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Portable electrochemical aptasensor for highly sensitive detection of 3,3',4,4'-tetrachlorobiphenyl.Biosensors & Bioelectronics Sep 2024Aptamer-based electrochemical sensors are frequently used as independent, surface-functionalized, passive electrodes. However, their sensitivity and detection limits...
Aptamer-based electrochemical sensors are frequently used as independent, surface-functionalized, passive electrodes. However, their sensitivity and detection limits become limited, particularly when the electrode area is reduced to facilitate miniaturization. A mobile phone-based microfluidic electrochemical aptamer sensing platform for 3,3',4,4'-tetrachlorobiphenyl (PCB77) detection was developed in this work. This aptamer sensor utilized Exonuclease I (Exo I) and DNA/AuNPs/horseradish peroxidase (DNA/AuNPs/HRP) nanoprobes as a merged signal amplification method, which resulted in an increase in the electrochemical sensing performance. Sensitive detection of PCB77 was accomplished by functionalizing the hierarchically structured Au@MoS/CNTs/GO modified working/sensing electrode with the specific aptamer. The aptamer sensor was tested with different concentrations of PCB77 within the microfluidic platform. Afterward, the differential pulse voltammograms were recorded using a wireless integrated circuit device. Subsequently, the collected data was transmitted to a smartphone using Bluetooth communication. A detection limit of 0.0085 ng/L was obtained for PCB77 detection, with a detection range from 0.1 to 1000 ng/L. In addition, the detection of PCB77 in spiked water samples validated the possibility of using this aptamer sensor in a real environment, and the aptamer sensor demonstrated high selectivity in distinguishing PCB77 from other potential interfering species. The merging of electrochemical aptamer sensors with purposefully engineered microfluidic and integrated devices in this study is a novel and promising method that provides a dependable platform for on-site applications.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Polychlorinated Biphenyls; Gold; Electrochemical Techniques; Limit of Detection; Metal Nanoparticles; Exodeoxyribonucleases; Horseradish Peroxidase; Nanotubes, Carbon; Molybdenum; Equipment Design; Water Pollutants, Chemical; DNA; Smartphone
PubMed: 38810414
DOI: 10.1016/j.bios.2024.116434 -
Biosensors & Bioelectronics Sep 2024To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro...
To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%-100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine.
Topics: Biosensing Techniques; Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Hepatitis B virus; DNA; Mycoplasma pneumoniae; CRISPR-Associated Proteins; Carcinoma, Non-Small-Cell Lung; Bacterial Proteins; Lung Neoplasms; Endodeoxyribonucleases; Pneumonia, Mycoplasma
PubMed: 38805891
DOI: 10.1016/j.bios.2024.116428 -
Nanoscale Jun 2024The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the...
The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.
Topics: Biosensing Techniques; Nanocomposites; Electrochemical Techniques; CRISPR-Cas Systems; Humans; CRISPR-Associated Proteins; Limit of Detection; Bacterial Proteins; Animals; Endodeoxyribonucleases; Biofouling
PubMed: 38804270
DOI: 10.1039/d4nr01618a -
Aging May 2024Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that...
Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.
Topics: Animals; Male; Female; Werner Syndrome; Liver; Mice; Proteomics; Aging; Werner Syndrome Helicase; Sex Characteristics; Disease Models, Animal
PubMed: 38795389
DOI: 10.18632/aging.205866 -
International Journal of Molecular... May 2024The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent...
The myostatin () gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between and age-related diseases. It is unclear how and age-associated muscle loss in other animals are related. In this study, we utilized gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to and muscle cell senescence. The expression of was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that influenced three prime repair exonuclease 1 () expression through the SMAD2/3 complex. The downregulation of contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of in cell senescence in large animals.
Topics: Animals; Myostatin; Cattle; Cellular Senescence; Exodeoxyribonucleases; Signal Transduction; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphoproteins; Cells, Cultured
PubMed: 38791317
DOI: 10.3390/ijms25105277 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They... (Review)
Review
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Topics: Gene Editing; CRISPR-Cas Systems; Transcription Activator-Like Effector Nucleases; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Engineering; Humans
PubMed: 38783797
DOI: 10.13345/j.cjb.230615 -
Analytica Chimica Acta Nov 2023Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and...
BACKGROUND
Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and sensitive detection methods is vital to prevent and manage foodborne diseases. In this study, we used tetraethylenepentamine (TEPA)-functionalized magnetic nanoparticles (MNPs) and a loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a-based biosensor to concentrate and detect, respectively, L. monocytogenes. LAMP enables DNA amplification at a constant temperature, providing a highly suitable approach for point-of-care testing (POCT). The ability of CRISPR/Cas12a to cleave ssDNA reporter, coupled with TEPA-functionalized MNPs effective attachment to negatively charged bacteria, forms a promising biosensor.
RESULTS
The LAMP assay was meticulously developed by selecting specific primers and designing crRNA sequences targeting a specific region within the hly gene of L. monocytogenes. We selected primer and refined the amplification conditions by systematically exploring a temperature range from 59 °C to 69 °C, ensuring the attainment of optimal performance. This process was complemented by systematic optimization of LAMP-CRISPR/Cas12a system parameters. In particular, we successfully established the optimal ssDNA reporter concentrations (0-1.2 μM) and Cas12a-mediated trans-cleavage times (0-20 min), crucial components that underpin the effectiveness of the LAMP-CRISPR/Cas12a-based biosensor. For optimizing parameters in capturing L. monocytogenes using TEPA-functionalized MNPs, capture efficiency was significantly enhanced through adjustments in TEPA-functionalized MNPs concentration, incubation times, and magnetic separation duration. Large-volume (20 mL) magnetic separation exhibited a 10-fold sensitivity improvement over conventional methods. Utilizing TEPA-functionalized MNPs, the LAMP-CRISPR/Cas12a-based biosensor achieved detection limits of 10 CFU mL in pure cultures and 10 CFU g in enoki mushrooms.
SIGNIFICANCE
The integration of this novel technique with the LAMP-CRISPR/Cas12a-based biosensor enhances the accuracy and sensitivity of L. monocytogenes detection in foods, and it can be a promising biosensor for POCT. The 10-fold increase in sensitivity compared to conventional methods makes this approach a groundbreaking advancement in pathogenic bacteria detection for food safety and public health.
Topics: Listeria monocytogenes; Biosensing Techniques; Nucleic Acid Amplification Techniques; CRISPR-Cas Systems; Magnetite Nanoparticles; Limit of Detection; Food Microbiology; Bacterial Proteins; Endodeoxyribonucleases; Molecular Diagnostic Techniques; CRISPR-Associated Proteins
PubMed: 38783743
DOI: 10.1016/j.aca.2023.341905 -
Gut May 2024Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to...
OBJECTIVE
Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH.
DESIGN
C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated.
RESULTS
We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract).
CONCLUSION
Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.
PubMed: 38777573
DOI: 10.1136/gutjnl-2023-331447 -
Analytica Chimica Acta Jun 2024CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step...
BACKGROUND
CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA.
RESULTS
Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %.
SIGNIFICANCE
The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.
Topics: SARS-CoV-2; DNA, Single-Stranded; Nucleic Acid Amplification Techniques; Humans; RNA, Viral; COVID-19; Limit of Detection; CRISPR-Cas Systems; Endodeoxyribonucleases; CRISPR-Associated Proteins; Bacterial Proteins
PubMed: 38772660
DOI: 10.1016/j.aca.2024.342693 -
BMC Biology May 2024Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be...
BACKGROUND
Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging.
RESULTS
Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control.
CONCLUSIONS
Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.
Topics: Gene Editing; Humans; Exodeoxyribonucleases; CRISPR-Cas Systems; HEK293 Cells; DNA Repair Enzymes
PubMed: 38769511
DOI: 10.1186/s12915-024-01918-w