Patterned Co-culture of Live Cells on a Microchip by Photocrosslinking with Benzophenone.

The patterned coculture of different types of living cells in a microfluidic device is crucial for the analysis of cellular interactions and cell-cell communication. In the present study, cell patterning was achieved by photocrosslinking benzophenone derivatives in a microfluidic channel. Optimization of UV irradiation conditions enabled successful fixation of live cells. In addition, patterning and co-culture of non-adherent K562 cells and adherent RF-6A cells was achieved by successive rounds of patterning. The present approach is expected to be useful for the development of in vitro methods for studying cell signaling.

Bead-based padlock rolling circle amplification for single DNA molecule counting.

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.

Photochemical immobilization of cells onto a glass substrate for in situ DNA analysis.

A simple and robust method to immobilize cells onto a glass substrate is presented. The method employs a photochemical reaction of benzophenone, which is modified on the substrate using a standard silane coupling agent, with cells. Cells were immobilized to an area irradiated with UV light from a standard light source under an inverted microscope. The dependence of immobilization on the light power intensity and irradiation time was investigated. In situ DNA analysis within the immobilized cells was demonstrated using target-primed rolling circle amplification and fluorescent detection.

Selective cell capture and analysis using shallow antibody-coated microchannels.

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 et al., 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.