CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

Abstract

CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high transcleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive smallmolecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of smallmolecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.

Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives

“…In a recent paper, biotin (among other analytes) was measured in blood plasma of healthy volunteers living in a low-altitude area who had been transported to high altitude using ultrahigh-performance liquid chromatography—tandem mass spectrometry (UHPLC-MS/MS) [ 133 ]. New analytical methods have also been described in the recent literature, including an artificial intelligence-aided, competitive massive parallel spectroscopy (MPS) bioaffinity assay [ 134 ], a chemiluminescent binding assay based on avidin and biotinylated quinone [ 135 ], an impedimetric immunosensor assay [ 136 ], a competitive immunoassay based on magnetic beads and gold nanoparticle probes [ 137 ], a method based on column-switching liquid chromatography–tandem mass spectrometry [ 138 ], as well as two types of CRISPR/Cas12a-powered competitive immunoassays [ 139 , 140 ].…”

CRISPR/Cas12a-Derived Photoelectrochemical Aptasensor Based on Au Nanoparticle-Attached CdS/UiO-66-NH2 Heterostructures for the Rapid and Sensitive Detection of Ochratoxin A

“…Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection technology has been applied for aptasesnor construction, owing to its excellent specificity and sensitivity. 18,19 Particularly, it possesses trans-cleavage activity and can indiscriminately cleave any ssDNA. 20,21 For instance, Li's team reported a CRISPR/Cas12a-assisted electrochemical aptasensor for nucleic acid analysis based on Au NPs/MXene electrodes.…”

“…Considering the high homology and low concentration in real samples, exploring an effective target recognition and amplification strategy to improve the sensitivity and selectivity of aptasensors is essential. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection technology has been applied for aptasesnor construction, owing to its excellent specificity and sensitivity. , Particularly, it possesses trans-cleavage activity and can indiscriminately cleave any ssDNA. , For instance, Li’s team reported a CRISPR/Cas12a-assisted electrochemical aptasensor for nucleic acid analysis based on Au NPs/MXene electrodes . The target nucleic acid was used as an “activator” to specifically bind to programming CRISPR-RNA (crRNA) and trigger the trans-cleavage activity, leading to the cleavage of ssDNA and the changes in sensing interface characteristics that can be employed for target quantification.…”