High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

Agronomic and Genetic Strategies to Enhance Selenium Accumulation in Crops and Their Influence on Quality.

Selenium (Se) is an essential trace element that plays a crucial role in maintaining the health of humans, animals, and certain plants. It is extensively present throughout the Earth's crust and is absorbed by crops in the form of selenates and selenite, eventually entering the food chain. Se biofortification is an agricultural process that employs agronomic and genetic strategies. Its goal is to enhance the mechanisms of crop uptake and the accumulation of exogenous Se, resulting in the production of crops enriched with Se. This process ultimately contributes to promoting human health. Agronomic strategies in Se biofortification aim to enhance the availability of exogenous Se in crops. Concurrently, genetic strategies focus on improving a crop's capacity to uptake, transport, and accumulate Se. Early research primarily concentrated on optimizing Se biofortification methods, improving Se fertilizer efficiency, and enhancing Se content in crops. In recent years, there has been a growing realization that Se can effectively enhance crop growth and increase crop yield, thereby contributing to alleviating food shortages. Additionally, Se has been found to promote the accumulation of macro-nutrients, antioxidants, and beneficial mineral elements in crops. The supplementation of Se biofortified foods is gradually emerging as an effective approach for promoting human dietary health and alleviating hidden hunger. Therefore, in this paper, we provide a comprehensive summary of the Se biofortification conducted over the past decade, mainly focusing on Se accumulation in crops and its impact on crop quality. We discuss various Se biofortification strategies, with an emphasis on the impact of Se fertilizer strategies on crop Se accumulation and their underlying mechanisms. Furthermore, we highlight Se's role in enhancing crop quality and offer perspective on Se biofortification in crop improvement, guiding future mechanistic explorations and applications of Se biofortification.

Coupling of different antioxidative systems in rice under the simultaneous influence of selenium and cadmium.

Selenium (Se) elevates the antioxidant ability of rice against cadmium (Cd) stress, but previous studies only focused on the variation in antioxidant enzymes or nonenzymatic substances induced by Se under Cd stress and ignored the relationships between different antioxidant parameters during the interaction. Here, hydroponic experiments with rice were performed by adding both Cd and Se at doses in the range of 0-50 µM to explore the physiological responses of rice and their relationships in the presence of different levels of Se and Cd. Exogenous Cd markedly promoted the activity of antioxidant enzymes with the exception of catalase (CAT) and the concentration of nonenzymatic substances in aerial parts. Se enhanced the antioxidant capacity by improving the activities of all the enzymes tested in this study and increasing the concentrations of nonenzymatic compounds. The couplings among different antioxidant substances within paddy rice were then determined based on cluster and linear fitting results and their metabolic process and physiological functions. The findings specifically highlight that couplings among the ascorbic acid (AsA)-glutathione (GSH) cycle, glutathione synthase (GS)-phytochelatin synthetase (PCS) coupling system and glutathione peroxidase (GPX)-superoxide dismutase (SOD) coupling system in aerial parts helps protect plants from Cd stress. These coupling systems form likely due to the fact that one enzyme generated a product that could be the substrate for another enzyme. Noticeably, such coupling systems do not emerge in roots because the stronger damage to roots than other organs activates the ascorbate peroxidase (APX)-GPX-CAT and PCS-GS-SOD systems with distinct functions and structures. This study provides new insights into the detoxification mechanisms of rice caused by the combined effect of Se and Cd.

Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.

Rolling Circle Amplification as an Efficient Analytical Tool for Rapid Detection of Contaminants in Aqueous Environments.

Environmental contaminants are a global concern, and an effective strategy for remediation is to develop a rapid, on-site, and affordable monitoring method. However, this remains challenging, especially with regard to the detection of various contaminants in complex water environments. The application of molecular methods has recently attracted increasing attention; for example, rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants. In this paper, we describe the fundamental principles of RCA and the advantages and disadvantages of RCA assays. Then, we discuss the recently developed RCA-based tools for environmental analysis to determine various targets, including heavy metals, organic small molecules, nucleic acids, peptides, proteins, and even microorganisms in aqueous environments. Finally, we summarize the challenges and outline strategies for the advancement of this technique for application in contaminant monitoring.

Exogenous selenium (cadmium) inhibits the absorption and transportation of cadmium (selenium) in rice.

Antagonism between selenium (Se) and cadmium (Cd) has been demonstrated in plants. However, a mutual suppression threshold for Se and Cd has not been identified in previous studies using Cd or Se individually. To fill this knowledge gap, we determined the levels of Se and Cd in various tissues of rice under concentration gradients of Se and Cd with different Se application times via hydroponic experiments. The results showed that the application of exogenous Se or Cd reduced the uptake and transport of the other. When the molar ratio of Se/Cd (R (Se/Cd)) was higher than 1, the concentration and transfer factor of Cd (TF-Cd) in all parts of rice simultaneously reached the lowest values. The minimum Se absorption in rice was obtained at R (Cd/Se) greater than 20, while no inhibition threshold was found for Se transport. In addition, approximately 1:1 R (Se/Cd) was observed in roots and the addition of exogenous Cd or Se promoted the enrichment of the other element in roots. These data suggested a mutual inhibition of Se and Cd in their absorption, transportation and accumulation in rice, which might be related to the formation of insoluble Cd-Se complexes in roots. This study provided new insights into a plausible explanation of the interactions between Se and Cd and contributed to the remediation and treatment of combined Se and Cd pollution in farmland systems.

Nanomaterial-based aptamer sensors for analysis of illicit drugs and evaluation of drugs consumption for wastewater-based epidemiology.

The abuse of illicit drugs usually associated with dramatic crimes may cause significant problems for the whole society. Wastewater-based epidemiology (WBE) has been demonstrated to be a novel and cost-effective way to evaluate the abuse of illicit drugs at the community level, and has been used as a routine method for monitoring and played a significant role for combating the crimes in some countries, e.g. China. The method can also provide temporal and spatial variation of drugs of abuse. The detection methods mainly remain on the conventional liquid chromatography coupled with mass spectrometry, which is extremely sensitive and selective, however needs advanced facility and well-trained personals, thus limit it in the lab. As an alternative, sensors have emerged to be a powerful analytical tool for a wide spectrum of analytes, in particular aptamer sensors (aptasensors) have attracted increasing attention and could act as an efficient tool in this field due to the excellent characteristics of selectivity, sensitivity, low cost, miniaturization, easy-to-use, and automation. In this review, we will briefly introduce the context, specific assessment process and applications of WBE and the recent progress of illicit drug aptasensors, in particular focusing on optical and electrochemical sensors. We then highlight several recent aptasensors for illicit drugs in new technology integration and discuss the feasibility of these aptasensor for WBE. We will summarize the challenges and propose our insights and opportunity on aptasensor for WBE to evaluate community-wide drug use trends and public health.

Paper-based microfluidics for rapid diagnostics and drug delivery.

Paper is a common material that is promising for constructing microfluidic chips (lab-on-a-paper) for diagnostics and drug delivery for biomedical applications. In the past decade, extensive research on paper-based microfluidics has accumulated a large number of scientific publications in the fields of biomedical diagnosis, food safety, environmental health, drug screening and delivery. This review focuses on the recent progress on paper-based microfluidic technology with an emphasis on the design, optimization and application of the technology platform, in particular for medical diagnostics and drug delivery. Novel advances have concentrated on engineering paper devices for point-of-care (POC) diagnostics, which could be integrated with nucleic acid-based tests and isothermal amplification experiments, enabling rapid sample-to-answer assays for field testing. Among the isothermal amplification experiments, loop-mediated isothermal amplification (LAMP), an extremely sensitive nucleic acid test, specifically identifies ultralow concentrations of DNA/RNA from practical samples for diagnosing diseases. We thus mainly focus on the paper device-based LAMP assay for the rapid infectious disease diagnosis, foodborne pathogen analysis, veterinary diagnosis, plant diagnosis, and environmental public health evaluation. We also outlined progress on paper microfluidic devices for drug delivery. The paper concludes with a discussion on the challenges of this technology and our insights into how to advance science and technology towards the development of fully functional paper devices in diagnostics and drug delivery.

Nanomaterial-based aptamer sensors for arsenic detection.

Arsenic (As) is a highly toxic contaminant in the environment and a serious carcinogen for the human being. The toxicity of arsenic significantly threatens environmental and human health. The effective removing technology for arsenic remains challenging, and one of the reasons is due to the lack of powerful detection method in the complex environmental matrix. There is thus an urgent need to develop novel analytical methods for arsenic, preferably with the potential for the field-testing. To combat arsenic pollution and maintain a healthy environment and eco-system, many analytical methods have been developed for arsenic detection in various samples. Among these strategies, biosensors hold great promise for rapid detection of arsenic, in particular, nanomaterials-based aptamer sensors have attracted significant attention due to their simplicity, high sensitivity and rapidness. In this paper, we reviewed the recent development and applications of aptamer sensors (aptasensors) based-on nanomaterial for arsenic detection, in particular with emphasis on the works using optical and electrochemical technologies. We also discussed the recent novel technology in aptasensors development for arsenic detection, including nucleic acid amplification for signal enhancement and device integration for the portability of arsenic sensors. We are hoping this review could inspire further researches in developing novel nanotechnologies based aptasensors for possible on-site detection of arsenic.