Amplification-free detection of telomerase activity at the single-cell level via Cas12a-lighting-up single microbeads (Cas12a-LSMBs).

Telomerase overexpresses in almost all cancer cells and has been deemed a universal biomarker for cancer diagnosis and therapy. However, simple and ultrasensitive detection of telomerase activity in single-cells is still a huge challenge. Herein, we wish to report Cas12a-lighting up single microbeads (Cas12a-LSMBs) for ultrasensitive detection of telomerase activity without nucleic acid amplification. In this platform, single-strand DNA reporter (ssDNA reporter)-functionalized single-microbeads (functionalized-SMBs) are employed as a reactor for the trans-cleavage of telomerase-activated CRISPR/Cas12a as well as a reporting unit for fluorescence signal enrichment and visualization. Due to the space-confined effect and signal enrichment mechanism on the surface of the functionalized SMBs, the Cas12a-LSMBs can accurately detect telomerase activity in crude cell lysates with high specificity. Importantly, we have demonstrated that the Cas12a-LSMBs are a reliable and practical tool to detect telomerase activity in single cells and investigate cellular heterogeneity of telomerase activity from cell-to-cell variations.

Single-microbead space-confined digital quantification strategy (SMSDQ) for counting microRNAs at the single-molecule level.

Quantification of microRNAs (miRNAs) at the single-molecule level is of great significance for clinical diagnostics and biomedical research. The challenges lie in the limits to transforming single-molecule measurements into quantitative signals. To address these limits, here, we report a new approach called a Single Microbead-based Space-confined Digital Quantification (SMSDQ) to measure individual miRNA molecules by counting gold nanoparticles (AuNPs) with localized surface plasmon resonance (LSPR) light-scattering imaging. One miRNA target hybridizes with the alkynyl-modified capture DNA probe immobilized on a microbead (60 µm) and the azide-modified report DNA probe anchored on AuNP (50 nm), respectively. Through the click reaction between the alkynyl and azide group, a single microbead can covalently link the AuNPs in the confined space within the view of the microscope. By digitally counting the light-scattering spots of AuNPs, we demonstrated the proposed approach with single-molecule detection sensitivity and high specificity of single-base discrimination. Taking the advantages of ultrahigh sensitivity, specificity, and the digital detection manner, the approach is suitable for evaluating cell heterogeneity and small variations of miRNA expression and has been successfully applied to direct quantification of miRNAs in one-tenth single-cell lysates and serum samples without RNA-isolated and nucleic acid amplification steps.

CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.

The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.

One-step and highly sensitive quantification of fusion genes with isothermal amplification initiated by a fusion-site anchored stem-loop primer.

As causative oncogenes and drug targets, fusion genes play critical roles in tumorigenesis, development, and treatment and thus are regarded as tumor-specific molecular biomarkers. Specific identification and sensitive quantification of fusion genes are of great significance in cancer diagnosis, classification, and prognosis as well as minimal residual disease (MRD) monitoring. Herein, we proposed a specific and sensitive method for the quantitative detection of fusion transcripts by designing stem-loop primers to directly track fusion junctions of fusion genes and subsequently initiate reverse transcript loop-mediated isothermal amplification (LAMP). Benefitting from the specific and direct mechanism of stem-loop primers and the high amplification efficiency of LAMP, the proposed method can sensitively measure fusion gene transcripts with a detection limit of 100 aM (1 zmol) and achieve a wide linear dynamic range spanning at least six orders of magnitude (100 aM-100 pM). Significantly, the whole fusion transcript assay can be accomplished in one step under isothermal conditions, greatly simplifying the operation and detection processes. Meanwhile, the one-step analysis method in one tube may effectively eliminate false-positive results from product cross-contamination during multiple experimental operations and cover-opening measurements. We have demonstrated that the proposed method is practical and accurate for the quantitation of fusion transcripts in biological samples. Owing to the outstanding features of high sensitivity, excellent specificity, and simple operation, the new strategy may provide a robust and attractive platform for the quantification of fusion genes.

Light Scattering Technology-Combined Ligation-Dependent Loop-Mediated Isothermal Amplification (LL-LAMP) for Sensitive Detection of RNA.

Loop-mediated isothermal amplification (LAMP) has been widely used in nucleic acid assay because of its high specificity, sensitivity, and isothermal property. However, the complexity of amplification product detection is still a major challenge for its wide applications. Herein, we developed a light scattering technology-assisted, low-cost, and simple detection manner of LAMP products without expensive reagents and complicated instruments. Only needing to add a kind of strong acid to the amplification products, the amplification products can aggregate into large particles in a strongly acidic medium, and large particles can produce strong light scattering, which shows a good proportional relationship with the number of amplification products in a wide range. The proposed method shows excellent sensitivity and high specificity that can quantify RNA as low as 100 aM with a single-base resolution.