Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

Abstract

Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.

High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil

“…36 Similarly, Li et al adopted PbS and CdS quantum dots to construct electrochemical probes and transduce trans-cleavage signals to ratiometric signals, which achieved detection limits of 1.12 and 1.25 cells/mL for Neu5Gc and Neu5Ac, respectively. 37 However, the two strategies are used separately, and it remains unclear whether the methods can be combined to achieve cascade amplification, which may further improve the sensitivity.…”

Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis

“…Cas12a (CRISPR-associated protein 12a) is a nucleic acid endonuclease from the V-A-type clustered regularly interspaced palindromic repeats (CRISPR) system for genome editing by processing CRISPR RNAs (crRNAs) from arrays using its dedicated RNase functional domain ( 1 , 2 ). It can indiscriminately cleave single-stranded DNA (ssDNA) after activation of intrinsic nuclease activity by recognizing its target site, which has been used for the detection of nucleic acids ( 3 , 4 ). However, there are still many challenges to overcome.…”

DNA Gate-Based CRISPR-Cas Exponential Amplification System for Ultrasensitive Small Extracellular Vesicle Detection to Enhance Breast Cancer Diagnosis

“…CRISPR-associated (CRISPR-Cas) effector protein system with nonspecific transcleavage activity enables amplified nucleic acid detection. 14,15 Among CRISPR/Cas family, the CRISPR/Cas12a system can cleave DNA labeled with a fluorophore-quencher (FQ) reporter and generate fluorescence signals for target detection. 16−18 Thus, the trans-cleavage ability of Cas12a activated by specific targets provides a versatile strategy to develop fluorescence biosensors.…”

“…Extracellular vesicles (EVs) are secreted by most mammalian cells and widely found in various body fluids with sizes varying from approximately 30 to 100 nm. , Among these, small EVs (sEVs) with diameters under 200 nm can carry much biological information, such as functional nucleic acids and proteins from parental cells to recipient cells. , Notably, tumor-derived sEVs (tEVs) with multiple biomarkers play significant roles in the initiation and progression of cancers. , Therefore, tEVs have emerged as promising biomarkers in cancer diagnosis. , Nevertheless, the ultrasensitive phenotyping and identification of tEVs remain challenging due to their high heterogeneity and low abundance. , Recent advances have demonstrated the features and applications of different nuclear acid amplification strategies, such as hybridization chain reaction (HCR), , rolling cycle amplification, clustered regularly interspaced short palindromic repeats (CRISPR) amplification, − and so on. CRISPR-associated (CRISPR-Cas) effector protein system with nonspecific trans -cleavage activity enables amplified nucleic acid detection. , Among CRISPR/Cas family, the CRISPR/Cas12a system can cleave DNA labeled with a fluorophore-quencher (FQ) reporter and generate fluorescence signals for target detection. − Thus, the trans -cleavage ability of Cas12a activated by specific targets provides a versatile strategy to develop fluorescence biosensors. − However, the sensitivity of CRISPR/Cas12a system alone is insufficient to detect trace biomarkers in the early stage of diseases. , To improve its sensitivity, cascade amplification that combines CRISPR/Cas12a with other signal amplification methods is necessary. Thus, some CRISPR/Cas12a-based methods have been developed for tEV detection. − For example, Xing et al developed a dual amplification system based on HCR and CRISPR/Cas12a for nucleolin + or programmed death ligand 1+ (PD-L1+) tEV detection with a limit of detection (LOD: 10 2 particles/μL) through corresponding aptamer targeted recognition, respectively .…”