CRISPR/Cas12a-based molecular diagnostics are considered a promising next-generation approach for nucleic acid detection due to their high specificity and programmable recognition. However, current amplification-free CRISPR assays suffer from limited sensitivity and a lack of accurate quantification, hindering their application in the areas such as early infectious disease screening and cancer liquid biopsy.

In a study published in Analytical Chemistry, researchers led by Prof. ZHU Ling from Hefei Institutes of Physical Science of the Chinese Academy of Sciences developed a digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) hydrogel fluorescence-enhancing microsphere sensing system (CrisprDEM) which enables amplification-free relative quantification of nucleic acids.

The system combines signal enrichment with digital imaging and automated data analysis in a single workflow. It uses hydrogel microspheres as confined microreactors. The spatial confinement effect significantly concentrates fluorescent signals generated by CRISPR/Cas12a collateral cleavage activity, resulting in strong signal enhancement. A microfluidic chip was designed to distribute microspheres into a monolayer, reducing signal overlap and aggregation and ensuring stable imaging.

For quantitative analysis, researchers introduced an unsupervised K-means clustering algorithm to automatically distinguish positive and negative microspheres. They then established a new metric termed the Positive Bead Ratio, which enables robust relative quantification of target nucleic acids without pre-amplification.

The system developed in this study provides a rapid and quantitative approach for detecting low-abundance nucleic acids, with applications in early pathogen screening and point-of-care diagnostics.