Amplification of the Fluorescence Signal with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a Based on Au Nanoparticle-DNAzyme Probe and On-Site Detection of Pb²⁺ Via the Photonic Crystal Chip

Abstract

Although great headway has been made in DNAzyme-based detection of Pb 2+ , its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb 2+ and its suitability for precise detection of Pb 2+ in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb 2+ detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb 2+ with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb 2+ fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb 2+ in complex biological or environmental samples, which is of great promise for widespread applications.

Mesoporous Nanozyme-Enhanced DNA Tetrahedron Electrochemiluminescent Biosensor with Three-Dimensional Walking Nanomotor-Mediated CRISPR/Cas12a for Ultrasensitive Detection of Exosomal microRNA

“…Nowadays, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) enzymes, including Cas12a, Cas13a, and Cas14, have been proven as an emerging field for genome editing and molecular diagnostics. − Specifically, the Cas12a, as guided by CRISPR RNA (crRNA), can not only specifically recognize the target double-strand DNA (dsDNA) or single-stranded DNA (ssDNA) but also precisely cleave target dsDNA (namely, cis -cleavage) and indiscriminately cleave any ssDNA (namely trans -cleavage). , Compared with other members of the CRISPR family, Cas12a possesses the simpler design of crRNA . Therefore, Cas12a has been widely employed for sequence-specific DNA detection in biosensing field, originating from its simple, specific, and applicable benefits. − However, there remains some challenges to detect exomiRNA directly, resulting from unsatisfactory sensitivity and limited detection species (only target dsDNA or ssDNA) . Therefore, incorporating a nucleic acid amplification strategy before the CRISPR/Cas12a system is indispensable for improving sensitivity and biological conversion of the input target exomiRNA.…”

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice

“…Particularly, we found that the 5′-part of the cleaved substrate by DNAzyme-catalyzing bears a 2′3′-cyclic phosphate at the 3′-terminus, which can act as the activator for RNase activity of Csm6, a much simpler multisubunit single effector from type III-A CRISPR/Cas systems than Cas12 and Cas13. − The Cas13a tandem Csm6 assay is the only application of Csm6 so far; , therefore, the discovery of DNAzymes capable of cascading Csm6 substantially broadens the Csm6 toolbox. In recent years, assays based on CRISPR/Cas12a and DNAzyme have been used for amplified detection of metal ions, but the indispensable separation step made them incapable of a one-tube homogeneous reaction, and the design of guide RNAs and DNAzyme probes could be complex and needs careful optimization. − …”

Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution

“…Cas12a (Cpf1), a class 2 type V-A CRISPR-associated enzyme, has crRNA-guided collateral cleavage activity, which can indiscriminately cleave any nearby single-stranded DNA substrates after activation by a specific target double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). , Taking advantage of collateral cleavage activity and selective target DNA recognition, Cas 12a has been used to develop sensitive assays for nucleic acid diagnostics with different signal outputs (e.g., fluorescence, electrochemistry, colorimetry, surface plasmon resonance (SPR), etc. ). ,− Very recently, a series of innovative Cas12a-based methods has been constructed to detect analytes beyond nucleic acids, ,, such as heavy metal ions, small molecules, − and proteins. − Since Cas12a cannot directly recognize non-nucleic acid molecules, an effective strategy to bridge target binding events with collateral cleavage activity of Cas12a is needed. Bacterial allosteric transcription factors (aTFs) and functional DNAs (e.g., aptamers and DNAzymes) , have been used as recognition elements in the Cas12a-based methods for SM analysis.…”

“…). ,− Very recently, a series of innovative Cas12a-based methods has been constructed to detect analytes beyond nucleic acids, ,, such as heavy metal ions, small molecules, − and proteins. − Since Cas12a cannot directly recognize non-nucleic acid molecules, an effective strategy to bridge target binding events with collateral cleavage activity of Cas12a is needed. Bacterial allosteric transcription factors (aTFs) and functional DNAs (e.g., aptamers and DNAzymes) , have been used as recognition elements in the Cas12a-based methods for SM analysis. However, these assays may suffer from complicated probe designs and the need for immobilization and separation in some assay formats.…”

“…24,25 Taking advantage of collateral cleavage activity and selective target DNA recognition, Cas 12a has been used to develop sensitive assays for nucleic acid diagnostics with different signal outputs (e.g., fluorescence, electrochemistry, colorimetry, surface plasmon resonance (SPR), etc.). 19,21−27 Very recently, a series of innovative Cas12a-based methods has been constructed to detect analytes beyond nucleic acids, 19,22,23 such as heavy metal ions, 28 small molecules, 29−31 and proteins. 32−34 Since Cas12a cannot directly recognize nonnucleic acid molecules, an effective strategy to bridge target binding events with collateral cleavage activity of Cas12a is needed.…”