| 1. |
- Jang, Kihoon, et al.
(författare)
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Selective cell capture and analysis using shallow antibody-coated microchannels
- 2012
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 6:4, s. 044117-
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Tidskriftsartikel (refereegranskat)abstract
- Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath , Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.
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| 2. |
- Kazoe, Yutaka, et al.
(författare)
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Quantitative characterization of liquids flowing in geometrically controlled sub-100 nm nanofluidic channels
- 2023
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Ingår i: Analytical Sciences. - : Springer Science and Business Media LLC. - 0910-6340 .- 1348-2246. ; 39:6, s. 779-784
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Tidskriftsartikel (refereegranskat)abstract
- With development of nanotechnologies, applications exploiting nanospaces such as single-molecule analysis and high-efficiency separation have been reported, and understanding properties of fluid flows in 101 nm to 102 nm scale spaces becomes important. Nanofluidics has provided a platform of nanochannels with defined size and geometry, and revealed various unique liquid properties including higher water viscosity with dominant surface effects in 102 nm spaces. However, experimental investigation of fluid flows in 101 nm spaces is still difficult owing to lack of fabrication procedure for 101 nm nanochannels with smooth walls and precisely controlled geometry. In the present study, we established a top-down fabrication process to realize fused-silica nanochannels with 101 nm scale size, 100 nm roughness and rectangular cross-sectional shape with an aspect ratio of 1. Utilizing a method of mass flowmetry developed by our group, accurate measurements of ultra-low flow rates in sub-100 nm nanochannels with sizes of 70 nm and 100 nm were demonstrated. The results suggested that the viscosity of water in these sub-100 nm nanochannels was approximately 5 times higher than that in the bulk, while that of dimethyl sulfoxide was similar to the bulk value. The obtained liquid permeability in the nanochannels can be explained by a hypothesis of loosely structured liquid phase near the wall generated by interactions between the surface silanol groups and protic solvent molecules. The present results suggest the importance of considering the species of solvent, the surface chemical groups, and the size and geometry of nanospaces when designing nanofluidic devices and membranes. Graphical abstract: [Figure not available: see fulltext.].
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| 3. |
- Sato, Kae, et al.
(författare)
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Microbead-based rolling circle amplification in a microchip for sensitive DNA detection
- 2010
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:10, s. 1262-1266
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Tidskriftsartikel (refereegranskat)abstract
- The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.
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| 4. |
- Shirai, Kentaro, et al.
(författare)
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Graft linker immobilization for spatial control of protein immobilization inside fused microchips
- 2009
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 30:24, s. 4251-4255
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Tidskriftsartikel (refereegranskat)abstract
- Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CHx (x = 1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.
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| 5. |
- Tanaka, Yo, et al.
(författare)
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Single-Molecule DNA Patterning and Detection by Padlock Probing and Rolling Circle Amplification in Microchannels for Analysis of Small Sample Volumes
- 2011
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 83:9, s. 3352-3357
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Tidskriftsartikel (refereegranskat)abstract
- The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.
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