A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits

The collateral cleavage function is only a corollary of programmable nuclease activity of certain Cas effectors, such as Cas12 and Cas13, but it can be utilised to amplify fluorescence signals in various CRISPR/Cas-based biosensing systems. In this work, this special signal amplification capability of CRISPR/Cas12a ribonucleoproteins has been employed to increase the sensitivity of a broad class of commercial ELISA systems with undisclosed chemistry except for the use of horseradish peroxidase (HRP), a common signal reporting molecule. We demonstrated that such ELISA systems with HRP on the detection antibody, can be amplified by 2 orders of magnitude using an example commercial IFN-gamma ELISA kit where the detection limit was decreased from 312.5 pg/ mL to 1.2 pg/mL. The detection range was simultaneously increased from 2 orders to 3 orders of magnitude. Our CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. The CES-Amplifier can be directly integrated into commercial ELISA kits to replace their original last step, without any additional changes of the ELISA kit reagents or setup. In this way, our CES-Amplifier provides a versatile and affordable approach for expanding CRISPR/Casbased biosensing to a wide range of non-nucleic acid analytes.

Automated summary

The study demonstrates the use of CRISPR/Cas12a ribonucleoproteins to enhance the sensitivity of commercial ELISA systems by amplifying fluorescence signals. The approach, known as CES-Amplifier, involves integrating a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme into commercial ELISA kits, allowing for expansion of CRISPR/Cas-based biosensing to a wide range of non-nucleic acid analytes.