Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT–based method. We believe this ht-G3/ThT–based label-free strategy could be widely applied for RNA detection.
Yaqin Zhang,Yingjie Cheng,Chenfei Zhao,Jianai Chen,Zhina Wu,Jiasi Wang,Di Wang
Simultaneous analysis of multiple protein and nucleic acid biomarkers in a single clinical sample can yield clinically valuable results for diagnosing, surveillance, and managing various diseases. However, the current diagnostic platforms depend largely on protein- or nucleic acid-independent assay, which is time-consuming and laborious. Here, we propose a novel assay termed MCD assay that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene RNA and SARS-CoV-2 S protein. This assay was based on magnetic separation technology, combined with the clustered regularly interspaced palindromic repeats (CRISPR) /CRISPR-associated proteins (Cas) system for accessible point-of-care detection of nucleic acids and proteins. The assay could recognize target molecules through nucleic acid hybridization or antigen–antibody reactions and integrated with the CRISPR/Cas-based enzymatic detection; the lateral flow test strips enabled visualization of the assay results. The limit of detection of MCD was 1000 copies/μL for viral nucleic acid and 10 pg/mL for viral protein. The combined evaluation of multiple biomarkers from diverse pathways can enhance the accuracy and specificity of disease diagnosis. It is also crucial to use a single platform for detecting biomarkers across various dimensions to evaluate and manage major diseases.
Upconversion nanoparticles (UCNPs) are promising nanoprobes in DNA/RNA detection, such as respiratory viral RNAs, and siRNA in cancer. However, recent studies have indicated that the sensitivity of UCNP-based biosensors is restricted, ranging from picomolar to femtomolar level. Moreover, most of existing UCNP-based probes are only able to detect short-chain oligonucleotides, which are not suitable for detection of long-chain oligonucleotides in many real applications. In this work, we introduced a new UCNP-based fluorescence resonance energy transfer (FRET) nanoprobe design composed of NaGdF 4 :Yb 3+ , Er 3+ @NaGdF 4 core-shell UCNPs (csUCNPs) linking with Au–Au dimer to detect long-chain oligonucleotides of SARS-CoV-2 N-gene. Compared with typical single gold nanoparticles (AuNPs) in FRET biosensors, our theoretical investigation shows that a stronger electromagnetic field is generated in the Au–Au dimer where the plasmon resonance can enhance FRET efficiency and increase the working distance. Thus, the synergetic effect of plasmonic resonance and FRET enables a greater quenching efficiency (QE) of Au–Au dimer to UCNPs, which leads to more remarkable upconversion luminescence (UCL) recovery for each target gene recognition. Importantly, our design significantly improved the limit of detection (LOD) to attomolar level, with a linear response ranging from 2 aM to 2 fM. Moreover, the clinical detection with inactivated SARS-CoV-2 samples was successfully performed with excellent specificity within 30 min using the developed UCNPs biosensors incorporated with Au–Au dimer. This UCNP biosensor based on Au–Au dimer strategy with ultra-sensitivity and good selectivity opens a new path for clinical diagnosis without target amplification and plays an instructive role in other virus diagnosis.
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