A CRISPR-Cas12a-based electrochemical biosensor for the detection of microphthalmia-associated transcription factor.

A novel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reported for the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.

Electrochemical biosensing for E.coli detection based on triple helix DNA inhibition of CRISPR/Cas12a cleavage activity.

BACKGROUND: Escherichia coli (E.coli) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract, posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. RESULTS: We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli. Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. SIGNIFICANCE: With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 102 - 1 × 107 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.

GPX8 regulates pan-apoptosis in gliomas to promote microglial migration and mediate immunotherapy responses.

Introduction: Gliomas have emerged as the predominant brain tumor type in recent decades, yet the exploration of non-apoptotic cell death regulated by the pan-optosome complex, known as pan-apoptosis, remains largely unexplored in this context. This study aims to illuminate the molecular properties of pan-apoptosis-related genes in glioma patients, classifying them and developing a signature using machine learning techniques. Methods: The prognostic significance, mutation features, immunological characteristics, and pharmaceutical prediction performance of this signature were comprehensively investigated. Furthermore, GPX8, a gene of interest, was extensively examined for its prognostic value, immunological characteristics, medication prediction performance, and immunotherapy prediction potential. Results: Experimental techniques such as CCK-8, Transwell, and EdU investigations revealed that GPX8 acts as a tumor accelerator in gliomas. At the single-cell RNA sequencing level, GPX8 appeared to facilitate cell contact between tumor cells and macrophages, potentially enhancing microglial migration. Conclusions: The incorporation of pan-apoptosis-related features shows promising potential for clinical applications in predicting tumor progression and advancing immunotherapeutic strategies. However, further in vitro and in vivo investigations are necessary to validate the tumorigenic and immunogenic processes associated with GPX8 in gliomas.