MicroRNA Detection Using Plasmon-Enhanced Fluorescence Nanoparticles and Atomic Force Microscopy

Abstract

Chapter I. Detection Methods for MicroRNA MicroRNA (miRNA), a short (19~25 nt) non-coding single strand RNA is a regulator on the corresponding target messenger RNAs (mRNAs), and modulates the post-transcription. miRNAs control the expression level of target proteins, and play critical roles in multiple biological processes, including cell differentiation, proliferation, and apoptosis as well as disease processes such as cancers, Alzheimer’s disease, and myocardial disease. Therefore, precise quantitative analysis of miRNAs is important for clinical diagnosis and prognosis of disease. Conventional methods for the quantitative analysis of miRNA such as northern blot, Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and microarray were introduced in this chapter. Drawbacks such as low sensitivity, low specificity, and poor signal intensity, and lengthy analysis time are observed in these methods. Therefore, more sensitive and specific diagnostic assays are urgently needed to detect miRNAs without time-consuming amplification processes and purification steps. In order to overcome such hurdles, emerging tools in combination of nanomaterials are being developed. Especially, specifically designed nanoparticles suggest a possible pathway for the improvement. Due to unique electrical or optical properties of the nanostructure, nanomaterials have been widely used as conductors and optical emitters to obtain the improved detection. The approach offers multiple advantages such as high sensitivity, good specificity, multiplexing capability, and easy operation, and is regarded as an attractive platform for the miRNA detection.

Chapter Ⅱ. Microarray-Based Detection of MicroRNA with Plasmon-Enhanced Fluorescence Nanoparticles One of the most common methodologies for the miRNA detection is the microarray, and high throughput and multiplexing capability are characteristics. However, a relatively low sensitivity limits the application, and the shortcoming can be only overcome only when it is combined with the amplification step. Here, we report a new microarray-based platform utilizing a plasmon-enhanced fluorescence nanoparticle, a core-shell nano-rod in which fluorophores maintain a specific distance from the metal surface. In this application, we selected miRNA-134 as a target miRNA, known as a brain-specific one that regulates the dendritic spine development. Also, a substrate with finely controlled surface, S9.6 antibody, and goat anti-mouse secondary antibody-tethered plasmon-enhanced fluorescence nanoparticle (FS-FANC-4) were employed to improve the sensitivity. The latter particles were coated with a silica shell for higher stability, and the antibody was conjugated on the shell surface. Coating glass slides with dendrons provided an ideal surface for DNA-microarray, resulting in single nucleotide discrimination efficiency as high as that in solution, and such coating plays a critical role to realize 1:1 interaction reproducibly in the force measurement with an atomic force microscope. After allowing to form DNA/miRNA duplexes through the hybridization, mouse monoclonal antibody S9.6 was used to bind to the duplexes. The secondary antibody tethered FANC resulted in ca. 186 fluorescence enhancement with respect to fluorescence intensity of free Alexa FluorTM 647-modified thiolated oligonucleotide solution. Through combination of the components described above ca. 103-fold higher sensitivity in comparison with other microarray approaches was observed. In other words, the limit of detection was improved to 100 aM ~ 1 pM.

Chapter Ⅲ. Analysis of Nanoparticle Population using Atomic Force Microscopy Atomic force microscopy (AFM) has been widely employed for multi-parametric nanoscale imaging. Here, we report a new approach to analyze and validate the microarray results using the AFM imaging. We measured total area of the surface-captured nanoparticles for each target concentration by AFM. Importantly, it was observed that the total area was proportional with the fluorescence intensity. Whereas the expected LOD with AFM is lower than that with fluorescence, it is found that two LODs are same, because the limit comes from the nonspecific binding of the nanoparticles on the blank spot. Our microarray and nanoparticle-based approach can be applicable to various miRNAs, and such lower limit of detection should be welcome in the relevant community.