Reading Single DNA with DNA Polymerase Followed by Atomic Force Microscopy

The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). In our approach, four surface conjugated nucleotides were examined sequentially with a DNA polymerase immobilized AFM tip. By observing the specific rupture events upon examination of a matching nucleotide, we could determine the template base bound in the polymerase's active site. The subsequent incorporation of the complementary base in solution enabled the next base to be read. Additionally, we observed that the DNA polymerase could incorporate the surface-conjugated dGTP when the applied force was controlled by employing the force-clamp mode.X1114Ysciescopu

Covalent Positioning of Single DNA Molecules for Nanopatterning

Bottom-up micropatterning or nanopatterning can be viewed as the localization of target molecules to the desired area of a surface. A majority of these processes rely on the physical adsorption of ink-like molecules to the paper-like surface, resulting in unstable immobilization of the target molecules owing to their noncovalent linkage to the surface. Herein, successive single nick-sealing facilitated the covalent immobilization of individual DNA molecules at defined positions on a dendron-coated silicon surface using atomic force microscopy. The covalently-patterned ssDNA was visualized when the streptavidin-coated gold nanoparticles bound to the biotinylated DNA. The successive covalent positioning of the target DNA under ambient conditions may facilitate the bottom-up construction of DNA-based durable nanostructures, nanorobots, or memory system

Micro-/Nanotechnology-Based Isolation and Clinical Significance of Circulating Tumor Cells

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Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability

We independently controlled surface topography and wettability of polystyrene (PS) films by CF4 and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF4 plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of similar to 11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10 degrees, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering. (C) 2012 Elsevier B.V. All rights reserved.X1119sciescopu

Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates

Prediction of hemorrhagic transformation in acute ischemic stroke: role of diffusion-weighted imaging and early parenchymal enhancement

BACKGROUND AND PURPOSE: MR imaging may help in predicting hemorrhagic transformation (HT) in acute ischemic stroke. Our purpose was to determine whether the lesion volumes on diffusion-weighted (DW) imaging, apparent diffusion coefficient (ADC) values, and early parenchymal enhancement are predictive of HT and to investigate the mechanism of the enhancement. METHODS: We retrospectively examined 55 patients with acute ischemic stroke who underwent gadolinium-enhanced MR imaging within 6 hours of symptom onset and follow-up CT or MR imaging within 72 hours. Intravenous thrombolysis was performed in 15 patients. DW imaging lesion volumes and ADC values were compared between patients with and those without HT. ADCs and perfusion parameters were compared between lesions with and those without parenchymal enhancement. RESULTS: Nineteen (34.5%) patients had HT (14 with hemorrhagic infarction, five with parenchymal hematoma). Patients with HT had decreased mean ADCs and large lesion volumes on DW imaging, but differences were not significant (P > .05). HT occurred in five patients (100%) with parenchymal enhancement, which corresponded to the site of HT. In enhancing lesions, the ADC ratio (0.76 +/- 0.06) was slightly higher and the delay in time to peak (0.10 +/- 2.79) was less than respective values in the rest of the ischemic lesion (0.66 +/- 0.06 and 8.79 +/- 4.86, respectively; P = .068). CONCLUSION: Early parenchymal enhancement is highly specific for HT and may be associated with early reperfusion and damage to the blood-brain barrier in ischemic tissue. DW imaging lesion volumes and ADC values had no strong relationship with HT

PDMS-ENCAPSULATED CRACK SENSOR INTEGRATED WITH SILICON RUBBER CANTILEVER FOR USE IN CELL CULTURE MEDIA

In this study, we propose a novel cantilever structure integrated with a high-sensitive and-reliable crack sensor that monitors the contractile behavior of cardiomyocytes in media. Si rubber with excellent performance in cell adhesiveness was used as the cantilever material. The proposed crack sensor formed on the Si rubber cantilever is chemically bonded with the PDMS thin layer to produce a sandwich structure. The protection layer greatly improves the reliability and stability of the crack sensor in the electrolyte solution. The high-sensitive crack sensor stably measures the contractility of cardiomyocytes without changing a gauge factor for up to 26 days.N

Biomechanical Characterization of Cardiomyocyte Using PDMS Pillar with Microgrooves

This paper describes the surface-patterned polydimethylsiloxane (PDMS) pillar arrays for enhancing cell alignment and contraction force in cardiomyocytes. The PDMS micropillar (μpillar) arrays with microgrooves (μgrooves) were fabricated using a unique micro-mold made using SU-8 double layer processes. The spring constant of the μpillar arrays was experimentally confirmed using atomic force microscopy (AFM). After culturing cardiac cells on the two different types of μpillar arrays, with and without grooves on the top of μpillar, the characteristics of the cardiomyocytes were analyzed using a custom-made image analysis system. The alignment of the cardiomyocytes on the μgrooves of the μpillars was clearly observed using a DAPI staining process. The mechanical force generated by the contraction force of the cardiomyocytes was derived from the displacement of the μpillar arrays. The contraction force of the cardiomyocytes aligned on the μgrooves was 20% higher than that of the μpillar arrays without μgrooves. The experimental results prove that applied geometrical stimulus is an effective method for aligning and improving the contraction force of cardiomyocytes

Following the DNA Ligation of a Single Duplex Using Atomic Force Microscopy

Nick-sealing of a single DNA duplex was studied with the use of atomic force microscopy (AFM). To form a nick between a 47mer DNA and a 24mer DNA, the complementary 71mer template DNA immobilized on an AFM tip was hybridized with the 47mer DNA and brought into contact with the 24mer DNA on a substrate surface. The AFM tip and substrate surface were modified with dendron molecules to ensure the formation of a single DNA duplex. When a single nick in the DNA duplex was sealed by DNA ligase during a pause, an increase in the unbinding force was observed after the pause. The change from 24.0 ± 4.4 piconewtons (pN) to 62.8 ± 14.6 pN matched well with the resulting DNA length (71 bp). Additionally, a 30 s pause showed a 3-fold higher nick-sealing probability (60%) than a 10 s pause, while the probability did not increase with a 120 s pause. In the presence of free 47mer DNAs in solution, the single nick-sealing event could be repeated at other positions