A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification

Oct 2, 2025Mikrochimica acta

Improving CRISPR/Cas12a tests with a new three-part probe and quick paper-strip detection using a special branched molecule

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Abstract

The CRISPR/Cas12a-tripod-LFT strategy achieved a detection limit of 1.4 pM for the DNA target of Salmonella Typhimurium.

A novel DNA probe with multiple fluorescein labels was engineered to improve detection capabilities.
The cleaved parts of the probes were detected using a lateral flow test designed for specificity and speed.
The highest signal-to-noise ratio was found with a tripod-branched probe synthesized through a specific chemical modification.
A rapid magnetic separation strategy effectively removed uncleaved probes within 5 minutes.
The system demonstrated excellent sensitivity without the need for preamplification.
Preliminary tests indicated the ability to detect as few as 0.3 cells per reaction when combined with isothermal amplification.

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CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

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A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification

Abstract

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