A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay.

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.

Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.

Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target a different site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing a fluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16 pM, which is a decrease of the LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.

Nucleic Acid Amplification-Free Digital Detection Method for SARS-CoV-2 RNA Based on Droplet Microfluidics and CRISPR-Cas13a.

Most of the methods currently developed for RNA detection based on CRISPR were combined with nucleic acid amplification. As a result, such methods inevitably led to certain disadvantages such as multiple operations, expensive reagents, and amplification bias. To solve the above problems, we developed a highly sensitive and specific nucleic acid amplification-free digital detection method for SARS-CoV-2 RNA based on droplet microfluidics and CRISPR-Cas13a. In this assay, thousands of monodisperse droplets with a size of 30 µm were generated within 2 min by a negative pressure-driven microfluidic chip. By confining a single target RNA recognition event to an independent droplet, the collateral cleavage products of activated Cas13a could be accumulated in one droplet. By combining the droplet microfluidics and CRISPR-Cas13a, SARS-CoV-2 RNA could be easily detected within 30 min with a detection limit of 470 aM. The performance of this assay was verified by specificity experiments and spiking and recovery experiments with human saliva. Compared with many developed methods for SARS-CoV-2 RNA detection, our method is time- and reagent-saving and easy to operate. Taken together, this digital detection method based on droplet microfluidics and CRISPR-Cas13a provides a promising approach for RNA detection in clinical diagnostics.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Removal of calcium and magnesium ions from reverse osmosis concentrate using a two-stage precipitation with carbonation process.

The reverse osmosis (RO) technique has been extensively employed in the advanced treatment of industrial water and wastewater. However, this process results in the production of a significant quantity of reverse osmosis concentrate (ROC), which contains high levels of salinity and organic contaminants, thereby posing serious environmental problems. This study reported a two-stage precipitation process utilizing quicklime (CaO) and caustic soda (NaOH) in conjunction with air blowing (carbonation) for the removal of Ca2+ and Mg2+ from real brackish water ROC of factory. In stage I, the CaO precipitation-carbonation process was employed to eliminate the majority of Ca2+ from the ROC, while leaving Mg2+ virtually unaffected, yielding high-purity CaCO3 precipitates. In stage II, the NaOH precipitation method was utilized to eliminate the remaining Ca2+ and Mg2+ from the ROC. It was demonstrated that under optimal conditions, the removal rates of Ca2+ and Mg2+ exceeded 97%. Finally, the characterization of precipitates demonstrated the generation of high-purity CaCO3 precipitates in stage I, as well as the formation of CaCO3 and Mg(OH)2 precipitates in stage II. The results confirmed the feasibility of employing the two-stage precipitation with carbonation process to economically treat ROC and enable its reuse, offering valuable insights for the treatment of industrial wastewater.