The enzyme-linked immunosorbent assay (ELISA) is a basic
technique
used in analytical and clinical investigations. However, conventional
ELISA is still not sensitive enough to detect ultralow concentrations
of biomarkers for the early diagnosis of cancer, cardiovascular risk,
neurological disorders, and infectious diseases. Herein we show a
mechanism utilizing the CRISPR/Cas13a-based signal export amplification
strategy, which double-amplifies the output signal by T7 RNA polymerase
transcription and CRISPR/Cas13a collateral cleavage activity. This
process is termed the CRISPR/Cas13a signal amplification linked immunosorbent
assay (CLISA). The proposed method was validated by detecting an inflammatory
factor, human interleukin-6 (human IL-6), and a tumor marker, human
vascular endothelial growth factor (human VEGF), which achieved limit
of detection (LOD) values of 45.81 fg/mL (2.29 fM) and 32.27 fg/mL
(0.81 fM), respectively, demonstrating that CLISA is at least 102-fold more sensitive than conventional ELISA.
A novel strategy was developed for microRNA-155 (miRNA-155) detection based on the fluorescence quenching of positively charged gold nanoparticles [(+)AuNPs] to Ag nanoclusters (AgNCs). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were introduced as fluorescent probes, and DNA-RNA heteroduplexes were formed upon the addition of target miRNA-155. Meanwhile, the (+)AuNPs could be electrostatically adsorbed on the negatively charged single-stranded DNA (ssDNA) or DNA-RNA heteroduplexes to quench the fluorescence signal. In the presence of duplex-specific nuclease (DSN), DNA-RNA heteroduplexes became a substrate for the enzymatic hydrolysis of the DNA strand to yield a fluorescence signal due to the diffusion of AgNCs away from (+)AuNPs. Under the optimal conditions, (+)AuNPs displayed very high quenching efficiency to AgNCs, which paved the way for ultrasensitive detection with a low detection limit of 33.4 fM. In particular, the present strategy demonstrated excellent specificity and selectivity toward the detection of target miRNA against control miRNAs, including mutated miRNA-155, miRNA-21, miRNA-141, let-7a, and miRNA-182. Moreover, the practical application value of the system was confirmed by the evaluation of the expression levels of miRNA-155 in clinical serum samples with satisfactory results, suggesting that the proposed sensing platform is promising for applications in disease diagnosis as well as the fundamental research of biochemistry.
An efficient electrochemical approach is developed for ultrasensitive profiling of the methylation status of the p53 tumor suppressor gene based on a label-free biosensor in combination with bisulfite conversion.
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