Recent findings indicate that microRNA (miRNA) signatures have significant potential as diagnostic, prognostic, and treatment-response biomarkers for various diseases 1, 2. miRNAs, acting as key regulators, have the ability to influence the expression of numerous genes in various tissues 3. This regulation extends to almost all cellular processes at the transcriptional level 4, encompassing cellular development, proliferation, migration, survival, metabolism, homeostasis, and regeneration. Prior studies have shown that a miRNA-based biomarker can be used to effectively guide the selection of nursing techniques for patients undergoing surgery 5, 6. Therefore, a precise analysis of the expression level of miRNAs can offer vital insights into their regulation and their use as biomarkers for the early diagnosis and treatment of malignancies. Hence, there is a significant need to create simple and reliable techniques for miRNA detection.
The majority of current techniques, such as northern blotting, microarray 7, 8, and PCR-based methods 9, 10, used signal amplification, target conversion, or enrichment strategy to achieve a relatively high sensitivity and selectivity because of the low abundance, short sequence, and high sequence homology of miRNAs. PCR-based techniques have gained significant popularity due to their excellent performance and broad applicability. However, the use of multiple enzymes, particularly in reverse transcription PCR-based techniques, leads to increased experimental costs and design complexities, as well as the requirement for a thermal cycler to accurately regulate the reaction temperature 11. These approaches cannot be implemented in point-of-care settings or locations with limited resources, in addition to the aforementioned disadvantages. Enzyme-assisted isothermal amplification approaches have demonstrated significant potential for “on the spot” miRNA testing 12–14. The polymerase/endonuclease assisted chain displacement technique has gained significant attention because of its excellent amplification efficiency and reproducibility. However, the polymerase/endonuclease assisted chain displacement approach often necessitates integration with fluorescence transducers and suffers from insufficient selectivity. The colorimetric approach with naked-eye recognition offers significant advantages over the fluorescence transducer for miRNA detection 15, 16. Due to their robust distance-dependent optical characteristics and the ability to change color when aggregated, gold nanoparticles (AuNPs) have been extensively applied in colorimetric assays 17, 18. This single-stranded DNA (ssDNA)-regulated state alteration of AuNPs is typically achieved through DNA hybridization, which is a one-to-one signal transduction process; its sensitivity is inadequate for the detection of miRNA, which has stringent sensitivity requirements, despite its considerable achievements. Hence, increased selectivity and simplicity are necessary for a viable alternative.
Therefore, we have devised a novel and efficient colorimetric method that ingeniously employs a dumbbell probe to enhance the specificity of polymerase/endonuclease assisted chain displacement, together with silver ions (Ag+) to facilitate the color reaction (Fig. 1). The sensing technique involves the designing of a dumbbell probe that consists of five distinct functional portions. Within this probe, the "1" segment contains sites that bind to the target miRNA, the "2" fragment aids in the recycling of the target, the "3" fragment is capable of transcribing sites that are recognized by the endonuclease, and the "5" fragment serves as a primer to initiate the color reaction. When the target miRNA is present, it hybridizes with the "1" fragment and slowly unravels the "stem" region in the hairpin structure, revealing the "2" fragment to serves as a recognition site for the "6" fragment in the assistance probe. The "6" fragment substitutes for the target miRNA from the dumbbell probe by binding to the "2" fragment. The target microRNA that was released forms a signal cycle by binding with a next dumbbell probe. The "6" fragment serves as a primer to commence the chain extension with the assistance of DNA polymerase, encoding the "8" fragment using "3" as the template. The endonuclease identifies the "8" segment and creates a nicking site. Through the collaboration of DNA polymerase and endonuclease, a substantial quantity of ssDNA sequences, consisting of “9” and “10” fragments are generated. The H probe is formed when the ssDNA products assemble into a hairpin structure; this structure is responsible for inducing the Ag + aptamer based color response. The aptamer was utilized to bind silver ions (Ag+) through the interaction between Ag + and the N3 of cytosine (C), which connects two cytosine residues to create a strong and stable "C–Ag+-C" hairpin structure. Specifically, the “10” fragment binds with the Ag + aptamer that is fixed on the surface of magnetic bead (Ag + aptamer@MB), and works as a primer to encode a complementary sequence under the assistance of the DNA polymerase. The Ag+ aptamer’s capacity to chelate Ag + is hindered by the creation of a double-stranded DNA structure (dsDNA). Therefore, Ag + was unable to chelate with the aptamer and instead underwent a reaction with TMB, resulting in the color reaction. Under these circumstances, the solution containing oxidized TMB (TMBox) had a blue color and a distinctive UV–vis absorption peak at 652 nm. If miRNA is not present, the formation of dsDNA product will not take place, and the aptamer will remain intact and can be extracted using a magnetic rack. Through this procedure, Ag+ ions can attach to the aptamer that has been magnetically enhanced by interacting with cytosine to create a hairpin structure known as "C–Ag+-C". As a result, the solution loses its color in the absence of TMBox.