CRISPR/Cas12a-Amplified Aptamer Switch Microplate Assay for Small Molecules.

The presence of small molecule contaminants such as mycotoxins and heavy metals in foods and the environment causes a serious threat to human health and huge economic losses. The development of simple, rapid, sensitive, and on-site methods for small molecule pollutant detection is highly demanded. Here, combining the advantages of structure-switchable aptamer-mediated signal conversion and CRISPR/Cas12a-based signal amplification, we developed a CRISPR/Cas12a-amplified aptamer switch assay on a microplate for sensitive small molecule detection. In this assay, a short DNA strand complementary to the aptamer (cDNA) is immobilized on a microplate, which can capture the aptamer-linked active DNA probe (Apt-acDNA) in the sample solution when the target is absent. With the addition of the Cas12a reporter system, the captured Apt-acDNA probes activate Cas12a to indiscriminately cleave fluorescent DNA substrates, producing a high fluorescence signal. When the target is present, the Apt-acDNA probe specifically binds to the target rather than hybridizing with cDNA on the microplate, and the fluorescence signal is reduced. The analytical performance of our method was demonstrated by the detection of two highly toxic pollutants, aflatoxin B1 (AFB1) and cadmium ion (Cd2+), as examples. The assay exhibited good selectivity and high sensitivity, with detection limits of 31 pM AFB1 and 3.9 nM Cd2+. It also allowed the detection of targets in the actual sample matrix. With the general signal conversion strategy, this method can be used to detect other targets by simply changing the aptamer and cDNA, showing potential practical applications in broad fields.

CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin.

BACKGROUND: Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. RESULTS: Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. SIGNIFICANCE: This assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.

CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.

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

Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.

Sol-gel based metal-organic framework zeolite imidazolate framework-8 fibers for solid-phase microextraction of nitro polycyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbons in water samples.

In this study, three kinds of Zeolite imidazolate framework-8 (ZIF-8), synthesized by solvothermal, stirring and ball-milling method, were fabricated on the stainless steel wire via sol-gel technique. These fibers were used as solid phase microextraction (SPME) coating materials and applied for analyzing 16 polycyclic aromatic hydrocarbons (PAHs) and 11 nitro polycyclic aromatic hydrocarbons (NPAHs) in environmental water samples by gas chromatography-tandem mass spectrometry (GC-MS). The optimal pH, ionic strength, extraction time, extraction temperature, desorption temperature and desorption time were 6.0, without salt addition, 45 min, 35 °C, 260 °C and 5 min, respectively. The extraction mechanism of the ZIF-8 fiber might be the hydrophobicity, molecular penetration and π-π stacking interactions. Under the optimized conditions, the as-proposed fiber provides a wide linearity range from 10 to 20,000 ng L-1 and a low detection limit of 0.3-27.0 ng L-1 for PAHs and NPAHs analysis. The single fiber and fiber to fiber relative standard deviations were observed in the range of 3.0%-13.9% and 3.5%-12.3%, respectively. The method shows great potential in environmental analysis field.

Transient Receptor Potential Vanilloid Subtype 1 Inhibits Inflammation and Apoptosis via the Release of Calcitonin Gene-Related Peptide in the Heart after Myocardial Infarction.

OBJECTIVE: A high mortality rate occurs with silent myocardial infarction (MI), particularly in aging and diabetic populations due to defects in the transient receptor potential vanilloid (TRPV1)-positive sensory nerve function. We have previously shown that TRPV1 deficiency markedly enhances post-MI inflammation and remodeling. However, the mechanisms remain unknown. The objective of this study was to clarify whether calcitonin gene-related peptide (CGRP) release was associated with the protective role of TRPV1 against postmyocardial inflammation and apoptosis. METHODS: TRPV1 gene knockout (TRPV1KO) and wild-type (WT) mice were subjected to left anterior descending ligation or sham operation. The concentration of CGRP in the myocardium was measured at 30 min, 1, 6 and 24 h post-MI. Mice received saline vehicle, CGRP or the CGRP antagonist CGRP8-37 before ligation. Inflammation was evaluated by ELISA assay and histological staining. Apoptosis was assessed by Western blot and TUNEL assay. RESULTS: Post-MI, both TRPV1KO and WT mice displayed elevated CGRP levels in myocardium when compared to sham controls. However, the levels of CGRP were significantly lower in TRPV1KO mice than in WT mice at 30 min after MI. Exogenous CGRP downregulated the levels of tumor necrosis factor-α and interleukin-6 expression in TRPV1KO mice post-MI. Moreover, exogenous CGRP decreased the neutrophil infiltration in TRPV1KO mice, whereas inhibition of CGRP by CGRP8-37 increased the neutrophil infiltration in WT mice. Western blotting data indicated that CGRP attenuated caspase-3 and caspase-9 expression, and enhanced Bcl-2 expression in TRPV1KO mice post-MI. CGRP8-37 upregulated caspase-3 and caspase-9 expression and downregulated Bcl-2 expression in WT mice. CONCLUSION: Our data suggest a protective role of TRPV1 activation against inflammation and apoptosis in mice post-MI, possibly through CGRP release. These findings elucidate a neurogenic mechanism in mice post-MI, which may participate in sensory neurotransmitter-mediated protection in TRPV1 activation.