found to be more effective than the SSNs-based nanopores in terms of the translocating time, current blockades, and pore size. Furthermore, DNA detection using α-HL and MspA (biological nanopores) require the longest translocation time and very high voltage compared to that performed using another biological nanopore, including SSNs-based nanopore technology. The DNA translocation can be easily monitored using graphene- and GO-based nanopore at the minimum voltage, pore size, and good conductance [13,28,169,178]. For instance, the ssDNA or dsDNA biomolecules move via a graphene and GO nanostructure (GO-laminates) at velocities of approximately 1 base for 10 nanoseconds in most cases, which makes them much easier to verify means of the magnitude variation of signals, such as current and voltage arising from the individual bases. In other words, the single-base tyresolution is a major task to be accomplished. Novel

10. Comparative studies

At present, the nanopore technology seems to be the most effective option for the single-molecule study and rapid detection. In terms of sensitivity, specificity, and real-time sensing response, nanopore has several benefits and properties for fast and accurate analysis of biomolecules. Protein pores of the biological origin were the first to be used in sensing mechanism of nucleic acid molecules (DNA and RNA) in label-free studies [108,147]. These channel devices allow for selective translocation and stochastic sensing of entities such as DNA, RNA, proteins, and metal ions based on their molecular recognition capability and can identify the molecules by changes in their electrochemical character by detecting variations in the volumes of molecules as they pass through the nanopore [147,197,198]. Thus, these devices enable molecular recognition and ionic current blockades for selective multi-detection. Despite of showing much promise, they are limited by their high sensitivity issue to temperature and pH, need for applying a bias across the nanopore-channel, and the extremely fragile nature of cell membranes. In other words, the 610

proteins peptides

oligonucleotide segments (dA2, dA3, dA4, dA5, dA10) and dA5 by Exo I DNAs (D5, D16, and D30) and RNA (miR-155)

ramos cells (human Burkitt’s lymphoma cells) Guanidium chloride (Gdm-HCl) with MalEwt and MalE219

–

1.5 nm

–

–

– 1.5 nm 1.5 nm –

–

˜1.4 nm 0.8 nm

2.5-3 nm 1.4 nm

˜1.0-1.4 nm

1.4 nm

1.0 to 1.7 nm

˜1.7±0.17 nm

α-HL

α-HL

α-HL

α-HL

α-HL α-HL α-HL α-HL

α-HL

α-HL MspA nanopore

engineered ClyA aerolysin

aerolysin

aerolysin

aerolysin

aerolysin

E. coli DNA adenine methyltransferase activity and kinetics electrolytes homopolymers and DNA

VEGF, thrombin, and cocaine microRNAs (lung cancer) cocaine free radicals

SC4, SC4:V2+-trans-Az, SC4:V2+-trans-Az with additional 1.6 μM SC4 1:1 and 1:2 complex (SC6 and MV2+)

peptides

Poly(dA)60, β-amyloid 42

Anthrax lethal factor DNA (aLF1 and aLF-2, with aLF DNA)

1.5 nm

mutant α-HL

Biological nanopores

Detected analyte

Pore Dia.

Corresponding name

Nature of nanopore

Table 1 Detection modality of the myriad of analytes with choice of nanopore selected.

611

a 0.21 ± 0.03 to 2.8 ±0.4 μM−1s−1 as voltage increased + 20 mV to 80 mV with different pH in trans chamber/b0.83 ms to 40 ms at voltage +10 ˜ + 80 mV with trans pH 3.4 ˜ 2.1 for DNA and 0.26 ± 0.09 ms, 2.9 ± 0.5 ms, 4.1 ± 0.5 ms for D5, D16, D30, respectively d 0.92 ± 0.01 and 0.25 to 0.8 for major one labeled and minor one labeled, respectively/e 0.25 to 10 ms for major one labeled b 79 ± 12 % (MalEwt), 72 ± 11 % (MalEwtMalEwt)/e93 ± 15 μs (tshort(MalEwt)), 613 ± 38 μs (tlong(MalEwt)) and 62 ± 6 μs (tshort(MalEwt-MalEwt)), 1000 ± 62 μs (tlong(MalEwt-MalEwt))

d

a

femto per sec (rates of water flow)/b˜10−3 s 31.6 + 3.0 % (5’-poly(dA)) is greater than 20.1 ± 2.3% (5’-poly(dT)) and 21.5 ± 0.9% (3’-poly(dT)) is greater than 16.7 ± 0.9% (3’-poly(dC)) d 26 ± 1 pA b 0.92, 0.94, 0.91, 0.61, 0.68, 0.88 ms (T1) and 0.14, 0.23, 0.39, 0.48, 0.25, 0.16 ms (T2)/d -17.0, -17.5, -18.1, -26.3, -23.8, -17.6 pA (I1) and -31.5, -32.5, -33.0, -38.1, -42.6, -35.0 pA (I2) for Fmoc-D2A10K2, FmocD2A14K2, Fmoc-D2A18K2, Fmoc-D2A22K2, FmocD3A14K2, Fmoc-DA14K, and D2A10K2 d 0.37 (Ib/Io), 0.49 (Ib/Io) and 0.67 (Ib/Io) for dA5 with Exo I, dA4 with Exo I and dA3 with Exo I, respectively

b 0.13 ms/d33.4 pA (PI), 77.5 pA (PII) by integration method and 23.7 pA (PI), 76.7 pA (PII) by conventional method b 0.19, 0.34, 0.54, 0.65, 0.21, 0.25 ms (T2)/d-62.0, -65.1, -69.3, -78.1, -68.0, -74.5 pA (I2) for FmocD2A10K2, Fmoc-D2A14K2, Fmoc-D2A18K2, FmocD2A22K2, Fmoc-D3A14K2, Fmoc-DA14K, and D2A10K2 b 0.09 ± 0.04 ms (SC4), 2.41 ± 0.14 ms (SC4:V2+-trans-Az), and 0.86 ± 0.11 ms (SC4:V2+-trans-Az with additional 1.6 μM SC4) d 0.2 to 0.85 (Ib/Io for SC6) and 0.6 (Ib/Io for complex)/e0.15 ± 0.01 ms (for SC6), 0.16 ± 0.01 ms (for 1:1 complex), and 0.28 ± 0.02 ms (for 1:2 coplex) c 0.998 (using for magnetic beads) b 270 ± 30 μs d 80 ± 5% (CBA in 500 ms)/e25 s (at 3 μgmL−1) slopes values is 1.61 ± 0.09 N min-1 (0.01 μM), 3.02 ± 0.12 N min−1 (0.1 μM), 4.15 ± 0.15 N min−1 (1 μM), 24.70 ± 0.09 N min−1 (10 μM), and 58.68 ± 2.01 N min−1 (100 μM) e 150 min

b 342 + 35 ms, 57.4 ± 2.5 ms, and 63.620 ± 63 ms/d7.9 + 0.3 vs & 8.2 ± 0.4 pA/e˜ 1 min

a Capture rate/bmean translocation time/cvalue of R2/dblockade current/eresponse time/fsensitivity

ionic current

Ionic current

Ionic current

ionic current/c0.5 nM Ionic current/dRRD = 0.08 a DNA Aptamer a ionic current

Ionic current/d1.8 ± 0.1 nS (−35 mV) ionic current

a

a

ionic current

(continued on next page)

[121]

[120]

a

ionic current/cas low as 5 cell

[119]

[111]

[108] [85]

[104] [107]

[103]

[88] [91] [92] [98]

[87]

[86]

[85]

[81]

[17]

Reference

a ionic current/e0.06 (16 folds) at trans pH 3.7 and 0.02 (50 folds) at trans pH 3.2

a

a

ionic current

ionic current ionic current a

a

0.03 U/ml

c

a

a

a

a

a

a ionic current/c15 nM and 100 pM for the hybridization of target Anthrax lethal factor and cDNA probe a ionic current

a Sensing Modality/bSNR/cdetection limit/dconductions/epermeability

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Solid-state nanopores

Nature of nanopore

Table 1 (continued)

612 KCl concentrations Cocaine

10 nm

self-aligned plasmonic nanopore Quartz capillaries nanoporous alumina with upconversion nanoparticles (BaGdF5:Yb/Er) and gold nanoparticles SiNx single nanopore

dsDNA translocation

7 to 200 nm –

2.5 nm

SiN nanopore

15 nm –

100, 200, 500 nm

SSN (Si3N4)

DNA depurination velocity of the DNA molecules or change in drag force protein

DNA bare and fully RecA protein coated DNA dsDNA nucleocapsid protein 7 (NCp7) protein HCV IRES motif

ssDNA (dA50dN50)

ssRNA homopolymers poly (A), (C), and (U) and ds A-RNA Free and intercalator-bound DNA or dye complex (Et+, Pr2+, EtHD2+)

alcohol-soluble molecules human telomere repeat sequence (T8) employ as a probe for nanopore characteristic unfolded DNA

polynucleotides (unlabeled random ssDNA) ions (K+, Cl−, H+, and OH−) Genomic DNA from N. brasiliensis Single protein

Detected analyte

Single molecule DNA dsDNA Ebola virus oligonucleotides

5-6 nm 10 nm

SiN nanopore

SiN nanopore SiN nanopore

2.2 nm

SiN nanopore

3.5 nm < 6 nm and 7-15 nm ˜3 nm

˜3.5 nm

SiN nanopore

SiN nanopore SiN nanopore

10 nm

SiN nanopore

6.7-9.6 nm 30 nm

5-6 nm

SiN nanopore

SiN nanopore SiN nanopore

6, 6.8, and 9 nm 1.7 nm to 12.1 nm

SSN SiNx

˜4 nm –

bacteria channel OmpF MinION R9 10-30 nm

1.0 to 1.7 nm

aerolysin

bilayer-coated SSN

Pore Dia.

Corresponding name

a

b Less than 400 μs for all possible orientations using protein b less than 33 μs b 0.89 (I/Io)

70 to 130 MΩ (resistance)

d

depends on the different pore size 7.03 ± 0.04, 6.56 ± 0.04, and 5.69 ± 0.05 nA for 0 M, 1 nM, and 1 μM

a

f

gradient is 0.11 nM−1s-1 mV−1 0.88 (quenching efficiency)

b

d below 10 μs (dwell time), free RNA 0.1-10 ms, and RNA plus drug < 0.1 ms b ˜70-100 μs b 1.9 to 4.8 ms corresponding 3.98 to 1.58 bpμs−1 (velocity) average signal intensity are 54 ± 8 au (5 min) and 45 ± 5 au (30 min) d up to 85 % (ionic conductance blockade)

a 0.57 ±0.02, 0.65 ±0.02, 0.52 ±0.02 s−1nM−1 (for free DNA) and 0.13 ± 0.01, 0.12 ±0.01, 0.15 ± 0.01 s−1nM−1 (for drug-bound) respectively/b0.11 ± 0.01, 0.09 ± 0.02, 0.10 ± 0.01 ms (for free DNA) and 0.44 ± 0.04, 0.55 ± 0.06, 0.52 ± 0.04 ms (for drug-bound)/ d ˜1 nA (Single molecule) b 0.11 ± 0.03 ms (both before and after for dA60 as negative control) and 0.25 ± 0.04 (before) to 7.1 ± 1.0 ms (after for dA50dN50)/ d ˜0.58 nA (both before and after for dA50) and 0.96 nA (after binding dA50dN50) d 2 - 0.3 nS (dsDNA), 6 –15 nS (ssDNA b 1.44 ± 0.07 and 1.45 ± 0.18 ms similar at 60 mV for example a ˜ 1s−1/b20 ms/c 0.97 –

b 118 ± 12, 46 ± 8, 29 ± 4 μs at pH 6.0, 7.0, and 8.0 respectively –

a

−

ionic current 7 pM

ionic current/ d30 pF 1nM c

a

c

a

[154] [155]

[152] [153]

[151]

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a ionic conductance/b˜ 70 at 100 kHz/d9.7 ± 0.4 nS at 1 M KCl @ 23 °C (conductivity is 10.8 Sm−1 and ionic conductance is 10.8) a ionic current

[150]

[149]

˜85-95% (mobile fraction) and ˜ 1.5 μm2/s

a

[144] [145]

[143]

[140] [141]

[138] [139]

[136]

electro kinetic translocation/ 1 nS electric field

a

a

d

ionic current electric field/d10-16 nS at 10-15 mV

residual ion current

ionic current resistive Pulse technique/d4.5-13.3 nS a ionic current

a

a

a

a

[136]

[135]

a

ionic conductance/ ˜1.6 nS all homopolymers at 100 mV,1.6 + 0.5 nS at 100 mV for ds B-RNA a residual ion current

[11] [130]

[132] d

16.5 Sm (for bulk LiCl) ionic current

[10]

[123] [126]

[122]

Reference

ionic current

a

a

−1

ionic current

ionic current ionic current

d

a

−

a

d 0.51 for Oligo-1 (Ires/Io) and 0.6 to 0.9 for Oligo-2 (Ires/Io)

ionic current

a Sensing Modality/bSNR/cdetection limit/dconductions/epermeability

a Capture rate/bmean translocation time/cvalue of R2/dblockade current/eresponse time/fsensitivity

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613

DNA and DNA-protein complex DNA DNA

Water separation envelope glycoprotein ectodomain λ-DNA DNA nucleotides (such as dAMP, dGMP, dCMP, dTMP) Protons and cations aniline detection

˜8.9 nm 23 nm 5 nm and 20 nm ˜0.7 (average ratio of pore opening, i.e., final/ untreated pore diameter) 8.5 nm 2.5 nm, 5 nm and 10 nm 2.2 nm 6.5 nm

6.5 nm

9 nm

10 nm

Invisible nanopore 0.88 nm

14-19 nm –

multilayered graphene-Al2O3 nanopore multilayered graphene-Al2O3 nanopore MoS2 nanopore (˜6.5 Å)

multi-layered graphene films (˜3-4 nm) to completely cover our nano-pipette

MoS2 nanopores MoS2 nanopores

MoS2 nanopores

MoS2 nanopores

graphene nanopore

GO nanosheets membrane GO nanosieve

DNA origami inserted in SSN graphene nanoroad embedded in hexagonal boron nitride (h-BN) PC15-gA-W

MOF

MOF nanopore

graphene nanopore MoS2 nanopores

7.5 nm 1.25 nm

DNA

1.45 nm ˜1.0-1.5 nm 2-7 nm

Slowing down dsDNA transport and single CpG site in dsDNA fragment IgG (antibody) subclasses (IgG2 and IgG3)

dsDNA (10 kb)

nanopower generator naked and Methylated dsDNA fragments

Ion selectivity λ-dsDNA

ion sieving and desalination ssDNA translocation (20 A, 20C, 20 G and 20 T) nucleic acids DNA DNA

graphene nanopore in SiO2 graphene nano-gap nanopore graphene nanopores

GO membrane graphene nanopore

dsDNA

Polystyrene nanoparticles dsRNA, dsDNA

250 nm 22 nm Micropore on the top of graphene nanogap 1.35 nm 1.0, and 1.1 nm

DNA

3 nm /14 nm

SiN Cylindrical nanopores /conical pores in glass nanocapillaries conical shaped GNMs SiN nanopore

graphene nanogaps

Detected analyte

Pore Dia.

Corresponding name

Hybrid nanopores

Graphene and GO nanopore

Nature of nanopore

Table 1 (continued)

10 mm s−1/b0.37 ( ± 0.02) ms/d59.3 ( ± 3.6) pA

nine kinds of common interfacing gases.

f

–

f

-60 pA 12.6% (dAMP), 40.9% (dGMP), 23.3% (dCMP), and 17.9% (dTMP)

d

a

impedance (9.4 ± 2.1 MΩ) 53 ± 3 μs, 36 ± 4 μs and 32 ± 2 μs at 50 mV, 80 mV, 100 mV/ b282 ± 22 pA, 312 ± 13 pA, and 376 ± 16 pA at 50 mV, 80 mV, 100 mV b 188 ± 17 μs, 118 ± 8 μs, and 83 ± 6 μs at 500, 700, and 1000 mV/d1.27 ± 0.24 nA, 1.7 ± 0.41 nA, and 2.35 ± 0.43 nA at 500, 700, and 1000 mV d -276 ± 20 pA for naked DNA and shallow at −251 ± 39 pA and deeper at −600 ± 66 pA for endMethDNA/MBD1x/ b 91 ns (IgG2) and 111 ns (IgG3), 2.54 ns (hinge disulfide bridge)/dblockade duration (˜17 and ˜28 ns for IgG2 and IgG3, respectively) a 80-276 l/m2-h-MPa e 12 s/f0.87 mAmM−1 cm−1

d

– b 130 ms (for λ-DNA) and 1.4 ms (2 M KCl)

a

b

˜1 ms

– 3.6 μs per nucleotide at 30 mV 1.6, 3.7, and 27 ns corresponding to 4.3, 2.5, 0.8 V respectively and n-charge (25 ns), p-charge (15 ns)/d11 nA (DNA), 19 nA (dsDNA), 26 nA (exist dsDNA) b 1.81 + 2.77 ms at 400 mV and 2.66 ± 4.08 ms at 250 mV d ˜18 nA b

b

d

ionic current ˜1 nS (RT ionic liquida) and 210 nS (concentration gradient) a ionic current a ionic current

ionic current

a

a Ionic conductivity and MD simulations/d0.4 (Nacl/ Kcl)/e0.3-0.64 a Change in frequency/cbetter than 1.4 part per million

a

a

ionic current ionic current

[188]

[195]

[8] [12]

[178] [184]

water flux and salt rejection ionic current a

a

[176]

[175]

[175]

[173] [175]

[170] [172]

[169]

[167]

[164]

[164]

[49] [160] [163]

[26] [28]

[13]

[159] [196]

[157]

Reference

ionic current (4.5-5.5 nA) and hinge region ionic current (0.93Io)

a

ionic current

a

d

a

less than 10/ ˜5 nS (unfold), ˜10 nS (partially fold), ˜10 nS (fully fold) a ionic conductivity/d(7 ± 0.8 pS)

ionic current

a

b

ionic current

a

a

b

20 G > 20C for 1.0 nm (two layer)

a

0.7 ms

−

b

ionic current ionic current (20C > 20 G AND 20 T) for 1.0 nm (two layer) a electric Current/Density functional theory a transverse conductance a ionic current

a ionic current/b(15/25)/dbetween 0.04 and 0.15 nS (3 nm), 1.7 nS (14 nm), and 19 nS (smaller than 14 nm pore) a resistive Pulse Counter a optical tweezers/d-2.0 ± 0.4 nS for dsRNA and -1.6 ± 0.5 nS (100 mV) d 3 nS

–

b ˜3.7 % (at 200 mV) –

a Sensing Modality/bSNR/cdetection limit/dconductions/epermeability

a Capture rate/bmean translocation time/cvalue of R2/dblockade current/eresponse time/fsensitivity

A. Nehra et al.

Sensors & Actuators: B. Chemical 284 (2019) 595–622

Pore Size

1.5 nm

–

1.5 nm

–

–

–

1.5 nm

1.5 nm

– –

˜1.4 nm

0.8 nm

2.5-3 nm

1.4 nm

˜1.0-1.4 nm

˜1.0-1.4 nm

1.4 nm

1.0 to 1.7 nm

Nanopore

mutant α-HL

α-HL

α-HL

α-HL

α-HL

α-HL

α-HL

α-HL

α-HL α-HL

α-HL

MspA

engineered ClyA

aerolysin

aerolysin

aerolysin

aerolysin

aerolysin

Nature of the Nanopore

Biological Nanopore

Table 2 Nanopore-based biosensing device parameters.

614 lipid bilayer membrane (50 μm)

1,2-diphytanoyl-sn-glycero-3-phosphocholine (3.0 mg) in decane (100 μl) for lipid solution (dia50 μm) 1,2-diphytanoyl-sn-glycero-3-phosphocholine (3.0 mg) in decane (100 μl) for lipid solution (dia50 μm) teflon film (100-150 μm) with planar lipid bilayer of 1,2-diphytanoyl-sn-glycerophosphatidylcholine

the MspA nanopore, containing residues 75 − 120, embedded with an 8 × 8 nm2 patch of 2-oleoyl-1pamlitoyl-sn-glyecro-3-phosphocholine bilayer. planar lipid bilayers (0.01 − 0.1 ng of oligomeric ClyA) planar lipid membrane

membrane–channel biomimetic system teflon film with lipid 1,2-Diphytanoyl-sn-glycero3-phosphocholine soft-walled electrostatic block model of the type αHL nanopore

teflon film (100-150 nm) with a bilayer of 1,2diphytanoy-sn-glycerophosphatidylcholine (Avanti Polar Lipids) –

diphytanoylphosphatidyl-choline in decane (30 mgmL−1) to a orifice (50 mm) in a 1 mL Delrin cup integrated into a lipid bilayer chamber teflon film with a bilayer of 1,2-diphytanoyl-snglycero-3-phosphochline teflon film with a bilayer of 1,2-diphytanoyl-snglycero-3-phosphochline

1,2-diphytanoyl-sn-glycero-3-phosphocholine in decane (30 mgmL−1) as a bilayer across a orifice (150 μm) in a lipid bilayer chamber planar lipid membrane

teflon septum (150 μm) with a bilayer of 1,2diphytanoylphosphatidylcholine

Supporting material

+ 80 mV to + 120 mV and + 100 mV

+ 40 mV, + 80 mV and + 100 mV

+ 100 mV

+ 100 mV

+ 100 mV

1.0 M KCl, 10 mM Tris, 1.0 mM DTT and 10 mM MgCl2 (pH 9.5) 1.0 M KCl in buffered with 8 mM K2HPO4 and 2 mM KH2PO4 with different pH in the cis (pH 7.4, 3.4, 3.2, 2.6, 2.3 and 2.1) and constant pH in trans (pH 7.4) 1 ml of buffer (cis: 0.5 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.8; trans: 3 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.8)

150 mM NaCl, 15 mM Tris-HCl (pH 7.5), 1.0 M KCl in 10 mM phosphate buffer (pH 8.0) 1.0 M KCl, 10 mM Tris and 1.0 mM EDTA, pH 8.0

+ 60 mV to – 90 mV

180 mV

−8 to 8 V

– + 150 mV

1.0 M KCl, 10 mM PBS, and 1 mM EDTA, pH 7.4 1 M KCl (pH 7.98) 10 mM Tris, 1.0 mM EDTA, 1.0 M NaCl, pH 7.4 1 M NaCl and 1 M KCl solutions or commonly used salts gradient of 0.2 M cis/1 M trans NaCl 1 M KCl

1 M KCl (same in both chamber)

1.0 M KCl buffered with 10 mM Tris-HCl (pH = 8.0) 3 M KCl, 10 mM Tris at pH 8.0 or pH 7.2

1.0 M KCl in 10 mM phosphate buffer (pH 8.0) 1.0 M KCl and 10 mM TrisEDTA (pH 8.0)

1 M NaCl (same in both chamber) and 3 M NaCl (trans) /0.15 M NaCl (cis) for hybridization of target aLF and cDNA probe 1 M KCl, 10 mM Tris−HCl, and 1 mM EDTA (pH 8.0)

Electrolyte solutions in cis and trans chamber

+ 100 mV

+ 100 mV to + 180 mV

+ 160 mV

−60 mV

−70 mV to–140 mV

+ 100 mV

+ 100 mV

+ 120 mV and + 180 mV for hybridization of target aLF and cDNA probe

Applied voltage

[98] [103]

free radicals/ 0.1 to 100 μM E.coli DNA adenine methyltransferase activity and kinetics/b0 - 50.0 U/ml a electrolytes

(continued on next page)

ramos cells (human Burkitt’s lymphoma cells)/b100 pmole of out DNA

a

[120]

[119]

a

DNAs (D5, D16, and D30) and RNA (miR155)/b1 μM

[111]

[111]

a dA5 by Exo I/b2,000 U of Exo I with 100 μM of dA5

oligonucleotide segments (dA2, dA3, dA4, dA5, dA10)/b100 μM for each sample

[85]

peptides/b10 μL a

a

[108]

[107]

proteins/b1 nM

homopolymers and DNA

[104]

a

a

a

b

[92] a

a

[88]

[87]

cocaine/b1 μM (˜300 ng/mL)

[86]

[91]

2+

[85]

[81]

poly(dA)60, β-amyloid 42

peptides/b10 μL

[17]

References

aLF

2+

Detection/bAnalyte concentration

SC4, SC4:V -trans-Az, SC4:V -trans-Az with additional 1.6 mM SC4/b0.8 μM to 8.0 μM a 1:1 and 1:2 complex/b(SC6 and MV2+)/[MV2+]/[SC6] = 0.2 to 5 a VEGF, Thrombin, and cocaine/b0.5-200 nM for VEGF, 5-100 nM for Thrombin, and 5-500 μM for cocaine a microRNAs (lung cancer)

a

a

a

a

a

A. Nehra et al.

Sensors & Actuators: B. Chemical 284 (2019) 595–622

Solid-state nanopore

Nature of the Nanopore

Table 2 (continued)

615

˜3.5 nm

2.2 nm

6.7-9.6 nm

30 NM

3.5 nm

< 6 nm and 7-15 nm

˜3 nm

5-6 nm

10 nm

100, 200, 500 nm

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

SiN nanopore

∼20 nm thick silicon nitride freestanding membranes supported by a Si chip using a JEOL 2010 FEG TEM 5–6 nm free-standing SiN membrane drilled by a helium ion microscope 20 nm thick free-standing SiN membranes drilled by TEM made by molecular assembly photolithographic lift-off (MAPL) nanopore approach with functionalization by Poly-L-lysine-graft-poly (ethylene glycol) and supported by Si chip

20 nm thin, free standing SiN membrane SiN window (25-nm thick, 25× 25 μm2) supported by silicon chip SiN nanopores drilled by using a FEI Tecnai F20 S/ TEM in a STEM mode

SiN nitride window (20-nm thick) with cyanine dye SYBR Green II (SSII) 20 nm thick, low-stress silicon nitride (SiN) membrane

freestanding 20 nm thick silicon nitride membranes drilled by using a 300 keV transmission electron microscope and fitted within a custom-built Teflon flow cell containing two reservoirs Si-chip device containing a 20-nm-thick silicon nitride window

10 nm

SiN nanopore

–

+ 100 mV –

200 mM NaCl, 5 mM NaH2PO4, pH 7.0 (cis) and 1 M NaCl, 5 mM NaH2PO4, pH 7.0 (trans) 0.3 M KCl electrolyte tris-buffered to pH 8.0

1 M KCl with pH 8 (both chamber) 1 M KCl and buffer 10 mM Tris-HCL, pH) 8.0 and 1 mM EDTA) 1 M KCl containing 100 mM Tris-HCl at pH 8.0 4 M KCl (trans)/0.2 M (trans)

1 M KCl with pH 8 (both chamber)

10 mM Tris buffer, 1 mM EDTA (pH 8) 4 M LiCl

+ 400 mV

+ 200 mV

+ 200 mV

˜1× 105 V cm−1

60 mV

240 mV

300 mV

300 mV

+ 100 mV up to 600 mV

1 M KCl 10 mM phosphate-HCl at pH 6.0, 7.0, or 8.0. 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 M KC

−400 to + 400 mV

5-6 nm

SiN nanopore

1 M KCl (pH 10)

+8 V

fabricated by focused ion beam (FIB) and focused the electron beam (FEB) and sealed with glue inbetween two polytetrafluoroethylenes (PTFE) flow cells 45 nm SiN membranes drilled by TEM with chemically (APTMS) functionalized SSNs

1.7 nm to 12.1 nm

SiNx

3 M LiCl

200 – 800 mV

–

6, 6.8, and 9 nm

SS nanopore

2 M KCL

–

10-30 nm

bilayer-coated SSN

+ 100 mV-150 mV

˜ 4 nm

bacteria channel OmpF

planar membranes (70-100 μM) on a 15-μm-thick teflon partition using diphytanoyl phosphatidylcholine fluid lipid bilayer coating of the nanopore

+ 100 mV

lipid bilayer membrane (50 μm)

1.0 to 1.7 nm

aerolysin

1 M KCl (same in both chamber)

Electrolyte solutions in cis and trans chamber

1.0 ml of buffer (1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH = 8.0) 2 M KCl

+ 110 mV, + 80 mV and + 130 mV

planner lipid bilayer membrane

˜1.7 ± 0.17 nm

aerolysin

Applied voltage

Supporting material

Pore Size

Nanopore

Detection/bAnalyte concentration

unfolded DNA/b250 ng

ssDNA/b7 kb

[149]

(continued on next page)

velocity of the DNA molecules or change in drag force protein/baccording to functionalization of nanopore surface a

[145]

[144]

DNA depurination/b˜40 ng/μl a

a

[143]

HCV IRES motif (subdomain IIa)/b0.8 μM

[141]

[140]

[139]

[138]

[136]

[136]

[135]

[132]

a

a

nucleocapsid protein 7 (NCp7) protein/b500 nM for small pore and 1 μM for large pore

a

a DNA-repair protein RecA/bRecA-coated dsDNA a dsDNA/b3.8 pM 400 bp DNA

a

drug-bound (dsDNA and ssDNA/dye complex)/b400 bp DNA, ∼103-104 DNA copies a dsDNA (dA50dN50)

a

ssRNA homopolymers poly (A), (C), and (U) and ds A-RNA

a

a

[130]

[11]

a

alcohol-soluble molecules and α-zein/ at least in μM range a human telomere repeat sequence (T8) employ as a probe for nanopore characteristic b

[123]

[10]

ions (K+, Cl−, H+, and OH−)

[122]

[121]

References

single protein

a

a

Guanidium chloride (Gdm-HCl) concentration of 0-1.5 μM for MalEwt/b0.35 μM and aGdm-HCl concentration of 0.7 M for MalE219/b0.35 μM a polynucleotides (unlabeled random ssDNA)/b2.0 μM

a

a

A. Nehra et al.

Sensors & Actuators: B. Chemical 284 (2019) 595–622

Graphene and GO nanopore

Nature of the Nanopore

Table 2 (continued)

−2 V to +2 V

−1 to + 1 V

–

quartz capillaries (inner and outer the diameter of 0.3 and 0.5 mm) pulled with a P-2000 laser pipet puller and assembled into a PDMS cell

174.3 ± 23.5 nm (base dia) and 30.2 ± 5.1 nm (tip dia) 3 nm /14 nm

Single nanopore

616

1.45 nm

˜1.0-1.5 nm

graphene nanopore in SiO2

graphene nano-gap nanopore graphene nanopores multilayered graphene-Al2O3 nanopore MoS2 nanopore (˜6.5 Å) multi-layered graphene films (˜34 nm) to completely cover our nanopipette graphene nanopore

˜0.7 (average ratio of pore opening, i.e., final/untreated pore diameter) 8.5 nm

5 nm and 20 nm

2-7 nm 23 nm

1.0, and 1.1 nm

graphene nanopore

GO membrane

graphene layer coated on 150-nm diameter aperture in a 300 nm thick low-stress LPCVD silicon nitride (SiNx) membrane using established wet transfer techniques

multi layers of graphene nanopore fitted in the simulation domain is 9.26 nm × 9.26 nm × 27.0 nm all ribbons constructed with hydrogen-terminated armchair edges and width of 3.3 nm in the ydirection while being continuous in the x-direction nano-gap fabricated by nanolithography with an STM – Nanopore drilled in the Al2O3 dielectric layers 1 and 2 in the graphene-Al2O3 stack by fieldemission gun TEM MoS2 flakes exfoliated onto substrates with 270 nm SiO2 pipettes (dia 25 ± 2 nm) dipped into the GNF dispersions (1, 1.5, and 3 mg)

−150 mV to + 150 mV

200 mV to 400 mV

200 to 400 mV

+ 4.3, 2.5, and 0.8 V + 500 mV

1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8

2 M KCl, 10 mM Tris, 1 mM EDTA, pH 7.4 0.1 M KCl at pH 8.0

1 M KCl 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8

–

−2 V to + 2V

30mV

–

+ 1.1 to 1.4 V

0.01 M KCl solution containing 0.1% Triton X-100, pH-6.9 1 M KCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.01% triton-X100 0.1 M KCl, 10 mM Tris, and 1 mM EDTA

1 M KCl, 1 mM Tris, and 0.1 mM EDTA buffer at pH 8

LiCl (12% RH), MgCl2 (33% RH), NaCl (75% RH) and KCl (84% RH) RH = relative humidities 1.0 M NaCl

–

˜ 20 mV

Nanogaps fabricated by a novel capillary-force induced graphene nanogap formation technique onto the SiO2/Si wafer GO fabricated by y dispersing millimetre-sized graphite oxide flakes

micropore on the top of graphene nanogap 1.35 nm

+ 100 mV

20 nm SiN membrane

35 nm to 3.5 nm

200 mV

GNMs (glass nanopore membrane)

375 nm/ 250-nmradius

graphene nanogaps

SiN cylindrical nanopores /conical pores in glass nanocapillaries conical-shaped glass nanopores

maximum voltage (1 V)

SiNx (20 nm thick) supported by homemade lids

7 to 200 nm

SiNx

10 mM Tris-HCl (pH 8), 1 mM EDTA KCl (10 mM, 100 mM, and 1 M) 0.1 M KCl, 1 M KCl, and 0.01 M PBS

15 nm

+ 600 mV

1 M KCl

±100 mV

Self-aligned plasmonic nanopore quartz capillaries

10 nm

1:1 M KCl

0.5 V

Si3N4 film (50 × 50 μm2) with 5 μm SiO2/500 μm Si Wafer free-standing silicon-nitride (30 × 30 μm, 20 nm thick) membranes drilled by TEM nanocapillaries created by laser based pulle

2.5 nm

SiN nanopore

Electrolyte solutions in cis and trans chamber

Applied voltage

Supporting material

Pore Size

Nanopore

Detection/bAnalyte concentration

dsDNA

a

ion selectivity/b1 M KCl

DNA

g

DNA/b1-10 ng/μL

(continued on next page)

[170]

[169]

[167]

a

a

[160]

[49]

[163] [164]

DNA

nucleic acids

[28]

[26]

[13]

DNA DNA and DNA-protein complex/b100 ng/ μL

a

a

a

ssDNA translocation (20 A, 20C, 20 G and 20 T)

a

a ion sieving and desalination/bLiCl (12% RH), MgCl2 (33% RH), NaCl (75% RH) and KCl (84% RH), RH = relative humidi-ties

a

[196]

a

dsRNA molecules

[159]

polystyrene (PS) nanoparticles (80- and 160-nm-radius)/b107 to 1011 particles/mL

a

DNA/b0.75 nM

[157]

[155]

cocaine/ 1 nM – 10 μM

a

[154]

a

b

ions

a

[152]

dsDNA/b1.6 nM (5, 10, and 20 kbp)

[151]

[150]

References

a

a dsDNA translocation /bds 15 kbp and 400 bp a single molecule DNA/b5 ng/μL

a

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Sensors & Actuators: B. Chemical 284 (2019) 595–622

617

2.2 nm 6.5 nm

6.5 nm

9 nm

10 nm

Invisible nanopore

MoS2 nanopores MoS2 nanopores

MoS2 nanopores

MoS2 nanopores

graphene nanopore

GO nanosheets membrane

–

MOF

commercial polycarbonate track-etched membrane PVP treated by ethanol percolation and coated gA-W ultrathin ZIF-8/GO membrane growing the plasma-treated seeding layer on the AAO support via the counter diffusion method MOF fabricated by MOF-5

14-19 nm

–

graphene nano-road embedded in a sheet of hexagonal boron nitride (h-BN)

1.25 nm

graphene nano-road embedded in hexagonal boron nitride (h-BN) PC15-gA-W

ZIF-8/GO/AAO

SiN membranes fabricated by an FEI Tecnai F20 TEM equipped with a field emission gun (FEG)

7.5 nm

GO fabricated by vacuum filtration unit

MoS2 fabricated by electron irradiation under TEM MoS2 monolayer coated on a 12 mm × 12 mm substrate structure consisting of 300 nm thick SiNx and 20 nm thick Al2O3 by plasma enhanced chemical vapor deposition and atomic layer deposition method MoS2 monolayer coated on a 12 mm × 12 mm substrate structure consisting of 300 nm thick SiNx and 20 nm thick Al2O3 by plasma enhanced chemical vapor deposition and atomic layer deposition method MoS2 monolayer coated on a 12 mm × 12 mm substrate structure consisting of 300 nm thick SiNx and 20 nm thick Al2O3 by plasma enhanced chemical vapor deposition and atomic layer deposition method nanopore (10.0 nm) drilled in the center of a 20 nm × 20 nm single-layer graphene LBL method

thin layers of MoS2 grown on the SiO2 and suspended on SiNx membranes drilled by HRTEM

Supporting material

DNA origami inserted in SSN

0.88 nm

2.5 nm, 5 nm and 10 nm

MoS2 nanopores

GO nanosieve

Pore Size

Nanopore

20 mM NaCl, 10 mM Na2SO4, 7.5 mg/L Rhodamine-WT and methylene blue phosphate buffer

–

1 M NaOH, KOH, HCl

−0.2 to + 0.2 V

–

–

NaCl or KCl

–

–

20 nm filtered buffer solution of 0.5 × TBE, 5.5 mM MgCl2, and 1 M KCl. –

+ 100 mV

0 - 2.0 V

0.5 M NaCl

0.6 M KCl (cis)/3 M KCl (trans)

+ 200 mV

+ 180 mV

1 M KCl containing 10 mM Tris and 1 mM EDTA at pH 7.2

RT ionic liquids (cis)/2 M KCl solution buffered with 10 mM Tris-HCl and 1 mM EDTA at pH 7.0 and BminPF6 (trans) 1 mM to 1 M KCl 0.6 M KCl containing 10 mM Tris and 1 mM EDTA at pH 7.4

Electrolyte solutions in cis and trans chamber

+ 50, 80, 100 mV

± 600 mV + 500, 700, 1000 mV

+ 400 mV

Applied voltage

nanopower generator dsDNA (10 kb)

naked and methylated dsDNA fragments

a

a

a

a

aniline detection/b1.4 to 50 per per million

metal ions

protons and cations

DNA nucleotides (such as dAMP, dGMP, dCMP, dTMP)

a

water separation/ 20 mM NaCl, 10 mM Na2SO4, 7.5 mg/L Rhodamine-WT and methylene blue a envelope glycoprotein ectodomain/b0.1 μM to 670 fM a λ-DNA/b1 nM

[188]

[187]

[195]

[12]

[8]

[184]

[178]

[176] b

IgG (antibody) subclasses (IgG2 and IgG3)

a

a

[175]

[175]

slowing down dsDNA transport and single CpG site in dsDNA fragment

a

a

a

[173] [175]

[172]

λ-dsDNA/b10 μL of λ-dsDNA with BminPF6

a

References

Detection/bAnalyte concentration

a

Abbreviations: APTMS–3-(aminopropyltrimethoxysilane; CBA-cocaine-binding aptamer; cDNA–complementary deoxyribonucleic acid; DTT-DTT is a skin and eye irritant and may cause respiratory irritation; EDTAEthylenediaminetetraacetic acid, EtBr-ethidium bromide, EtHD2+; -ethidium homodimer, GNMs–glass nanopore membranes; Exo I, exonuclease I; ; HCV IRES-hepatitis C virus internal ribosome entry site; I1 and I2current intensity during bumping event (small iblock and translocation event (large iblock; IgG–immunoglobulin; MalE219-destabilized variant; MV2+;, methyl viologen; NC–nucleocapsid; PM–polycarbonate membrane; Pr2+; -propidium, RRD-relative residual conductance; SC4;, Sulfonato-calix [4]arene; SC6, para-sulfonatocalix [6]arenes; SiN–Silicon nitride; T1 and T2- duration of current blockade during bumping event (long tblock and translocation event (small tblock; Tris, Tris(hydroxymethylaminomethane; VEGF, vascular endothelial growth factor, V2+-Az, light-sensitive 4, 4′-dipyridinium-azobenzene.

MOF nanopore

Hybrid nanopores

Nature of the Nanopore

Table 2 (continued)

A. Nehra et al.

Sensors & Actuators: B. Chemical 284 (2019) 595–622

Sensors & Actuators: B. Chemical 284 (2019) 595–622

A. Nehra et al.

biosensing techniques (e.g., alternative read-out methods to notice the ionic current blockade, to employ the intrinsic conductivity of carbonbased nanomaterial, and so on) and alternative nanopore materials (e.g., other two dimensional nanomaterials namely MoS2, MOF, graphene, GO, and so on) are thus required [207]. Lastly, the grapheneand GO-based nanopore is the most excellent nanomaterial for the fluidic electrochemical biosensors for sensing of the translocation time, blockades current, and pore size, as summarized in Table 1. Herein, it is evident from Table 2 that nanopore device used in sensing/biosensing practices employing parameters, such as pore size, apply voltage, and matrices for the biosensing of several biomolecules, are excellent (up to pico-Siemens conductivity and minimum concentration of analytes) for multi-layer graphene film [169].

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Hall, Solid-state nanopore analysis of alcohol-soluble molecules, Analyst 142 (2017) 1676–1681, https://doi.org/10.1039/C7AN00022G. [12] F.A.L. Souza De, R.G. Amorim, W.L. Scopel, R.H. Scheicher, Electrical detection of nucleotides via nanopores in a hybrid graphene/h-BN sheet, Nanoscale 9 (2017) 2207–2212, https://doi.org/10.1039/C6NR07154F. [13] H.N. Patel, I. Carroll, R. Lopez, S. Sankararaman, C. Etienne, S.R. Kodigala, M.R. Paul, H.W.C. Postma, DNA-graphene interactions during translocation through nanogaps, PLoS One 12 (2017) e0171505, , https://doi.org/10.1371/ journal.pone.0171505. [14] J.S. Kim, K.Y. Chun, C.S. Han, Ion channel-based flexible temperature sensor with humidity insensitivity, Sens. Actuators A 271 (2018) 139–145, https://doi.org/10. 1016/j.sna.2018.01.025. [15] S. Ahlawat, A. Nehra, V. Pandey, K.P. 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Sweeti Ahlawat received her M.Sc. from D.A.V. College in 2010 under Biotechnology discipline. Afterward, she joined in a UGC funded project for the tenure of 3 years in 2011. She is currently joined Kumaun University for pursuing her Ph.D. degree in Biotechnology field. Her research interests include biosensors, electrochemical microfluidic sensors, and nanoporous based sensing devices.

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K.P. Singh received his M.Sc., M.Phil., and PhD degree in Chemistry (Bio-membrane Science) from Aligarh Muslim University in 1991, 1993, and 1997 respectively. He has also served as a director, Uttarakhand Council for Biotechnology, Govt. of Uttarakhand, India. Currently, he is working as Vice-chancellor of Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India since 2016, and additionally he is also Vice-Chancellor of Maharana Pratap Horticultural University, Karnal, Haryana, India since 2017. His research interest includes nano-biotechnology, nanobiosensor, nanomedicine, targeted drug delivery, nanofertilisers, and bioenergy where he has authored several publications in terms of articles, books, patents, policy papers, international scientific and strategic documents. Currently, he is working on nanopore based nanobiosensing devices for the environment, medical and agriculture fields.

Anuj Nehra received his B.Sc degree in physical science and M.Sc degree in Physics both from Chaudhary Charan Singh University, Meerut. He is currently a Ph.D. student in Uttarakhand Technical University. His research interests include nanomaterial characterization, synthesis, nanobiosensor, an electrochemical and nanopore-based biosensor.

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