Increasing the sensitivity and selectivity of a GONS quenched probe for an mRNA assay assisted with duplex specific nuclease

Abstract

The authors report a new graphene oxide nanosheet (GONS) based fluorescence method for mRNA assay with duplex-specific nuclease (DSN)-assisted signal amplification.

Hairpin-structured probe conjugated nano-graphene oxide for the cellular detection of connective tissue growth factor mRNA

“…They were absorbed on the surface of nano-GO sheets via noncovalent interactions including π-π stacking, hydrogen bonding, hydrophobic interaction, and van der Waals forces, forming the nano-GO/HP probe assembly. [21,22,19] The sensing principle of the assembly was based on the two characteristics of nano-GO sheets: 1) the high fluorescence quenching efficiency of nano-GO sheets; 2) the distinct difference on the affinity of nano-GO sheets towards single-stranded and double-stranded DNA. [18] The fluorescence quenching was confirmed by monitoring the fluorescence change of the HP solution with the continuous addition of nano-GO.…”

“…Once inside a cell, nano-GO can protect the absorbed probes from nuclease unspecific digestion. [19] Moreover, the excellent fluorescence quenching ability of nano-GO would provide a low background and thus enable the sensitive response to analytes. [20] Although nano-GO has been applied for many applications such as apoptosis monitoring [21], cancer diagnosis [22], and induced pluripotent stem cell generation [23], it has not been adapted for in skin cell identification.…”

Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA

“…[8,9] Moreover, as protein expression levels are mirrored in mRNA concentration, mRNA biomarkers are also useful for detecting cell abnormalities. [10,11] Finally, microRNA (miRNA) is a class of naturally occurring RNA of short length, [12] and the abundance of specific sequences can be correlated with various diseases such as cancer, diabetes, heart failure and diabetes mellitus amongst others. [2,[13][14][15] Thus, the development of a simple, rapid and sensitive RNA detection platform that can be performed with minimal training and in limited resource settings is key for early disease diagnosis, prognosis, and treatment especially in low income countries where infectious diseases are more prevalent.…”

“…[29,30] Next the cDNA is liberated from the RNA by digestion or nicking enzymes followed by amplification by polymerase. [11,28] These sequential steps to form the cDNA and then amplify it often lead to the use of multiple temperatures even when isothermal amplification methods are employed [31]. To overcome these problems and present a complementary approach to RT-polymerase based methods, we describe a simple, instrument-free method for detecting RNA that is completely performed at room temperature, uses only one enzyme (a ligase) and colorimetric detection.…”