The importance of studying systems at the nanoscale. Motivations for choosing the investigated subject. [309782]

The importance of studying systems at the nanoscale. Motivations for choosing the investigated subject.

The use of transmembrane pores in the "single molecule" study of the interaction between molecules of interest and a pore inserted into an artificial lipid membrane is a popular research technique in the field of biophysics.

α-hemolysin protein was extensively used in recent years as a molecular nanosensor. [anonimizat], make it an ideal candidate for molecular and biochemical tool in study at a unimolecular level.

When applying a certain difference of potential, a stable and well known amplitude current is established through the α-hemolysin pore. The detection applications are based on the fact that a molecule that enters into its lumen produces a blockade in the ionic current. [anonimizat]. [anonimizat], [anonimizat].

In this work we have investigated the discrimination between aminoacids from selected cationic peptides through electrophysiology unimolecular experiments…

I. INTRODUCTION

I.1. [anonimizat].

The new approaches in nanotechnology and protein engineering provide the possibility to investigate the matter at single molecule level. [anonimizat] “stochastic sensing” [anonimizat], enantiomers, pathogens, [anonimizat], [anonimizat] [1-5]. The nanopore analysis uses a voltage to drive the molecule of interest through a nanopore inserted in a [anonimizat] [1].

DNA sequencing using nanopores

Much of the nanopore field's development can be ascribed as DNA sequencing platforms. It turns out that the nanopore technologies for DNA and RNA sequencing are faster and cheaper than the usual genome sequencing methods. Fig.1. highlights some of the current directions of DNA sequencing with nanopores. Fig.1(a) demonstrates two systems for transverse electronic detection of the DNA sequence. [anonimizat]`s procedure [1], illustrates the translocation of DNA through a graphene pore when electrons pass through the graphene ribbon [2-3]. [anonimizat]. [anonimizat] [4-6].

Fig.1(b) [anonimizat]. A DNA molecule to be sequenced in this system is first transformed into a bigger sequence and hybridized with reporter samples. Next, the structures are unzipped using an array of small nanopores mounted on a cleverly designed fluorescence microscope [7], and each time a [anonimizat]or that match the reporter's identity. The device [8] has already measured the sequence of all four bases. While the basic conversion steps are rather tedious, for many sequences they can be performed in parallel.

Fig.1. Using nanopores for DNA sequencing: (a) Electronic transverse sequencing: two methods: drawing [1] indicates a single DNA strand translocating through a single layer graphene nanopore while electrons pass through the graphene ribbon [2-3], and DNA is translocated through an electrode gap and the resulting currents are registered [4-6]. (b) DNA sequencing based on optipore: each base of the DNA is transformed into a longer sequence fitted with optical reporters through biochemical preparation, and an array-based readout is provided by unzipping the sequence [8]. (c) Sequencing with exonuclease-pore complex: an exonuclease attached to an adapted α-hemolysin channel degrades the strand base by base and then the base enters the pore and the residual current is registered. (d) Single molecule DNA sequencing nanopore/mass spectrometer device: a DNA strand situated under vacuum is fragmented by irradiation and the nucleotides are identified using a mass spectrometer. (e) Ion current-based polymerase sequencing. The DNA-polymerase complex and a template strand are absorbed into the pore and the template strand is shifted upwards each time a base is incorporated [15]. In combination with the polymerase motor method [16-17], improved current resolution of a single base by a protein-tethered MspA channel can help in the readout. [18]

In Fig.1(c), exonuclease is attached to the adapted α-hemolysin channel. This complex is strong because a strand close the pore and it can be degraded by exonuclease and then it can be identified in the cyclodextrin-adapted α-hemolysin. The presented trace demonstrates the high capacity of the adapted α-hemolysin channel to discriminate the four nucleotides [9-10]. In Fig.1(d), degradation-based nucleotide detection technique is discussed, in which a DNA molecule is allowed to translocate through a nanopore into a vacuum where it is chemically fragmented using some irradiation-based method (e.g. photofragmentation) and then identified using a mass spectrometer [11]. Fig.1(e) displays ion current-based sequencing using a polymerase system. This model is based on the capacity to use an immobilized strand at the pore to read base information [12-14]. First, by applying voltage, a complex of DNA polymerase and a template strand / primer duplex is absorbed into a pore. The template strand is shifted upwards [15] each time a base is integrated, which offers a mechanism for driving a strand through one base at a point of time. The figure also shows an MspA channel which, in combination with the polymerase motor method, can assist in the readout by providing a better resolution [16-18].

Detection of microRNAs through a nanopore

The nanopore offers a sensitive single-molecule platform to explore a wide range of issues of life sciences. [19] While nanopores are not only frequently produced for fast and low-cost gene sequencing [20-23], epigenetic modifications such as DNA methylation [24] and gene damage have also been found to be analyzable. The nanopore sensor has lately been intended in this rapidly evolving domain to electrically detect microRNAs (miRNAs) [25-26], a class of small but highly significant regulatory RNA molecules. Since miRNAs are biomarkers of cancer, maybe a precise nanopore sensor for circulating miRNA detection would provide a potential non-invasive tool for disease screening and diagnosis [19].

Moreover, due to the complexity of clinical samples, translating the nanopore sensor into a clinically usable technology faces difficulties. Generally speaking, the clinical samples used for miRNA testing are extractions of RNA from biofluids such as plasma from a patient, and constitute a complex collection of different species of RNA: miRNAs, mRNAs, tRNAs, etc. When using the nanopore to detect target miRNA, any free nucleic acids in the mixture of RNA may also interact with the pore in a non-specific manner. These interactions consist in intensive "contaminative" signals that significantly affect the determination of the target miRNA, and should be eliminated [19].

Fig.2. miRNAs detection in the nanopore: a capture domain (PNA, green) with a polymer lead (peptide, blue) consists of the sample. With the target miRNA (red), the capture domain hybridizes. The miRNA sample complex is taken into the nanopore by a transmembrane voltage, whereas the electric field pitch at the opening of a pore takes adverse (gray) charges and electrophoretically moves away from the pore by any free nucleic acids without the binding probe [19].

The nanopore can selectively capture and detect the target miRNA by using a polycationic probe as a carrier (Fig.2.). The sample consists of a series of peptide nucleic acids (PNA) combined with a polycationic lead peptide. The PNA is designed to capture the target miRNA specifically. The positively charged peptide result and the negatively loaded miRNA form a dipole together after hybridization. A big electrical field gradient around the nanopore opening can drive this structure into the nanopore. Simultaneously, any free nucleic acids without probe hybridization would carry negative charge and migrate away from the opening of the pore. Accordingly, only the miRNA complex and probe signatures alone in nanopore are recognized, with no interference signal from free nucleic acids [19].

Slowing down the translocation time through the nanopore and discrimination between amino acids from polypeptides

An impediment in nanopore molecular detection is the high rate of translocation of the molecule through the pore, resulting in a low detection resolution. Thus, various methods have been developed to slow down the passage of analytes through nanopores, such as: modification of the pH of the electrophysiological solution, genetic changes of the primary structure of the α-HL protein, concentration imbalance between the two cuvettes, cis and trans, polypeptide chains having oppositely electrically charged amino acid ends that determine the voltage-controlled braking of polymer transport through nanometer-scale pore, etc. [27, 28].

To improve the resolution of discrimination between amino acids in the primary structure of a peptide by volumetric ionic current measurements, it was used a dipole-type peptide having a central region comprising the amino acids of interest (in the present case: isoleucine, non-polar amino acid and serine, polar amino acid) and oppositely electrically charged ends: at neutral pH, one end of positive charged arginine amino acid, qR12 = 12 | e- |, and one end of negative charged glutamic acid, qE12 = – 12 | e- |. The translocation time and the capture rate can be preferentially controlled by the applied transmembrane voltage [28].

Fig.4. Placing the electrically charged amino acids opposite the ends of a polypeptide to slow down its nanoporous translocation. a) The driving force acting on the peptide near the pore is the electrophoretic force acting on the tail containing Arg (), which is greater than the force acting on the end containing the Glu ( , because the electric field is much more intense in the immediate vicinity of and inside the nanoparticle. When the resultant electric force acting opposite to the ends of the peptide is approximately zero b), the peptide is in a metastable state of equilibrium, and the middle section of the peptide is located near the constriction zone, the most sensitive area of ​​volumetric detection of the nanoparticle. When the resultant forces become different from 0 due to thermal fluctuations, the peptide will move in the direction of the higher electrophoretic force that either will ensure the translocation through the pore, or it will return it to the trans side. The supplementary fluctuations in blockade currents due to reversible peptide interactions with nanopore provide information on the identity of a group of ~ 3 amino acid residues [28].

Discrimination of certain amino acid groups takes place in the constriction zone of the wild-type HL nanopore, which is the most sensitive region for molecule detection, through single-molecule volumetric measurements [28, 29]. By means of molecular dynamics simulation, it has been shown that blocking of the nanopore is affected by the occupied volume of amino acids, their hydrophobicity and net electrical charge [28].

The constriction zone is located between the amino acids Met-113, Lys-147 and Glu-111, and is ~ 0.6 nm in length and 1.4 nm in diameter, providing a spatial resolution theoretical of ~ 1.6 amino acids. Due to the thermal agitation that causes the peptide to perform a forward-backward movement within the pore, we can say that a group of ~ 3 amino acid residues of the protein sequence can be discriminate when is situated in the constriction region of ​​the wild-type α-HL nanopore [28].

Fig.3. Discrimination of a group of ~ 3 neutral amino acids with the α-HL protein nanoparticle. a) The most sensitive region for molecular detection is the constriction area, colored with green; b) Using the wild-type α-HL nanopore, it can be distinguished between groups of ~ 3 amino acid residues based exclusively on their molecular volumes. Each block I1 reflects a group of ~ 3 amino acids (either alanine, Ala, or tryptophan, Trp); substate I2 occurs due to the partial exit from the constriction region of an amino acid from the group followed by the entry of another such amino acid. The relative amplitude of the substrate lock I1 is correspondingly greater for the tryptophan-containing peptide, Trp, compared to the alanine-containing peptide, Ala, due to the greater volume of Trp than Ala; the substate I2 in amplitude is greater for the Trp-containing peptide because of its large dimensions and low mobility [28].

A protein nanopore-based technique for real-time detection of selected Gram-negative bacteria

Another study focuses on the interaction between Gram-negative bacteria (Pseudomonas aeruginosa or Escherichia coli) and the α-hemolysin protein nanopore in order to develop a method of detecting bacterial cells by molecular electrophysiology experiments. In the same study, hybrid antimicrobial peptides were used to allow discrimination between different bacterial species (E. coli and P. aeruginosa) by altering the electrostatic profile of external membranes.

These experiments have demonstrated, conceptually, the possibility of using protein nanopores and antimicrobial peptides as novel, rapid and simple detection systems and the identification of pathogenic organisms [30].

Fig.4. a) The experimental principle of single-bacteria detection using the α-HL nanopore. The trans-added negatively charged bacteria are electrophoretically driven towards the lumen entrance of the protein by the electric field lines generated by the transmembrane potential. It was shown that the mean association time of E. coli to the α-HL is roughly one order of magnitude faster than that of P. aeruginosa. In b) and c) panels is represented the role of CMA3 peptide adsorption to P.a., on the bacteria association with the pore: a) the control case when there is no CMA3 peptide in solution, just the P.a., and b) the CMA3 peptide with charge = + 8 |e−| at pH=7 is adsorbed to P.a., which leads to a decrease in the net charge of the bacterial outer membrane (Q), which produce an increase in association rate of the P.a. with the α-HL, due to the smaller value of electrostatic repulsive forces between the negative bacteria and the entrance in the lumen of the α-HL. The addition of CMA3 decreased the average time values measured between successive blockade events [30].

The study of Protein Unfolding with Nanopores

Single-molecule technologies based on nanopores offer the possibility to explore the folding and dynamics of a single protein, to characterize regions of the energy landscape and conformational space that are experimentally inaccessible using traditional bulk experiments. Protein unfolding through protein nanopore may occure due to the denaturating agents or an applied electric field [31]. The events frequency is related to the number of chains captured by the nanopore and to interactions between molecules and the lumen`s entrance.

Fig.5. Protein unfolding through protein nanopore may occure due to (a) the denaturating agents as guanidium or (b) an applied electric field as the transport of unfolded protein MalEwt through an aerolysin nanopore by applied transmembrane potential [31].

The measurements of dwell time of proteins inside the nanopore, the time between two successive events and the amplitude of blockade allow the clear discrimination between native, unfolded, and partly folded conformations [31, 32]. The interaction between proteins from bulck and the nanopore depend on many factors, as the transmembrane potential, the pH of electrophysiology solution, the salt concentration, the net charge of protein and of the nanopore, the conformational changes in polymer structure.

Fig.6. Protein unfolding through solid-state nanopore of 15 nm in diameter due to the transmembrane electrical field. The fraction of unfolded protein is shown in function of the applied voltage for the 3 proteins: WT and two unstable mutants, V388A and L322A [31].

I.2. The Coulter-Counter method as an inspiration for nanopore-based sensing and quantification of analytes

Wallace H. Coulter proposed in 1949 a method to detect and count particle suspended in a fluid medium, to simplify the blood cell analysis and counting. He started from an aperture made with a heated needle in a plastic film from a pack of cigarette and he developed a technique in which the molecules of interest are driven through a pore by an electric field produced by an external applied potential. These molecules disturb the electric field and the value of the disturbance is linked with the molecule`s dimensions [33, 34].

Fig.7. The Coulter Counting principle: the molecules of interest are driven through a pore by an electric field produced by an external applied potential. These molecules disturb the electric field and the value of the disturbance is linked with the molecule`s dimensions [34].

I.2.1. How particles are sized and counted

This principle is based on the passage of particles or cells through an aperture with specific features: a glass tube equipped with a bored ruby disk managing the fluid flow through the tube, maintaining also controlled, to an established value, the current. For apertures situated between two electrodes, a resistance is measured, due to the presence of the current of a low concentration electrolyte, generating, in this case, a sensing zone. In this case, a small volume of electrolyte is passing through the sensing zone creating a change in its impedance, being possible to measure the changed resistance from the tension pulse and the current corresponding to the immersed volume [33].

To determine the number and the volume of particles passing through the aperture, a counter and a pulse circuit are used. The physical properties of the particles, such as color, refractive index or transparency, do not influence the measurements; these are depending only in the diameter of the particles [33].

I.2.2 Coincidence

A fact that has to be taken in account during these measurements is the coincident particle passage. The meaning of it is two or more particles are passing through the sensing zone in the same time. This is related to the concentration of the volume of particles and to the measuring devices. This can represent an error on the particles distribution and also on the counts, but some mathematical corrections can be done (for coincidence up to 10%) or they can be automatically achieved by the software [33].

Fig.8. Left: Effect of primary coincidence. Right: Effect of secondary coincidence [33].

I.2.3 Particle path through the aperture

When particles are drawn through the aperture, they come from all angles around the aperture. Some particles pass through the aperture middle straight, while other particles move closer to the aperture edge. All particles that pass through the aperture displace an electrolyte solution mass and produce a maximum voltage. However, those particles that do not pass through the middle of the aperture will generate a slightly bigger peak than their displaced volume would have been anticipated. This slightly bigger pulse of the "artifact" leads the distribution of particle size to be skewed to the bigger dimensions, as in Fig.9 [33].

The velocity of the liquid (related directly to the velocity of the particle) also plays a part in the particle size distribution. The velocity at the aperture walls is lower, generating pulses that, in amplitude are wider and higher. The large, wide pulses skew the size distribution to the bigger dimensions [33].

Fig.9. The shape of the pulse relies on the stream of particles through the aperture [33].

I.2.4 Particle sizing response

The electrical signal will rely on both particle and aperture parameters for an aperture with a length-to-diameter ratio of about 1:1 or 2:1. For particle diameters between 2% and 60% of the aperture diameter, the voltage response is guaranteed to be linear. Aperture diameter varies from 10 microns to 2,000 microns for normal tubes. Each aperture can be used to monitor particles between 2% and 60% of their nominal diameter, with the alternative of extending the variety for all apertures up to 80%. It is therefore feasible to have an general particle size variety of 0.2 micron to 1,600 microns. For instance, an aperture of 30 microns can measure particles from about 0.6 to 24 microns in diameter, while an aperture of 140 microns can measure particles from ~2.8 microns to 112 microns safely [33].

I.2.5 Electrolytes

The electrolyte depends on the particles that are to be suspended. The solution should be chemically consistent with the sample material and permit correct dispersion of the sample. To suspend some bigger particles, a thickening agent such as glycerol or sucrose may need to be added to increase the solution's viscosity and density. As they pass through big diameter apertures (d > 560 microns), a thickening agent will also assist decrease the noise produced by turbulent flow of low viscosity electrolyte solutions [33].

I.3. Types of nanopores used in single-molecule nanopore-based sensing techniques.

I.3.a. Biological nanopores

Translocation of ions, DNA, RNA, polypeptides and different macromolecules across the membrane inside or between cells is a key and universal procedure. The transportation procedure includes a wide selection of passive pores, active ion channels, and viral motors with stylish and highly-ordered structures. The tale and refined structure of the transport machineries have roused the advancement of nanopores for single molecule detection [35].

Nanopore based investigation is at present an area of extraordinary enthusiasm for some controls with the potential for unfathomably adaptable applications. These incorporate sensing small particles, for example, ions, nucleotides, enantiomers, and medications, just as bigger polymers, for example, PEG, RNA, DNA, and polypeptides. The detection can be done with high affectability within the sight of large number of contaminants [35].

The stochastic nanopore technique depends on the working principle of the traditional Coulter Counter or the 'resistivepulse' routine [36], which exhibited estimating of micron measured particles with a micron sized aperture. In the nanopore technique, charged polymers are electrophoretically driven through a nanometer sized aperture (normally a couple of nm to several nm) implanted in a thin membrane. The nanopore is situated in an electrochemical chamber isolated into cis-and trans-compartments, each containing conducting buffers. Under an applied voltage, electrolyte ions movement through the nanopore is estimated as ionic current in the electrical circuit. The current, regularly in the pico-Ampere scale, is estimated utilizing a patch clamp setup alongside related ultra-delicate hardware, housed inside a Faraday cage [35].

Throughout the years, moderately bigger channels contrasted with ion channels have been investigated for the purpose behind nanopore detection. The substrate of decision for all biological pores is planar lipid layers, liposomes or polymer membranes housed inside an electrochemical chamber. Huge scale production and purification of different channel proteins are conceivable by utilizing standard molecular biology techniques. In greater part of the cases, the purified channel pores are homogeneous from various groups. Likewise, express designing of the channel pores through site directed mutagenesis is conceivable because of accessible precious crystal structure of a few channel proteins. The defining parts of three very much studied biological nanopores are discussed below.

MspA channel

MspA (Mycobacterium smegmatis porin A) is a funnel shaped octameric channel pore which permits the transport of water soluble molecules crosswise over bacterial cell bilayers. It contains a single constriction ∼1.2 nm wide and 0.6 nm long (Fig.1).

Fig.10. Octameric MspA porin from Mycobacterium smegmatis [37].

MspA channels can unexpectedly insert into a planar bilayer to frame a nanopore [37], like α-hemolysin. MspA is vigorous and holds channel-framing activity at pH 0—14 after extraction at 100 ◦C for 30 min or even incubation at 80 ◦C in nearness of 2% SDS [37, 38]. Since the crystal structure is available [39], site-coordinated mutagenesis can be done to chemically reengineer mutant channels for wanted applications [37, 40, 41].

Phi29 connector channel

The bacterial virus phi29 DNA-bundling nanomotor contains an exquisite and elaborate channel made out of twelve duplicates of the protein gp10, which enclose to shape a dodecamer channel [42-48] that goes about as a way for the translocation of double-stranded DNA. The length of the connector is ∼7 nm, while the cross-sectional region of the channel is 10 nm2 (3.6 nm in diameter) at the tight end and 28 nm2 (6 nm in diameter) at its more extensive end (Fig. 1C) [13-15]. The mode of connector insertion and anchoring inside the viral capsid is intervened through protein—protein interactions [45, 49, 50].

Fig.11. Dodecameric connector channel from bacteriophage phi29 [51].

The connector is first reconstituted into lipid vesicles during the rehydration step, trailed by vesicle combination with a planar bilayer [51]. The conductance of each pore is practically indistinguishable and is splendidly straight as for the applied voltage. The connector channel is steady under a wide scope of test conditions, including high salt and extreme pH [52].

The most noteworthy preferred position of the phi29 framework, not quite the same as other well-contemplated frameworks, is that the phi29 connector has a bigger channel allowing for the passage of ssDNA, dsDNA, peptides and conceivably little proteins. The bigger pore size is likewise favorable in that it makes it simpler for channel adjustments to either make a sharper detection region for accomplishing single nucleotide goals or for the insertion or conjugation of chemical groups for detecting and diagnostic applications [53].

Alpha-hemolysin protein pore

α-hemolysin (α-HL) is a toxin secreted by Gram-positive Staphylococcus Aureus bacteria in monomers shape with the property of penetrating lipid membranes, where it is self-assembled into heptametrical structures in the form of transmembrane pores. The protein is toxic to a large variety of cells, primarily erythrocytes (hence the origin of the name) where it acts as a mechanism for transforming host tissues into nutrient sources for bacteria. The secreted monomers are soluble in water, having a molecular weight of ~33 kDa, are inserted into the membranes of the target cells, and then by oligomerization, permeate the membrane, leading to release of the cellular material (Fig.12) [54].

Fig.12. Schematic representation of the process of insertion and oligomerization of α-HL monomers in lipid membranes [54].

Electron microscopy [55, 56] and subsequent X-ray crystallography [57] elucidated the crystalline structure of the fully assembled heptameric nanopore, long ~10 nm, which highlighted three distinct regions: (1) the outer head (‘head’), exposed to the aqueous medium, (2) the edge of the head (‘rim’) which is in contact with the outer surface of the cell membrane, and (3) the transmembrane (‘stem’) region with ‘β-barrel’ structure. As shown in Fig.13.a, the nanopore has an ~2.6 nm aperture in the extra-membrane region, which continues with a nanocavity, having a maximum diameter of ~3.6 nm, called the vestibule. The transition to the transmembrane region is made by a constriction with a diameter of 1.5 nm, followed by a lumen with a constant diameter of 2.2 nm [55, 56].

Fig.13. (a) Lateral view of the heptameric protein α-HL with fully assembled structure and inserted into membrane with the main marked areas and (b) a longitudinal section which highlights the regions of the internal cavity portion [54, 57]

Determination of the primary structure of α-HL monomers and subsequently of the entire protein, allowed the understanding of the membrane binding process in the physical-chemical properties that make possible to exploit and use protein-based nanopore analysis techniques. As can be seen in Fig.14., a series of non-polar amino acids cover the outer region of the transmembrane domain, making it possible to establish hydrophobic bonds with hydrocarbonated tails of membrane phospholipids. These hydrophobe bonds, along with the polar and cationic interactions between lipid polar heads and water or between polar head-polar head, provide nanopore stability in the target cell membranes [57, 58].

Fig.14. Section through α-HL with the representation of amino acid residues at pH = 7.5: cationic (blue), anionic (red), hydrophobic (green) and hydrophilic (gray). [59]

The electrostatic profile of the inner cavity (due to residues of charged amino acids) confers an anionic selectivity to the pore, which is maintained even at high salt concentrations (4 M KCl) [60, 61]. Applying a potential difference of 100 mV through pore, to a salt concentration of 1 M KCl results in a current of ~ 100 pA. The current increases linearly with increasing potential difference, which gives the pore a conductivity of ~ 1 nS at 22° C [62, 63]. Both selectivity and pore conductance are sensitive to pH changes and salt concentration due to changes in the degree of electrical charge of amino acids in the α-HL structure. [64, 65] For all that, the pore structure may remain functionally stable at temperatures close to 100° C and in a wide pH range (pH = 2 ÷ 12) [66].

All of these properties make the α-HL reference nanopore within stochastic analysis techniques at the single molecule level. The α-HL nanopore was successfully used to identify and characterize DNA and RNA chains [67, 68], peptides [69], detection of metal ions [70] and characterization of induced changes in the structure of some organic molecules [71, 72].

I.3.b. Solid-state nanopores

Solid-state nanopore was recommended in the mid-21st century, which complemented the impediments of the natural nanopore, for example, its unmodifiable pore measure, and mechanical and substance instability [2,16,17]. Ordinary structure of the solid-state nanopore imitates the structure of the biological nanopore: a nanopore a couple of nanometers in size perforated in a 10–20 nm thick silicon nitride (SiN) detached bilayer, upheld by a silicon (Si) substrate. The solid-state nanopore with customizable pore size and sturdiness has widened the range of the target biomolecules for nanopore-based sensing, the structure of the device, and materials utilized for fabrication. As far back as its first appearance almost two decades prior, different fabrication techniques and applications of the solid-state nanopore have been effectively contemplated and talked about by scientists from diverse disciplines, for example, materials science, biomedical, electrical and mechanical engineering, biophysics, chemistry and medicine. [solid-state article]

Principles of Biomolecule Sensing Using the Solid-State Nanopore

Fig.15. represents a schematic of the experimental setup and sign creation in the solid-state nanopore. As shown in Fig.15.a, a nanopore is built on a membrane material bolstered by a substrate, and an electrolyte fills two chambers on the both sides of the nanopore. Usually, the device is made of Si and SiN as the substrate and the membrane materials separately, where the Si substrate is anisotropically wet etched to expose the freestanding SiN membrane. The nanopore acts as the main way for the particles and the biomolecules to translocate between the cis and the trans chambers.[3] Stream cells are usually used to create the electrolyte chambers [57], which are each connected with the ground and headstage electrodes. Guided by the electric potential difference across the bilayer, ions and biomolecules travel through the nanopore and produce ionic current signals as shown in Fig.15.b [3,84]. A translocating DNA molecule somewhat obstructs the ionic movement through the channel, temporarily decreasing the ionic current level. The ionic current recoups to its initial dimension after the translocation [2, 66, 85].

Fig.15. Principles of the solid-state nanopore tests: a) a case of the test settings for solid-state nanopore. The names show each piece of the stream cell get together. Biomolecules move from the cis chamber to the trans chamber, toward the path demonstrated by the white arrow. This picture isn't in scale; b) diagram of a DNA being electrophoretically translocated (orange coil) through the solid-state nanopore, and resultant ionic current regulation from a real experiment. ΔI, td, and event frequency are graphically demonstrated. This picture isn't in scale. [article]

The fundamental principle of the nanopore-based detection is that biomolecules of various sizes and conformations produce the current drop signals of various magnitude and duration. This idea has been demonstrated in the examinations utilizing dsDNA of various lengths [37,66,86], proteins [7], and diverse polynucleotides[8,56] or single nucleotides [53].

Nonetheless, the distinctions in the structures of various nucleotides are little, to such an extent that the nanopore sign of the four nucleotides are hard to determine with a solid-state nanopore. The key in the solid-state nanopore is to upgrade this resolution; one approach to accomplish it is desing and fabricate appropiate devices with such high goals.

Fabrication of the Solid-State Nanopore

The device fabrication of the solid-state nanopore comprises of two sections: creating the unattached membrane on a supporting substrate and penetrating the nanopore.[2,25] The traditional fabrication sequence of the silicon substrate nanopore chip is to some degree standardized utilizing Si substrate and applying semiconductor fabrication technique.[2,17,47,87] To create the nanopore membrane, membrane material—basically SiN—is kept onto the substrate, trailed by characterizing and uncovering the membrane.

Fig.16. outlines the representative nanopore perforation methods. In the pore fabrication procedure, FIB showed up as the primary technique, which works by sputtering the SiN membrane to uncover a nanopore over the cavity.[16,67,90] Another method for the formation of a nanopore named controlled dielectric breakdown (CDB) was accounted for, which uses a solid electric field (on the request of 100 MV m−1) applied across the nanomembrane to precisely perforate the membrane.[87] Kwok et al. suggested that the system behind the aperture of the nanomembrane is like the dielectric breakdown component, in which the name of CDB procedure is originated (condensed in Figure 3c).[87,97]

The nanopores perforated utilizing electron bar (e-beam) lithography [94] and laser-pulled glass nanopipettes are in a asymmetric conical shape (Figure 3d).[71,89,100] As a rule, the dimension of the tightest constriction are effectively characterized and observed utilizing electron microscopes in the symmetric nanopores. In the glass nanocapillary, the compelling pore measurements are assessed from the deliberate ionic conductance.[74] Nevertheless, the uneven pore structure throws perceptible asymmetry of the force field and in this way contrast in the DNA translocation elements in 2 different directions.

Forming the nanopore is the most significant advance of the solid-state nanopore creation in light of the fact that the affectability and reliability of the biomolecule sensing are determined by the size and the uniformity of the nanopore structures.[8,56,101] On the other hand, perforating such a little nanopore in a uniform size is an extremely troublesome undertaking, and the throughput of the pore formation is restricted in the at present accessible innovations, particularly in the TEM-based technique. Subsequently, a stable pore fabrication method ought to be created and streamlined for large scale production of the solid-state nanopore device with uniform structure and device efficiency.

Fig.16. Solid-state nanopore fabrication. a) Schematics of the TEM-based electron beam pore drilling technology. The sample pore diameter is 3 nm. b) Graph of current (blue line) and voltage (red) versus time during the CDB process. c) Mechanism of the controlled dielectric breakdown method for pore formation. d) Images of laser-pulled glass nanocapillaries. The scale bars all indicate 1 mm. a) Reproduced with permission.[2] Copyright 2007, Nature Publishing Group; b) Reproduced under the terms of the under Creative Commons Attribution License (CC-BY).[87] Copyright 2014, The Authors, published by Public Library of Science; c) Reproduced under the terms of the under Creative Commons Attribution License (CC-BY).[97] Copyright 2015, The Authors, published by Nature Publishing Group; d) Reproduced under the terms of the under Creative Commons Attribution License (CC-BY).[89] Copyright 2016, The Authors, published by Public Library of Science.

Limitations of the Solid-State Nanopore

The solid-state nanopore device and its efficienty have been persistently improved as far back as the principal report in 2001, yet issues still exist and confine the stage regarding the utilization in genuine biological applications.[21] The restrictions are basically resolution and reliability issues: the farthest point in spatial resolutions, both lateral and vertical,[47,102] and in temporal resolution [12,36,42] and pore clogging during trial brought about by nonspecific adsorption of biomolecules.[103,104]

II. Materials and Methods

II.1. Materials used in our study

II.2. Montal and Mueller technique for the formation of planar lipid bilayers and the experimental set-up

Monitoring of ionic current through a single proteic nanopore shows multiple difficulties both in the implementation of the system and in the recording and processing of the signal. Making a stable physical support for nanopore, which does not affect the psysico-chemical properties of the pore or the analyst wishing to be investigated, are the first steps in the preparation of a precise and efficient detection system. In practice, a biological support such as an artificial lipid bilayer can be used in which the desired nanopore will be subsequently inserted or a solid support. The most common biological supports are planar bilayers which are easy to perform and provide well-defined experimental conditions. On the other hand, these systems are mechanically and chemically fragile, have a limited lifetime and signal recording through these membranes can be affected by electrical noise. Nanopores can be synthetically created by micro- and nanotechnology techniques [54,2,3]. These structures are characterized by high mechanical stability in time on a large plateau of measurement parameters (temperature, pH, ionic strength) and can be more easily integrated into detection devices [4].

In these experiments, we used a planar lipid support formed in the aperture of ~100 µm diameter, made in a Teflon film. The film is placed between two cells of the experimental cuvette, forming a reliable seal using a silicone-based vaseline (Dow Corning) so that the aperture is the only access pathway between the two cells. The bilayer is formed after the film was treated with a hydrophobic solution of hexadecane dissolved in pentane, using the Montal-Mueller technique (Fig.17.). The phospholipid used is DPhPC (di-phthaloanoyl-phosphatidyl choline) solubilized in n-pentane at 1 mg/mL concentration. From this solution an amount of ~4 µL was added in the both compartments, over the electrophysiological solution (KCl at appropriate salt concentration to the desired pH). The use of KCl is due to similar ionic mobility of K+ and Cl- that prevents an imbalance in recorded ionic current. The proper formation of the bilayer is signaled by the significant increase in resistance and electrical capacity measured in the system.

Fig.17. Schematic representation of Montal-Mueller technique for forming planar lipid bilayers: a) the electrophysiological solution is added to both cells under the aperture; b) the lipids are added to both cells and after evaporation of the pentane, they are arranged as a monolayer at the surface of the liquid; c), d) successive lifting of the liquid levels leads to bilayer organization of the lipids in the aperture [54].

In the cuvette are inserted two electrodes Ag/AgCl connected to the amplifier through which a potential difference is applied and the resulting ionic current is recorded. At the level of the electrodes is a uniform transition of the electron flow, from the copper wire, into the ion flux of the solution. The electron flow transforms AgCl into Ag atoms and Cl- ions which are hydrated and transferred to the solution, and in reverse order, the Ag atoms give off an electron and combine with Cl ions form the solution resulting in the formation of AgCl insoluble at the surface of the electrodes (Reaction 1).

(1)

Note, however, that Ag/AgCl electrodes are working properly only in electrophysiological solutions containing Cl- ions (NaCl, KCl). Additionally, due to the transfer of ions between the solution and the surface of the electrode, the ionic current can lead to AgCl depletion, which would lead to direct contact of pure Ag with the aqueous medium. Ag ions can leak into solution resulting in environmental contamination and compromise experimental results [6,1]. For this reason, Ag/AgCl electrodes should be cleaned from the salt deposited after each experiment and frequently rechlorinated by placing them in dilute sodium hypochlorite solution.

Ag/AgCl electrodes are connected to a patch-clamp amplifier (AxoPatch 200B) operated in voltage-clamp mode, which allows the storage of a fixed potential difference between the electrodes (the control electrode and the electrode connected to the ground). Measurements of the ionic current is done indirectly by measuring the potential drop on the amplifier feed-back resistor and then converted into current through an algebraic operation [7]. The electrical signal is then filtered over a low-pass Bessel filter at a cut-off frequency of 10 kHz. Filtration is intended to eliminate unwanted signals and noise from recording [1].

Fig.18. Schematic representation of the experimental device used to perform electrophysiological measurements at the single molecule level through the α-HL [54].

Data acquisition is done by digitizing the analog signal using a 16-bit (NI PCI 6014, National Instruments, Inc.) acquisition card (DAC) and represented in a LabView command graphical interface (National Instruments, Inc., USA). Data processing was performed using a specific program for electrophysiology signal AxonTM pClampTM 10 (Molecular Devices, LLC, USA) and the analysis was performed in Origin 6.0 (OriginLabCorporation,USA).

II.3. The principle of single-molecule nanopore-based sensing techniques. Transport of a charged particle from solution toward nanopore`s entrance.

The dimensions of the α-hemolysin pore, high stability under extreme environmental conditions (ionic strength, temperature up to 100 °C, pH 2-12) and the possibility of modification by genetic engineering techniques make it an ideal candidate in molecular and study detection applications of biochemical reactions at the unimolecular level, such as detection and quantitation of cations and organic molecules (biosensor role), detection, sequencing and characterization at the single molecule level of DNA and genomic RNA, investigation of the way of protein packaging [7, 8].

When applying a transmembrane potential difference, an ionic current with constant amplitude, known value from the literature, is established through the α-HL pore. Molecular detection applications are based on the fact that a molecule that enters into nanopore`s lumen produces a blockade in channel-mediated ionic current. When the molecule leaves the pore, the ionic current returns to its original value. From an electrical point of view, the pore resistance increases once the molecule occludes the ionic channel and thus, according to Ohm's law, the current intensity decreases [4, 7].

Fig.19. Schematic representation of the principle of a stochastic biosensor based on a protein nanopore inserted into an artificial lipid membrane. When applying a transmembrane potential difference, a stable and well known amplitude current is established by the α-HL nanopore. When a molecule enters in its lumen, there is a blockage in the channel-mediated ionic current. When the molecule leaves the pore, the ionic current returns to its original value [8].

The frequency, amplitude and duration of these blockades provide information on the physio-chemical properties of the analytes (such as peptides, proteins, DNA, RNA, PNA, dendrimers, metal ions, etc.). Hence, the applications of nanopore sensing include detection and quantification of the analyte concentration in solution, the analysis of the analytes dimensions and charge, discrimination of the primary structure of polymers, detection of conformational changes in structure, the monitoring of the binding interactions between the nanopore and the analyte which can give kinetic and equilibrium information, [7, 8].

Fig. 20. Capture rate of analytes by the α-HL nanopore and the frequency, amplitude, and dwell time of the blockades provide information on the physio-chemical properties of the analytes. For instance, the concentration of analyte in solution can be obtained by measuring the capture rate (τon), and the diffusion coefficient, by measuring the dwell time (τoff). Protein sequencing and conformation changes detection can be achieved by analyzing the event amplitude. Overall charge of molecule can be determined through dwell time and changes of pH [8].

As described in the literature, the interaction process of an electrically charged molecule (DNA, RNA, peptide) with a nanopore is influenced by the weak, non-uniform electric field extending from the mouth of the pore in the entire solution. The potential associated with this field will act on a loaded molecule spaced at a distance from the pore. To determine the value of this potential, based on the potential difference applied, the formalism described by Grosberg and Rabin [1-35] from the electrical potential to the distance of a spherical electrode with diameter, in a medium characterized by conductivity [2]. The voltage drop between two concentric layers and is:

(2)

Where the term represents the electrical resistance of a spherical thick layer. Thus we can write:

(3)

That the integration provides the solution:

(4)

Fig. 21. Schematic representation of the dV potential between two hemispherical regions and around a pore [2].

As the potential difference between infinity and the surface of the electrode, located at distance, is:

(5)

The potential at distance becomes:

(6)

Turning to the case of a pore inserted in a membrane, the "electrode" is the circular opening of the pore on one side of the membrane, which makes the value of the current only half of that resulting from the relation (5) (Fig. 21). The potential difference between pore and infinity can be determined by considering the distribution of potential differences on each component of the environment, ignoring the potential drop in the electrodes. Considering the regions of both sides of the pore as symmetrical we can say that:

(7)

Pore resistance of length and diameter is , making the current equal to:

(8)

The current value reduced by half is obtained:

(9)

From the relations (6) and (9) we obtain the value of the potential at the distance from the por:

(10)

Under static conditions, the potential difference would not be able to carry a charged electrostatically charged molecule from the solution to the pore mouth. In reality, however, the phenomenon occurs outside the equilibrium and the potential V (r) extends more in the solution as a result of the external current [1-35].

Fig.22. The distribution of the equipotential lines (a) and the electric field lines (b) at the nanopore level due to the application of a potential difference. The distribution was determined by finding solutions to the Laplace equation. [3-36, 2]

The process of capturing a molecule by the pores and effecting the interaction is characterized by a catch rate , which is the fraction of molecules approaching the pore that manage to block the registered ionic current. This rate depends on the size of the molecule being transported, its concentration, the pore geometry, the salt concentration, the pH and the potential difference applied. At a large distance from the pore, the charged molecule is subjected to free diffusion in the solution and can be considered as a punctiform object, characterized by the diffusion coefficient and electrophoretic mobility . As the molecule approaches the pores, at the distance it is captured irreversibly by the electric field, and electrophoretically transported along the field lines to the pore mouth.

To understand the transport and subsequent interaction of the pore with molecules, as the basis of detection, first consider the unidimensional variation of the concentration of molecules loaded along the pore axis at time.

(11)

Where is the net flux of molecules at time in point.

(12)

– the velocity of the molecules at time in point . In the absence of pressure gradients the velocity comprises the components of two processes: the action of the electrophoretic force which determines and the force action due to the local gradients of the free energy. [4-37]

(13)

By replacing the relation (13) in relation (12) we obtain for the flow of molecules the relation:

(14)

By explicitly representing the electric potential V (x), the relation (14) becomes:

(15)

The three terms cumulate the contributions of diffusion, drift and free energy that contribute to the movement of molecules to the pore mouth. Solving the Smoluchowski equation (15) allows the determination of the capture rate of the molecules by pore, if the dominant processes of the association are known.

II.4. Single-channel ion current recordings and data analysis. Random variables of interest.

The recording of electric current fluctuations mediated by membrane ion channels through molecular electrophysiology techniques is essential to directly visualize the molecular transitions made by a single ion channel and to study the ion transport phenomena through each conductive substrate. These transitions are purely stochastic so that in describing the behavior of these molecules it is important to estimate the exact number of molecular states ("closed" and "open") through which an ionic channel can pass, determining the transition rates between these states as and the analysis of ionic transport processes mediated by those channels. [1]

We consider a transmembrane ionic channel that performs stochastic molecular transitions between only two conductive states, "closed" and "open". Fluctuations of ionic current through such a channel have the form shown in Figure 1, where the closed state is marked C (“closed”) and open state with O (“open”).

Fig.20. Current fluctuations through an ionic channel that execute stochastic transition between two conductive states, “closed (C) and “open” (O). represents the time that the ion channel is in the “closed state” and represents the time that the ion channel is in the “open” state. [1]

This process can be modelled by a simple kinetic model whose parameters are given by and reaction constants:

(16)

and times, as the protein is in one of the two conductive states, are random sizes. The moment when the ion channel is transitioning to a certain state, as well as the time spent in that state, is impossible to specify. We can, however, statistically quantify the stochastic distribution of the numbers represented by the and time intervals, and we can determine the probability density function (pdf – “probability density function”) for these stochastic magnitudes. With the probability density function, quantitative assessments can be made on the and reaction constants.

Considering that the molecular transitions of such an ionic channel are described by Markov`s stochastic processes, for the time spent by the protein in the closed state it can be written the relation:

(17)

where represents the probability that the studied protein is in the “closed” state at time , knowing that she was in the state at time with the probability and remained in the state “closed” after the time interval , which is the probability . [2 – 107]

The probability that an ionic channel will make a transition from the "closed" state to the "open" condition in time, when is given by:

(18)

Similarly, the probability that a channel will make a transition from the "open" state to the "closed" state during time, when is given by:

(19)

Therefore, the probability that an ionic channel remains in the "closed" state during time, when, is given by:

(20)

The equation (20) can be rewritten as follows:

(21)

(22)

Knowing that at the moment, the probability of the protein being in the “closed” state is, it follows that and the final solution of the equation becomes:

(23)

Expression (23) provides a quantitative value of the probability of the protein being in the "closed" state at time . Under these conditions, the ion channel can either remain in the "closed" state or perform a molecular transition in the "open" state.

The probability of the protein still remaining in the "closed" state is given by the difference to certainty:

By deriving this relationship from time to time, we obtain:

(24)

Taking into account the definition of the probability density function for stochastic size represented by [2-107], we find the relation:

(25)

By analogy to the above, the stochastic magnitudes represented by the time intervals are statistically described by a probability density function given by:

(26)

According to the relations obtained for the probability density functions, the time intervals and are described by an exponential distribution function.

Knowing the pdf of the stochastic variables allows quantitative estimation of the average value of those numbers. Thus, for the average time intervals spent by the ionic channel in the "closed" or "open" state, we can write:

(27)

Thus, by measuring discrete values for time intervals as the studied ionic channel is in the "closed" or "open" state, and, we can determine, by arithmetic mean of these time intervals, estimated values for and .

According to (27), calculating the inverse of these mean values, we can determine estimative values for the and reaction constants. The estimated values are all the more precise as the number of measurements of these time intervals is greater.

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[2-107] aceeasi mai sus – Luchian Tudor, Electrofiziologie moleculară. Teorie și aplicații, 2006, Ed. Sedcom Libris, Iași