Abstract

The presented dataset contains data and protocols produced in the framework of the Nano-ImmunoEra project within Task 4.2 (Integrated Sample to Answer Device for Ab quantification) of Work Package 4 (Development of CRISPR-based POC for Ab monitoring), led by Feral, and focuses on the optimization of a CRISPR/Cas-based lateral flow assay (LFA) for nucleic acid and antibody detection. The primary aim was to develop a reliable, sensitive, and user-friendly point-of-care diagnostic tool by refining all components and experimental protocols to deliver robust results.​ The LFA system is designed using gold nanoparticles functionalized with anti-FITC (Mouse) antibodies, serving as a visual signal. The test strip features two main lines: a streptavidin-coated test line and an anti-antiFITC (Anti-Mouse) antibody-coated control line. In the absence of target DNA, the CRISPR-Cas system remains inactive, and the intact linear reporter (LR) —a biotin- and FITC-tagged oligonucleotide — accumulates at the test line, resulting in a distinct signal. When target DNA is present, activation of the Cas enzyme cleaves the reporter, distributing its fragments to both lines and enabling clear discrimination through their differential intensities.​ The optimization workflow began with membrane selection and visual assessment, focusing on achieving the cleanest background and uniform, intense test and control lines. No quantitative data were produced at this stage, and decisions were driven solely by visual inspection. Subsequent experimental phases targeted three variables: Tween-20 concentration in the buffer (to suppress nonspecific binding, ajdust the flow rate and enhance signal quality), optimal incubation time for CRISPR-Cas activity, and systematic assessment of detection performance as a function of target DNA and linear reporter concentrations, with the latter kept constant.​ Experimental highlights include precise conjugation of antibodies to gold nanoparticles using EDC/sulfo-NHS chemistry and the identification of a buffer system providing the best membrane quality. Test and control lines were optimized at 1 mg/mL streptavidin and 0.5 mg/mL anti-anti-FITC antibody, respectively. The resulting platform achieved robust and reproducible results, with reliable detection at target DNA concentrations as low as 50 pM, monitored using a dedicated instrument for signal readout. Overall, the project established a strategic and reproducible framework for rapid visual optimization and subsequent quantitative fine-tuning of CRISPR/Cas-based lateral flow diagnostics.