A universal ratiometric method for Micro-RNA detection based on the ratio of electrochemical/electrochemiluminescence signal, and toehold-mediated strand displacement amplification.

An ultra-selective and reproductive ratiometric platform was introduced based on the ratio of Ru(phen)32+ electrochemiluminescence (ECL) signal and methylene blue (MB) electrochemistry (EC) signal, which was amplified using a specific and efficient toehold-mediated strand displacement (TMSD). The stable DNA nanoclews (NCs) were efficiently loaded with MB (MB-NCs) as EC signal tags after being synthesized utilizing a simple rolling circle amplification reaction. Besides, Ti3C2-based nanocomposite could apply as a superb carrier for both Ru(phen)32+ and gold nanoparticles (Ti3C2-Au-Ru), resulting in a nearly constant ECL internal reference to eliminate the possible interferences. The Ti3C2-Au-Ru was attached to the surface of the electrode using Nafion, which exhibited excellent conductivity, and hairpin DNAs (hDNAs) were fixed on AuNPs via an Au-S bond. The designed biosensor was finally applied for miRNA-18a detection as a target model. The TMSD method made it possible to concurrently convert and amplify a single miRNA-18ainput into a large amount of output DNAs with high selectivity. These output DNAs were designed to unfold the stem-locked area of hDNAs. The opened hDNAs then hybridized with the MB-NCs to produce an EC signal. In the proposed biosensing system, by raising the target concentration of miRNA, the EC signal gradually rose, the ECL signal remained nearly constant, and the ratiometric detection method markedly promoted biosensor accuracy. Linear correlations of the ratio value of the EC/ECL with miRNA-18a concentrations between 20 aM and 50 pMwere observed, with the limit of detection of 9 aM. The biosensor was applied to detect miRNA-18a in real serum samples with satisfactory results.

A rapid and simple method for simultaneous determination of three breast cancer related microRNAs based on magnetic nanoparticles modified with S9.6 antibody.

Cancer progression is typically associated with the simultaneous changes of multiple microRNA (miR) levels. Therefore, simultaneous determination of multiple miR biomarkers exhibits great promise in early diagnosis of cancers. This research seeks to discuss a simple biosensing method for the ultrasensitive and specific detection of the three miRs related to the breast cancer based on S9.6 antibody coated magnetic beads, titanium phosphate nanospheres, and screen-printed carbon electrode. To prepare signaling probes, three hairpin DNAs (hDNAs) were labeled with three encoding titanium phosphate nanospheres with large quantities of different heavy metal ions (zinc, cadmium, lead), which have been utilized to discriminate the signals of three microRNA targets in relation with the corresponding heavy metal ions. After that, these hairpin structures hybridize with miR-21, miR-155 and miR-10b to form miR-21/hDNA1, miR-155/hDNA2 and miR-10b/hDNA3 complexes, which were captured by S9.6 antibodies (one anti-DNA/RNA antibody) pre-modified on magnetic bead surface. Therefore, the specific preconcentration of targets from complex matrixes can be carried out using magnetic actuation, increasing the sensitivity and specificity of the detection. The biosensor was suitably applied for direct and rapid detection of multiple microRNAs in real sample. It was observed that there were no significant differences between the results obtained by the suggested method and qRT-PCR as a reference method. So, this method makes an ultrasensitive novel platform for miRNAs expression profiling in clinical diagnosis and biomedical research.

A novel signal amplification tag to develop rapid and sensitive aptamer-based biosensors.

Determination of microRNAs (miRNAs) as valuable blood-borne biomarkers has attracted many scientific attentions. However, analytical methods are still restricted by miRNAs intrinsic characteristics. In this study, for the first time, novel blackberry-like magnetic DNA/FMMA nanospheres were synthesized and mounted on a gold stir-bar as signal amplification probes. To produce this strong electrochemical signal label, double strand DNAs were immobilized on gold coated magnetic nanospheres through a hybridization chain reaction followed by reversible addition-fragmentation chain-transfer polymerization, which brought a great quantity of the electroactive tags (FMMA) on the nanosphere surface. These nanospheres were then fixed on the gold stir-bar as signal probes. The magnetic DNA/FMMA nanosphere probes can be released by substituting with the newly emerging DNA fragments of catalyzed hairpin assembly products. Eventually, these signal probes were magnetically enriched on the electrode surface to produce electrochemical signal and finally, the biosensor was developed to detect miRNA-106a (model target). The suggested aptamer-based biosensor demonstrated considerable selectivity, acceptable storage stability, high specificity, and excellent performance in real sample analysis without any pretreatments. As a result, current study reveals that the developed strategy has a great potential for the early diagnosis of gastric cancer and additionally the clinical monitoring of any miRNA sequences.