RESUMO
Reactive oxygen species (ROS) including the superoxide anion (O2â¢-) are typically studied in cell cultures using fluorescent dyes, which provide only discrete single-point measurements. These methods lack the capabilities for assessing O2â¢- kinetics and release in a quantitative manner over long monitoring times. Herein, we present the fabrication and application of an electrochemical biosensor that enables real-time continuous monitoring of O2â¢- release in cell cultures for extended periods (> 8 h) using an O2â¢- specific microelectrode. To achieve the sensitivity and selectivity requirements for cellular sensing, we developed a biohybrid system consisting of superoxide dismutase (SOD) and Ti3C2Tx MXenes, deposited on a gold microwire electrode (AuME) as O2â¢- specific materials with catalytic amplification through the synergistic action of the enzyme and the biomimetic MXenes-based structure. The biosensor demonstrated a sensitivity of 18.35 nA/µM with a linear range from 147 to 930 nM in a cell culture medium. To demonstrate its robustness and practicality, we applied the biosensor to monitor O2â¢- levels in human leukemia monocytic THP-1 cells upon stimulation with lipopolysaccharide (LPS). Using this strategy, we successfully monitored LPS-induced O2â¢- in THP-1 cells, as well as the quenching effect induced by the ROS scavenger N-acetyl-L-cysteine (NAC). The biosensor is generally useful for exploring the role of oxidative stress and longitudinally monitoring O2â¢- release in cell cultures, enabling studies of biochemical processes and associated oxidative stress mechanisms in cellular and other biological environments.
Assuntos
Técnicas Biossensoriais , Superóxido Dismutase , Superóxidos , Humanos , Superóxidos/metabolismo , Superóxidos/análise , Técnicas Biossensoriais/métodos , Superóxido Dismutase/metabolismo , Células THP-1 , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Lipopolissacarídeos/farmacologia , Limite de DetecçãoRESUMO
Per- and polyfluoroalkyl substances (PFAS) pose a significant threat to the environment due to their persistence, ability to bioaccumulate, and harmful effects. Methods to quantify PFAS rapidly and effectively are essential to analyze and track contamination, but measuring PFAS down to the ultralow regulatory levels is extremely challenging. Here, we describe the development of a low-cost sensor that can measure a representative PFAS, perfluorooctanesulfonic acid (PFOS), at the parts per quadrillion (ppq) level within 5 min. The method combines the ability of PFOS to bind to silver nanoparticles (AgNPs) embedded within a fluorine-rich Ti3C2-based multilayered MXene, which provides a large surface area and accessible binding sites for direct impedimetric detection. Fundamentally, we show that MXene-AgNPs are capable of binding PFOS and other long-chain PFAS compounds, though the synergistic action of AgNPs and MXenes via electrostatic and F-F interactions. This binding induced concentration-dependent changes in the charge-transfer resistance, enabling rapid and direct quantification with extremely high sensitivity and no response to interferences. The sensor displayed a linear range from 50 ppq to 1.6 ppt (parts per trillion) with an impressively low limit of detection of 33 ppq and a limit of quantification of 99 ppq, making this sensor a promising candidate for low-cost screening of the PFAS content in water samples, using a simple and inexpensive procedure.
Assuntos
Ácidos Alcanossulfônicos , Técnicas Eletroquímicas , Fluorocarbonos , Nanopartículas Metálicas , Prata , Fluorocarbonos/química , Fluorocarbonos/análise , Nanopartículas Metálicas/química , Prata/química , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Ácidos Alcanossulfônicos/análise , Ácidos Alcanossulfônicos/química , Limite de Detecção , Poluentes Químicos da Água/análiseRESUMO
Two-dimensional (2D) layered materials that integrate metallic conductivity, catalytic activity and the ability to stabilize biological receptors provide unique capabilities for designing electrochemical biosensors for large-scale detection and diagnostic applications. Herein, we report a multifunctional MXene-based 2D nanostructure decorated with enzyme mimetic cerium oxide nanoparticle (MXCeO2) as a novel platform and catalytic amplifier for electrochemical biosensors, specifically targeting the detection of oxidase enzyme substrates. We demonstrate enhanced catalytic efficiency of the MXCeO2 for the reduction of hydrogen peroxide (H2O2) and its ability to immobilize oxidase enzymes, such as glucose oxidase, lactate oxidase and xanthine oxidase. The designed biosensors exhibit high selectivity, stability, and sensitivity, achieving detection limits of 0.8 µM H2O2, 0.49 µM glucose, 3.6 µM lactate and 1.7 µM hypoxanthine, when the MXCeO2 and their respective enzymes were used. The MXCeO2 was successfully incorporated into a wearable fabric demonstrating high sensitivity for lactate measurements in sweat. The unique combination of MXenes with CeO2 offers excellent conductivity, catalytic efficiency and enhanced enzyme loading, demonstrating potential of the MXCeO2 as a catalytically active material to boost efficiency of oxidase enzyme reactions. This design can be used as a general platform for increasing the sensitivity of enzyme based biosensors and advance the development of electrochemical biosensors for a variety of applications.
Assuntos
Técnicas Biossensoriais , Dispositivos Eletrônicos Vestíveis , Técnicas Biossensoriais/métodos , Suor , Peróxido de Hidrogênio , Glucose Oxidase/química , Lactatos , Técnicas Eletroquímicas/métodosRESUMO
Nanoelectrochemistry allows for the investigation of the interaction of per- and polyfluoroalkyl substances (PFASs) with silver nanoparticles (AgNPs) and the elucidation of the binding behaviour of PFASs to nanoscale surfaces with high sensitivity. Mechanistic studies supported by single particle collision electrochemistry (SPCE), spectroscopic and density functional theory (DFT) calculations indicate the capability of polyfluorooctane sulfonic acid (PFOS), a representative PFAS, to selectively bind and induce aggregation of AgNPs. Single-particle measurements provide identification of the "discrete" AgNPs agglomeration (e.g. 2-3 NPs) formed through the inter-particles F-F interactions and the selective replacement of the citrate stabilizer by the sulfonate of the PFOS. Such interactions are characteristic only for long chain PFAS (-SO3 - ) providing a means to selectively identify these substances down to ppt levels. Measuring and understanding the interactions of PFAS at nanoscale surfaces are crucial for designing ultrasensitive methods for detection and for modelling and predicting their interaction in the environment.
Assuntos
Fluorocarbonos , Nanopartículas Metálicas , Poluentes Químicos da Água , Alcanossulfonatos , Citratos , Fluorocarbonos/química , Prata , Poluentes Químicos da Água/análiseRESUMO
BACKGROUND: Genome-wide sequencing has emerged as a promising strategy for the timely diagnosis of rare diseases, but it is not yet available as a clinical test performed in Canadian diagnostic laboratories. We describe the protocol for evaluating a 2-year pilot project, Genome-wide Sequencing Ontario, to offer high-quality clinical genome-wide sequencing in Ontario, Canada. METHODS: The Genome-wide Sequencing Ontario protocol was codesigned by the Ontario Ministry of Health, the Hospital for Sick Children in Toronto and the Children's Hospital of Eastern Ontario in Ottawa. Enrolment of a prospective cohort of patients began on Apr. 1, 2021. Eligible cases with blood samples available for the index case and both parents (i.e., trios) are randomized to receive exome sequencing or genome sequencing. We will collect patient-level data and ascertain costs associated with the laboratory workflow for exome sequencing and genome sequencing. We will compare point estimates for the diagnostic utility and timeliness of exome sequencing and genome sequencing, and we will determine an incremental cost-effectiveness ratio (expressed as the incremental cost of genome sequencing versus exome sequencing per additional patient with a causal variant detected). INTERPRETATION: Findings from this work will provide robust evidence for the diagnostic utility, cost-effectiveness and timeliness of exome sequencing and genome sequencing, and will be disseminated via academic publications and policy briefs. Findings will inform provincial and cross-provincial policy related to the long-term organization, delivery and reimbursement of clinical-grade genome diagnostics for rare disease.
Assuntos
Doenças Raras , Criança , Humanos , Ontário/epidemiologia , Projetos Piloto , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Doenças Raras/diagnóstico , Doenças Raras/genética , Sequenciamento do ExomaRESUMO
Current advancements in the development of functional nanomaterials and precisely designed nanostructures have created new opportunities for the fabrication of practical biosensors for field analysis. Two-dimensional (2D) and three-dimensional (3D) nanomaterials provide unique hierarchical structures, high surface area, and layered configurations with multiple length scales and porosity, and the possibility to create functionalities for targeted recognition at their surface. Such hierarchical structures offer prospects to tune the characteristics of materials-e.g., the electronic properties, performance, and mechanical flexibility-and they provide additional functions such as structural color, organized morphological features, and the ability to recognize and respond to external stimuli. Combining these unique features of the different types of nanostructures and using them as support for bimolecular assemblies can provide biosensing platforms with targeted recognition and transduction properties, and increased robustness, sensitivity, and selectivity for detection of a variety of analytes that can positively impact many fields. Herein, we first provide an overview of the recently developed 2D nanostructures focusing on the characteristics that are most relevant for the design of practical biosensors. Then, we discuss the integration of these materials with bio-elements such as bacteriophages, antibodies, nucleic acids, enzymes, and proteins, and we provide examples of applications in the environmental, food, and clinical fields. We conclude with a discussion of the manufacturing challenges of these devices and opportunities for the future development and exploration of these nanomaterials to design field-deployable biosensors.
Assuntos
Técnicas Biossensoriais , Nanoestruturas , Anticorpos , Técnicas EletroquímicasRESUMO
Importance: Pharmacogenomic (PGx) testing provides preemptive pharmacotherapeutic guidance regarding the lack of therapeutic benefit or adverse drug reactions of PGx targeted drugs. Pharmacogenomic information is of particular value among children with complex medical conditions who receive multiple medications and are at higher risk of developing adverse drug reactions. Objectives: To assess the implementation outcomes of a PGx testing program comprising both a point-of-care model that examined targeted drugs and a preemptive model informed by whole-genome sequencing that evaluated a broad range of drugs for potential therapy among children in a pediatric tertiary care setting. Design, Setting, and Participants: This cohort study was conducted at The Hospital for Sick Children in Toronto, Ontario, from January 2017 to September 2020. Pharmacogenomic analyses were performed among 172 children who were categorized into 2 groups: a point-of-care cohort and a preemptive cohort. The point-of-care cohort comprised 57 patients referred to the consultation clinic for planned therapy with PGx targeted drugs and/or for adverse drug reactions, including lack of therapeutic benefit, after the receipt of current or past medications. The preemptive cohort comprised 115 patients who received exploratory whole-genome sequencing-guided PGx testing for their heart conditions from the cardiac genome clinic at the Ted Rogers Centre for Heart Research. Exposures: Patients received PGx analysis of whole-genome sequencing data and/or multiplex genotyping of 6 pharmacogenes (CYP2C19, CYP2C9, CYP2D6, CYP3A5, VKORC1, and TPMT) that have established PGx clinical guidelines. Main Outcomes and Measures: The number of patients for whom PGx test results warranted deviation from standard dosing regimens. Results: A total of 172 children (mean [SD] age, 8.5 [5.6] years; 108 boys [62.8%]) were enrolled in the study. In the point-of-care cohort, a median of 2 target genes (range, 1-5 genes) were investigated per individual, with CYP2C19 being the most frequently examined; genotypes in 21 of 57 children (36.8%) were incompatible with standard treatment regimens. As expected from population allelic frequencies, among the 115 children in the whole-genome sequencing-guided preemptive cohort, 92 children (80.0%) were recommended to receive nonstandard treatment regimens for potential drug therapies based on their 6-gene pharmacogenetic profile. Conclusions and Relevance: In this cohort study, among both the point-of-care and preemptive cohorts, the multiplex PGx testing program provided dosing recommendations that deviated from standard regimens at an overall rate that was similar to the population frequencies of relevant variants.
Assuntos
Testes Genéticos/estatística & dados numéricos , Pediatria/estatística & dados numéricos , Testes Farmacogenômicos/estatística & dados numéricos , Testes Imediatos/estatística & dados numéricos , Medicina de Precisão/métodos , Medicina de Precisão/estatística & dados numéricos , Atenção Terciária à Saúde/estatística & dados numéricos , Adolescente , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Ontário , Projetos PilotoRESUMO
Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF (n = 175) or related CHD. We adapted a burden test originally developed for de novo variants to assess ultra-rare variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets and for multiple testing. For truncating variants, the gene burden test confirmed significant burden in FLT4 (Bonferroni corrected p-value < 0.01). For missense variants, burden in NOTCH1 achieved genome-wide significance only when restricted to constrained genes (i.e., under negative selection, Bonferroni corrected p-value = 0.004), and showed enrichment for variants affecting the extracellular domain, especially those disrupting cysteine residues forming disulfide bonds (OR = 39.8 vs. gnomAD). Individuals with NOTCH1 ultra-rare missense variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in CHD had more modest statistical support in gene burden tests. Gene-set burden tests for truncating variants identified a cluster of pathways corresponding to VEGF signaling (FDR = 0%), and of mouse phenotypes corresponding to abnormal vasculature (FDR = 0.8%); these suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5). Results for the most promising genes were driven by the TOF subset of the cohort. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF, accounting for 11-14% of individuals in the TOF cohort. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder.
RESUMO
MXenes are recently developed 2D layered nanomaterials that provide unique capabilities for bioanalytical applications. These include high metallic conductivity, large surface area, hydrophilicity, high ion transport properties, low diffusion barrier, biocompatibility, and ease of surface functionalization. MXenes are composed of transition metal carbides, nitrides, or carbonitrides and have a general formula Mn+1Xn, where M is an early transition metal while X is carbon and/or nitrogen. Due to their unique features, MXenes have attracted significant attention in fields such as clean energy production, electronics, fuel cells, supercapacitors, and catalysis. Their composition and layered structure make MXenes attractive for biosensing applications. The high conductivity allows these materials to be used in the design of electrochemical biosensors and the multilayered configuration makes them an efficient immobilization matrix for the retention of activity of the immobilized biomolecules. These properties are applicable to many biosensing systems and applications. This review describes the progress made on the use and application of MXenes in the development of electrochemical and optical biosensors and highlights future needs and opportunities in this field. In particular, opportunities for developing wearable sensors and systems with integrated biomolecule recognition are highlighted.
Assuntos
Técnicas Biossensoriais , Nanoestruturas , Elementos de TransiçãoRESUMO
PURPOSE: This study investigated the diagnostic utility of nontargeted genomic testing in patients with pediatric heart disease. METHODS: We analyzed genome sequencing data of 111 families with cardiac lesions for rare, disease-associated variation. RESULTS: In 14 families (12.6%), we identified causative variants: seven were de novo (ANKRD11, KMT2D, NR2F2, POGZ, PTPN11, PURA, SALL1) and six were inherited from parents with no or subclinical heart phenotypes (FLT4, DNAH9, MYH11, NEXMIF, NIPBL, PTPN11). Outcome of the testing was associated with the presence of extracardiac features (p = 0.02), but not a positive family history for cardiac lesions (p = 0.67). We also report novel plausible gene-disease associations for tetralogy of Fallot/pulmonary stenosis (CDC42BPA, FGD5), hypoplastic left or right heart (SMARCC1, TLN2, TRPM4, VASP), congenitally corrected transposition of the great arteries (UBXN10), and early-onset cardiomyopathy (TPCN1). The identified candidate genes have critical functions in heart development, such as angiogenesis, mechanotransduction, regulation of heart size, chromatin remodeling, or ciliogenesis. CONCLUSION: This data set demonstrates the diagnostic and scientific value of genome sequencing in pediatric heart disease, anticipating its role as a first-tier diagnostic test. The genetic heterogeneity will necessitate large-scale genomic initiatives for delineating novel gene-disease associations.
Assuntos
Cardiopatias/genética , Criança , Mapeamento Cromossômico , Exoma , Humanos , Mecanotransdução Celular , Transposição dos Grandes VasosRESUMO
A quenching based apta-sensing platform was developed for the detection of Patulin. Three different aptamer sequences were studied to screen the aptamer with the maximum affinity towards Patulin. Carboxyfluorescein (CFL) was used as a fluorescent dye while -COOH functionalized multiwall carbon nanotubes (MWCNTs) were applied as novel nanoquenchers. Aptamer tagged at the 3' end with 40 nucleotide bases exhibited the maximum affinity towards Patulin and caused substantial fluorescence recovery. Interestingly, the limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.13 µg L-1and 0.41 µg L-1 respectively. Commonly occurring mycotoxins in food were also tested to confirm the selectivity of apta-assay. The developed apta-assay was applied to a spiked apple juice sample and toxin recoveries were observed ranging from 96% to 98% (n = 3). These results demonstrated the potential of the developed apta-assay for the selective detection and quantification of Patulin in food samples.
Assuntos
Fluoresceínas/química , Nanotubos de Carbono , Fluorescência , Sucos de Frutas e Vegetais/análise , Limite de Detecção , Malus/química , Patulina/análiseRESUMO
In the present work, an aptasensing platform was developed for the detection of a carcinogenic mycotoxin termed patulin (PAT) using a label-free approach. The detection was mainly based on a specific interaction of an aptamer immobilized on carbon-based electrode. A long linear spacer of carboxy-amine polyethylene glycol chain (PEG) was chemically grafted on screen-printed carbon electrodes (SPCEs) via diazonium salt in the aptasensor design. The NH2-modified aptamer was then attached covalently to carboxylic acid groups of previously immobilized bifunctional PEG to build a diblock macromolecule. The immobilized diblocked molecules resulted in the formation of long tunnels on a carbon interface, while the aptamer was assumed as the gate of these tunnels. Upon target analyte binding, the gates were assumed to be closed due to conformational changes in the structure of the aptamer, increasing the resistance to the charge transfer. This increase in resistance was measured by electrochemical impedance spectroscopy, the main analytical technique for the quantitative detection of PAT. Encouragingly, a good linear range between 1 and 25 ng was obtained. The limit of detection and limit of quantification was 2.8 ng L-1 and 4.0 ng L-1, respectively. Selectivity of the aptasensor was confirmed with mycotoxins commonly occurring in food. The developed apta-assay was also applied to a real sample, i.e., fresh apple juice spiked with PAT, and toxin recovery up to 99% was observed. The results obtained validated the suitability and selectivity of the developed apta-assay for the identification and quantification of PAT in real food samples.
Assuntos
Aptâmeros de Nucleotídeos/química , Sucos de Frutas e Vegetais/análise , Malus/química , Patulina/análise , Técnicas Biossensoriais/métodos , Carbono/química , Eletrodos , Contaminação de Alimentos , Limite de DetecçãoRESUMO
Small molecule toxins such as mycotoxins with low molecular weight are the most widely studied biological toxins. These biological toxins are responsible for food poisoning and have the potential to be used as biological warfare agents at the toxic dose. Due to the poisonous nature of mycotoxins, effective analysis techniques for quantifying their toxicity are indispensable. In this context, biosensors have been emerged as a powerful tool to monitors toxins at extremely low level. Recently, biosensors based on fluorescence detection have attained special interest with the incorporation of nanomaterials. This review paper will focus on the development of fluorescence-based biosensors for mycotoxin detection, with particular emphasis on their design as well as properties such as sensitivity and specificity. A number of these fluorescent biosensors have shown promising results in food samples for the detection of mycotoxins, suggesting their future potential for food applications.
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Técnicas Biossensoriais , Micotoxinas/análise , FluorescênciaRESUMO
OBJECTIVE: To characterise sleep quality and assess degree of daytime sleepiness among medical students of Karachi. METHODS: The cross-sectional study was conducted between August and December 2013 and subjects were recruited from five haphazardly selected medical colleges in Karachi. A convenience sample of medical students underwent two validated self-administered questionnaires i.e. Pittsburgh Sleep Quality Index and Epworth Sleepiness Scale. SPSS 17 was used for statistical analysis. RESULTS: Of the 650 students approached, 504(77.5%) subjects completely filled out the questionnaires. Of them, 300(59.5%) were females and 204(40.5%) were males. Overall mean age was 20±1.4 years. Of them, 199(39.5%) were classified as "Poor Sleepers". Poor sleep quality was associated with female gender (p <0.05), excessive daytime sleepiness (p <0.05), total hours slept (p <0.001) and sleep disturbances (p <0.001). Bed-timing analysis showed 365(72%) students went to bed after midnight. CONCLUSIONS: Sleep quality among Pakistani medical students was significantly poor. Efforts must be directed towards proper sleep hygiene education.