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Nanopores have emerged as highly sensitive biosensors operating at the single-molecule level. However, the majority of nanopore experiments still rely on averaging signals from multiple molecules, introducing systematic errors. To overcome this limitation and obtain accurate information from a single molecule, the molecular ping-pong methodology provides a precise approach involving repeated captures of a single molecule. In this study, we have enhanced the molecular ping-pong technique by incorporating a customized electronic system and control algorithm, resulting in a recapture number exceeding 10,000. During the ping-pong process, we observed a significant reduction in the variance of translocation characteristics, providing fresh insights into single-molecule translocation dynamics. An inhomogeneous translocation velocity of folded DNA has been revealed, illustrating a strong interaction between the molecule and the solid-state nanopore. The results not only promise heightened experimental efficiency with reduced sample volume but also increase the precision in statistical analysis of translocation events, marking a significant stride toward authentic single-molecule nanopore sensing.

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"Molecular ping-pong," emerging as a control strategy in solid-state nanopore technology, presents a highly promising approach for repetitive measurements of single biomolecules, such as DNA. This paper introduces a high-precision, high-speed nanopore molecular ping-pong control system consisting of a home-built trans-impedance amplifier (TIA), a control system based on a Field Programmable Gate Array (FPGA), and a LabVIEW program operating on the host personal computer. Through feedback compensation and post-stage boosting, the TIA achieves a high bandwidth of about 200 kHz with a gain of 100 MΩ, along with low input-referred current noise of 1.6 × 10-4 pA2/Hz at 1 kHz and 1.1 × 10-3 pA2/Hz at 100 kHz. The FPGA-based control system demonstrates a minimum overall response time (tdelay) of 6.5 µs from the analog input current signal trigger to the subsequent reversal of the analog output drive voltage signal, with a control precision of 1 µs. Additionally, a LabVIEW program has been developed to facilitate rapid data exchange and communication with the FPGA program, enabling real-time signal monitoring, parameter adjustment, and data storage. Successful recapture of individual DNA molecules at various tdelay, resulting in an improvement in capture rate by up to 2 orders of magnitude, has been demonstrated. With unprecedented control precision and capture efficiency, this system provides robust technical support and opens novel research avenues for nanopore single-molecule sensing and manipulation.

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Understanding the dynamic behavior of a nanostructure translocating through a nanopore is important for various applications. In this paper, the characteristics in ion current traces of tetrahedral DNA nanostructures (TDN) translocating through a solid-state nanopore are examined, by combined experimental and theoretical simulations. The results of finite element analysis reveal the correlation between orientation of TDN and the conductance blockade. The experimentally measured fluctuations in the conductance blockade, expressed as voltage-dependent histogram profiles, are consistent with the simulation, revealing the nature of a random distribution in orientation and weak influence of electrostatic and viscous torques. The step changes in orientation of a TDN during translocation are further explained by the collision with the nanopore, while the gradual changes in orientation illustrate the impact of a weak torque field in the nano-fluidic channel. The results demonstrate a general method and basic understanding in the dynamic behavior of nanostructures translocating through solid-state nanopores.

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Detection and characterization of an individual cisplatin adduct on a single DNA molecule is a demanding task. We explore the characteristic features of cisplatin adducts in the nanopore sequencing signal in aspects of dwell time, genome anchored current trace, and basecalling accuracy. The offset between the motor protein and the nanopore constriction region is revealed by dwell time analysis to be about 14 bases in the nanopore device as we examined. Characteristic distortions due to cisplatin adducts are illustrated in genome anchored current trace analysis, constituting the fingerprint for identification of cisplatin adduct. The sharp increase in odds ratio at the location of adducting sites provides additional feature in the detection of the adduct. By these combined methods, single cisplatin adducts can be detected with high fidelity on a single read of the DNA sequence. The study demonstrates an effective method in the detection and characterization of single cisplatin adducts on DNA at the single-molecule level and with single nucleotide spatial resolution.

RESUMEN

The interaction between DNA and the nanopore structure plays an important role in nanopore DNA sequencing. Differential interaction forces between each base type and the nanopore structure are obtained by examining the correlation between translocation dwell time and the sequence. The viscous drag force and the intermolecular interaction are identified with single-nucleotide resolution. Active hydrogen donors and acceptors on the inner wall of the nanopore structure are revealed at various offset coordinates. The differential forces as demonstrated in this study have great potential in probing active hydrogen bond interaction in a single protein molecule with subnanometer spatial resolution.

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