Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA

Sulaiman, Dana Al; Gatehouse, Alfie; Ivanov, Aleksandar P.; Edel, Joshua B.; Ladame, Sylvain

doi:10.1021/acsami.1c01145

Cited by 13 publications

(9 citation statements)

References 37 publications

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“…To further explore the SNR enhancement achieved by using CsBr, we studied a shorter, 500 bp fragment of dsDNA (Supporting Figure 12) to compare the effects of CsBr, KCl, and LiCl in the PEG electrolyte bath. The detection of short dsDNA (<1000 bp) can be challenging with glass solid-state nanopores because the SNR is often very small due to a poor analyte to pore ratio and the translocation of short DNAs through the nanopore is too fast to be detected ,, even with state-of-the-art electronics . In order to ensure that the signals obtained were not due to slight deviations of nanopore size during fabrication, the same nanopipette filled with the 500 bp dsDNA was used for translocation experiments in all three salt PEG baths.…”

Section: Resultsmentioning

confidence: 99%

“…Salt gradients can also be used to improve the translocation frequency across a nanopore, but this affects neither the speed nor the current magnitude of the single molecule events . Alternatively, the nanopore surface can be chemically modified to slow down the translocation of analytes, − but this method is difficult to generalize as it needs to be tailored for each analyte.…”

Section: Introductionmentioning

confidence: 99%

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Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

et al. 2022

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Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with singlemolecule resolution. The conformation of biomolecules, such as RNA, is highly dependent on the electrolyte composition, but solid-state nanopore systems often require high salt concentration to operate, precluding analysis of macromolecular conformations under physiologically relevant conditions. Here, we report the implementation of a polymer−electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer− electrolyte bath governs the translocation dynamics of the analyte which correlates with the physical properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer−electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiologically relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the cotranscriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer−electrolyte solidstate nanopore system will further enable structural and conformational analyses of individual biomolecules under physiologically relevant conditions.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

et al. 2022

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“…Resultantly, the dwell time was approximately three times longer, and the median well time of the signals was 0.28 ms ( n = 346) (Supporting Information Figure S8). These results indicated that the translocation speed of poly(dA) 50 was reduced by the hydrogel effect because the formation of the hydrogel at the aperture reduced the translocation speed of the analytes through the nanopores. ,, The duration time (>80% current blockage) was about 100 times shorter than that in the previous report . This is because the nucleotide number was one-third, and only the inner solution of nanopipettes was gelled in our system.…”

Section: Resultsmentioning

confidence: 56%

Nanopore Probes Using Hydrogel-Filled Nanopipettes as Sensors for Chemical Imaging

Yoshihara

Nomi

Shoji

2022

ACS Appl. Nano Mater.

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Scanning ion conductance microscopy (SICM) using biological nanopores is a powerful analytical tool that can provide spatially resolved topography and chemical information. However, the low spatial resolution caused by the large tip diameter of the probe has limited the practical application of the SICM system. Although the tip-dip method has conventionally been used to form a lipid bilayer at the tip of pipettes, an alternative method to form the lipid bilayer is required because the lifetime of lipid bilayers formed by the tip-dip method is too short to map chemical information by stochastic nanopore sensing. Here, we propose a method to prepare lipid bilayers using a hydrogel-filled nanopipette. The lipid bilayer is formed at the tip of the pipette by inserting the pipette into a layered solution of an oil/lipid mixture and an aqueous electrolyte. Since the hydrogel supports the lipid bilayer, the lifetime of the lipid bilayers prepared by this method is improved over that formed by the tip-dip method. Furthermore, the hydrogel at the pore aperture reduces the translocation speed of analytes through nanopores, indicating that the hydrogel-filled nanopipette system can offer highly sensitive chemical sensing. Finally, we measure local single-strand deoxyribonucleic acid concentrations in the concentration gradient generated by a microhole using the biological nanopore probe. We believe that the SICM system using the hydrogel-filled nanopipette-based biological nanopore probe will offer a powerful analytical system for biological phenomena, including cell communication and signal transduction.

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“…Cell-free DNA (cfDNA) are DNA fragments of variable length between 50 and 250 base pairs, which dissociated in the plasma [ 38 ]. cfDNA has become an attractive research subject as a non-invasive biomarker in diseases.…”

Section: Discussionmentioning

confidence: 99%

Plasma Cell-Free DNA as a Novel Biomarker for the Diagnosis and Monitoring of Atherosclerosis

Qian

Lou

et al. 2022

Cells

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Atherosclerosis (AS) is the leading cause of cardiovascular diseases (CVDs) with a high rate of mortality worldwide. Plasma cell-free DNA (cfDNA), mainly originating from apoptosis, necrosis, and active secretion, has been recognized as a promising biomarker for the diagnosis and prognosis of multiple cancers, whereas there are no reports about cfDNA in CVDs. Here, we found an increased quantity and decreased integrity of cfDNA (cfDI) in the serum from AS patients compared with normal controls. Moreover, the reduced cfDI is inversely correlated with serum LDL levels, carotid plaque size, and carotid plaque thickness in the progression of AS. Consistently, in vivo experiments confirmed that the release and cleavage of cfDNA were increased concomitantly with the development and progression of AS in ApoE−/− mice. Our study sheds light on the potential of cfDNA and cfDI as molecular biomarkers for detecting and monitoring AS.

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Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA

Cited by 13 publications

References 37 publications

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Nanopore Probes Using Hydrogel-Filled Nanopipettes as Sensors for Chemical Imaging

Plasma Cell-Free DNA as a Novel Biomarker for the Diagnosis and Monitoring of Atherosclerosis

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