BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).

• Klíčová slova
• COVID-19, CRISPR, Cas12b, Diagnostics, Dual-detection, One-pot detection, RT-LAMP, SARS-CoV-2, Thermophillic, Variants of concern,
• MeSH
• Brevibacillus * genetika   MeSH
• COVID-19 * diagnóza   MeSH
• lidé MeSH
• SARS-CoV-2 genetika   MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• vodící RNA, systémy CRISPR-Cas MeSH

In the current research, we present a single-step, one-pot, room temperature green synthesis approach for the development of functional poly(tannic acid)-based silver nanocomposites. Silver nanocomposites were synthesized using only tannic acid (plant polyphenol) as a reducing and capping agent. At room temperature and under mildly alkaline conditions, tannic acid reduces the silver salt into nanoparticles. Tannic acid undergoes oxidation and self-polymerization before the encapsulating of the synthesized silver nanoparticle and forms silver nanocomposites with a thick capping layer of poly(tannic acid). No organic solvents, special instruments, or toxic chemicals were used during the synthesis process. The results for the silver nanocomposites prepared under optimum conditions confirmed the successful synthesis of nearly spherical and fine nanocomposites (10.61 ± 1.55 nm) with a thick capping layer of poly(tannic acid) (~3 nm). With these nanocomposites, iron could be detected without any special instrument or technique. It was also demonstrated that, in the presence of Fe3+ ions (visual detection limit ~20 μM), nanocomposites aggregated using the coordination chemistry and exhibited visible color change. Ultraviolet-visible (UV-vis) and scanning electron microscopy (SEM) analysis also confirmed the formation of aggregate after the addition of the analyte in the detection system (colored nanocomposites). The unique analytic performance, simplicity, and ease of synthesis of the developed functional nanocomposites make them suitable for large-scale applications, especially in the fields of medical, sensing, and environmental monitoring. For the medical application, it is shown that synthesized nanocomposites can strongly inhibit the growth of Escherichia coli and Staphylococcus aureus. Furthermore, the particles also exhibit very good antifungal and antiviral activity.

• Klíčová slova
• antibacterial, antifungal, antiviral activity, green synthesis, poly(tannic acid), silver nanocomposites, visual detection of ferric,
• Publikační typ
• časopisecké články MeSH

Herein, a self-assembled hierarchical structure of hematite (α-Fe2O3) was synthesized via a one-pot hydrothermal method. Subsequently, the nanomaterial was doped to obtain M x Fe2-x O3 (M = Mn-Co; x = 0.01, 0.05, and 0.1) at precise concentrations. An electrode was fabricated by coating the resulting nanocomposite onto a nickel foam (NF) substrate. Electrochemical characterization demonstrated the excellent performance of cobalt-doped α-Fe2O3, among which Co0.05Fe0.95O3 (CF5) exhibited a superior performance, showing a two-fold increase in sensitivity of 1364.2 μA mM-1 cm-2 (±0.03, n = 3) in 0.5 M KOH, a limit of detection (LOD) of ∼0.17 mM, and a limit of quantification (LOQ) of ∼0.58 mM. The Density Functional Theory (DFT) was performed to understand the doping prompting in the reduced bandgap. The fabricated electrode displayed a rapid response time of 2 s and demonstrated 95% stability, excellent reproducibility, and selectivity, as confirmed by tests with several interfering species. A comprehensive evaluation of the electrode's performance using human blood serum highlighted its robustness and reliability for cholesterol detection in clinical settings, making it a promising tool for clinical and pharmaceutical applications.

• Publikační typ
• časopisecké články MeSH

Nanomolar-level detection of priority toxic pollutant 4-nitrophenol (4-NP) in environment using a novel ternary nanocomposite based electrochemical sensor and its photocatalytic degradation is reported in this paper. A non-toxic and renewable natural biopolymer, chitosan wrapped carbon nanofibers was embedded with Ag doped spinel Co3O4 to prepare the bi-functional ternary nanocomposite. Economical and ecofriendly sonochemical method was employed in preparation of this porous nanocomposite. We used one-pot aqueous solution approach to synthesize Ag-Co3O4 nanoflowers and ultrasound-assisted method was utilized to prepare CS-CNFs. Morphological and structural properties of synthesized materials were analyzed using different characterization techniques. Electrochemical investigations using cyclic voltammetry and differential pulse voltammetry carried out with prepared ternary nanocomposite modified carbon electrode revealed its outstanding electrocatalytic activity in 4-NP quantification. The developed 4-NP sensor showcased excellent sensitivity of 55.98 μAμM-1cm-2 and nanomolar detection limit of 0.4 nM. Moreover, reproducibility, repeatability, stability, and selectivity were evaluated to confirm reliability of developed sensor. Further, real sample analyses were conducted using domestic sewage, underground water, and tomato to affirm the practical feasibility of 4-NP detection using the proposed sensor.

• Klíčová slova
• 4-Nitrophenol, Carbon nanofibers, Chitosan, Silver, Ternary nanocomposite, Toxic pollutant,
• MeSH
• biopolymery MeSH
• elektrochemické techniky * MeSH
• elektrody MeSH
• kobalt MeSH
• oxid hlinitý MeSH
• oxid hořečnatý MeSH
• oxidy MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biopolymery MeSH
• cobalt oxide MeSH Prohlížeč
• cobalt tetraoxide MeSH Prohlížeč
• kobalt MeSH
• oxid hlinitý MeSH
• oxid hořečnatý MeSH
• oxidy MeSH
• spinell MeSH Prohlížeč

Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.

• Klíčová slova
• COVID-19, CRISPR, MOF, biomedicine, biosensor, gene delivery,
• MeSH
• buňky Hep G2 MeSH
• buňky PC12 MeSH
• COVID-19 diagnóza   MeSH
• CRISPR-Cas systémy MeSH
• dusík chemie   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• jednovláknová DNA MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• limita detekce MeSH
• nanokompozity MeSH
• nanostruktury MeSH
• porézní koordinační polymery chemie   MeSH
• poréznost MeSH
• porfyriny chemie   MeSH
• povrchové vlastnosti MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 MeSH
• senzitivita a specificita MeSH
• testování na COVID-19 MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dusík MeSH
• jednovláknová DNA MeSH
• porézní koordinační polymery MeSH
• porfyriny MeSH
• RNA virová MeSH
• vodící RNA, systémy CRISPR-Cas MeSH

Borane and heteroborane clusters have been known as neutral or anionic species. In contrast to them, several ten-vertex monocationic nido and closo dicarbaborane-based systems have recently emerged from the reaction of the parent bicapped-square antiprismatic dicarbaboranes with N-heterocyclic carbenes followed by the protonization of the corresponding nido intermediates. The expansion of these efforts has afforded the very first closo-dicationic octahedral phosphahexaborane along with new closo-monocationic pnictogenahexaboranes of the same shapes. All are the products of the one-pot procedure that consists in the reaction of the same carbenes with the parent closo-1,2-Pn2B4Br4 (Pn = As, P). Whereas in the case of phosphorus such a monocation appears to be a mixture of stable intermediates, and arsenahexaboranyl monocation has occurred as the final product, all of them without using any subsequent reaction. The well-established DFT/ZORA/NMR approach has unambiguously confirmed the existence of these species in solution, and computed electrostatic potentials have revealed the delocalization of the positive charge in these monocations and in the very first dication, namely within the octahedral shapes in both cases.

• Publikační typ
• časopisecké články MeSH

RATIONALE: Silver tellurides find applications in the development of infrared detection, imaging, magnetics, sensors, memory devices, and optic materials. However, only a limited number of silver tellurides have been described to date. Laser ablation synthesis (LAS) was selected to generate new Ag-Te clusters. METHODS: Isothermal adsorption was used to study the formation of silver nano-particles-tellurium aggregates. Laser desorption ionization quadrupole ion trap time-of-flight mass spectrometry (LDI-QIT-TOFMS) was used for the generation and analysis of Agm Ten clusters. Scanning electron microscopy and energy-dispersive X-ray spectroscopy were used to visualize the structure of materials. The stoichiometry of the generated clusters was determined by computer modeling of isotopic patterns. RESULTS: A simple, one-pot method for the preparation of Ag-Te nano-composite was developed and found suitable for LAS of silver tellurides. The LDI of Ag-Te nano-composite leads to the formation of 11 unary and 52 binary clusters. The stoichiometry of the 34 novel Agm Ten clusters is reported here for the first time. CONCLUSIONS: LAS with TOFMS detection was proven to be a powerful technique for the generation of silver telluride clusters. Knowledge of the stoichiometry of the generated clusters might facilitate the further development of novel high-tech silver tellurium nano-materials.

• MeSH
• hmotnostní spektrometrie metody   MeSH
• ionty chemie   MeSH
• mikroskopie elektronová rastrovací MeSH
• nanokompozity chemie   MeSH
• stříbro chemie   MeSH
• telur chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ionty MeSH
• stříbro MeSH
• tellurous acid MeSH Prohlížeč
• telur MeSH

Synthesis of complex polymeric architectures (CPAs) via reversible-deactivation radical polymerization (RDRP) currently relies on the rather inefficient attachment of monofunctional initiation/transfer sites onto CPA precursors. This drawback seriously limits the overall functionality of the resulting (macro)initiators and, consequently, also the total number of installable polymeric chains, which represents a significant bottleneck in the design of new polymeric materials. Here, we show that the (macro)initiator functionality can be substantially amplified by using trichloroacetyl isocyanate as a highly efficient vehicle for the rapid and clean introduction of trichloroacetyl groups (TAGs) into diverse precursors. Through extensive screening of polymerization conditions and comprehensive NMR and triple-detection SEC studies, we demonstrate that TAGs function as universal trifunctional initiators of copper-mediated RDRP of different monomer classes, affording low-dispersity polymers in a wide molecular weight range. We thus unlock access to a whole new group of ultra-high chain density CPAs previously inaccessible via simple RDRP protocols. We highlight new opportunities in CPA synthesis through numerous examples, including the de novo one-pot synthesis of a novel "star-on-star" CPA, the preparation of β-cyclodextrin-based 45-arm star polymers, and facile grafting from otherwise problematic cellulose substrates both in solution and from surface, obtaining effortlessly ultra-dense, ultra-high-molecular weight bottle-brush copolymers and thick spatially-controlled polymeric coatings, respectively.

• Publikační typ
• časopisecké články MeSH

Sweet basil (Ocimum basilicum L.) is an aromatic plant that is cultivated as a pot plant in greenhouses or in fields in the Czech Republic. The plants are intended for direct consumption or for drying. In April of 2012, the first large chlorotic from the middle necrotic spots occurred gradually on leaves of pot plants O. basilicum cv. Genovese in greenhouses in Central Bohemia. The characteristic gray to brown furry growth of downy mildew appeared on abaxial surfaces of leaves in the place of chlorotic spots within 3 to 4 days. The infested leaves fell off in the late stages of pathogenesis. The infestation gradually manifested itself in ever-younger plants and in July, cotyledons and possibly the first true leaves were already heavily infected and damaged and these plants rapidly died. The plant damage reached 80 to 100%, so it was necessary to stop growing the plants in the greenhouse at the end of July. The causal agent was isolated and identified as Peronospora belbahrii Thines by means of morphological and molecular characters (2,3). Conidiophores were hyaline, straight, monopodial, 280 to 460 μm, branched three to five times, ended with two slightly curved branchlets with a single conidia on each branchled tip. The longer branchlets measured 13 to 24 μm (average 18.2 μm), the shorter one 4 to 15 μm (average 9.7 μm). Conidia were rounded or slightly ovoid, from brownish to dark brownish, measured 22 to 31 × 20 to 28 μm (length/width ratio 1.2). A pathogen-specific sequence was detected with the help of the pathogen ITS rDNA specific primers in symptomatic leaves (1). DNA from plant tissues was isolated using the DNeasy plant Mini Kit (Qiagen, Germany) following the standard protocol. PCR was performed using KAPA2G Robust HotStar kit (Kapa Biosystems, United States) according to the conditions recommended in Belbahri et al. (1). The specific products were visualized by electrophoresis through 1.5% agarose gels. Leaves of 20-day-old potted plants O. basilicum 'Genovese' were inoculated by spraying with 5 × 105 conidia/ml of the pathogen. Each pot contained 10 plants. Sterilized distilled water was applied to control plants. Plants were covered with polyethylene bags during the entire incubation period to maintain high humidity, and kept at a temperature of 22 to 24°C. Typical disease symptoms appeared on leaves 5 to 9 days after inoculation. Control plants were symptomless. P. belbahrii was re-isolated from the lesions of inoculated plants, thus fulfilling Koch's postulates. Downy mildew on sweet basil was reported in countries in Africa, Europe, and South and North America (4). To our knowledge, this is the first report of downy mildew on sweet basil in the Czech Republic. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) Y.-J. Choi et al. Mycol. Res. 113:1340, 2009. (3) M. Thines et al. Mycol. Res. 113:532, 2009. (4) C. A. Wyenandt et al. HortScience 45:1416, 2010.

• Publikační typ
• časopisecké články MeSH

The reaction pathways connecting methanol to methane and formaldehyde are among the most emblematic in chemistry because of their outstanding interest in the fields of energy, synthesis, and bio- and geo-chemistry. Despite of its fundamental nature, the one-pot synthesis of formaldehyde and methane stemming from methanol has never been reported before. Here we present a study, based on ab initio molecular dynamics and free-energy methods, in which the simultaneous oxidation and reduction (i.e., the disproportionation) of liquid methanol into methane and formaldehyde has been achieved at ambient temperature through the application of a static electric field. Because strong electric fields can be generated in the proximity of field emitter tips, this finding shows that the challenge of experimentally disproportionating methanol into formaldehyde and methane could be attempted. We show that the methanol "solvent" molecules play a major role in this process and that the chemical pathway connecting methanol to the detected products in the bulk liquid phase is very different from its reproduced gas-phase counterpart. Finally, we demonstrate that switching on an external electric field drastically modifies the reaction network of methanol, lowering some activation barriers, stabilizing the methane and formaldehyde products, and opening otherwise difficult-to-achieve chemical routes.

• Publikační typ
• časopisecké články MeSH