A dual-signaling electrochemical ratiometric strategy combining "signal-off" and "signal-on" approaches for detection of MicroRNAs.

A dual-signaling electrochemical ratio metric strategy was developed for detection microRNA-18a based on the duplex-specific nuclease-assisted target recycling and electrochemical atom transfer radical polymerization signal amplification. In the presence of target microRNA, RNA/DNA duplexes are formed, which become the substrate of the duplex-specific nuclease-assisted target recycling. Hence only the DNA strand is cleaved by duplex-specific nuclease enzyme, resulting in the throw away of methylene blue (MB) from the electrode (signal off) accompanied by releasing of target microRNA, which can be recycled in the next hybridization. The remaining piece of capture DNAs on the electrode surface hybridize with the Azide labeled-signal DNAs. "Click reactions" were carried out between 3-Butynyl-2-bromoisobutyrate and Azide to initiate the electrochemical atom transfer radical polymerization reaction. This process could bring a great number of ferrocenylmethyl methacrylate (FMMA) on the surface of electrode (signal on). The IFMMA/IMB value was proportionate to the microRNA-18a concentration and measured by square wave voltammetry. The promising potential of the proposed biosensor in clinical analyses was exhibited by its remarkable features such as strong performance, high specificity, agreeable storage stability, and notable selectivity in real sample evaluation with no pretreatment or amplification. Finally, our biosensing method offers such an application to be used for the early clinical diagnosis of Pancreatic Cancer.

An enzyme-free electrochemical biosensor for simultaneous detection of two hemophilia A biomarkers: Combining target recycling with quantum dots-encapsulated metal-organic frameworks for signal amplification.

A sensitive and selective electrochemical method for simultaneous detection of two hemophilia A-related microRNAs (miR-1246 and miR-4521) was developed. This detection tactic was based on gold nanoparticles (AuNPs), heavy metals quantum dots-encapsulated metal-organic frameworks (QDs@ZIF-8), and catalytic hairpin assembly (CHA) for signal application. Primarily, hairpins H1 and H2 were hybridized with targets miR-1246 (T1) and miR-4521 (T2) for forming H1-T1 and H2-T2 duplex stranded DNAs (dsDNAs) that were able to open the hairpins H3 and H4 for the formation of H1-H3 and H2-H4 dsDNAs. Meanwhile, lots of H1-H3 and H2-H4 dsDNAs were created by releasing the target to take part in the next cycle for signal amplification. And then single stranded fragments of H1-H3 and H2-H4 dsDNAs were utilized for hybridizing the PbS@ZIF-8-S1 and CdS@ZIF-8-S2 in order to amplify the electrochemical signal. The diagnosis of corresponding target miRs using differential pulse voltammetry has been possible by releasing Pb (II) and Cd (II) ions from PbS@ZIF-8 and CdS@ZIF-8 tags by HCI leaching. In this context, encapsulation of heavy metals quantum dots (QDs) was done in zeolitic imidazolate framework-8 (ZIF-8) to form QDs@ZIF-8 muti-core-shell particles by in situ growth of ZIF-8 in the presence of QDs. Since the quantity of QDs tagged to each target miRs grows massively, being resulted from a huge number of QDs that encapsulated in each QDs@ZIF-8 label, the sensitivity of the biosensor using QDs@ZIF-8 particles as signal tags is about 15 times that of a biosensor using QDs as signal tags. Several conditions of determination like incubation time for labeling and capture probe, HCl leaching time, and reaction time of CHA were optimized. Under the optimized conditions, this assay allowed the detection of target miRs in the range of 1 fM to 1  µM with detection limits of 0.19 fM and 0.28 fM for miR-1246 and miR-4521 (S/N = 3). The biosensor can discriminate complementary, 1-base mismatched and non-complementary sequences quite well, according to the catalytic hairpin assembly. Furthermore, the biosensor was utilized efficiently for quick and direct analysis of microRNAs in human serum. Thus, this tactic presents an innovative platform for microRNAs expression profiling in biomedical research and clinical diagnosis.

[Tumor escape mechanism involving Fas and Fas-L molecules in human colorectal tumors].

OBJECTIVES: The interaction between Fas and its ligand (Fas-L) leads to Fas-positive cell apoptosis. Our objective was to study a new mechanism of tumor escape involving these molecules, the so-called "counterattack". METHODS: We used flow cytometry to analyze Fas expression and apoptosis sensitivity in different human colorectal tumor cell lines. The presence of Fas-L mRNA was analyzed by RT-PCR. We studied apoptosis rate in peripheral blood lymphocytes and lymph node lymphocytes from patients with colorectal cancer by flow cytometric cell cycle analysis after in vitro culture with or without tumor cells. RESULTS: We found differences in Fas expression and sensitivity to Fas-induced apoptosis between different colorectal tumor cell lines. Interferon-gamma was also found to affect Fas expression and apoptosis sensitivity induced by an anti-Fas antibody. Actinomycin-D decreased Fas expression and apoptosis sensitivity in certain cell lines. Our data confirmed the tumor cell "counterattack" hypothesis by showing their capacity to induce apoptosis in lymphocytes from patients with colorectal cancer. CONCLUSION: Fas expression and apoptosis sensitivity in colorectal tumor cell lines can be modulated by actinomycin-D or interferon-gamma. These data may suggest new therapeutic options based on increased Fas expression in tumor cells induced by interferon-gamma, or on apoptosis induction in tumor cells with a local intratumoral treatment with actinomycin-D.