Novel Point-of-care dIagnostics with CRISPR and Isothermal amplification and an aptamer based detection method for a cancer marker osteopontin

Abstract

The detection of infectious diseases is imperative in biomedicine and safety. Thus, rapid, sensitive and specific diagnostics remains to be accelerated and prioritized. CRISPR-Cas system has been currently exploited in diagnostics based on Cas enzymes (Cas12a/b, Cas13a/b, and Cas14) collateral cleavage of target and non-target ssDNA in the vicinity, an effect which offers a fluorescent readable result from cleavage of a fluorophore-labelled reporter ssDNA. Based on this unique trans-cleavage property, we aimed to construct novel reporter and activator DNA sequences for LbaCas12a rapid and precisely detect cognate nucleic acids. Moreover, we combined loop-mediated isothermal amplification (LAMP) to increase the sensitivity of the assay. We furthermore designed a novel DNA probe-based lateral flow biosensor (LFB) for visual detection and monitoring of reporter sequences trans-cleavage. Under optimized conditions, the target recognition by LbaCas12a/b-based system induced entire trans-cleavage of ssDNA reporters, resulting in short sequences undetectable by the LFB test line capture probe. By targeting different bacterial and viral pathogens (HPV, P. aeruginosa, African swine fever virus and Epstein-Barr virus), 1×10-18 M (1.2 copies) and 3.1×10-18 M (1.8 copies) sensitivities for P. aeruginosa and HPV, respectively were obtained due to the LAMP pre-amplification. With PCR pre-amplification prior to Cas12a reaction, 2.5×10-15 M (~1554.46 copies/µl) and 7.1×10-14 M (~42,000 copies/µl) and sensitivities were achieved for African swine fever virus and Epstein-Barr virus, respectively. Most importantly, our CRISPR based assays showed uncompromised specificity –in the presence of related/unrelated strains- at low-cost in less than an hour for CIALFB. Furthermore, this assay demonstrated applicability in complex samples such as serum, milk and clinical samples. This system promise robust, versatile and reprogrammable tool for Point-of-Care diagnosis, treatment, and study of pathogens. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance. Furthermore, we presented herein the rapid determination of human osteopontin (OPN) protein, a potential cancer biomarker, which holds substantial promise for point-of-care diagnostics and biomedical applications. To date, most reported platforms for OPN detection are apparatus-dependent, time-consuming, and expensive. Herein, we established a lateral flow biosensor (LFB) for OPN detection. A biotinylated aptamer was used for OPN pre-capture from samples, an antibody for OPN was immobilized on the test line for a second specific target identification, and streptavidin-modified gold nanoparticles were sprayed on the conjugation pad for color detection. This LFB achieved as low as 0.1 ng mL-1 OPN Abstract V sensitivity with a good dynamic detection between 10-500 ng mL-1 within 5 minutes. Intriguingly, the LFB allowed a qualitative and semi-quantitative detection of OPN in serum at clinically cut-off levels as in cancer patients, and can discriminate OPN from interfering proteins with high specificity. Thus, it is a promising alterative approach for point-of-care OPN screening and detection. Keywords: CRISPR/Cas12a/b, Nucleic acid Amplification, Lateral flow biosensor, Diseases prognosis, Aptamer