Construction of a Quencher-Free Cascade Amplification System for Highly Specific and Sensitive Detection of Serum Circulating miRNAs

Abstract

Circulating miRNAs are a newly emerging class of noninvasive biomarkers, and the accurate quantification of their expression is essential to the biological research and early clinic diagnosis. Herein, we demonstrate the construction of a quencher-free cascade amplification system for highly sensitive detection of serum circulating miRNAs. The target miRNA can hybridize with the linear probe to induce the cyclic strand displacement amplification (SDA) (cycle I) for the production of the binding probes. The binding probe can subsequently react with the 2-aminopurine (2-AP)-hairpin probe to induce the recycling exonuclease cleavage of 2-AP-hairpin probes (cycle II), releasing the triggers and 2-AP molecules simultaneously. The released trigger can hybridize with the free linear probe to start new cycles I and II amplifications. Through multiple rounds of cascade amplifications, a large number of 2-AP molecules are released, generating an enhanced fluorescence signal. This method exhibits a large dynamic range of 8 orders of magnitude and a detection limit of 0.16 aM. It can differentiate a single-base mismatch in miR-486-5p, quantify miR-486-5p in lung cancer cells at various stages, and even discriminate the expressions of serum circulating miR-486-5p in healthy persons from that in nonsmall-cell lung carcinoma (NSCLC) patients. Moreover, this assay can be rapidly carried out in one step under isothermal condition in a label-free manner, holding promising applications in point-of-care diagnosis and prognosis of lung cancers.

Improving the Sensitivity of the miRNA Assay Coupled with the Mismatched Catalytic Hairpin Assembly Reaction by Optimization of Hairpin Annealing Conditions

“…The calibration curve obtained under the optimized conditions obeyed a double rectangular hyperbola equation with the formula ( R 2 0.9979), where a , b , c , and d were 2.03 × 10 6 , 0.12, 5.86 × 10 7 , and 365, respectively, and may be used to calculate the miRNA-141 concentration in the samples of interest. The curves with similar behaviors were previously reported for assays coupled with CHA/mCHA. ,, The analysis of the calibration curve showed that when the target concentration changed from 1 to 100 pM, the chemiluminescence intensity increased by 8 times, while in many of the miRNA assays reported previously, the signal value changed only by 3–4 times with changing the analyte concentration by 6–7 orders of magnitude. − …”

A Versatile Electrochemical Biosensor for the Detection of Circulating MicroRNA toward Non‐Small Cell Lung Cancer Diagnosis

“…A significant reduction in the expression of miR‐486‐5p was detected in the NSCLC cell lines, compared with normal cells (MRC5), consistent with previous reports that there is an inverse correlation between miR‐486‐5p expression and NSCLC progression. [ 27 ] We further used the standard RT‐qPCR method to quantify the miR‐486‐5p concentrations in the NSCLC cell lines, and the obtained values were shown in Figure S4 (Supporting Information). As shown in Figure 3B, the miR‐486‐5p concentrations in the NSCLC cell lines was much less, decreased in the order of H460 > H1299 > H1975 > H226 cells.…”

A self-quenching fluorescence probe-mediated exponential isothermal amplification system for highly sensitive and specific detection of microRNAs

“…With the unique design in probe structure, and the cascade amplification in double-cycle system, the SqPIA approach could achieve the goals that with an ultrasensitive detection limit of 0.08 aM, an ability of effective distinction in a single-base difference of homologous miRNAs and triplex detection of miRNAs. Furthermore, compared with previous studies in miRNA assays, 30,31 the SqPIA strategy has its significant advantages: (i) the sensitivity and the signal-tobackground ratio in miRNA detection was greatly improved; (ii) usage of self-quenching probes instead of dye could make multiple targets detection in single sample more feasible; (iii) the probes used with special design in structure and strict controlling in amount can effectively reduce non-specific amplification. More importantly, the proposed method can get rid of the dependence on sophisticated equipment, when compared with the traditional PCR.…”