Quantification of DNA and protein biomarkers without amplification using AFM

In the following we propose to study quantification of DNA and protein biomarkers using atomic force microscopy (AFM). The primary goals are 1) quantification of DNA biomarkers of chronic myeloid leukemia (LIMK1) under 10 copies without amplification, 2) quantification of protein biomarkers in a single neuronal cell, 3) examining two types of AFM, NanoWizard III of JPK and TREC of Keysight (Agilent), and characterizing and optimizing two approaches. Parks group (Pohang University of Science and Technology (Postech), Republic of Korea) demonstrated that the detection limit of AFM can be dramatically enhanced when the whole area of a capture spot is scanned with the force-mapping mode, and the observed detection limit for the translocated gene (b2a2) is the single copy. Also, Parks group demonstrated previously that quantification of miR-134 in a single neuronal cell is possible with the same mode. The group will move forward to quantify a corresponding protein biomarker (LIMK1) in the single cell. For this end, a small spot (5 microns in diameter) of the capture antibody will be fabricated, and a subsection of the spot will be scanned to count the captured LIMK1. Parks group will examine various parameters of Nanowizard III, whereas Hinterdorfers group (Johannes Kepler University Linz (JKU), Austria) will study biomarker distributions at the nano-scale using various high-resolution AFM techniques developed in his lab, such as molecular recognition imaging (TREC) and single molecular force spectroscopy, on functional nanoarrays. The accessibility of the affinity tags will be verified by single-molecule force spectroscopy studies, while simultaneous topography and recognition imaging (TREC) of the nanoarray will result in high-resolution maps of the molecular binding sites with a positional accuracy of few nanometres. The combination of high- resolution atomic force microscope recognition imaging with single molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events, and allows to identify and localize specific binding sites with unprecedented resolution. The combined effort of the two groups will provide understanding on the molecular recognition process in depth and establish AFM as a tool for the highly sensitive biomarker quantification without amplification. PR_Abstract_eng

Our overall aim was: (i) to establish atomic force microscope as a tool to quantify DNA and protein biomarkers without amplification or fluorescence labeling, (ii) to understand the molecular recognition process during lateral mapping, and (iii) to find the most effective mapping modes for high reliability and short scanning time. For this, we utilized and tested the methods established in Park's group in Korea and in Hinterdorfer's group in Austria, and developed these strategies further in a collaborative and synergistic approach. Finally, the combined efforts of the two groups provided understanding on the molecular recognition process in depth, established AFM as a tool for the high sensitive biomarker quantification without amplification, and clarified the effective and reliable approaches for the use in a wider community with the implementation of our modifications.