Structure-Switchable Hairpin-Powered Exponential Replications for Sensing Attomolar microRNA-Related Single Nucleotide Polymorphisms in Human Cancer Tissues with Zero Background.

MicroRNA (miRNA)-related single-nucleotide polymorphisms (miR-SNPs) are a novel class of genetic variations involved in multiple cellular functions, and they have emerged as promising biomarkers for cancer diagnostics and prognostics. Herein, we demonstrate for the first time the structure-switchable hairpin-powered exponential replications for sensing attomolar miR-SNPs in human cancer tissues with zero background. In the presence of target miR-196a2T, hairpin probes (i.e., HP1 and HP2) are splinted together to construct the dumbbell-shaped probe (DSP) with SplintR ligase catalysis. Once the DSP is formed, the self-primed polymerization extension and linear FIP/BIP-primed strand displacement DNA synthesis (SDS) are automatically repeated to activate self-circulated exponential amplification, producing large amounts of double-stranded DNAs (dsDNAs) which can be real-time monitored using SYBR Green I. This nanodevice can detect miR-196a2T with an ultralow detection limit of 2.46 aM; distinguish rare miR-196a2 SNP with a selectivity factor of 0.001%; and even profile miR-196a2T in human tissues for nonsmall cell lung cancer (NSCLC) diagnosis, risk assessment, and cancer type prediction. Notably, this nanodevice can be rapidly and homogeneously used in one tube in a real-time and label-free manner, providing a powerful point-of-care platform for noninvasive diagnostics and prognostics of miR-SNPs-related human cancers.

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

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.

Development of an in Vitro Autocatalytic Self-Replication System for Biosensing Application.

Molecular self-replication is a fundamental function of all living organisms with the capability of templating and catalyzing its own synthesis, and it plays important roles in prebiotic chemical evolution and effective synthetic machineries. However, the construction of the self-replication system in vitro remains a great challenge and its application for biosensing is rare. Here, we demonstrate for the first time the construction of an in vitro enzymatic nucleic acid self-replication system and its application for amplified sensing of human 8-oxoguanine DNA glycosylase (hOGG1) based on autocatalytic self-replication-driven cascaded recycling amplification. In this strategy, hOGG1 excises 8-oxoguanine (8-oxoG) to unfold the hairpin substrate, activating the autonomous biocatalytic process with molecular beacons (MBs) as both the fuels for producing nucleic acid templates and the generators for signal output, leading to the continuous replication of biocatalytic nucleic acid templates and the repeated cleavage of MBs for an enhanced fluorescence signal. This strategy exhibits an extremely low detection limit of 4.3 × 10-7 U/µL and a large dynamic range of 5 orders of magnitude from 1 × 10-6 to 0.05 U/µL. Importantly, it can be applied for the detection of enzyme kinetic parameters, the screening of hOGG1 inhibitors, and the quantification of hOGG1 activity in even 1 single lung cancer cell, providing a new approach for biomedical research and clinical diagnosis.