Commercial Reagents Research Articles

An efficient and cost-effective method for disrupting genes in RAW264.7 macrophages using CRISPR-Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are widely used for genome editing in cultured cell lines. However, the implementation of genome editing is still challenging due to the complex and often costly multi-step process associated with this technique. Moreover, the efficiency of genome editing varies across cell types, often limiting utility. Herein, we describe pCRISPR-EASY, a vector for simplified cloning of single guide RNAs (sgRNAs) and its simultaneous introduction with CRISPR-Cas9 into cultured cells using a non-viral delivery system. We outline a comprehensive, step-by-step protocol for genome editing in RAW264.7 macrophages, a mouse macrophage cell line widely used in biomedical research for which genome editing using CRISPR-Cas9 has been restricted to lentiviral or expensive commercial reagents. This provides an economical, highly efficient and reliable method for genome editing that can easily be adapted for use in other systems.

Quality-by-Design Approach to Process Intensification of Bioinspired Silica Synthesis.

Characterizing nanomaterials is challenging due to their macromolecular nature, requiring suites of physicochemical analysis to fully resolve their structure. As such, their synthesis and scale-up are notoriously complex, especially when compared to small molecules or bulk crystalline materials, which can be provided a unique fingerprint from nuclear magnetic resonance (NMR) or X-ray diffraction (XRD) alone. In this study, we address this challenge by adopting a three-step quality-by-design (QbD) approach to the scale-up of bioinspired silica nanomaterials, demonstrating its utility toward synthesis scale-up and intensification for this class of materials in general. First, we identified material-specific surface area, pore-size distribution, and reaction yield as critical quality attributes (CQAs) that could be precisely measured and controlled by changing reaction conditions. We then identified the critical process parameters (CPPs) controlling bioinspired synthesis properties, exploring different process routes, incorporating commercial reagents, and optimizing reagent ratios, comparing silica properties against original CQA values to identify acceptable limits to each CPP. Finally, we intensified the synthesis by increasing reagent concentration while simultaneously incorporating the optimized CPPs, thereby modifying the bioinspired silica synthesis to make it compatible with existing manufacturing methods. We increased the specific yield from ca. 1.1 to 38 g/L and reduced the additive intensity from ca. 1 to 0.04 g/g product, greatly reducing both synthesis cost and waste production. These results identify a need for mapping the effects of critical process parameters on material formation pathways and CQAs to enable accelerated scale-up and transition from the lab to the market.

A strategy for the enhanced development of latent fingermarks with minimally destructive to DNA based on fluorescence Eu-Tb metal–organic framework

Fingermarks identification have always been the most effective and reliable way for individuals determination among the forensic science. The fingermarks at the scene of the case as the valuable physical evidence to analyze the trajectory of criminal behavior and portray the characteristics of the suspect play an irreplaceable role in the process of investigation. To reach its pivotal biometric individualization, the luminescent metal–organic framework (Eu@Tb-LL) was prepared via a simple optimized solvothermal method for visualizing the latent fingermarks with minimally destructive to DNA in this work. Eu@Tb-LL exhibited ideal optical stability and fluorescence intensity at the wavelength of 530 and 620 nm during revealing latent fingermarks. Meanwhile, Eu@Tb-LL is more effective than indandione (commonly used in the site investigation) in revealing aged fingermarks. More significantly, the Eu@Tb-LL superimposed on commercial manifestation reagents (Hfb) enhanced the effect of Hfb for revealing blood latent fingermarks on the non-porous objects. In addition, the STR testing results performed after the visualization process proved the Eu@Tb-LL was minimally destructive to DNA with a confidence interval of 95 %, exhibiting promising prospects for practical criminal investigation applications.

A multilevel core-shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for DNA extraction

DNA extraction is greatly significant in biological detection fields, including species identification, pathogen detection, environmental monitoring, biosafety, and food safety. Scientific research and practical application must conduct high-quality trace DNA separation, purification and extraction. Herein, a two-step strategy successfully prepares a multilevel core–shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for trace DNA extraction. The morphologies, structures, elemental compositions, components, and properties of the materials are systematically characterized. Fe3O4@SiO2@InGaO@SiO2 magnetic particles in the chaotropic salt solutions are used to directly target DNA binding due to the existence of Si-O–H bond. The maximum DNA adsorption rate can highly reach 83.1 % after 700 °C annealing in the air for 30 min, far exceeding the adsorption rate of commercial reagent kit, which is attributed to the increased specific surface area. The excellent result is conducive to rapid DNA extraction in practical applications, especially in daily criminal cases, further promoting social security and stability.

An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Peptide Self-Assembly Facilitating DNA Transfection and the Application in Inhibiting Cancer Cells.

Non-viral vectors have been developing in gene delivery due to their safety and low immunogenicity. But their transfection effect is usually very low, thus limiting the application. Hence, we designed eight peptides (compounds 1-8). We compared their performances; compound 8 had the best transfection efficacy and biocompatibility. The transfection effect was similar with that of PEI, a most-widely-employed commercial transfection reagent. Atomic force microscope (AFM) images showed that the compound could self-assemble and the self-assembled peptide might encapsulate DNA. Based on these results, we further analyzed the inhibitory result in cancer cells and found that compound 8 could partially fight against Hela cells. Therefore, the compound is promising to pave the way for the development of more effective and less toxic transfection vectors.

Facile molybdenum and aluminum recovery from spent hydrogenation catalyst

Facile molybdenum and aluminum recovery from spent hydrogenation catalyst

Point-of-care image-based quantitative urinalysis with commercial reagent strips: Design and clinical evaluation

Urinalysis is a useful test as an indicator of health or disease and as such, is a part of routine health screening. Urinalysis can be undertaken in many ways, one of which is reagent strips used in the general evaluation of health and to aid in the diagnosis and monitoring of kidney disease. To be effective, the test must be performed properly, and the results interpreted correctly. However, different light conditions and colour perception can vary between users leading to ambiguous readings. This has led to camera devices being used to capture and generate the estimated biomarker concentrations, but image colour can be affected by variations in illumination and inbuilt image processing. Therefore, a new portable device with embedded image processing techniques is presented in this study to provide quantitative measurements that are invariant to changes in illumination. The device includes a novel calibration process and uses the ratio of RGB values to compensate for variations in illumination across an image and improve the accuracy of quantitative measurements. Results show that the proposed calibration method gives consistent homogeneous illumination across the whole image. Comparisons against other existing methods and clinical results show good performance with a correlation to the clinical values. The proposed device can be used for point-of-care testing to provide reliable results consistent with clinical values.

Protein Structure-Based Organic Chemistry-Driven Ligand Design from Ultralarge Chemical Spaces.

Ultralarge chemical spaces describing several billion compounds are revolutionizing hit identification in early drug discovery. Because of their size, such chemical spaces cannot be fully enumerated and require ad-hoc computational tools to navigate them and pick potentially interesting hits. We here propose a structure-based approach to ultralarge chemical space screening in which commercial chemical reagents are first docked to the target of interest and then directly connected according to organic chemistry and topological rules, to enumerate drug-like compounds under three-dimensional constraints of the target. When applied to bespoke chemical spaces of different sizes and chemical complexity targeting two receptors of pharmaceutical interest (estrogen β receptor, dopamine D3 receptor), the computational method was able to quickly enumerate hits that were either known ligands (or very close analogs) of targeted receptors as well as chemically novel candidates that could be experimentally confirmed by in vitro binding assays. The proposed approach is generic, can be applied to any docking algorithm, and requires few computational resources to prioritize easily synthesizable hits from billion-sized chemical spaces.

The prevalence of antibiotic resistance genes of carbopenemases of the metallo-β-lactamase group in enterobacteria strains isolated from patients of a multidisciplinary hospital in a region with a developed petrochemical industry

The research work included strains of Klebsiella pneumonaie (n = 128) isolated from patients of a multidisciplinary hospital in a large industrial city. The antibiotic sensitivity of the isolated pure cultures was studied in relation to the most popular antibacterial drugs in clinical practice (in the amount of 21), by the standard disco-diffusion method. Identification of resistance genes was carried out by PCR using commercial reagent kits «AmpliSens MDR-MBL-FL», «AmpliSens MDR-KPC/OXA-48‑FL». The results of determining the sensitivity of isolates isolated from the lower respiratory tract revealed low activity of amoxicillin/clavulanic acid, cephalosparins of the III and IV generation (ceftazidim, cefotaxime, cefepim). 86.7 % of isolates are resistant to the aminoglycoside group — amikacin, and 100 % are resistant to gentamicin. Meropenem was the most active of the carbapenem group — 26.6 %, about 7 % are sensitive to ertapenem. From the group of fluoroquinolones, the sensitivity varied from 20 to 30 %. The greatest activity of all drugs was shown by ticarcillin / clavulanate — 33.3 %. A high proportion of strains of Kl. pneumoniae with a phenotype of multiple resistance has been established. The identification of resistance genes made it possible to establish their diversity in antibiotic-resistant isolates, a high proportion (26.6 %) of multi-resistant strains with the absence of the studied genes, and the combination of VIM + NDM + OXA-48 genes, identified in every second studied strain, prevails. Thus, the analysis of the prevalence of antibiotic resistance genes of carbopenemases of the metallo- β-lactamase group in enterobacteria strains isolated from patients of a multidisciplinary hospital in a region with a developed petrochemical industry showed the predominance of isolates (53.9 %) with the production of a combination of three antibiotic resistance genes (VIM + NDM + OXA-48). Isolates containing two genes simultaneously (NDM + OXA-48 and VIM + OXA-48) identified in 7.8 and 6.3 %. Isolates containing a single gene (carbapenemase Kl. Pneumoniae) amounted to 5.4 %.

Enhanced gene transfection and induction of apoptosis in melanoma cells by branched poly(β-amino ester)s with uniformly distributed branching units

Melanoma, one of the most devastating forms of skin cancer, currently lacks effective clinical treatments. Delivery of functional genes to modulate specific protein expression to induce melanoma cell apoptosis could be a promising therapeutic approach. However, transfecting melanoma cells using non-viral methods, particularly with cationic polymers, presents significant challenges. In this study, we synthesized three branched poly(β-amino ester)s (HPAEs) with evenly distributed branching units but varying space lengths through a two-step “oligomer combination” strategy. The unique topological structure enables HPAEs to condense DNA to form nano-sized polyplexes with favorable physiochemical properties. Notably, HPAEs, especially HPAE-2 with intermediate branching unit space length, demonstrated significantly higher gene transfection efficiency than the leading commercial gene transfection reagent, jetPRIME, in human melanoma cells. Furthermore, HPAE-2 efficiently delivered the Bax-encoding plasmid into melanoma cells, leading to a pronounced pro-apoptotic effect without causing noticeable cytotoxicity. This study establishes a potent non-viral platform for gene transfection of melanoma cells by harnessing the distribution of branching units, paving the way for potential clinical applications of gene therapy in melanoma treatment.

Synthesis of 4H-Pyrazolo[3,4-d]pyrimidin-4-one Hydrazine Derivatives as a Potential Inhibitor for the Self-Assembly of TMV Particles.

Tobacco mosaic virus coat protein (TMV-CP), as a potential target for the development of antiviral agents, can assist in the long-distance movement of viruses and plays an extremely important role in virus replication and propagation. This work focuses on the synthesis and the action mechanism of novel 4H-pyrazolo[3,4-d] pyrimidin-4-one hydrazine derivatives. The synthesized compounds exhibited promising antiviral activity on TMV. Specifically, compound G2 exhibited high inactivating activity (93%) toward TMV, slightly better than commercial reagent NNM (90%). The action of mechanism was further explored by employed molecular docking, molecular dynamics simulation, microscale thermophoresis, qRT-PCR, and transmission electron microscopy. Results indicated that G2 had the capability to interact with amino acid residues such as Trp352, Tyr139, and Asn73 in the active pocket of TMV-CP, creating strong hydrophobic interactions and thus obstructing the virus's self-assembly.

A novel microfluidic chip-based digital PCR method for enhanced sensitivity in the early diagnosis of colorectal cancer via mSEPT9

BackgroundTo enhance the sensitivity of plasma methylated Septin9 gene (mSEPT9) detection in colorectal cancer (CRC) screening, we developed a microfluidic chip-based digital PCR (dPCR) method suitable for low-concentration samples, aiming to apply it for mSEPT9 detection in CRC diagnosis. MethodsOur microfluidic chip-based dPCR method utilized specific primers and probes with locked nucleic acids (LNAs) modifications for mSEPT9 detection. We evaluated its performance, including detection limit, specificity, and linear range, comparing it with a commercial qPCR reagent kit using the same samples (95 CRC, 23 non-CRC). ResultsThe LNAs-modified dPCR method showed a linear range of 100–104 copies/μL and a detection limit of 100 copies/μL. Clinical testing revealed that our dPCR method exhibited a sensitivity of 82.11 % and specificity of 95.65 % for CRC diagnosis, outperforming the commercial qPCR kit (sensitivity: 58.95 %, specificity: 91.30 %), particularly in Stage I with a diagnostic sensitivity of 90.91 %. Combining mSEPT9 and carcinoembryonic antigen (CEA) improved diagnostic sensitivity to 91.49 %. ConclusionsOur accurate microfluidic chip-based dPCR method, especially in combination with CEA, holds promise for effective CRC screening and timely interventions, offering enhanced mSEPT9 quantification over conventional qPCR.

Copper-Catalyzed Synthesis of Masked (Hetero)Aryl Sulfinates.

Catalysis using substoichiometric copper facilitates the synthesis of masked (hetero)aryl sulfinates under mild, base-free conditions from aryl iodides and the commercial sulfonylation reagent sodium 1-methyl 3-sulfinopropanoate (SMOPS). The development of a tert-butyl ester variant of the SMOPS reagent allowed the use of aryl bromide substrates. The sulfones thus generated can be unmasked and functionalized in situ to form a variety of sulfonyl-containing functional groups.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

Quaternary ammonium fluorides and difluorosilicates as nucleophilic fluorination reagents.

TBAT (tetrabutylammonium difluorotriphenylsilicate) is an excellent homogeneous nucleophilic fluorination reagent, but a high excess of the reagent was reported to be essential. We hence optimized the reaction conditions and compared its nucleophilic fluorination reactivity with that of other common commercial nucleophilic fluorination reagents, such as anhydrous TBAF and TASF (tris(dimethylamino)sulfonium difluorotrimethylsilicate). As the substrates, we employed a standard set of primary and secondary octyl substrates under identical conditions. To eliminate the possibility of hydrogen fluoride elimination in the above reagents, we prepared four quaternary ammonium fluorides lacking β-elimination possibility in the hydrocarbon chain, transformed them to the corresponding difluorotriphenylsilicates, and compared their reactivity with that of the commercial reagents. Furthermore, attempts to isolate analogous tetrabutylammonium difluoromethyldiphenylsilicate or difluorodimethylphenylsilicate failed, as was confirmed by comparison of the published experimental data with computed 19F NMR spectra. Finally, we studied the transition states of decomposition of various tetramethylammonium methylphenyldifluorosilicates by DFT methods and found that their relative energies increase with an increasing number of phenyl groups. The formation of difluorosilicates is a nearly barrierless process.

Enriching new transplantable RGC-like cells from retinal organoids for RGC replacement therapy

Optic neuropathies, such as glaucoma, are due to progressive retinal ganglion cells (RGCs) degeneration, result in irreversible vision loss. The promising RGCs replacement therapy for restoring vision are impeded by insufficient RGC-like cells sources. The present work was enriched one new type RGC-like cells using two surface markers CD184 and CD171 from human induced pluripotent stem cells (hiPSCs) by FACS sorting firstly. These new kind cells have well proliferation ability and possessed passage tolerance in vitro 2D or 3D spheroids culture, which kept expressing Pax6, Brn3b and βIII-Tubulin and so on. The transplanted CD184+CD171+ RGC-like cells could survive and integrate into the normal and optic nerve crush (ONC) mice retina, especially they were more inclined to across the optic nerve head and extend to the damaged optic nerve. These data support the feasible application for cell replacement therapy in RGC degenerative diseases, as well as help to develop new commercial cells sorting reagents and establish good manufacturing practice (GMP) grade RGC-like donor cells for further clinical application.

Enhanced enrichment of extracellular vesicles for laboratory and clinical research from drop-sized blood samples.

In the realm of biomedical advancement, extracellular vesicles (EVs) are revolutionizing our capacity to diagnose, monitor, and predict disease progression. However, the comprehensive exploration and clinical application of EVs face significant limitations due to the current isolation techniques. The size exclusion chromatography, commercial precipitation reagents, and ultracentrifugation are frequently employed, necessitating skilled operators and entailing challenges related to consistency, reproducibility, quality, and yields. Notably, the formidable challenge of extracellular vesicle isolation persists when dealing with clinical samples of limited availability. This study addresses these challenges by aiming to devise a rapid, user-friendly, and high-recovery EVs isolation technique tailored for blood samples. The NTI-EXO precipitation method demonstrated a 5-fold increase in the recovery of serum EVs compared to current methodologies. Importantly, we illustrate that a mere two drops of blood (∼100 µL) suffice for the recovery of enriched EVs. The integrity and quality of these isolated EVs were rigorously assessed for the size, purity, and contaminants. This method was validated through the successful isolation of EVs from organ transplant recipients to detect disease-specific exosomal markers, including LKB1, SARS-CoV-2 spike protein, and PD-L1. In conclusion, NTI-EXO method can be used for small clinical samples, thereby advancing discoveries in the EV-centric domain and propelling the frontiers of biomedical research and clinical applications.

Development of NIR-II small animal living fluorescence imaging system

Fluorescence imaging technology can dynamically monitor gene and cell changing in live animals in real-time, with advantages such as high sensitivity, high resolution, and non-invasion. In recent years, it has been widely used in tumor research, gene expression research, drug development research, etc. The imaging wavelength of traditional fluorescence imaging technology falls in the visible and near-infrared-I region. Due to the absorption and scattering effects of light propagation in biological tissues, and the inherent fluorescence of biological tissues, traditional fluorescence imaging techniques still have significant limitations in penetration depth and image signal-to-noise ratio. In this work, a highly integrated near-infrared-II (NIR-II, 900—1880 nm) small animal living fluorescence imaging system is developed by taking the advantages of NIR-II fluorescence imaging technology, such as low absorption, low scattering, and deep penetration depth in biological tissues. And a method of enhancing and correcting fluorescence image is proposed to optimize fluorescence images. In this work, the biological tissue simulation experiments and live animal experiments are conducted to test the performance and imaging effect of the system. The experimental results show that the system has the advantages of deep penetration depth, high signal-to-noise ratio, and high sensitivity. Combined with commercial indocyanine green reagents and aggregation-induced emission dyes, this system can monitor the distribution of blood vessels in real time and continuously monitor deep tissues and organs in mice, and conduct the dynamically monitoring research in living mice in a conscious state. This helps to promote tumor research and drug development research in the field of biomedical imaging to enter a new stage.

Evaluation of commercial immunodiffusion reagents for detecting serum anti-Paracoccidioides antibodies.

Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.