In situ biosensor for detection miRNA in living cells based on carbon nitride nanosheets with catalytic hairpin assembly amplification

Abstract

In this study, an ultrasensitive fluorescence turn-on assay for in situ sensing of intracellular microRNA (miRNA) was developed utilizing a carbon nitride nanosheet (CNNS) and a catalytic hairpin assembly (CHA). The CHA showed favourable signal amplification for low-level biomarkers, and CNNS was an excellent candidate as a fluorescence quencher and gene vector.Moreover, the hairpin DNA of CHA could be adsorbed onto the surface of CNNS. An enzymefree fluorescence biosensor for ultrasensitive sensing of intracellular miRNA in cells based on CHA and CNNS was designed. When faced with target miRNA, the fluorescence was recovered due to the miRNA, which could trigger cycling of CHA circuits, leading to the production of a marked enhanced fluorescence signal. Compared with traditional methods, the proposed method is convenient, with low cytotoxicity, and high specificity and ultrasensitivity. It has promising potential for detection low-level biomarkers. KEYWORDScarbon nitride nanosheet, catalytic hairpin assembly, intracellular signal amplification, microRNA, nanoprobe

Applications of Catalytic Hairpin Assembly Reaction in Biosensing

“…are labeled onto the hairpins, which is the most simple and direct method. Additionally, the fluorophores are separated from the quencher or nano materials (graphene oxide, Au) through fluorescence quenching ability to release signals . This strategy has been employed to develop robust and user‐friendly strip biosensor for visual, rapid and sensitive detection of microRNAs (miRNA) .…”

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges

“…Due to the advantages of unique optical properties, large surface area and good biocompatibility, several nanomaterials, such as carbon nitride nanosheet [97], GO [98] and MoS 2 nanosheets [99], can be used as efficient fluorescence quenchers for the fluorophore-labeled probe based miRNA detection. Intriguingly, fluorescent metal nanoclusters (<1 nm), collected from two to tens atoms of gold or silver [100], have been developed as a new class of fluorophores and widely applied in the area of miRNA bioassays owing to their facile synthesis and tunable fluorescence emission [101].…”

“…Fluorescence-labelled probes can be divided into fluorescence-labelled ssDNA/ssRNA probes and fluorescence-labelled MBs. The former ones are typically used in nanomaterial-based biosensors in which their fluorescence is quenched when immobilized and restored upon capturing the target miRNA [27,[97], [98], [99]]. The latter ones rely on the distance-dependent fluorescence quenching property, because they can be disrupted to linear forms in the presence of target miRNA, thus restoring their fluorescence [13,31,32].…”

Chemiluminescence assay for kanamycin based on target recycling strategy

“…[12][13][14] Catalytic hairpin assemblies (CHA), as a mature enzyme-free DNA amplification technique with high efficiency and target cycle, have been applied in the field of signal transduction and amplification related to nucleic acid analysis and biosensor. [15,16] Chemiluminescence (CL) is a phenomenon that produces light radiation through chemical reactions. [17,18] It has the strengths of high sensitivity, wide linear range, low detection limit, and no need for an excitation light source.…”

“…[ 12–14 ] Catalytic hairpin assemblies (CHA), as a mature enzyme‐free DNA amplification technique with high efficiency and target cycle, have been applied in the field of signal transduction and amplification related to nucleic acid analysis and biosensor. [ 15,16 ]…”