Metal–Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers

Abstract

CRISPR/Cas12a shows excellent potential in disease diagnostics. However, insensitive signal conversion strategies hindered its application in detecting protein biomarkers. Here, we report a metal–organic framework (MOF)-based DNA bio-barcode integrated with the CRISPR/Cas12a system for ultrasensitive detection of protein biomarkers. In this work, zirconium-based MOF nanoparticles were comodified with antibodies and bio-barcode phosphorylated DNA as an efficient signal converter, which not only recognized the protein biomarker to form the sandwich complex but also released the bio-barcode DNA activators after MOF dissociation to activate the trans-cleavage activity of Cas12a. Due to the obvious advantages, including numerous loaded oligonucleotides, a convenient release process, and the nontoxic release reagent, this MOF-DNA bio-barcode strategy could amplify the CRISPR/Cas12a system to achieve simple and highly sensitive detection of tumor protein biomarkers with detection limits of 0.03 pg/mL (PSA) and 0.1 pg/mL (CEA), respectively. Furthermore, this platform could detect PSA directly in clinical serum samples, offering a powerful tool for early disease diagnosis.

CRISPR-Powered Strategies for Amplification-Free Diagnostics of Infectious Diseases

“…Therefore, compared to Cas9, Cas12 provides more opportunities for targeting AT-rich sequences . In addition, experiments also showed that PAM binding promotes strand separation of the target dsDNA and consequently R-loop formation, while trans -cleavage activity is activated by crRNA-target strand DNA hybridization, rather than PAM binding. , Due to its trans -cleavage activity to cleave the fluorophore-quencher (FQ)-labeled ssDNA or DNA G-triplex/G-quadruplex reporter, Cas12 can be used not only for gene editing but also for high-sensitive detection. ,− Nowadays, many diagnostic methods that combine the CRISPR/Cas12 system with isothermal nucleic acid amplification methods have also been developed. For example, Chen et al reported a method termed DETECTR to detect HPV subtypes with attomole sensitivity .…”

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

“…Otherwise, the preamplification and subsequent test reaction system can be optimized to be compatible with one tube to save space and time . For instance, biobarcode, a complex consisting of a nanocarrier (e.g., metal–organic framework) and numerous ssDNA activators, can perform both preamplification and signal conversion during detection . Utilizing biobarcodes to convert miRNA input into quantitatively amplified ssDNA activators, Zhao et al have detected different miRNAs down to the attomolar level …”

“…109 For instance, biobarcode, a complex consisting of a nanocarrier (e.g., metal−organic framework) and numerous ssDNA activators, can perform both preamplification and signal conversion during detection. 120 Utilizing biobarcodes to convert miRNA input into quantitatively amplified ssDNA activators, Zhao et al have detected different miRNAs down to the attomolar level. 121 Deeper Dig into Mechanism.…”

Ultrasensitive Detection of miRNA via CRISPR/Cas12a Coupled with Strand Displacement Amplification Reaction

“…Owing to its characteristic of highly efficient collateral cleavage, the CRISPR/Cas12a system is often used as a tool for signal amplification in biosensors. It has been extensively used for detection of nucleic acids, − metal ions, , and proteins , and shows great promise in the development of POC diagnostic devices. In addition, many amplification techniques such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and recombinase polymerase amplification (RPA) have been reported to be combined with the CRISPR/Cas12a system to further improve the sensitivity.…”