Determination of adenosine by CRISPR-Cas12a system based on duplexed aptamer and molecular beacon reporter linked to gold nanoparticles.

Abstract

Adenosine as a potential tumor marker is of great value for clinical disease diagnosis. Since the CRISPR-cas12a system is only capable of recognizing nucleic acid targets we expanded the CRISPR-cas12a system to determine small molecules by designing a duplexed aptamer (DA) converting g-RNA recognition of adenosine to recognition of aptamer complementary DNA strands (ACD). Tofurther improve the sensitivity of determination, we designed a molecule beacon (MB)/gold nanoparticle (AuNP)-based reporter, which has higher sensitivity than traditional ssDNA reporter. In addition, the AuNP-based reporter enables more efficient and fast determination. The determination of adenosine under 488-nm excitation can be realized within 7 min, which is more than 4 times faster than traditional ssDNA reporter. The linear determination range of the assay to adenosine was 0.5-100 μM with the determination limit of 15.67 nM. The assay was applied torecovery determination of adenosine in serum samples with satisfactory results. The recoveries were between 91 and 106% and the RSD values of different concertation were below 4.8%. This sensitive, highly selective, and stable sensing system is expected to play a role in the clinical determination of adenosine and other biomolecules.