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Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of ß-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

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Detecting ß-estradiol (E2) in environmental monitoring is a complex task due to its status as a significant environmental contaminant. The detection methods require precision, sensitivity, and the ability to be conducted on-site without expensive instrumentation. Herein, we developed a novel approach using E2 aptamer assembled spherical nucleic acids (SNAs), which combines the sensitivity of fluorescence and the simplicity of colorimetry. Initially, a fluorescein (FAM)-labeled DNA aptamer is attached to the surface of gold nanoparticles (AuNPs) through hybridization with thiol-labeled DNA, resulting in fluorescence quenching. However, when E2 is present, the aptamer specifically binds to it, displacing from the thiol-DNA and releasing from the AuNP's surface. This leads to the recovery of fluorescence, allowing for quantitative detection of E2 by measuring the increase in fluorescence signal. Additionally, E2 detection can also be achieved visually using ultraviolet light. For colorimetric analysis, we introduce another set of AuNPs modified with thiol-DNA complementary to the DNA remaining on the surface of the previous AuNPs. When E2 triggers the release of the aptamer, the DNA on both AuNPs hybridized to each other, causing the aggregation of AuNPs and resulting in a distinct color change from red to purple. The detection limits for fluorescence and colorimetric analyses are 1 nM and 5 nM, respectively. We successfully applied this biosensing strategy to determine E2 concentrations in tap water and serum samples. Furthermore, our assay exhibits high selectivity towards E2 over other estrogens. Overall, this innovative approach provides an effective and versatile method for convenient on-site monitoring of E2.

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Triclocarban (TCC) is an antimicrobial ingredient that commonly incorporated in many household and personal care products, raising public concerns about its potential health risks. Previous research has showed that TCC could cross the blood-brain barrier, but to date our understanding of its potential neurotoxicity at human-relevant concentrations remains lacking. In this study, we observed anxiety-like behaviors in mice with continuous percutaneous exposure to TCC. Subsequently, we combined lipidomic, proteomic, and metabolic landscapes to investigate the underlying mechanisms of TCC-related neurotoxicity. The results showed that TCC exposure dysregulated the proteins involved in endocytosis and neurodegenerative disorders in mouse cerebrum. Brain energy homeostasis was also altered, as evidenced by the perturbation of pyruvate metabolism, TCA cycle, and oxidative phosphorylation, which in turn caused mitochondrial dysfunction. Meanwhile, the changing trends of sphingolipid signaling pathway and overproduction of mitochondrial reactive oxygen species (mROS) could enhance the neural apoptosis. The in vitro approach further demonstrated that TCC exposure promoted apoptosis, accompanied by the overproduction of mROS and alteration in the mitochondrial membrane potential in N2A cells. Together, dysregulated endocytosis, mROS-related mitochondrial dysfunction and neural cell apoptosis are considered to be crucial factors for TCC-induced neurotoxicity, which may contribute to the occurrence and development of neurodegenerative disorders. Our findings provide novel perspectives for the mechanisms of TCC-triggered neurotoxicity.

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Active clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) systems possess both cis-cleavage (targeted) and trans-cleavage (collateral) activities, which are useful for genome engineering and diagnostic applications. Both single- and double-stranded DNA can activate crRNA-Cas12a ribonucleoprotein (RNP) to achieve cis- and trans-cleavage enzymatic activities. However, it is not clear whether RNA can activate the CRISPR/Cas12a system and what is critical to the trans-cleavage activity. We report here that RNA can activate the CRISPR/Cas12a system and trigger its trans-cleavage activity. We reveal that the activated crRNA-Cas12a RNP favors the trans-cleavage of longer sequences than commonly used. These new findings of the RNA-activated trans-cleavage capability of Cas12a provided the foundation for the design and construction of CRISPR nanorobots that operate in living cells. We assembled the crRNA-Cas12a RNP and nucleic acid substrates on gold nanoparticles to form CRISPR nanorobots, which dramatically increased the local effective concentration of the substrate in relation to the RNP and the trans-cleavage kinetics. Binding of the target microRNA to the crRNA-Cas12a RNP activated the nanorobots and their trans-cleavage function. The repeated (multiple-turnover) trans-cleavage of the fluorophore-labeled substrates generated amplified fluorescence signals. Sensitive and real-time imaging of specific microRNA in live cells demonstrated the promising potential of the CRISPR nanorobot system for future applications in monitoring and modulating biological functions within living cells.

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Arsenic pollution in paddy fields has become a public concern by seriously threatening rice growth, food security and human health. In this review, we delve into the biogeochemical behaviors of arsenic in paddy soil-rice system, systemically revealing the complexity of its migration and transformation processes, including the release of arsenic from soil to porewater, uptake and translocation of arsenic by rice plants, as well as transformation of arsenic species mediated by microorganism. Especially, microbial processes like reduction, oxidation and methylation of arsenic, and the coupling of arsenic with carbon, iron, sulfur, nitrogen cycling through microbes and related mechanisms were highlighted. Environmental factors like pH, redox potential, organic matter, minerals, nutrient elements, microorganisms and periphyton significantly influence these processes through different pathways, which are discussed in this review. Furthermore, the current progress in remediation strategies, including agricultural interventions, passivation, phytoremediation and microbial remediation is explored, and their potential and limitations are analyzed to address the gaps. This review offers comprehensive perspectives on the complicated behaviors of arsenic and influence factors in paddy soil-rice system, and provides a scientific basis for developing effective arsenic pollution control strategies.

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The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.

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Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

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Real-time monitoring of different types of intracellular tumor-related biomarkers is of key importance for the identification of tumor cells. However, it is hampered by the low abundance of biomarkers, inefficient free diffusion of reactants, and complex cytoplasmic milieu. Herein, we present a stable and general method for in situ imaging of microRNA-21 and telomerase utilizing simple highly integrated dual tetrahedral DNA nanostructures (TDNs) that can naturally enter cells, which could initiate to form the three-dimensional (3D) higher-order DNA superstructures (DNA nanofireworks, DNFs) through a reliable target-triggered entropy-driven strand displacement reaction in living cells for remarkable signal amplification. Importantly, the excellent biostability, biocompatibility, and sensitivity of this approach benefited from (i) the precise multidirectional arrangement of probes with a pure DNA structure and (ii) the local target concentration enhanced by the spatially confined microdomain inside the DNFs. This strategy provides a pivotal molecular toolbox for broad applications such as biomedical imaging and early precise cancer diagnosis.

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DNAzyme motor systems using gold nanoparticles (AuNPs) as scaffolds are useful for biosensing and in situ amplification because these systems are free of protein enzymes, isothermal, homogeneous, and sensitive. However, detecting different targets using the available DNAzyme motor techniques requires redesigns of the DNAzyme motor. We report here a toehold-exchange translator and the translator-mediated DNAzyme motor systems, which enable sensitive responses to various nucleic acid targets using the same DNAzyme motor without requiring redesign. The translator is able to efficiently convert different nucleic acid targets into a specific output DNA that further activates the pre-silenced DNAzyme motor and consequently initiates the autonomous walking of the DNAzyme motor. Simply adjusting the target-binding region of the translator enables the same DNAzyme motor system to respond to various nucleic acid targets. The translator-mediated DNAzyme motor system is able to detect as low as 2.5 pM microRNA-10b and microRNA-21 under room temperature without the need of separation or washing. We further demonstrate the versatility of the translator and the DNAzyme motor by successful construction and operation of four logic gates, including OR, AND, NOR, and NAND logic gates. These logic gates use two microRNA targets as inputs and generate amplified fluorescence signals from the operation of the same DNAzyme motor. Incorporation of the toehold-exchange translator into the DNAzyme motor technology improves the biosensing applications of DNA motors to diverse nucleic acid targets.

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CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

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Protein coronae formed with nanoparticles confer several useful properties. However, the non-specific nature of protein corona formation makes it difficult to deliver specific proteins for therapeutic applications. Herein, we report on the construction of a new type of protein corona, termed binding-mediated protein corona. This new corona enables the efficient and controllable delivery of functional proteins, which is otherwise challenging for conventional protein coronae. We show the design and delivery of the ribonucleoprotein corona for the CRISPR/Cas9 system. Successful gene editing in human cell lines (Hela and HEK293) demonstrates the efficient delivery, high stability, low cytotoxicity, and well-controlled activity of the Cas9-guide RNA ribonucleoprotein. The binding-mediated protein corona strategy opens up new opportunities for therapeutic protein delivery.

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We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

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Binding of nucleic acid aptamers to specific targets and detection with fluorescence anisotropy (FA) or fluorescence polarization (FP) take advantage of the complementary features of aptamers and the fluorescence techniques. We review recent advances in affinity binding assays using aptamers and FA/FP, with an emphasis on studies of molecular interactions and identification of binding sites. Aptamers provide several benefits, including the ease of labelling fluorophores on specific sites, binding-induced changes in aptamer structures, hybridization of the aptamers to complementary sequences, changes in molecular volume upon binding of the aptamer to its target, and adsorption of aptamers onto nanomaterials. Some of these benefits have been utilized for FA/FP assays. Once the aptamer binds to its target, the resulting changes in molecular volume (size), structure, local rotation of the fluorophore, and/or the fluorescence lifetime influence changes to the FA/FP values. Measurements of these fluorescence anisotropy/polarization changes have provided insights into the molecular interactions, such as the binding affinity and the site of binding. Studies of molecular interactions conducted in homogeneous solutions, as well as those with separations, e.g., capillary electrophoresis, have been summarized in this review. Studies on mapping the position of binding in aptamers at the single nucleotide level have demonstrated a unique benefit of the FA/FP techniques and pointed to an exciting direction for future research.

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Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

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The RNA-guided CRISPR/Cas9 system is a powerful genome-editing technology with broad applications. Improving delivery efficiency and controllable activity of the CRISPR/Cas9 system is an area of intense research. We report the design, construction, and application of a CRISPR/Cas9 nanomachine (LACM), activated by a near-infrared (NIR) laser, which enables efficient delivery of single-guide RNA (sgRNA) into living cells and achieves controlled release of the sgRNA for the CRISPR/Cas9 activity. The LACM was constructed using a gold nanorod (AuNR) as a carrier that was decorated with dozens of protector DNAs stably hybridizing with the target binding domain of sgRNA. The DNA assembly on the AuNR protected the sgRNA. Irradiation with a NIR laser generated heat on the AuNR, resulting in controlled release of sgRNA, which guided the CRISPR/Cas9 genome editing. Successful editing of the EGFP and EMX1 genes in A549 and HEK293T cells, as well as knocking down of the PLK1 gene to induce apoptosis of the target cells, highlights the promising potential of the LACM for diverse applications.

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Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, ROX) is an arsenic-containing compound widely used as a feed additive in poultry industries. ROX excreted in chicken manure can be transformed by microbes to different arsenic species in the environment. To date, most of the studies on microbial transformation of ROX have focused on anaerobic microorganisms. Here, we isolated a pure cultured aerobic ROX-transforming bacterial strain, CZ-1, from an arsenic-contaminated paddy soil. On the basis of 16S rRNA gene sequence, strain CZ-1 was classified as a member of the genus Enterobacter. During ROX biotransformation by strain CZ-1, five metabolites including arsenate (As[V]), arsenite (As[III]), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and a novel sulfur-containing arsenic species (AsC9H13N2O6S) were detected and identified based on high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), HPLC-ICP-MS/electrospray ionization mass spectrometry (ESI-MS) and HPLC-electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS) analyses. N-AHPAA and 3-AHPAA were the main products, and 3-AHPAA could also be transformed to N-AHPAA. Based on the results, we propose a novel ROX biotransformation pathway by Enterobacter. sp CZ-1, in which the nitro group of ROX is first reduced to amino group (3-AHPAA) and then acetylated to N-AHPAA.

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