Label-free sensing platform for miRNA-146a based on chromo-fluorogenic pyrophosphate recognition

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• MicroRNAs (miRNAs) are crucial regulators of gene expression.
• Dysregulation of miRNAs is linked to various diseases.
• Sensitive and selective detection of miRNAs is essential for diagnostics.

Purpose of the Study:

• To develop a novel dual-responsive chromo-fluorescent chelate (1C) for miRNA-146a detection.
• To establish a pyrophosphate (PPi) sensing strategy integrated with rolling circle amplification (RCA).
• To create a robust, selective, and sensitive label-free method for miRNA quantification.

Main Methods:

• Application of a dual-responsive chromo-fluorescent Cu²⁺ chelate (1C).
• Utilizing a pyrophosphate (PPi) sensing strategy within a rolling circle amplification (RCA) process.
• Employing modified T4 ligase and ϕ₂₉ buffer conditions for RCA at pH 7.0.

Main Results:

• The chelate 1C demonstrated selective recognition of Cu²⁺ and PPi ions with distinct color and fluorescence changes.
• Achieved attomolar (fluorogenic) and sub-micromolar (chromogenic) sensitivity for miRNA-146a detection.
• Developed a label-free RCA method for miRNA-146a detection in 40-50 minutes with atto-/nanomolar sensitivity.

Conclusions:

• The developed sensing strategy is highly robust, selective, and sensitive for miRNA-146a detection.
• The cost-effective, label-free approach offers a universal method for detecting miRNAs based on template length.
• This method holds promise for future miRNA-based diagnostics and research.