| 1. |
- Kuroda, Arisa, et al.
(författare)
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Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell
- 2014
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Ingår i: Analytical Sciences. - : Springer Science and Business Media LLC. - 0910-6340 .- 1348-2246. ; 30:12, s. 1107-1112
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Tidskriftsartikel (refereegranskat)abstract
- In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondria' DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 mu L, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.
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| 2. |
- Sato, Kae, et al.
(författare)
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Bead-based padlock rolling circle amplification for single DNA molecule counting
- 2013
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 437:1, s. 43-45
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Tidskriftsartikel (refereegranskat)abstract
- Padlock rolling circle amplification (RCA) is a powerful analytical method for ultrasensitive DNA detection. Although there are some advantages to bead-based RCA, a detailed study of the relationship between the bead material and the efficiency of bead-based RCA has not been reported. Here, we compared the reaction efficiencies of bead-based RCA performed on two types of bead material: agarose and polystyrene. Agarose was a more suitable material for on-bead RCA. The calibration curve showed linearity between 0.05 and 1 nM, and the limit of detection was 9 pM (9 amol) for Salmonella DNA determination.
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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. |
- Sato, Kae, et al.
(författare)
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Microfluidics for nano-pathophysiology
- 2014
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Ingår i: Advanced Drug Delivery Reviews. - : Elsevier BV. - 0169-409X .- 1872-8294. ; 74, s. 115-121
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Forskningsöversikt (refereegranskat)abstract
- Nanotechnology-based drug delivery systems hold promise for innovative medical treatment of cancers. While drug materials are constantly under development, there are no practical cell-based models to assess whether these materials can reach the target tissue. Recently developed microfluidic systems have revolutionized cell-based experiments. In these systems, vascular endothelial cells and interstitium are set in microchannels that mimic microvessels. Drug permeability can be assayed in these blood vessel models under fluidic conditions that mimic blood flow. In this review, we describe device fabrication, disease model development, nanoparticle permeability assays, and the potential utility of these systems in the future.
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| 5. |
- 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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| 6. |
- 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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| 7. |
- 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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