{"title":"PAIT effect: Padlock activator inhibits the trans-cleavage activity of CRISPR/Cas12a","authors":"","doi":"10.1016/j.bios.2024.116607","DOIUrl":null,"url":null,"abstract":"<div><p>The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a <u>p</u>adlock activator which could <u>i</u>nhibit the <em><u>t</u>rans</em>-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca<sup>2+</sup> sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca<sup>2+</sup> dependent. In the presence of Ca<sup>2+</sup>, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the <em>trans</em>-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca<sup>2+</sup> concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg<sup>2+</sup>, Mn<sup>2+</sup>, Cd<sup>2+</sup>, Cu<sup>2+</sup>, Na<sup>+</sup>, Al<sup>3+</sup>, K<sup>+</sup>, Fe<sup>2+</sup>, and Fe<sup>3+</sup> indicated high selectivity. Recovery ranged from 93.32% to 103.28% with <em>RSD</em>s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca<sup>2+</sup> in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.</p></div>","PeriodicalId":259,"journal":{"name":"Biosensors and Bioelectronics","volume":null,"pages":null},"PeriodicalIF":10.7000,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biosensors and Bioelectronics","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0956566324006122","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca2+ dependent. In the presence of Ca2+, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg2+, Mn2+, Cd2+, Cu2+, Na+, Al3+, K+, Fe2+, and Fe3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
期刊介绍:
Biosensors & Bioelectronics, along with its open access companion journal Biosensors & Bioelectronics: X, is the leading international publication in the field of biosensors and bioelectronics. It covers research, design, development, and application of biosensors, which are analytical devices incorporating biological materials with physicochemical transducers. These devices, including sensors, DNA chips, electronic noses, and lab-on-a-chip, produce digital signals proportional to specific analytes. Examples include immunosensors and enzyme-based biosensors, applied in various fields such as medicine, environmental monitoring, and food industry. The journal also focuses on molecular and supramolecular structures for enhancing device performance.