Keiichi Hatakeyama

Contributions of Phosphate to DNA Adsorption/Desorption Behaviors on Aminosilane-Modified Magnetic Nanoparticles

The adsorption and desorption behaviors of DNA on aminosilane-modified magnetic nanoparticles were investigated by altering both type of anions and solvation state to achieve eficient recovery of DNA useful for subsequent polymerase chain reaction (PCR) analysis. The effects of multiple anions in accordance with the Hofmeister ion series were determined to clarify the contribution of phosphate ions on the effective desorption of DNA from aminosilane surfaces. Efficient DNA desorption (85% recovery) occurred in the presence of 1 M phosphate buffer, however, little DNA desorption was observed using any other anions. This phenomenon indicates that desorption originated from the replacement of DNA by phosphate ions. Furthermore, the adsorption and desorption were significantly affected by the addition of both protic and aprotic solvents. Efficient recovery of adsorbed DNA was attained using deoxynucleotide triphosphates (dNTPs) in place of phosphate buffer and was suitable for subsequent PCR analyses. Therefore, the DNA adsorption/desorption process proposed in this study will be a promising, novel approach for DNA purification with a high recovery ratio that is suitable for subsequent enzymatic reactions, such as PCR or restriction enzyme digestion.

Characterization of magnetic nanoparticles modified with thiol functionalized PAMAM dendron for DNA recovery.

Magnetic nanoparticles (MNPs) modified with the thiol functionalized polyamidoamine (PAMAM) dendron were synthesized to estimate their DNA recovery capabilities. Aminosilane-modified MNPs and MNPs surrounded by a phospholipid (distearoylphosphatidylethanolamine (DSPE)) bilayer were used as core particles. Cystamine-core PAMAM dendrimers were reduced by dithiothreitol to dendron thiols and chemically conjugated to the core particles. Characterization of the synthesis revealed an increase of the surface amine charge from generation 1 (G1) to G6, starting with an aminosilane initiator. Particle size distribution analysis indicated that G6 PAMAM-modified MNPs exhibited monodispersity in an aqueous solution. G6 PAMAM-MNPs and G6 PAMAM-PE-MNPs synthesized by the proposed method have equivalent DNA recovery abilities to PAMAM-MNPs prepared by the conventional divergent synthesis method. In optimized conditions, 96% of λDNA was recovered using G6 PAMAM-PE-MNPs. Therefore, the method for preparing PAMAM-MNPs and PAMAM-PE-MNPs proposed in this study will be a novel approach for producing DNA carriers for efficient DNA purification by magnetic separation.

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk☆

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader™) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader™ consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader™ with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader™ for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.