Discrimination of Protein Amino Acid or Its Protonated State at Single‐Residue Resolution by Graphene Nanopores

Abstract

cellular processes at a single-molecule level by taking advantage of the electrochemical nanoconfinement of a nanopore. Thus nanopore is a powerful sensor for investigation of fundamental physical, chemical or biological issues, such as molecule gating [5] and cation-induced current changing, [6] at single-molecule resolution [8] compared to other traditional technologies. To date, nanopores have been exploited for expanded applications, including detection of nanoparticles, [9][10][11] biopolymers, [1,12] proteins, [13][14][15] DNA-origami, [16] DNA-protein complexes, [17][18][19][20] and so on. One of the most potential applications of nanopore technology is nucleic acid sequencing, which is characterized as low cost and high throughput. Most recently, a lot of works have shown that each type of DNA nucleotide can induce correlated modulation of ionic current as they translocate through a nanopore. [12,[21][22][23] With the help of enzyme [24] or polymerase, [25] some biological nanopores [26,27] have shown the ability to determine the nucleotide sequence of a singlestranded DNA at single-base resolution.Different from DNA molecules, proteins consist of more than 20 amino acids (AAs) and are irregularly and slightly charged. Besides, proteins usually keep in folded state in natural environment. Thus, investigation of proteins is much more challenging than DNA. Unraveling the mechanisms of protein folding and unfolding and even realizing protein sequencing are essential for cellular behaviors and diseases. [28] Measurement of tunneling current [29,30] was reported to recognize single amino acids, however to make the tunneling device be a protein sequencing tool it should be coupled with a means that can thread the peptide chain through the gap in a controllable way, which could be possibly realized if the tunneling device is coupled to a nanopore. In the past two decades, most interests in nanopores have focused on DNA sequencing. Only in recent years, nanopore technology has been extended to protein sequencing as nanopores could sequence full-length proteins with high temporal and spatial resolution while traditional sequencing methods such as mass spectrometry [31] and Edman degradation [32] only rely on digestion of proteins into short peptides. Many experiments have shown that protein molecules can be electrophoretically driven through nanopores. [33,34] Some experiments showed that proteins can be unfolded using nanopores by adjusting pH values of the solution, [35] The function of a protein is determined by the composition of amino acids and is essential to proteomics. However, protein sequencing remains challenging due to the protein's irregular charge state and its high-order structure. Here, a proof of principle study on the capability of protein sequencing by graphene nanopores integrated with atomic force microscopy is performed using molecular dynamics simulations. It is found that nanopores can discriminate a protein sequence and even its protonation state at single-residue resolution. Both the pull...

Nanopore‐Based Protein Sequencing Using Biopores: Current Achievements and Open Challenges

“…Both these observations suggest that charged homopeptides leave more room for the electrolyte passage and may tentatively explain the smaller value of the nanopore steric exclusion estimator with respect to residues of the same size. A similar result was recently reported in a computational work by Si et al, [137] where ionic currents of different short homo-and hetero-peptides translocating through a graphene nanopore are studied via nonequilibrium MD. In particular, it is reported that two different protonation states of the histidine residue show two significantly different current signals, the larger current being related to the charged state.…”

“…However, the local conductivity map does not take into account the effects of surface charge of the biomolecules, this may be one of the possible reasons for the different behavior of charged residues when compared with our study, [124] and Si et al work. [137] These recent studies, together with the constant increase in the computational performance, suggest that in the near future MD simulations will play an important role to elucidate the main mechanism responsible for the current blockades.…”

Detergent‐Assisted Braking of Peptide Translocation through a Single‐Layer Molybdenum Disulfide Nanopore

“…Some preliminary results have shown that the native structure of a protein is determined by the sequence which is developed by Oxford Nanopore Technologies, has been applied to study genomics in clinical laboratories. [22] However, expanding the applications of nanopore technology in identification, [23,24] fingerprinting, [25,26] and sequencing [17,27] of proteins is still challenging because proteins are composed of more than 20 amino acids and are more complicated than DNA molecules. Increasing bias voltage is usually applied for the purpose to increase the current blockade difference between residues.…”

“…Nanopore, which is a nanoconfinement [ 11–13 ] with great versatility, has been widely used for sensing single molecules, such as nanoparticles, [ 14 ] DNAs, [ 15 ] RNAs, [ 16 ] proteins, [ 17,18 ] and viruses. [ 19 ] Besides of single molecule detection, nanopores are also able to quantify analyte‐protein binding complexes.…”

“…To investigate the feasibility of detergents in assisting the slowdown of protein translocation through a MoS 2 nanopore in preparation for future nanopore protein sequencing by MoS 2 nanopores, which have good temporal and spatial resolution for biomolecule sensing, [ 34,58 ] we performed proof‐of‐principle all‐atom molecular dynamics (MD) simulations of PDC through a single‐layer MoS 2 nanopore with a diameter of 1.4 nm. The 1.4 nm nanopore was chosen according to our previous finding that the good signal‐to‐noise ratio and discrimination between amino acids could be obtained by using nanopore diameters ranging from 1.2 to 2.0 nm [ 17 ] which should be also considered by the experimentalists. Detergent DDM is selected because it is one of the popular detergents studied in many biochemical investigations [ 54,59 ] and is easily available.…”