Simultaneous detection of multiple microRNAs (miRNAs) is crucial for diagnosing major diseases. Conventional miRNA detection methods face limitations in multiplexing, detection cycle, sensitivity and specificity. Herein, we present a novel CRISPR-linked amplification-free miRNA photoelectrochemical (CLAMP) biosensor for the direct and simultaneous analysis of multiple miRNAs with high sensitivity and specificity. The biosensor employs multi-channel screen-printed electrodes with vertical reduced graphene oxide as the substrate for the working electrode. In particular, based on the surface plasmon resonance effect of Au nanoparticles, ZnO/Au heterojunctions with an excellent photoelectric response are constructed and modified on the surface of vertical graphene to generate photocurrent signals. The probes containing protospacer adjacent motif sites, immobilised on the Au nanoparticle surface, enable the dCas9/sgRNA complex to act as a clamp to specifically recognise and bind target sequences. This biosensor translates miRNA recognition into the effect of the dCas9/sgRNA complex on the ultrahigh photoelectric response of ZnO/Au heterojunction to “CLAMP” the target miRNAs, with no need for reverse transcription or amplification. The biosensor achieves multi-channel miRNA detection and an amol-level detection limit while demonstrating single-base recognition specificity as well as excellent stability and reproducibility during the direct detection of miRNA-10b, miRNA-155 and miRNA-21. Clinical tests revealed higher miRNA concentrations in malignant pleurisy than in non-malignant pleurisy. The CLAMP biosensor presents a substantial advancement in miRNA detection and is a promising tool for clinical diagnostics and the comprehensive analysis of multiple miRNA targets.
Single nucleotide polymorphisms (SNPs) present significant challenges in microbial detection and treatment, further raising the demands on sequencing technologies. In response to these challenges, we have developed a novel barcode-based approach for highly sensitive single nucleotide recognition. This method leverages a dual-head folded complementary template probe in conjunction with DNA ligase to specifically identify the target base. Upon recognition, the system triggers rolling circle amplification (RCA) followed by the self-assembly of CdSe quantum dots onto polystyrene microspheres, enabling a single-particle fluorescence readout. This approach allows for precise base identification at individual loci, which are then analyzed using a bio-barcode array to screen for base changes across multiple sites. This method was applied to sequence a drug-resistant mutation site inHelicobacter pylori(H. pylori), demonstrating excellent accuracy and stability. Offering high precision, high sensitivity, and single nucleotide resolution, this approach shows great promise as a next-generation sequencing method.
Shujuan Xu,Yong Shao,Kun Ma,Qinghua Cui,Guiying Liu,Fei Wu,Minjie Li
DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.
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