Recent advances and applications of ionic covalent organic frameworks in food analysis.

Ionic covalent organic frameworks with both crystallinity and charged sites have attracted significant attention from the scientific community. The versatile textural structures, precisely defined channels, and abundant charged sites of ionic COFs offer immense potential in various areas such as separation, sample pretreatment, ion conduction mechanisms, sensing applications, catalytic reactions, and energy storage systems. This review presents a comprehensive overview of facile preparation methods for ionic covalent organic frameworks (iCOFs), along with their applications in food sample pretreatment techniques such as solid-phase extraction (SPE), magnetic solid-phase extraction (MSPE), and dispersive solid-phase extraction (DSPE). Furthermore, it highlights the extensive utilization of iCOFs in detecting various food contaminants including pesticides, contaminants from food packaging, veterinary drugs, perfluoroalkyl substances, and poly-fluoroalkyl substances. Specifically, this review critically discusses the limitations, challenges, and future prospects associated with employing iCOF materials to ensure food safety.

Dual benefits of NBD-Cl fluorogenic action and sample pretreatment (SALLE) technology in the assessment of anticoagulant medication: Dabigatran in pharmaceutical capsules and plasma samples.

Dabigatran (DBG), marketed as Pradaxa, is an anticoagulant medication prescribed for the treatment and mitigation of blood clots and to lower the risk of stroke in individuals with the heart condition known as atrial fibrillation. This medication is specifically indicated for preventing blood clots post hip or knee replacement surgeries and in patients with a prior history of clots. Compared to warfarin, dabigatran serves as a viable alternative that does not necessitate routine blood monitoring tests. The complimentary benefits associated with SALL (salting-out assisted liquid-liquid extraction) and the fluorogenic capabilities of benzofurazan. These methods were combined to provide an affordable and sensitive DBG assaying method. The spectral strength of the yellow luminous product was examined at 533.8 nm and by adjustment of a wavelength of 474.7 nm for excitation. To assess its linearity, the calibration chart was tested across a DBG concentration range of 30-500 ng/ml. Via accurate computation based on ICH, the detection limit (LD) was determined to be 9.5 ng/ml, and the strategy can quantify the DBG to a limit of 28 ng/ml. To ensure success, various crucial parameters for method implementation have been extensively studied and adapted. The validation of the strategy adhered to the policies outlined by ICH, affirming its precision in quantifying DBG in capsules. Furthermore, the inclusion of SALLE steps facilitated accurate monitoring of DBG in plasma samples, introducing a unique and advanced methodology for analyzing this compound in biological samples.

Rapid detection of ricin at trace levels in complex matrices by asialofetuin-coated beads and bottom-up proteomics using high-resolution mass spectrometry.

Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry-based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays.

Microfluidic Sample Preparation for Multiplexed Single-Cell Proteomics Using a Nested Nanowell Chip.

Mass spectrometry-based single-cell proteomics has undergone rapid progress and has become an active research area. However, because of the ultralow amount of proteins in single cells, it is still highly challenging to achieve efficient sample preparation and sensitive LC-MS detection. Here, we provide a detailed protocol for isobaric labeling-based single-cell proteomics relying on a microfluidic droplet-based sample processing technology. The protocol allows for processing both single cells and carrier samples in separate microchips using a commercially available platform (cellenONE) with high sample recovery and high throughput. We also provide an optimized LC-MS method for sensitive and robust data collection.

A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Blood proteomics of COVID-19 infection: An update.

Despite significant progress in understanding SARS-CoV-2 and its impact, a deeper comprehension of the molecular changes in the host following infection is still lacking. Proteomic analysis of COVID-19 infected patients can provide valuable data about the events and mechanisms related to the initial, progression, and terminal stages of the disease. Such information can lead to better prevention, treatment, and convalescence strategies. In this review, we discuss blood proteomic studies based on mass spectrometry in COVID-19 patients. The analysis focuses on various blood components including plasma, platelets, serum, red blood cells, and peripheral blood mononuclear cells. Scientific papers published since 2022 are covered, with approaches categorised based on whether they use in-gel, in-solution, or on-beads/on-filter digestion modes. Additionally, candidate protein biomarkers related to COVID-19 are surveyed and discussed.

Exploiting the Affimer platform against influenza A virus.

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents. IMPORTANCE: Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

Microplastic loads in Eurasian otter (Lutra lutra) feces-targeting a standardized protocol and first results from an alpine stream, the River Inn.

Microplastics (MP) are omnipresent in a wide range of environments, constituting a potential threat for aquatic and terrestrial wildlife. Effects in consumers range from physical injuries to pathological reactions. Due to potential bioaccumulation of MP, predators are of particular concern for MP induced health effects. The Eurasian otter is an apex predator in (semi-)aquatic habitats feeding primarily on fish. Furthermore, the species is classified as "near threatened" on the IUCN Red List. Thus, the Eurasian otter is of conservation concern and may serve as a bioindicator for MP pollution. Feces can be used to detect pollutants, including MP. Initial studies confirmed the presence of MP in otter feces (= spraints). However, as specific, validated protocols targeting at an efficient and standardized extraction of MP from otter spraints are missing, experimental results reported from different groups are challenging to compare. Therefore, we (i) present steps towards a standardized protocol for the extraction of MP from otter feces, (ii) give recommendations for field sample collection of otter spraints, and (iii) provide a user-friendly step-by-step workflow for MP extraction and analysis. Applying this framework to field samples from five study sites along the River Inn (n = 50), we detected MP of different sizes and shapes (ranging from microfibers to road abrasion and tire wear) in all otter spraint samples.

Point-of-care isothermal nucleic acid amplification tests: progress and bottlenecks for extraction-free sample collection and preparation.

INTRODUCTION: Suitable sample collection and preparation methods are essential to enable nucleic acid amplification testing at the point of care (POC). Strategies that allow direct isothermal nucleic acid amplification testing (iNAAT) of crude sample lysate without the need for nucleic acid extraction minimize time to result as well as the need for operator expertise and costly infrastructure. AREAS COVERED: The authors review research to understand how sample matrix and preparation affect the design and performance of POC iNAATs. They focus on approaches where samples are directly combined with liquid reagents for preparation and amplification via iNAAT strategies. They review factors related to the type and method of sample collection, storage buffers, and lysis strategies. Finally, they discuss RNA targets and relevant regulatory considerations. EXPERT OPINION: Limitations in sample preparation methods are a significant technical barrier preventing implementation of nucleic acid testing at the POC. The authors propose a framework for co-designing sample preparation and amplification steps for optimal performance with an extraction-free paradigm by considering a sample matrix and lytic strategy prior to an amplification assay and readout. In the next 5 years, the authors anticipate increasing priority on the co-design of sample preparation and iNAATs.

Effect of short-term sample storage and preparatory conditions on losses of 18 per- and polyfluoroalkyl substances (PFAS) to container materials.

There is a lack of agreement on a suitable container material for per- and polyfluoroalkyl substances (PFAS) analysis, particularly at trace levels. In this study, the losses of 18 short- and long-chain (C4-C10) PFAS to commonly used labware materials (high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), polypropylene co-polymer (PPCO), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and glass were investigated. The influence of sample storage and preparation conditions, i.e., storage time, solvent composition, storage temperatures (4 °C and 20 °C), and sample agitation techniques (shaking and centrifugation) on PFAS losses to the container materials were investigated. The results showed higher losses for most of the considered PFAS (up to 50.9%) in 100% aqueous solutions after storage for 7 days regardless of the storage temperature compared to those after 3 days. Overall, the order of losses to different materials varied for individual PFAS, with the highest losses of long-chain PFAS observed to PP and HDPE after 7-day storage at room temperature. The addition of methanol to aqueous PFAS solutions reduced the losses of long-chain PFAS to all tested materials. The use of sample centrifugation and shaking did not influence the extent of losses for most of the PFAS in 80:20 water:methanol (%, v/v) to container materials except for 8:2 fluorotelomer sulfonic acid (8:2 FTS), 9-chlorohexadecafluoro-3-oxanone-1-sulfonic acid (9Cl-PF3ONS), perfluorodecanoic acid (PFDA) and 4:2 fluorotelomer sulfonic acid (4:2 FTS). This study demonstrates lower losses of both long- and short-chain PFAS to glass and PET. It also highlights the need for caution when deciding on sample preparatory steps and storage during the analysis of PFAS.

Liquid-liquid and solid-liquid extractions with low-temperature partitioning - A review.

The paper represents the first review of solvent extraction techniques utilizing the low-temperature partitioning/purification (LTP) approach. Initially conceived in the 1960s to purify extracts from fatty matrices, it wasn't until the 2000s that this approach received increasing attention for its efficacy in extracting organic compounds from diverse samples, often without additional cleanup steps. This review covers a brief history and proposes a mechanism for LTP-based solvent extraction. Furthermore, the principal practical issues of the technique are spotlighted, elucidating the factors influencing extraction efficiency. The advantages, limitations, and potential combinations with other extraction techniques of the LTP-based solvent extractions are analyzed. The versatility of the LTP approach is demonstrated by its applications in extracting various compounds from food, environmental, and biological samples, emphasizing its potential for rapid sample preparation with minimal steps, few chemicals, and minimal analyst intervention.

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

Detailed Protocol for Solid-Phase Extraction for Lipidomic Analysis.

Developing robust analytical techniques is a vital phase to facilitate understanding the roles and impacts of various omic profilings in cellular functions. The comprehensive analysis of various biological molecules within a biological system requires a precise sample preparation technique. Solid-Phase Extraction (SPE) has proven to be an indispensable method in lipidomic analysis, providing an uncomplicated and user-friendly technique for extraction and purification of lipid components from complex biological matrices. Of all the factors influencing the reliability and success of SPE, column or adsorbent materials, flow rate, and storage conditions are paramount in terms of their significance. In this chapter, we will discuss in detail the SPE steps for lipidomic analysis in biofluid samples (serum and plasma) and muscle tissues.

Preparing research samples for safe arrival at centers and facilities: recipes for successful experiments.

Preparation of biomacromolecules for structural biology studies is a complex and time-consuming process. The goal is to produce a highly concentrated, highly pure product that is often shipped to large facilities with tools to prepare the samples for crystallization trials or for measurements at synchrotrons and cryoEM centers. The aim of this article is to provide guidance and to discuss general considerations for shipping biomacromolecular samples. Details are also provided about shipping samples for specific experiment types, including solution- and cryogenic-based techniques. These guidelines are provided with the hope that the time and energy invested in sample preparation is not lost due to shipping logistics.

Py-GC-MS analysis for microplastics: Unlocking matrix challenges and sample recovery when analyzing wastewater for polypropylene and polystyrene.

Matrix interference and recovery when using pyrolysis gas chromatography (Py-GC-MS) to analyze wastewater for polystyrene (PS) and polypropylene (PP) microplastics (MP) was studied. Raw wastewater underwent a sample preparation train commonly applied for such matrix. The train consisted of six discrete steps to reduce the organic matter content without affecting MP in the sample. One large wastewater sample was collected, homogenized, and subdivided into 21 subsamples. Three samples were analyzed without further sample preparation. The remaining samples were divided in sets of three, and each set underwent an increasing number of steps of the procedure, up to the last set, which underwent the full treatment. The matrix effect on the determination of PS and PP was statistically evaluated by comparing in-matrix and external calibration curves at each step. Recovery of MP was assessed after each step by adding deuterated PS to the samples. A main finding was that there was no significant matrix effect for these polymers throughout the preparation train, suggesting that matrix components did not interfere with the analytical method. However, a significant loss of polymer mass was found between the steps, which may result in MPs falling below detection limits. Therefore, Py-GC-MS could be used for MP quantification before analysis by other techniques which require more extensive matrix removal. A downside of this approach is that analyzing such samples without matrix reduction will increase the need for instrumental maintenance.

Investigation of thermal effects of laser micromachining for APT and TEM specimen preparation: A modeling and experimental study.

Laser micromachining can serve as a coarse machining step during sample preparation for high-resolution characterization methods leading to swift sample preparation. However, selecting the right laser parameters is crucial to minimize the heat-affected zone, which can potentially compromise the microstructure of the specimen. This study focuses on evaluating the size of heat-affected zone in laser annular milling, aiming to ascertain a minimal scan diameter that safeguards the inner region of micropillars against thermal damage. A computational model based on the finite element method was utilized to simulate the laser heating process. To validate the simulation results, a picosecond pulsed laser is then used to machine the micropillars of Al and Si. The laser-machined samples were subjected to surface and microstructural analysis using Scanning Electron Microscope (SEM) and Electron Backscatter Diffraction (EBSD) scans. The length of heat affected zone obtained from simulations was approximately 6 µm for silicon and 12 µm for aluminum. The diameter of micropillars formed with laser machining was 10 µm for silicon 26 µm for aluminum. The core of the pillars was preserved with less than one degree of microstructural misorientations making it suitable for further processing for preparing specimens for techniques like APT and TEM. For silicon micropillars, the preserved central region has a diameter of 6 µm and for aluminum its around 20-24 µm. Additionally, the study determines the minimum scan diameter that can be achieved using the given laser machining setup across a range of common materials.

Cleaning validation for blister packaging machines in hospital-supplying pharmacies.

OBJECTIVE: To ensure quality-assured care for patients, validation of a cleaning process for blister machines is essential. Due to the high operating costs of maintaining high-performance liquid chromatography (HPLC) which is mainly used for this type of analysis, a new, quick and cost-effective analysis method using UV-Vis spectroscopy has been developed. METHOD: Marker substances (metamizole (dipyrone) and paracetamol tablets) were packed in blisters. Afterwards test tablets were packaged before and after cleaning the blister machine and examined for contamination using UV-Vis spectroscopy. RESULTS: UV-Vis spectroscopy has been shown to be superior to HPLC analysis for cleaning validation of blister machines, as it is much faster and cheaper, requires less equipment and personnel effort, while maintaining the same reliability and sensitivity. CONCLUSION: Unit-dose blistering is becoming increasingly popular in the daily routine of hospital pharmacies worldwide due to a variety of advantages. Therefore, cleaning validation of blistering machines has become a mandatory duty of care. The UV-Vis spectroscopic method presented here is the first innovative method suitable for the cleaning validation of blister machines to date.

Dual-modal analysis of cis-diols in traditional Chinese medicine via boronic acid incorporated metal organic frameworks.

In this study, a boronic acid incorporated metal organic frameworks (inBA-MIL-100) were prepared via metal-ligand-fragment co-assembly strategy. The prepared frameworks can be served either as enrichment sorbent or SALDI-MS matrix for cis-diol containing molecules. Thus, a dual-modal analysis of cis-diols in traditional Chinese medicine has been established. Several significant advantages of the proposed strategy have been experimentally demonstrated, including high selectivity, high binding capacity (70 mg/g), good generality (5-250 µg/mL for HPLC based sample preparation, 10-500 ng/mL for SALDI-MS), high sensitivity (LOD: 180 ng/mL for HPLC based sample preparation, 5 ng/mL for SALDI-MS) and reliable quantification (RSD<3 % for HPLC based sample preparation, RSD<12 % for SALDI-MS) performance. Finally, the successful analysis of various cis-diols (active component and mycotoxin) in various Chinese traditional medicine was also achieved.

An Update on Current Trend in Sample Preparation Automation in Bioanalysis: strategies, Challenges and Future Direction.

Automation in sample preparation improves accuracy, productivity, and precision in bioanalysis. Moreover, it reduces resource consumption for repetitive procedures. Automated sample analysis allows uninterrupted handling of large volumes of biological samples originating from preclinical and clinical studies. Automation significantly helps in management of complex testing methods where generation of large volumes of data is required for process monitoring. Compared to traditional sample preparation processes, automated procedures reduce associated expenses and manual error, facilitate laboratory transfers, enhance data quality, and better protect the health of analysts. Automated sample preparation techniques based on robotics potentially increase the throughput of bioanalytical laboratories. Robotic liquid handler, an automated sample preparation system built on a robotic technique ensures optimal laboratory output while saving expensive solvents, manpower, and time. Nowadays, most of the traditional extraction processes are being automated using several formats of online techniques. This review covered most of the automated sample preparation techniques reported till date, which accelerated and simplified the sample preparation procedure for bioanalytical sample analysis. This article critically analyzed different developmental aspects of automated sample preparation techniques based on robotics as well as conventional sample preparation methods that are accelerated using automated technologies.