A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids
ABSTRACT Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.
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- "Mathematical models were introduced for the quantitative analysis of the dynamics and stability of cellular status at the genome, transcriptome, proteome, and/or metabolome levels. This type of single-cell study showed that the mRNA          and protein expression         levels show extreme fluctuation in not only undifferentiated but also all kinds of cells. "
ABSTRACT: mRNA from single cells was quantified using real-time RT-PCR after recording the address and reporter protein activity with chemiluminescence, fluorescence, and electrochemical techniques, using luciferase, green fluorescent protein, and secreted alkaline phosphatase. mRNA copy number ranging from below 10(3) to 10(7) in single cells showed a lognormal distribution for both externally introduced reporter genes and internally expressed genes. The fluctuation in the gene expression decreased with the increase of the number of cells picked but did not decrease with the increase of mRNA copy number per cell. We found that the correlation coefficients for mRNA and protein expression in logarithmic plot at single-cell level were much lower than 1.00.FEBS letters 09/2010; 584(18):4000-8. DOI:10.1016/j.febslet.2010.08.008 · 3.17 Impact Factor
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ABSTRACT: Chemically and topographically patterned surfaces have high potential as model surfaces for studying cell and bacteria responses to surface chemistry and surface topography at a nanoscale level. In this work, we demonstrated the possibility to combine pulsed plasma polymerization and UV-irradiation to obtain topographical patterns and chemical patterns perfectly controlled at microlateral resolution and sub-micrometer depth level. Biological experiments were conducted using human osteoprogenitor cells and Escherichia coli K12. Proliferation and orientation of cells and bacteria were analyzed and discussed according to the size and the chemistry of the features. This work showed interesting opposite behavior of bacteria compared to eukaryotic cells, in response to the surface chemistry and to the surface topography. This result may be particularly useful on medical implants. From a methodological point of view, it highlighted the importance of working with versatile and well-characterized surfaces before and after sterilization. It also points out the relevance and the necessity of analyzing eukaryotic cell and bacteria adhesion in parallel way.Langmuir 07/2009; 25(14):8161-9. DOI:10.1021/la900457f · 4.46 Impact Factor
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ABSTRACT: Microfluidics have emerged as powerful processing and analytical tools. However, as they are usually closed systems, they are difficult to use with large samples and surfaces that cannot pass inside their conduits. Here, we extend our previously reported microfluidic probe (MFP) concept, in which a focused stream of liquid is scanned over a surface, for which heads were fabricated using one Si wafer and a molded poly(dimethylsiloxane) (PDMS) block. The new heads comprise a microfabricated Si lid, a chip for hydrodynamic flow confinement (HFC) and standard ports and fittings. The HFC chip carries the smallest structures needed for focusing the liquid on the surface; the Si lid is bonded to the HFC chip using a polyimide adhesive and has macroscopic vias for bonding standard ports and fittings. These heads are easy to fabricate at high yield, have high-resolution potential and are compatible with a large number of chemicals and solvents. We exemplify this by patterning proteins on a surface, directly developing a resist using a high-pH developer, and producing tapered structures in a resist. This work should foster the adoption of MFPs for processing, patterning and analyzing surfaces under microfluidic conditions.Journal of Micromechanics and Microengineering 09/2009; 19(11):115006. DOI:10.1088/0960-1317/19/11/115006 · 1.73 Impact Factor