A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker

Oct 27, 2025Analytical chemistry

A CRISPR-based fluorescent sensor using DNA and RNA parts for quick drug level detection

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Abstract

The aptasensor can detect vancomycin in various biological fluids with detection limits of 13.62 nM in serum, 7.56 nM in synovial fluid, and 6.75 nM in cerebrospinal fluid.

Combining an aptamer and RNA regulates CRISPR-Cas12a activity to enhance detection accuracy.
The dual regulatory mechanism reduces false-positive signals during target detection.
Detection of vancomycin occurs within 30 minutes using a one-pot format.
Clinical validation in 22 serum samples shows no significant difference from traditional enzyme-multiplied immunoassays.
Cross-validation with a quinine aptamer indicates the aptasensor's potential universality for different targets.

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The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (= 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection. R2

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A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker

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