Ultrasensitive Nucleic Acid Assays Based On Novel Amplification Rotocols And Surface-Enhanced Raman Spectroscopy

In this study, several signal amplification protocols were developed. The developed protocols include Rolling-Circle DNA polymerization and Rolling Circle Transcription combination amplification, triggered nonlinear hybridization chainreaction amplification, and entropy driven the transcription and nonlinear dendritic hybrid cycle of DNA amplification method. These protocols were further coupled with Surface-Enhanced Raman Scattering (SERS) to build sensitive and specific miRNA assays.1. A dual amplification protocol was established by combining Rolling-Circle DNA polymerization (RCP) and Rolling Circle Transcription (RCT); an interlinking stage was designed to achieve such combination. The dual amplification was further coupled with surface-enhanced Raman scattering (SERS) to build a miRNA assay. To implement this design, a single-stranded circular DNA structure was designed and built as the probe, which include the following functional fractions:"Trigger site"-specifically recognized the target miRNA; "Primer switch"-a dynamic segment that is complementary to the primer, which is "locked" at the initial state; this switch was designed to be coupled to the "recognizing trigger":opening of the latter would cause the opening of the former. "MG aptamer template", the DNA segment with complementary sequence to the RNA aptamer of MG; the "nicking switch"-half of the double-stranded nicking site of the nicking endonuclease; it can bind with its complementary strand to form a functional double-stranded nicking site; and "Promoter switch"-half of the functional double-stranded T7 promoter. Based on this probe, the reaction system in this assay started with a tailored triggering strand for target recognition, featured the combination of Rolling Circle Polymerization (RCP) and Rolling Circle Transcription (RCT) that provided dual signal amplification, and utilized the Surface-Enhanced Raman Scattering (SERS) from the malachite green bound on its aptamer as the signal. Specially, an interlinking stage was designed for the combination of RCP and RCT. Due to the design, most parts in the reaction system of the assay were automatic, thus the assay, except for the signal-producing stage, can be operated in one-pot mode. This assay can detect miRNA with high sensitivity, with a detection limit well below 10-16 M. It was further testified that the reported assay posed extra amplification over the RCT amplification itself; it was also testified that the interlinking stage was crucial in this enhancement of sensitivity, since it conveyed the amplification of RCP stage to the downstream stages. The assay was also testified with good specificity due to the trigger stage was sequence-specific to the target miRNA. It was also resistant to the interference in the analyte matrix to certain extent, through the fact that it was capable to detect miRNA in biological matrix or real samples, such as miRNA added in the serum, or miRNA in the RNA extracts from real cells.2. A dendritic hybridization chain reaction was coupled with SERS detection system is designed based on system. In this system, a trgger DNA initiated self-sustained assembly of two assembling units in the form of double strand DNA, resulting in a dendritic structure with ROX (Raman label). The Raman gold nanoparticles were then attached to enhance the Raman scattering signal, and thus the signal amplification was realised. This method required raw material for DNA and hybridization buffer only, without the participation of any enzyme (compared to the system of Rolling-Circle DNA polymerization and Rolling Circle Transcription amplification used joint SERS signal amplifier system); furthermore, the operation was simple and quick.3. Entropy-driven amplification was overlayed on the basis of the previous system, and then coupled with SERS to form a signal amplification method. By design, target miRNA tiggered entropy-driven cycle, resulting in the production of dsDNA in multiplied quantities. These.dsDNA products, by design, can serve as transcription template. Based on this template, transcription occurred, producing messenger RNA in multiplied quantities, which then led to triggered dendritic chain hybridization and assembling of ROX modified DNA strands. Gold nanoparticles were attached to the dendritic assembly to enhance the Raman signal of ROX, realizing the signal amplification.