Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection.

OBJECTIVE: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays. RESULTS: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations.

A new chemical derivatization reagent sulfonyl piperazinyl for the quantification of fatty acids using LC-MS/MS.

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80 % (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

Reagent storage and delivery on integrated microfluidic chips for point-of-care diagnostics.

Microfluidic-based point-of-care diagnostics offer several unique advantages over existing bioanalytical solutions, such as automation, miniaturisation, and integration of sensors to rapidly detect on-site specific biomarkers. It is important to highlight that a microfluidic POC system needs to perform a number of steps, including sample preparation, nucleic acid extraction, amplification, and detection. Each of these stages involves mixing and elution to go from sample to result. To address these complex sample preparation procedures, a vast number of different approaches have been developed to solve the problem of reagent storage and delivery. However, to date, no universal method has been proposed that can be applied as a working solution for all cases. Herein, both current self-contained (stored within the chip) and off-chip (stored in a separate device and brought together at the point of use) are reviewed, and their merits and limitations are discussed. This review focuses on reagent storage devices that could be integrated with microfluidic devices, discussing further issues or merits of these storage solutions in two different sections: direct on-chip storage and external storage with their application devices. Furthermore, the different microvalves and micropumps are considered to provide guidelines for designing appropriate integrated microfluidic point-of-care devices.

Direct radical functionalization of native sugars.

Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free 'cap and glycosylate' approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.

Cell Wall Modification Based on Combination Reagents to Improve Dimensional Stability of Wood with High Efficiency.

Although γ-methacryloxypropyltrimethoxysilane (MPS) was proved to be an effective reagent for improving the dimensional stability of wood, a bottleneck in ASE value (around 50%) existed. The reason was that MPS with low polarity opened few hydrogen bonds in the amorphous region of cellulose, while these hydrogen bonds could be reopened by water. Therefore, citric acid (CA) is chosen to cooperate with MPS to further enhance the dimensional stability of wood. In this paper, MPS and CA were used to modify wood individually (MW and CW) or with different combinations, that is, one-step modification (M/CW) and two-step modification with MPS first (M-CW) or CA first (C-MW). CA and MPS concentrations were optimized at 5 wt%. The ASE value for M/CW was only 25.74% at a weight percent gain (WPG) of 6.43%, which was only 0.6 times to MW or 0.7 times to CW. For M-CW, the ASE value gradually decreased with the soaking cycles, from 65.64% at a WPG of 9.05% to 51.20%. The C-MW had the best dimensional stability, with the ASE value 75.35% at a WPG of 11.50%. Although it decreased during the first soaking cycle, it stabilized at 62.20% at last. SEM and EDS images showed that the polymer mainly distributed in cell walls and few in cell lumen in C-MW. Thus, the enhanced dimensional stability of C-MW could be explained by CA opening the hydrogen bonds in the amorphous region of cellulose first, which provided more binding sites for MPS.

[Concerns of Performance Evaluation on Central Nervous System Infection Pathogen Metagenome Sequencing Reagent].

From the perspective of the performance evaluation, concerns of the range of pathogens, establishment of enterprise reference material, reaction system study and analytical performances evaluation of central nervous system infection pathogen metagenome sequencing reagent are briefly described, including study methods and quality control requirements. This study is intended to increase the research and development efficiency of products, and contribute to the development of associated industry.

[Thoughts on High-Quality Innovation and Development of in Vitro Diagnostic Reagents Industry in Shanghai].

As an important part of the Big Health Industry, in vitro diagnostic(IVD) reagents play a vital role in the prevention, diagnosis and treatment of diseases. In recent years, especially after the novel coronavirus infection, IVD industry has developed rapidly in China, but it still cannot meet the needs of clinical use. By conducting desk research, expert interview, manufacturer survey and hospital survey, this study analyzed the development status of IVD industry in Shanghai, summarized the problems encountered in the high-quality development from the aspects of raw materials, innovation ability, and clinical trials, etc., and proposed recommendations for promoting the high-quality innovation and development of IVD industry in Shanghai.

[Design and Application of an IVD Reagent Management Information Systems Based on Unique Code].

The management of in vitro diagnostic (IVD) reagents in hospitals often faces issues such as the lack of a unified coding system, unclear consumption patterns, and unknown cost-to-income ratios. It is necessary to employ information systems to achieve comprehensive, detailed, and traceable management of IVD reagents. An information management system for IVD reagents based on unique coding is introduced, which integrates admission, acceptance, and consumption processes through unique codes. The system calculates the income per experimental item based on the consumption of IVD reagents and the charge for each experimental item. The system enhances the efficiency of the IVD reagent supply chain management and promotes detailed oversight of IVD reagent usage.

Development of a fluorous trapping reagent for rapid detection of electrophilic reactive metabolites.

A cysteine-based fluorous trapping reagent, Rf8CYS, was developed. Rf8CYS formed adducts with soft and hard electrophilic reactive metabolites. These fluorous-tagged adducts were purified via both fluorous solid-phase extraction and the direct injection method. The highly sensitive mass spectrometric detection of an unprecedented adduct of the ticlopidine metabolite was realized.

Study on the methods of visualizing bloodstains after thermal exposure.

To establish the correlation between thermal conditions imposed on bloodstains and visualizing effect of enhancement techniques, infrared photography and four chemical enhancement reagents were used to visualize bloodstains following thermal exposure. A black tile was selected as the substrate to intensify the visualization challenge, with a Cone Calorimeter serving as the standardized heating source to control thermal conditions. Compared with standard photography, infrared photography is proven to be a valuable complement to chemical reagents, showing significant advantages in visualizing bloodstains after thermal exposure. However, it is worth noting that infrared image fell short of standard image when bloodstains displayed raised, embossed morphology or when bloodstains almost disappeared under specific conditions. The enhancement effectiveness was found to be strongly correlated with thermal conditions imposed on bloodstains, and the morphology evolution of bloodstains during heating affected the chemical enhancement effect additionally, especially when the bulge morphology was formed, and it was observed that reagents were more effective after removing the dense shell of the bulge. Among the four selected chemical enhancement reagents, fluorescein performed exceptionally well, maintaining its effectiveness even for bloodstains heated at 641°C for 10 min. TMB demonstrated its visualizing ability for bloodstains heated at 396°C for 5 min and heated at 310°C for 20 min. BLUESTAR® followed afterwards, while luminol performed worst. The correlation between thermal conditions imposed on bloodstains and the corresponding visualizing effectiveness of enhancement techniques provides important references for detecting bloodstains at fire scenes.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

Advances in the Synthesis of Bioorthogonal Reagents: s-Tetrazines, 1,2,4-Triazines, Cyclooctynes, Heterocycloheptynes, and trans-Cyclooctenes.

Aligned with the increasing importance of bioorthogonal chemistry has been an increasing demand for more potent, affordable, multifunctional, and programmable bioorthogonal reagents. More advanced synthetic chemistry techniques, including transition-metal-catalyzed cross-coupling reactions, C-H activation, photoinduced chemistry, and continuous flow chemistry, have been employed in synthesizing novel bioorthogonal reagents for universal purposes. We discuss herein recent developments regarding the synthesis of popular bioorthogonal reagents, with a focus on s-tetrazines, 1,2,4-triazines, trans-cyclooctenes, cyclooctynes, hetero-cycloheptynes, and -trans-cycloheptenes. This review aims to summarize and discuss the most representative synthetic approaches of these reagents and their derivatives that are useful in bioorthogonal chemistry. The preparation of these molecules and their derivatives utilizes both classical approaches as well as the latest organic chemistry methodologies.

Recent advances in asymmetric synthesis of chiral amides and peptides: racemization-free coupling reagents.

Over past decades, chiral amides and peptides have emerged as powerful and versatile compounds due to their various biological activities and interesting molecular architectures. Although some chiral condensation reagents have been applied successfully for their synthesis, the introduction of racemization-free methods of amino acid activation have shown lots of advantages in terms of their low cost and low toxicity. In this review, advancements in amide and peptide synthesis using racemization-free coupling reagents over the last 10 years are summarized. Various racemization-free coupling reagents have been applied in the synthesis of enantioselective amides and peptides, including ynamides, allenones, HSi[OCH(CF3)2]3, Ta(OMe)5, Nb(OEt)5, Ta(OEt)5, TCFH-NMI, water-removable ynamides, DBAA, DATB, o-NosylOXY, TCBOXY, Boc-Oxyma, NDTP, 9-silafluorenyl dichlorides, the Mukaiyama reagent, EDC and T3P. The racemization-free reagents described in this review provide an alternative greener option for the asymmetric synthesis of chiral amides and peptides. We hope that this review will inspire further studies and developments in this field.

An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Selective Macrocyclization of Unprotected Peptides with an Ex Situ Gaseous Linchpin Reagent.

Peptide cyclization has dramatic effects on a variety of important properties, enhancing metabolic stability, limiting conformational flexibility, and altering cellular entry and intracellular localization. The hydrophilic, polyfunctional nature of peptides creates chemoselectivity challenges in macrocyclization, especially for natural sequences without biorthogonal handles. Herein, we describe a gaseous sulfonyl chloride derived reagent that achieves amine-amine, amine-phenol, and amine-aniline crosslinking through a minimalist linchpin strategy that affords macrocyclic urea or carbamate products. The cyclization reaction is metal-mediated and involves a novel application of sulfine species that remains unexplored in aqueous or biological contexts. The aqueous method delivers unique cyclic or bicyclic topologies directly from a variety of natural bioactive peptides without the need for protecting-group strategies.

A new derivatizing reagent for the determination of 5-nitro-2-furaldehyde in trout muscle by liquid chromatography-tandem mass spectrometry.

The 5-nitro-2-furaldehyde (5-NF) is an aldehyde aromatic organic compound that has been envisaged as an alternative marker for detecting nitrofurazone treatment abuse and to avoid the false positive results induced by the semicarbazide. Analyzing 5-NF presents challenges, and its derivatization reaction with hydrazine reagents is required to enhance the capability of its detection and its identification. This study aims at developping an analytical method for 5-NF determination in trout muscle samples based on chemical derivatization prior to analysis by liquid chromatography-tandem mass spectrometry. Four commercially available hydrazine reagents, namely: N,N-Dimethylhydrazine (DMH), 4-Hydrazinobenzoic acid (HBA), 2,4-Dichlorophenylhydrazine (2,4-DCPH) and 2,6-Dichlorophenylhydrazine (2,6-DCPH) were proposed for the first time as derivatizing reagents in the analysis of 5-NF. The derivatization reaction was simultaneously performed along with the extraction method in acidic condition using ultrasonic assistance and followed by liquid extraction using acetonitrile. The efficiency of the chemical reaction with 5-NF was examined and the reaction conditions including the concentration of hydrochloric acid, pH, temperature, reaction time and the concentration of the derivatizing reagents were optimized. Experiments with fortified samples demonstrated that 2,4-DCPH derivatizing reagent at 20 mM for 20 min of ultrasonic treatment under acidic condition (pH 4) gave an effective sample derivatization method for 5-NF analysis. Under the optimized conditions, the calibration curves were linear from 0.25 to 2 µg kg-1 with coefficient of determination >0.99. The recoveries ranged from 89 % to 116 % and precision was less than 13 %. The limit of detection and quantification were 0.1 and 0.2 µg kg-1, respectively.

An indicator color chart for quick detection of Pigment Red 53 in cosmetic products in Indonesia.

BACKGROUND: Pigment Red 53 is a dangerous synthetic dye that is often added to cosmetics, even though its use in cosmetic products has been prohibited because of possible impacts on health. Faster and more sensitive detection of Pigment Red 53 is needed for onsite analysis to protect the community from illegal cosmetics that contain the dye. Indicator color charts are a kind of analytical method that can be used to detect Pigment Red 53 in cosmetic products, including lipstick, rouge, and eyeshadow. Such charts are practical, fast, and can be used for onsite analysis. METHODS: In this study, an indicator for Pigment Red 53 detection was obtained through a reagent reaction that caused a specific color change. An indicator color chart was then produced by setting out in paper form the series of colors which resulted from the reaction of specific chemical reagents and Pigment Red 53 solutions at concentrations of 10, 20, 40, 60, 80, and 100 ppm. RESULTS: The testing results showed that the indicator color chart may be used as an initial screening method for the detection of Pigment Red 53 in cosmetic products with a detectable minimum concentration of 10 ppm. Out of nine samples, only one (Eyeshadow 3) tested positive for Pigment Red 53. Further analysis was carried out on the indicator color chart and the results showed good agreement with TLC and UV-Vis spectrophotometry methods. CONCLUSION: The results reported in this paper demonstrate that the indicator color chart is a good prospective method for onsite analysis to detect Pigment Red 53 in cosmetic samples, with a lower detection limit compared to polymer-based indicators.

Further research into alternative carrier solvents for the detection of latent fingermarks.

A number of solvents, (Solstice PF, Opteon SF33 and Amolea AS-300), are compared to the recommended carrier solvent of HFE7100 for the ninhydrin and 1,2-indandione formulations. As the supply of HFE7100 will cease by the end of 2025, suitable alternatives are required in the short-term to ensure the detection of latent fingermarks on porous surfaces is still effective. Although these solvents, with the exception of Amolea AS-300, are classified as per- and polyfluoroalkyl substances (PFAS); they are not classed as hazardous. The alternatives in this study have a low global warming potential and atmospheric lifetime and are volatile, non-flammable and non-ozone depleting, in addition to other desirable properties such as a high wetting-index. During Phase 2 trials with deposited fingermarks, HFE7100 provided the best performing results followed by Opteon SF33, Solstice PF and Amolea AS-300. A significant difference with a negligible effect size was observed for ninhydrin formulations (p-value 0.00179; ε2 0.00418) while a significant difference with a weak effect size was observed for 1,2-indanedione formulations (p-value 2.095 ×10-10; ε2 0.0167). Furthermore, HFE7100 provided the least ink diffusion and the brightest 1,2-indanedione luminosity (significant difference with a moderate effect size p-value 1.772 ×10-13; ε2 0.0434) but the HFE formulation turned cloudy more quickly and needed regular replacements. Phase 3 pseudo-operational trials of 100 porous items followed a similar trend whereby HFE7100 formulations detected the highest number of marks followed by Opteon SF33 and Solstice PF. Although HFE7100 is still the best performing carrier solvent, this study demonstrates that, in the short-term, Opteon SF33 and Solstice PF may have potential as non-flammable replacement carrier solvents while developing the long-term goal of solvent-less methods.