Label‐Free Identification of Single Mononucleotides by Nanoscale Electrophoresis

Abstract

amounts of input material. [1,2] While the sequencing-by-synthesis technology dominates currently, this technology requires significant sample preparation and amplification steps, the use of costly biological reagents such as fluorescently labeled molecules, and expensive imaging and data handling instruments. [3][4][5][6][7] Singlemolecule sequencing, which does not require amplification and labeling steps, would simplify the entire sequencing process significantly reducing costs and time of acquiring sequencing data, and is thus more adaptable to clinical translation to enable precision medicine even from mass-limited samples such as those provided by liquid biopsies. [8] Nanopores offer a fast and low-cost sequencing platform that does not require labeling and sample amplification. [6,7,[9][10][11][12][13] The basic principle of nanopore sequencing is to electrically drive charged single molecules, either an intact DNA molecule in strand sequencing or individual nucleotides cleaved from the DNA in exonuclease sequencing, [10,[13][14][15] through a nanopore and determine the identity of each constituent nucleotide by monitoring small changes in the ionic current flowing through the pore while individual nucleotides temporarily reside within the pore (i.e., resistive pulse sensing, RPS). [13,16] Strand nanopore sequencing has demonstrated whole-genome sequencing [14] Nanoscale electrophoresis allows for unique separations of single molecules, such as DNA/RNA nucleobases, and thus has the potential to be used as single molecular sensors for exonuclease sequencing. For this to be envisioned, label-free detection of the nucleotides to determine their electrophoretic mobility (i.e., time-of-flight, TOF) for highly accurate identification must be realized. Here, for the first time a novel nanosensor is shown that allows discriminating four 2-deoxyribonucleoside 5'-monophosphates, dNMPs, molecules in a label-free manner by nanoscale electrophoresis. This is made possible by positioning two sub-10 nm in-plane pores at both ends of a nanochannel column used for nanoscale electrophoresis and measuring the longitudinal transient current during translocation of the molecules. The dual nanopore TOF sensor with 0.5, 1, and 5 µm long nanochannel column lengths discriminates different dNMPs with a mean accuracy of 55, 66, and 94%, respectively. This nanosensor format can broadly be applicable to label-free detection and discrimination of other single molecules, vesicles, and particles by changing the dimensions of the nanochannel column and in-plane nanopores and integrating different pre-and postprocessing units to the nanosensor. This is simple to accomplish because the nanosensor is contained within a fluidic network made in plastic via replication.

Electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) through α-phase phosphorene carbide nanochannel for electrophoretic detection

“…Excitingly, our findings are in good agreement with the experimental report by Choi et al , who also found a broad distribution when monitoring the TOF of dGMP by 5 μm nanochannel electrophoresis, but did not identify the reason. 37…”

“…The pattern of the electrophoretic migration velocity of nucleotides along the electric field direction is dTMP 4 dCMP 4 dAMP 4 dGMP, which is in agreement with experimental data. 37 Through the analyses, we found optimized electric field strengths of 0.7-0.8 V nm À1 which show considerably high identification accuracy among the four nucleotides. In addition, we found significant differences in the velocities corresponding to the two adsorption conformations of dGMP leading to the largest spreading of the signal in time, which explains the same experimental phenomenon.…”

High Sensitivity Extended Nano-Coulter Counter for Detection of Viral Particles and Extracellular Vesicles

“…We determined the average width of the current transient events at half-height and the average event amplitudes at voltages of 1 and 5 V. For these measurements, the current transients were measured using an in-house built current transimpedance amplifier (TIA) with the XnCC containing Ag/AgCl electrodes for recording electrical signals at the appropriate applied voltage. 28 Figure 3B shows a representative RPS data trace distributed over 100 s in 1× PBS blank and in Figure 3C is shown an expanded region (0.01 s); applied voltage = 1 V. An event was scored only if it had >1 data point per peak, an amplitude greater than a selected threshold, and a half-width ≥0.02 ms. As seen, the 1× PBS buffer resulted in no events exceeding the threshold.…”

Detection of DNA translocations in a nanopore series circuit using a current clamp

“…25 Afterward, Choi et al reported that the distance between two nanopores could influence the separation accuracy of molecules with different mobilities, showing an optimized length of 10 mm to discriminate four deoxynucleotide monophosphates. 26 These dual nanopore TOF sensors are actually a nanopore series circuit, while parallel nanopores are examined as well, termed ''Tug-of-War''. Zhang et al first fabricated this ''Tug-of-War'' nanofluidic device and reported its two features: (i) sensing and resensing the same DNA molecule in sequence by two independent nanopores; (ii) cocapture of one DNA molecule by two nanopores and significantly extending the translocation time.…”