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This article explores the development and application of innovative piezoelectric sensors in point-of-care diagnostics. It highlights the significance of bedside tests, such as lateral flow and electrochemical tests, in providing rapid and accurate results directly at the patient's location. This paper delves into the principles of piezoelectric assays, emphasizing their ability to detect disease-related biomarkers through mechanical stress-induced electrical signals. Various applications of piezoelectric chemosensors and biosensors are discussed, including their use in the detection of cancer biomarkers, pathogens, and other health-related analytes. This article also addresses the integration of piezoelectric materials with advanced sensing technologies to improve diagnostic accuracy and efficiency, offering a comprehensive overview of current advances and future directions in medical diagnostics.
- MeSH
- biologické markery analýza MeSH
- biosenzitivní techniky * MeSH
- elektrochemické techniky MeSH
- lidé MeSH
- vyšetření u lůžka MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The polymerase acidic (PA) subunit of the influenza virus, an endonuclease of the RNA-dependent RNA polymerase, represents a viable target for anti-influenza therapies, as evidenced by the efficacy of the FDA-approved drug Xofluza. A characteristic feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions within the enzyme's catalytic site. Previously, our studies identified luteolin and its C-8-glucoside orientin as potent endonuclease inhibitors. This report details our subsequent investigation into the structural modifications of the phenyl moiety attached to the C-8 position of luteolin. The inhibitory potencies (IC50 values) quantified with AlphaScreen technology indicated that substituting the C-8 glucose moiety of orientin resulted in compounds with comparable inhibitory potency. From a series of eighteen compounds, acid 12 with 3-carboxylphenyl moiety at the C-8 position was the most potent inhibitor with nanomolar potency.
- MeSH
- antivirové látky * farmakologie chemická syntéza chemie MeSH
- endonukleasy * antagonisté a inhibitory metabolismus MeSH
- inhibitory enzymů * farmakologie chemická syntéza chemie MeSH
- luteolin * farmakologie chemická syntéza chemie MeSH
- molekulární struktura MeSH
- racionální návrh léčiv * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Recent advances in optical sensing technologies underpin the development of high-performance, surface-sensitive analytical tools capable of reliable and precise detection of molecular targets in complex biological media in non-laboratory settings. Optical fibre sensors guide light to and from a region of interest, enabling sensitive measurements of localized environments. This positions optical fibre sensors as a highly promising technology for a wide range of biochemical and healthcare applications. However, their performance in real-world biological media is often limited by the absence of robust post-modification strategies that provide both high biorecognition and antifouling capabilities. In this study, we present the proof-of-concept antifouling and biorecognition performance of a polymer brush nano-coating synthesized at the sensing region of optical fibre long-period grating (LPG) sensors. Using a newly developed antifouling terpolymer brush (ATB) composed of carboxybetaine methacrylamide, sulfobetaine methacrylamide, and N-(2-hydroxypropyl)methacrylamide, we achieve state-of-the-art antifouling properties. The successful on-fibre ATB synthesis is confirmed through scanning electron microscopy (SEM), fluorescence microscopy, and label-free bio-detection experiments based on antibody-functionalized ATB-coated LPG optical fibres. Despite the challenges in handling optical fibres during polymerization, the resulting nano-coating retains its remarkable antifouling properties upon exposure to blood plasma and enables biorecognition element functionalization. These capabilities are demonstrated through the detection of IgG in buffer and diluted blood plasma using anti-IgG-functionalized ATB-coated sensing regions of LPG fibres in both label-based (fluorescence) and label-free real-time detection experiments. The results show the potential of ATB-coated LPG fibres for use in analytical biosensing applications.
This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.
- MeSH
- biosenzitivní techniky * přístrojové vybavení metody MeSH
- COVID-19 * diagnóza virologie MeSH
- design vybavení MeSH
- elektronické tranzistory MeSH
- fosfoproteiny MeSH
- grafit * chemie MeSH
- koronavirové nukleokapsidové proteiny izolace a purifikace MeSH
- lidé MeSH
- limita detekce MeSH
- nazofarynx virologie MeSH
- RNA virová * izolace a purifikace analýza MeSH
- SARS-CoV-2 * izolace a purifikace genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Ganoderma sp., the fungal agent causing basal stem rot (BSR), poses a severe threat to global oil palm production. Alarming increases in BSR occurrences within oil palm growing zones are attributed to varying effectiveness in its current management strategies. Asymptomatic progression of the disease and the continuous monoculture of oil palm pose challenges for prompt and effective management. Therefore, the development of precise, early, and timely detection techniques is crucial for successful BSR management. Conventional methods such as visual assessments, culture-based assays, and biochemical and physiological approaches prove time-consuming and lack specificity. Serological-based diagnostic methods, unsuitable for fungal diagnostics due to low sensitivity, assay affinity, cross-contamination which further underscores the need for improved techniques. Molecular PCR-based assays, utilizing universal, genus-specific, and species-specific primers, along with functional primers, can overcome the limitations of conventional and serological methods in fungal diagnostics. Recent advancements, including real-time PCR, biosensors, and isothermal amplification methods, facilitate accurate, specific, and sensitive Ganoderma detection. Comparative whole genomic analysis enables high-resolution discrimination of Ganoderma at the strain level. Additionally, omics tools such as transcriptomics, proteomics, and metabolomics can identify potential biomarkers for early detection of Ganoderma infection. Innovative on-field diagnostic techniques, including remote methods like volatile organic compounds profiling, tomography, hyperspectral and multispectral imaging, terrestrial laser scanning, and Red-Green-Blue cameras, contribute to a comprehensive diagnostic approach. Ultimately, the development of point-of-care, early, and cost-effective diagnostic techniques accessible to farmers is vital for the timely management of BSR in oil palm plantations.
The emergence of high-throughput methodologies such as next-generation sequencing and proteomics has necessitated significant advancements in biological databases and bioinformatic tools, therefore reshaping the landscape of research into parasitic peptidases. In this review we outline the development of these resources along the -omics technologies and their transformative impact on the field. Apart from extensive summary of general and specific databases and tools, we provide a general pipeline on how to use these resources effectively to identify candidate peptidases from these large datasets and how to gain as much information about them as possible without leaving the office chair. This pipeline is then applied in an illustrative case study on the endothelin-converting enzyme 1 homologue from Schistosoma mansoni and attempts to highlight the contemporary capabilities of bioinformatics. The case study demonstrate how such approach can aid to hypothesize enzyme functions and interactions through computational analysis alone effectively and emphasizes how such virtual investigations can guide and optimize subsequent wet lab experiments therefore potentially saving precious time and resources. Finally, by showing what can be achieved without traditional wet laboratory methods, this review provides a compelling narrative on the use of bioinformatics to bridge the gap between big data and practical research applications, highlighting the key role of these technologies in furthering our understanding of parasitic diseases.
BACKGROUND: Manufacturers and diagnostic companies often recommend on-site verification of analytical performance in the clinical laboratory. The validation process used by manufacturers is rarely described in detail, and certain information on analytical performance is missing from the product sheet, especially for immunoanalytical methods. We describe an approach to the detailed validation of an ELISA method for the measurement of proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma concentrations. We compared manufacturers' claims of analytical performance with data obtained in the field laboratory using several approaches. METHODS: We used the Human Proprotein Convertase 9/PCSK9 Quantikine ELISA diagnostic kit (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) and three levels of quality control solution Quantikine Immunoassay Control Group 235 (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) to verify precision. We measured the concentration of PCSK9 using the DS2 ELISA Reader (Dynex Technologies GmbH, Denkendorf, Germany). We used analysis of variance (ANOVA) and the R statistical package (R core team, version 1.4.5). Statistical analysis and terminology were performed according to protocol CLSI EP15-A3, and the reference interval was checked according to CLSI/IFCC C28-A3c. RESULTS: We found a significant difference between the manufacturer's claims of analytical performance and real data measured in the routine clinical laboratory. The calculated CV (%) for repeatability (calculated by simple estimation of the mean and SD, as used by the manufacturer) was between 5.5% and 7.4%, but the manufacturer's claim was between 4.1% and 6.5%. Using ANOVA, the true repeatability was between 5.0% and 6.9%. Similarly, ANOVA revealed values of CV (%) for within-laboratory imprecision between 6.5% and 9.1%, while the manufacturer's claims were between 4.1% and 5.9%. The recovery ranged from 105.5% to 121.8%. The manufacturer's recommended reference interval was checked and we didn't find any significant difference between men and women. CONCLUSIONS: We describe a comprehensive approach to verify the analytical performance of an ELISA method using the measurement of PCSK9 plasma concentration as an example. We found differences between the results of this approach based on the CLSI EP15-A3 protocol and data provided by the manufacturer. We recommend the verification of analytical performance by more complex statistical tools in laboratory practice.
- MeSH
- ELISA * normy metody MeSH
- lidé MeSH
- proproteinkonvertasa subtilisin/kexin typu 9 * krev imunologie MeSH
- reagenční diagnostické soupravy normy MeSH
- reprodukovatelnost výsledků MeSH
- řízení kvality MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Dietary exposure to aflatoxin B1 (AFB1) is harmful to the health and performance of domestic animals. The hepatic cytochrome P450s (CYPs), CYP1A1 and CYP2A6, are the primary enzymes responsible for the bioactivation of AFB1 to the highly toxic exo-AFB1-8,9-epoxide (AFBO) in chicks. However, the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1 metabolism remains unknown. Dual-luciferase reporter assay, bioinformatics and site-directed mutation results indicated that specificity protein 1 (SP1) and activator protein-1 (AP-1) motifs were located in the core region -1,063/-948, -606/-541 of the CYP1A1 promoter as well as -636/-595, -503/-462, -147/-1 of the CYP2A6 promoter. Furthermore, overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6. Moreover, bioactivation of AFB1 to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression, which was trans-activated owing to the upregulalion of AP-1, rather than SP1, stimulated by AFB1-induced reactive oxygen species. Additionally, nano-selenium could reduce ROS, downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6, thus alleviating the toxicity of AFB1. In conclusion, AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1 to AFBO in chicken liver, which could provide novel targets for the remediation of aflatoxicosis in chicks.
- MeSH
- aflatoxin B1 * metabolismus MeSH
- aktivace transkripce MeSH
- cytochrom P-450 CYP1A1 * genetika metabolismus MeSH
- cytochrom P450 CYP2A6 * metabolismus genetika MeSH
- játra * metabolismus MeSH
- kur domácí * metabolismus MeSH
- promotorové oblasti (genetika) * MeSH
- transkripční faktor AP-1 * metabolismus genetika MeSH
- transkripční faktor Sp1 * metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Enzymová syntéza představuje alternativu k chemické syntéze a poskytuje nové možnosti v přípravě farmaceuticky účinných látek. Cílem je minimalizovat neefektivnost chemického způsobu přípravy a zvýšení jeho účinnosti. Navzdory pokroku se biotechnologické procesy v syntéze nukleosidů, kde se využívá katalýzy nukleosidovými fosforylasami, průmyslově aktivně neuplatňují. Rozvoj enzymového způsobu syntézy by mohl přinést výhody jak z hlediska zlepšení účinnosti a jednoduchosti procesu, tak minimalizaci spotřeby organických rozpouštědel a tím i dopadu na životní prostředí. Využití enzymové syntézy ve výrobě kladribinu přináší jednoduchý a rychlý způsob výroby této účinné látky ve vysoké čistotě bez vzniku nežádoucích vedlejších produktů.
Enzymatic synthesis is an alternative to chemical synthesis and provides new possibilities in the preparation of pharmaceutically active substances. The aim is to minimize the inefficiency of the chemical method of preparation and increase its effectiveness. Despite advances, biotechnological processes in nucleoside synthesis using catalysis by nucleoside phosphorylases are not actively applied industrially. The development of an enzymatic synthesis route could bring benefits in terms of improved efficiency, process simplicity, and minimization of organic solvent consumption and thus environmental impact. The use of enzymatic synthesis in the production of cladribine provides a simple and rapid way of producing this active substance in high purity without the formation of undesirable by-products.
Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.
