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Bio-AFM for Single Molecular Level Analysis The nanotechnology for life sciences, nano-biotechnology, is the convergence of nanoscience/technology and biological science, leading to new “eyes and hands” for understanding and manipulating biological systems. In this field, atomic force microscopy (AFM) has attracted keen attentions, because it is compatible with non-conductive materials, does not require labeling, and can be operated under the physiological condition, while allowing single molecular level analysis. Therefore, AFM has become a central tool for nano-biotechnology. Also, imaging surfaces with the nanoscale resolution, probing local mechanical properties, and measuring a variety of interaction forces from nN to pN are possible with the instrument. In particular, the microscopy measuring the force of a picoNewton range has been utilized for a variety of single molecule measurements, and the examples include protein unfolding process, DNA-DNA, protein-protein, and ligand-receptor interaction. At the same time, a significant growth of the genome and proteome studies for drug discovery, as well as for disease diagnosis and prevention, has placed a strong demand for advanced biomolecular recognition probes with high sensitivity and enhanced specificity. It is therefore clear that this type of approach finds important applications not only within basic life science research, but also within emerging areas for the analysis of larger biomolecular libraries.

When the force measurement between individual biomolecules is carried out with AFM, the accuracy and the resolution of the force value is a critical factor for the reliability. In order to enhance the reliability, it is necessary to control density and orientation of the immobilized biomolecules on surface. It used to be hard to control small scale variations due to spatial differences in surface topography and chemistry, and such factor is some of the remaining bottle-necks to the future progress of such force-based approaches. To resolve such issues, NB POSTECH offers a new surface modification of AFM tips and substrates for measuring specific biomolecular interaction forces reliably (J. Am. Chem. Soc. 2007, 9349; Adv. Mater. doi:10.1002/adma.200801323). The dendron-modification of the surfaces can optimize the density of immobilized biomolecules, remove steric hindrance between interacting biomolecules, and avoid unwanted nonspecific binding and/or the formation of multiple biomolecular complexes. Therefore, dendron modified-AFM tip of NB POSTECH provides a superb option to the existing biomolecular surface immobilization methods. We believe that thus-modified AFM tips are ideal for studying the interactions between DNA and DNA/RNA, ligand and protein, protein and protein, as well as receptor on cell surface and ligand.

Bio-AFM Figure 1

Figure 1. A schematic drawing shows predominant 1:1 DNA interaction during the force-distance measurement with AFM

1) DNA-DNA Interaction at Single Molecule Level (J. Am. Chem. Soc. 2007, 9349) The dendron modified-AFM tip simplifies the force-distance curves for the specific biomolecular interaction, and enhances the reliability of the analysis. We confirmed effect of the spacing provided by the dendron layer on the force analysis by using dendrons of different generations. In case of DNA force measurement, the use of the 9-acid gave a narrow histogram in comparison with the cases of the lower generation dendrons (3-acid). It is clear that the control of the spacing between the biomolecules is essential for the fine analysis.


Figure 2. Effect of spacing on the adhesion forces. Distributions of adhesion forces (recorded with a retract velocity of 0.10 μm/s) obtained for 30 mer oligonucleotide functionalized with (a) the first generation dendron (3-acid), (b) the second generation dendron (9-acid). The sequence of the DNA is 5′-NH2-GCT GCT ATG GAG ACA CGC CCT GGA ACG AAG-3′, and its complementary DNA sequence is 5′-NH2-CTT CGT TCC AGG GCG TGT CTC CAT AGC AGC-3′.

The adhesive events were observed by a chance of 50 – 80 %, and the retraction traces with a single clean pull-off event were recorded by using a substrate and an AFM tip with the optimal lateral spacing. The adhesive force histograms displayed the narrow distribution in comparison with the case other immobilization methods provided.


Figure 3. Distribution of adhesive forces recorded at 0.10 μms-1 for all of the fully complementary sequences. The y-axis shows the probability of observing the specific force for each case.

2) Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling (J. Am. Chem. Soc. 2016, 7075) Polymerase chain reaction (PCR) is a highly sensitive and convenient diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. We developed a quantification method, using force–distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (< 2 μm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, a biomarker for chronic myeloid leukemia, we examined samples containing between one and 10 target copies. A high degree of correlation (r2 = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of the DNA biomarker without amplification, modification, or labeling.

Figure 4. High resolution mapping of the entire capture spot area visualizes all of individual captured DNA markers on surface

Table 1. Observed cluster number for the sample of various copy numbers

3) Direct detection of low abundance genes for liquid biopsy (the context will be released soon).