We have prepared solid substrates modified with a cone-shaped dendron that generates mesospacing (3.2 nm on average) on the surface. This nanoscale-controlled surface provided an ideal DNA microarray in which each probe DNA strand was given ample space for the incoming target DNA, resulting in selectivity as high as that in solution (100: < 1). In addition, high hybridization yield confirms that DNA probes on the mesospaced surface are sterically unhindered for the hybridization.

The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.

Single-stranded 50-mer, 100-mer, and 150-mer DNAs were immobilized on a surface, and force-based atomic force microscopy (AFM) was employed to examine their behavior. A complementary 20-mer probe DNA on an AFM tip was used for the measurements. High-resolution maps were generated, and relevant parameters, including the force, stretching distance, unbinding probability, cluster size, and degree of distortion, were analyzed. Due to thermal drift, the cluster shape became increasingly distorted as the scan speed was decreased and as the map area was reduced. The cluster radius increased with the number of base (N), and the radius was proportional to N(0.6) (r = 0.977) and N(0.53) (r = 0.991). Due to the effect of the pulling angle, the apparent values of the stretching distance and the unbinding force decreased as the AFM probe was moved away from the center position; these values can be described as a function of sin θ.

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 μL or 20 μL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system.

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