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Herein, a dual channel surface plasmon resonance imaging (SPRi) biosensor has been developed for the simultaneous and highly sensitive detection of multiplex miRNAs based on strand displacement amplification (SDA) and DNA-functionalized AuNP signal enhancement. In the presence of target miRNAs (miR-21 or miR-192), the miRNAs could specifically hybridize with the corresponding hairpin probes (H) and initiate the SDA, resulting in massive triggers. Subsequently, the two parts of the released triggers could hybridize with capture probes (CP) and DNA-functionalized AuNPs, assembling DNA sandwiches with great mass on the chip surface. A significantly amplified SPR signal readout was achieved. This established biosensing method was capable of simultaneously detecting multiplex miRNAs with a limit of detection down to 0.15 pM for miR-21 and 0.22 pM for miR-192. This method exhibited good specificity and acceptable reproducibility. Moreover, the developed method was applied to the determination of target miRNAs in a complex matrix. Thus, this developed SPRi biosensing method may present a potential alternative tool for miRNA detection in biomedical research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

A sensitive and specific bioassay based on cyclic enzymatic amplification was developed for determination of microRNA (miRNA) by taking advantage of the exodeoxyribonuclease activity of T7 exonuclease (T7 Exo). In the presence of miRNA, DNA/RNA duplexes are formed by hybridization of miRNA and capture probes (Cp). Then, the Cp is digested to release miRNA into the next amplification cycle assisted by T7 Exo. This leads to the digestion of numerous Cp molecules. The broken Cp does no longer hybridize with hairpin probes (Hp) to unveil G-quadruplex DNAzyme (GDNAs). However, in the absence of miRNA, the Hp hybridizes with Cp to unveil GDNAs. The generated GDNAs form assemblies with hemin to form the G-quadruplex/hemin DNAzyme complex which is capable of catalyzing the oxidation of the substrate ABTS by HO. Upon cyclic enzymatic amplification, the output signal is reduced accordingly, this resulting in a 'signal-off' signal best acquired at a wavelength of 418 nm. The lower detection limit is 0.69 pM (at an S/N ratio of 3). The assay involves efficient signal amplification, is homogeneous and isothermal, and enables visual detection. It provides a simple, rapid and sensitive platform for use in clinical diagnostics. [ABSTRACT FROM AUTHOR]

Exosomal miRNAs are potential tumor biomarkers for early diagnosis of non-small cell lung cancer (NSCLC). Herein, a surface plasmon resonance imaging (SPRi)-based biosensor was developed for simultaneous detection of multiplex NSCLC-associated exosomal miRNAs in a clinical sample using Au-on-Ag heterostructure and DNA tetrahedral framework (DTF). Exosomal miRNAs are captured by various DTF probes immobilized on the gold array chip. Subsequently, single-stranded DNA (ssDNA) functionalized silver nanocube (AgNC) hybridizes with the captured exosomal miRNAs and then the ssDNA-coated Au nanoparticles assembled on the surface of AgNC, forming Au-on-Ag heterostructures as essential labels to realize amplified SPR response. With the aid of DNA programmed Au-on-Ag heterostructure and DTF, the SPRi-based biosensor exhibits wide detection range from 2 fM to 20 nM, ultralow limit of detection of 1.68 fM, enhanced capture efficiency, and improved antifouling capability. Furthermore, the biosensor enables accurate discrimination of NSCLC patients based on detection results of exosomal miRNAs. Overall, this developed biosensor is a promising tool for multiplex exosomal miRNAs detection, providing a new possibility for early diagnosis of NSCLC. • DNA programmed heterostructure of Au-on-Ag endowed the SPRi biosensor with ultrahigh sensitivity. • The high-throughput biosensor was capable of simultaneously detecting four exosomal miRNAs in a clinical sample. • The biosensor showed enhanced antifouling capability, benefiting from DTF. • The biosensor could accurately diagnose NSCLC patients from the healthy ones. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are potentially useful biomarkers for early diagnosis of human diseases. Here, a simple surface plasmon resonance (SPR) biosensor has been developed for highly sensitive detection of miRNA by designing a new enzyme-free and isothermal amplification strategy, named multi component nucleic acid enzyme-mediated mismatched catalyzed hairpin assembly (MNAzyme-CHA). The partial MNAzymes co-recognized the target to form a stable active MNAzyme, which continued to digest multiple hairpin H0 substrates, concomitantly generating a lot of fragments. The H0 fragments could initiate the mismatched CHA cycles, resulting in the generation of massive hairpin H1–H2 complexes. As a result, the H1–H2 complexes and streptavidin were attached to the sensor surface, leading to a significantly amplified SPR signal readout. The established biosensor showed high sensitivity and selectivity with a wide dynamic range from 1 pM to 100 nM. It was also successfully applied to the determination of target miRNA spiked into human total RNA samples. Thus, this developed biosensing strategy presents a simple and stable platform toward sensitive and convenient miRNA detection, and has great potential in assays of many other nucleic acids analytes for biomedical research and early clinical diagnosis. [ABSTRACT FROM AUTHOR]