Sensitive Small Molecule Aptasensing based on Hybridization Chain Reaction and CRISPR/Cas12a Using a Portable 3D-Printed Visualizer

Abstract

Next-generation biosensing tools based on CRISPR/Cas have revolutionized the molecular detection. A number of CRISPR/Cas-based biosensors have been reported for the detection of nucleic acid targets. The establishment of efficient methods for non-nucleic acid target detection would further broaden the scope of this technique, but up to now, the concerning research is limited. In the current study, we reported a versatile biosensing platform for non-nucleic acid small-molecule detection called SMART-Cas12a (small-molecule aptamer regulated test using CRISPR/Cas12a). Simply, hybridization chain reaction cascade signal amplification was first trigged by functional nucleic acid (aptamer) through target binding. Then, the CRISPR/Cas system was integrated to recognize the amplified products followed by activation of the trans-cleavage. As such, the target can be ingeniously converted to nucleic acid signals and then fluorescent signals that can be readily visualized and analyzed by a customized 3D-printed visualizer with the help of a home-made App-enabled smartphone. Adenosine triphosphate was selected as a model target, and under the optimized conditions, we achieved fine analytical performance with a linear range from 0.1 to 750 μM and a detection limit of 1.0 nM. The satisfactory selectivity and recoveries that we have obtained further demonstrated this method to be suitable for a complex sample environment. The sample-to-answer time was less than 100 min. Our work not only expanded the reach of the CRISPR-Cas system in biosensing but also provided a prototype method that can be generalized for detecting a wider range of analytes with desirable adaptability, sensitivity, specificity, and on-site capability.

CRISPR/Cas System: The Accelerator for the Development of Non-nucleic Acid Target Detection in Food Safety

“…Moreover, the detection sensitivity was improved by combining isothermal amplification. As shown in Figure D, Ma et al reported a versatile biosensing for ATP using the HCR (hybridization chain reaction) and CRISPR/Cas . The HCR cascade signal amplification was first triggered by the target-binding functional nucleic acid (aptamer), followed by CRISPR/Cas12a recognizing the amplified products to activate trans -cleavage.…”

“…As shown in Figure 3D, Ma et al reported a versatile biosensing for ATP using the HCR (hybridization chain reaction) and CRISPR/Cas. 24 The HCR cascade signal amplification was first triggered by the target-binding functional nucleic acid (aptamer), followed by CRISPR/Cas12a recognizing the amplified products to activate trans-cleavage. The target was converted to fluorescent signals that can be easily visualized and analyzed by using a custom 3D-printed visualizer.…”

“…Furthermore, the CRISPR/Cas system could be used to detect non-nucleic acid targets via designing appropriate and compatible biometric recognition and transduction elements as well. , Interestingly, functional DNAs such as aptamers and DNAzymes provide an excellent measure for converting non-nucleic acid targets to nucleic acid targets with significant performance advantages, including a simple synthetic process, low cost, low toxicity, simple molecular modification, and multiple denaturation and regeneration cycles without loss of binding ability or activity to substrates . Aptamers are synthetic short single-stranded nucleic acid sequences, usually consist of 20 to 80 nucleotides, and are typically developed in vitro through “systematic evolution of ligands by exponential enrichment (SELEX)” .…”

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

“…have been constructed for the detection of nucleic acids and different non-nucleic acid targets, such as ions, small molecules, proteins, and pathogenic bacteria, ,− and detection of non-nucleic acid analytes needs an efficient means to convert targets quantification to nucleic acid analysis. For small molecule analysis, current methods mainly focus on precious designing of target-induced structure-switchable aptamer probes to regulate the activity of the CRISPR/Cas system; thus, rational engineering of the guide RNA (crRNA) and active DNA (acDNA) sequence for different targets is required. ,,,, The development of universal strategies for direct switching CRISPR/Cas systems for different small molecule detection is challenging and critical for expanding the CRISPR/Cas-based analytical application.…”

“…For small molecule analysis, current methods mainly focus on precious designing of target-induced structure-switchable aptamer probes to regulate the activity of the CRISPR/Cas system; thus, rational engineering of the guide RNA (crRNA) and active DNA (acDNA) sequence for different targets is required. 1,12,13,16,17 The development of universal strategies for direct switching CRISPR/Cas systems for different small molecule detection is challenging and critical for expanding the CRISPR/Cas-based analytical application.…”