Direct quantitative analysis of HCV RNA by atomic force microscopy without labeling or amplification

Abstract

Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force–distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30–60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

Label-Free Sensing of Adenosine Based on Force Variations Induced by Molecular Recognition

“…AFM-based SMFS can also be employed as a promising label-free biosensing technique with high sensitivity. Until now, there are a few reports on the detection of biomolecules with SMFS [ 23 , 24 , 25 , 26 ]. For example, Zhang and co-workers reported SMFS-based detection of DNA mismatched hybridization [ 23 ].…”

Imaging a specific mRNA in pollen with atomic force microscopy

“…For the enhanced specicity, the sequence of 34 bases was utilized instead of the previously employed length, 18 bases. 27 The interaction force between the DNA probe immobilized on the AFM tip and the 34-mer target RNA immobilized on a substrate is shown in Fig. 1A.…”

“…[22][23][24][25][26] We reported imaging of Pax6 mRNA distribution in mouse brain tissue, 23 and more recently, demonstrated its capability to quantify the concentration of hepatitis C virus (HCV) RNA without labeling or amplication. 27 In the current work, we investigate the subcellular localization and distribution of AtAMT1;4 mRNA, which encodes an ammonium transporter (AMT), 28 in the sperm cells of the model plant Arabidopsis. Ammonium is the main source of nitrogen for plant growth and development.…”

Principles and Applications of Force Spectroscopy Using Atomic Force Microscopy

“…The controlled spacing between surface‐immobilized molecules enabled reliable and reproducible force measurements . In addition, the capture of target molecules on a homogeneous surface with controlled density has led to the development of a sensitive quantification method using AFM . Individual biomarkers are visualized on a subsection of a probe surface; then, the average number of detected biomarkers in the area is used to estimate the total number captured on the entire probe surface.…”