Electrophoretic Extraction of microRNA from Clinical Samples using Fabricated Nanofilter Membrane

Kidan LEE,Jae-Hyun KANG,Hyun-Mi KIM,Junhyoung AHN,Hyungjun LIM,Jaejong LEE,Wan-Jin JEON,Jae-Hoon LEE,Ki-Bum KIM 한국생물공학회 2019 한국생물공학회 학술대회 Vol.2019 No.4

Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane

Lee Kidan,강재현,Kim Hyun-Mi,Ahn Junhyoung,Lim Hyungjun,이재종,Jeon Wan-Jin,이재훈,김기범 나노기술연구협의회 2020 Nano Convergence Vol.7 No.2

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 10 8 /cm 2 . The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRTPCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA

Nuttall, Philippa,Lee, Kidan,Ciccarella, Pietro,Carminati, Marco,Ferrari, Giorgio,Kim, Ki-Bum,Albrecht, Tim American Chemical Society 2016 The Journal of physical chemistry B Vol.120 No.9

<P>p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option.</P>

Investigation of constantly and transiently propagating cracks in functionally graded materials by dynamic photoelasticity

Kwang Ho Lee,Addis Kidane,Arun Shukla 대한기계학회 2022 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.36 No.9

In this study, the stress intensity factors (SIFs) for steady and transient propagation of cracks in transparent homogeneous functionally graded materials were analyzed by using the photoelasticity technique. The fracture analysis was carried out for the cracks propagating from a region with high elasticity towards low elasticity, as well as the cracks propagating from a region with low elasticity towards high elasticity. The analysis includes cracks propagating (1) at an almost steady speed, and (2) with the rapid increase, followed by a decrease in speed. For cracks with almost constant velocity, the SIFs were greater when a crack started from a high elasticity region, as compared to the cracks which initiated from a low elasticity region. For cracks propagating with rapid acceleration and deceleration, when the strain energy accumulated in the material due to an increase in load or stress was released at the moment of crack propagation, the SIF was momentarily lowered by approximately 45 %-50 % of the static SIF(before crack initiation), which subsequently increases by approximately 30 % eventually, the crack acceleration approaches nearly zero; the SIF decreases and increases respectively as the crack propagates in a material with decreasing and increasing modulus of elasticity.

Translocation of DNA and protein through a sequentially polymerized polyurea nanopore

Kim, Hyung-Jun,Choi, Ui-Jin,Kim, Hyein,Lee, Kidan,Park, Kyeong-Beom,Kim, Hyun-Mi,Kwak, Dong-Kyu,Chi, Seung-Wook,Lee, Jin Seok,Kim, Ki-Bum The Royal Society of Chemistry 2019 Nanoscale Vol.11 No.2

<P>Here, we investigated the translocation of biomolecules, such as DNA and protein, through a sequentially polymerized polyurea nanopore, with a thin (<10 nm) polymer membrane of uniform thickness. The polyurea membrane was synthesized by molecular layer deposition using <I>p</I>-phenylenediisocyanate (PDI) and <I>p</I>-phenylenediamine (PDA) as sequential precursors. The membrane exhibited a hydrophobic surface with a highly negative surface charge density (−51 mC m<SUP>−2</SUP> at pH 8). It was particularly noted that the high surface charge density of the membrane resulted in a highly developed electro-osmotic flow which, in turn, strongly influenced the capture probability of biomolecules, depending on the balance between the electro-osmotic and electrophoretic forces. For instance, the capture frequency of negatively charged DNA was demonstrated to be quite low, since these two forces more or less cancelled each other, whereas that of positively charged MDM2 was much higher, since these two forces were additive. We also identified that the mean translocation time of MDM2 through the polyurea nanopore was 26.1 ± 3.7 μs while that of the SiN nanopore was 14.2 ± 2.0 μs, hence suggesting that the enhanced electrostatic interaction between positively charged MDM2 and the negatively charged pore surface affects the translocation speed.</P>

The dynamics of electron beam scattering on metal membranes: nanopore formation in metal membranes using transmission electron microscopy

Kim Hyun-Mi,Park Kyeong-Beom,김형준,Chae Hongsik,Yu Jae-Seok,Lee Kidan,Kim Ki-Bum 나노기술연구협의회 2018 Nano Convergence Vol.5 No.32

The dynamics of nanopore formation in metal membranes using the highly focused and high energy electron beams (e-beams) of transmission electron microscopy instruments was investigated. Various metals such as Al, Ti, Cr, Cu, and Au were selected to investigate the effect of the atomic mass of the metal on nanopore drilling, namely, elastic versus inelastic scattering. We demonstrated that the effect of elastic scattering (pore formation by sputtering) decreased as the atomic mass of the metal increased. Furthermore, experimental cross-sections obtained from normalized drilled volume vs. electron dose curves (characteristic contrast curves) matched well the calculated atomic displacement cross-sections determined from elastic scattering data. The sputtering energies of Ti, Cr, and Cu were determined to be approximately 10, 9, and 7 eV, respectively, which were in good agreement with the reported range of sputtering energy values.

Detection of metal corrosion characteristics in chlorine solution using solid state nanopore

Kim, Hyung-Jun,Park, Kyeong-Bum,Kang, Jae-Hyun,Lee, Kidan,Kim, Hyun-Mi,Kim, Ki-Bum IOP 2019 Nanotechnology Vol.30 No.22

<P>Nanopore structures were originally proposed for detection of biomolecule translocation through nanometer-scale pores that perforate membranes by transient changes in ionic current. In this study, these changes are utilized to detect corrosion of different metals in aqueous chlorine media. The corrosion behaviors of Cu, Al, Ti, and Cr were analyzed by monitoring the changes in ion current resulting from ion concentration variations in solutions due to corrosion of the metals. We observed that the Cu layer passivated by CuO<I> <SUB>x</SUB> </I> was severely corroded when a drastic change of ion current was induced, from 10 to 30 nS to the level of 10<SUP>4</SUP> nS, as soon as the bias voltage was applied. In the case of Al passivated by thin AlO<I> <SUB>x</SUB> </I>, the conductance increased from 10–30 to 166?±?52 nS and became saturated. Highly localized pitting corrosion was observed on the periphery of the nanopore, where the electrical field was most concentrated. Finally, we observed that Ti and Cr passivated by oxide showed long-term stability without corrosion in 1 M KCl in the pH range of 4–11.</P>