단 분자 힘 분광법을 이용한 아토 몰 농도 미만 DNA의 정량 분석

Abstract

Single molecule force spectroscopy has been widely used in biological study such as molecular adhesion between DNA-DNA and ligand-receptor, mechanical properties of a cell and tissue surface, and dynamics of motor proteins. With nanometric resolution, atomic force microscopy (AFM) has been applied for quantifying individual biomolecules distributed on surface. However, the capability of the approach has not been fully explored. It has been suggested that use of a small capture spot and scanning the whole area or a sizeable part of it will enhance the detection limit below to attomolar concentrations. Such necessary component to realize the potential capacity has been developed, and dip-pen nanolithography (DPN) and FluidFM technology are examples. While real-time polymerase chain reaction (RT-PCR) has been widely used for its high sensitivity, the limitations are use of fluorescence dyes, DNA size restraint, and amplification distortion. Recently, a few approaches utilizing nanoparticles have been developed by amplifying the detection signal instead of the target molecule amplification, but, these require labeling of the target with nanoparticles. Here, we report a new detection platform combining the nanocontact printing and AFM force mapping. Through the DNA-DNA hybridization, the target DNAs were captured specifically, and individual DNAs were counted from the force map. Because spots of micron size were fabricated with the nanocontact printing, the whole part or a significant part of the spot can be scanned within a reasonable time. As a proof of concept, a DNA biomarker for chronic myeloid leukemia (CML), BCR-ABL gene, was examined. The label-free and amplification free-detection method showing the ultimate sensitivity may find many usage in clinical diagnosis that dodges PCR analysis. For example, a precise answer whether a CML patient is totally cured by the treatment of Gleevec will be obtained with the approach. Combined with high-speed AFM, our method can be faster in speed, and with the use of the multi probe AFM technology, the multiplexing can be even realized in the near future.