Sample Preparation Research Articles

Nano-microcrystals revealed on Tang dynasty gilded bronze using advanced TEM-SEM and synchrotron methods.

Over the years, numerous gold and silver artifacts have been excavated from the tombs of the Tang dynasty, which give evidence of the sophisticated metalworking techniques at that time. Few of the artifacts were thoroughly studied and their manufacturing processes were barely known. The present investigation concerns a metal headgear from a newly excavated tomb of a female in Xi'an of the Tang dynasty (618-907 A.D.), using advanced techniques in a complementary way, especially performing a detailed analysis of the corrosion products and alloying processes. The combined state-of-the-art methods and instrumentation used for the corrosion study included spectroscopy, diffraction, electron microscopy, synchrotron and their versions for specific measurements and sample preparation. The investigated headgear metal consists of a copper-based core, which is gilded by a thin gold layer, consisting of an Au-Hg alloy with a thin layer of about 400 nm. The technique used for shaping and hammered embellishments led to the creation of nanosized grains on the side that would eventually be the interior of the headgear. It was gilded using the mercury-amalgamation process, and the liquid diffusion caused the development of intermetallic compounds. This is the first recorded instance of these nano-scale and eutectic phases being observed on objects from an archaeological context. The crystallographic analysis offered valuable insights into the formation of needle-like malachite crystals growing on a layer of cuprite found on the surface of the corroded piece. The results highlight that the artisans utilized advanced methods in the creation of funerary items during the Tang dynasty.

Biomedical SERS - the current state and future trends.

Surface enhanced Raman spectroscopy (SERS) is meeting the requirements in biomedical science being a highly sensitive and specific analytical tool. By employing portable Raman systems in combination with customized sample pre-treatment, point-of-care-testing (POCT) becomes feasible. Powerful SERS-active sensing surfaces with high stability and modification layers if required are available for testing and application in complex biological matrices such as body fluids, cells or tissues. This review summarizes the current state in sample collection and pretreatment in SERS detection protocols, SERS detection schemes, i.e. direct and indirect SERS as well as targeted and non-targeted SERS, and SERS-active sensing surfaces. Moreover, the recent developments and advances of SERS in biomedical application scenarios, such as infectious diseases, cancer diagnostics and therapeutic drug monitoring is given, which enables the readers to identify the sample collection and preparation protocols, SERS substrates and detection strategies that are best-suited for their specific applications in biomedicine.

Nanoparticle Assisted Fabric Phase Sorptive Extraction for Azo Dye Determination in the Industrial Sewage.

Currently, one of the significant environmental problems is the presence of azo dye materials in water sources. In this study, for the first time, a fast and sensitive sample preparation approach using nanoparticle-assisted fabric phase sorptive extraction (NFPSE) followed by high-performance liquid chromatography was examined to remove some azo dyes such as methyl red and sunset yellow from aqueous solutions. Primarily, the significance of several parameters affecting NFPSE, such as fabric type, the kind of sorbent, the number of contacts with sol-gel and the time of contact, was investigated. In addition, experiments were performed to determine the effect of different adsorption parameters, such as sample volume, adsorption time, adsorbent value, desorption time, ionic strength and pH. It was found that the calibration curve was linear within two ranges of concentrations (0.05-0.1 and 0.5-15ng/L for methyl red; 0.05-0.5 and 0.5-15ng/L for sunset yellow) with correlation coefficients better than 0.9683. The limit of detection was 0.014ng/L for methyl red and 0.015ng/L for sunset yellow. Repeatability Relative Standard Deviation (RSD) with three replicated experiments was 1.5-10% for methyl red and 2.5-5.8% for sunset yellow. Relative recovery percentages of 88-96% for methyl red and 62-92% for sunset yellow were obtained in the samples. Moreover, the results have shown that acceptable accuracy, precision and linearity make the "fabric phase sorptive extraction" a proper method for the determination of dyes from industrial sewage samples.

Simultaneous Bulk‐Rock Elemental and Boron Isotope Ratio Measurement Using LA‐ICP‐MS on Silicate Micro‐Particulate Pellets

We have established a method for the simultaneous measurement of element abundances and boron isotopic compositions of bulk‐rock silicate samples by laser ablation coupled to two different ICP‐MS instruments. Fifteen silicate reference materials were prepared as micro‐particulate pressed powder pellets (MP) for analysis. Approximately 1/5th of the ablated material was transported to a single‐collector ICP‐MS for elemental measurement, while 4/5ths were sent to a multi‐collector ICP‐MS for boron isotope ratio measurement. Compositions of different milling materials (agate, zirconium oxide, tungsten carbide, sintered corundum and silicon nitride) were measured for the evaluation of potential contamination during sample preparation. Determined element mass fractions are in agreement with literature values within ±20%. For boron isotope ratios, the matrix‐dependent background elevation across the m/z from 9.9 to 11, and the effect of ablation mass was quantified. Measured δ11B values of most reference materials are consistent with literature values within ±1‰. The intermediate measurement precision is ±0.3‰ for JB‐2 with 30 μg g−1 B and ±0.4‰ for JA‐2 with 21 μg g−1 B. A publically available, web‐based streamlit app was developed for data processing (https://zenodo.org/doi/10.5281/zenodo.11150471). This paves the way for combined, accurate and precise elemental and boron isotope ratio measurements of geological materials.

Analysis of the utilization of inspectorate audit report provincial and regency/city inspectorates by BPK representative of North Sulawesi Province

This study aims to determine the implementation of the use of the Provincial and Regency/City Inspectorate Audit Report (LHP) by the BPK Representative of North Sulawesi Province in the period of 2021 to 2023, obstacles and efforts to overcome obstacles. This research uses a qualitative method. Data collection with unstructured observations, in-depth interviews, and documentation. The results of the study show that the LHP of the Regional Inspectorate has not fully made an active contribution to the preparation of the program, implementation, and control of audit activities by the BPK Representative of North Sulawesi Province. The LHP of the Regional Inspectorate has contributed when the BPK Audit Team carried out the audit task. The BPK Audit Team is active in requesting the Regional Inspectorate's LHP and the Regional Inspectorate's Annual Supervision Work Program (PKPT) during the implementation of the audit task used in examining problems and recommendations along with not continuing the Regional Inspectorate LHP. The results of the BPK Audit Team's study are used in the preparation of the business risk matrix, fraud risk matrix, sample selection strategy and preparation of additional/alternative procedures if necessary. The obstacle to the use of the Regional Inspectorate LHP for BPK is the lack of understanding of the obligation of the Regional Inspectorate to actively submit its LHP to the BPK, the lack of timely receipt of the Regional Inspectorate LHP by the BPK Audit Team so that an in-depth evaluation cannot be carried out, the scope of the Regional Inspectorate LHP object is less relevant to the needs of the Audit Team. The efforts that have been made by BPK are that the BPK Audit Team has made a written request for the list of LHPs along with the LHP of the Regional Inspectorate and the Badiklat PKN BPK RI has opened opportunities to improve the competence of Regional Inspectorate inspectors with training for Regional Inspectorate inspectors.

Standardizing Protein Corona Characterization in Nanomedicine: A Multicenter Study to Enhance Reproducibility and Data Homogeneity.

We recently revealed significant variability in protein corona characterization across various proteomics facilities, indicating that data sets are not comparable between independent studies. This heterogeneity mainly arises from differences in sample preparation protocols, mass spectrometry workflows, and raw data processing. To address this issue, we developed standardized protocols and unified sample preparation workflows, distributing uniform protein corona digests to several top-performing proteomics centers from our previous study. We also examined the influence of using similar mass spectrometry instruments on data homogeneity and standardized database search parameters and data processing workflows. Our findings reveal a remarkable stepwise improvement in protein corona data uniformity, increasing overlaps in protein identification from 11% to 40% across facilities using similar instruments and through a uniform database search. We identify the key parameters behind data heterogeneity and provide recommendations for designing experiments. Our findings should significantly advance the robustness of protein corona analysis for diagnostic and therapeutics applications.

RhizoMAP: a comprehensive, nondestructive, and sensitive platform for metabolic imaging of the rhizosphere

BackgroundElucidating the intricate structural organization and spatial gradients of biomolecular composition within the rhizosphere is critical to understanding important biogeochemical processes, which include the mechanisms of root-microbe interactions for maintaining sustainable plant ecosystem services. While various analytical methods have been developed to assess the spatial heterogeneity within the rhizosphere, a comprehensive view of the fine distribution of metabolites within the root-soil interface has remained a significant challenge. This is primarily due to the difficulty of maintaining the original spatial organization during sample preparation without compromising its molecular content.ResultsIn this study, we present a novel approach, RhizoMAP, in which the rhizosphere molecules are imprinted on selected polymer membranes and then spatially profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We enhanced the performance of RhizoMAP by combining the use of two thin (< 20 μm) membranes (polyester and polycarbonate) with distinct MALDI sample preparations. This optimization allowed us to gain insight into the distribution of over 500 different molecules within the rhizosphere of poplar (Populus trichocarpa) grown in rhizoboxes filled with mycorrhizae soil. These two membranes, coupled with three different sample preparation conditions, enabled us to capture the distribution of a wide variety of molecules that included phytohormones, amino acids, sugars, sugar glycosides, polycarboxylic acids components of the Krebs cycle, fatty acids, short aldehydes and ketones, terpenes, volatile organic compounds, fertilizers from the soil, and others. Their spatial distribution varies greatly, with some following root traces, others showing diffusion from roots, some associated with soil particles, and many having distinct hot spots along the plant root or surrounding soil. Moreover, we showed how RhizoMAP can be used to localize the origin of the molecules and molecular transformation during root growth. Finally, we demonstrated the power of RhizoMAP to capture molecular distributions of key metabolites throughout a 20 cm deep rhizosphere.ConclusionsRhizoMAP is a method that provides nondestructive, untargeted, broad, and sensitive metabolite imaging of root-associated molecules, exudates, and soil organic matter throughout the rhizosphere, as demonstrated in a lab-controlled native soil environment.

Development and validation of a multiresidue method for the determination of antibiotic residues in crawfish using LCMS/MS

ABSTRACT This study developed a rapid multi-class method for determining 29 antibiotic residues in crawfish using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A quick and affordable extraction method was employed, and sample preparation was performed using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique. The extraction process involved adding acetonitrile to crawfish samples. The sample was homogenised with a solution of sodium citrate buffer and sodium EDTA, followed by a cleanup step. The method was validated according to European Union (EU) guidelines 2002/657/EC. The limit of quantitation (LOQ) for all antibiotics was 5 µg/kg, except for flumequine and trimethoprim, which had LOQs of 2.5 µg/kg. The LOQ for the tetracycline group was 25 µg/kg, and for chloramphenicol, it was 0.015 µg/kg. The average recoveries at different concentration levels varied between 60–120%. The precision, expressed as repeatability and reproducibility, was calculated to be less than 16%. Thirty crawfish samples collected from local Egyptian market were monitored for antibiotic residues. The results revealed contamination with antibiotics belonging to three different groups. Of the thirty samples, 27% were contaminated with ciprofloxacin and enrofloxacin, 17% with chlortetracycline and oxytetracycline, 7% with oxolinic acid, and 3% with sulphamethazine. The applicability of the developed method was proven through successful proficiency testing. The developed method met performance criteria and is suitable for monitoring these antibiotics in crawfish.

Determination of metformin from micro-liter plasma and blood glucose test strip using eco-friendly micro-extraction procedures for point-of-care diagnostics

Metformin is commonly used in the treatment of type 2 diabetes and polycystic ovary syndrome. Beyond these common applications, it also holds promise in treating chronic pain, and as an antiaging and anticancer agent. In addition, it has cardiovascular protective and neuroprotective effects. However, elevated concentrations of metformin in the plasma, such as in patients with renal impairment or with conditions that can disrupt lactate clearance, could cause metformin-associated lactic acidosis. Moreover, metformin can cause a deficiency of vitamin-B12 and folic acid and impact one-carbon metabolism negatively. This study employs micro-extraction procedures to extract metformin from the human plasma and blood glucose test strips followed by detection using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Under the optimized micro-extraction procedures and by using an eco-friendly base and solvent, the proposed method required only 10 µL of the human plasma and a small piece of the blood glucose test strip for metformin analysis. The method exhibits a linear range of 0.05–5 μg mL−1 with a coefficient of determination (r2) of 0.999 and a limit of detection of 0.01 μg mL−1. The relative standard deviation (RSD) and relative error (RE) were both below 6.7 %. The test strip is a disposable product; its reuse before disposal could increase its additional applicability. Furthermore, major blood proteins and their modifications could be identified using this tiny volume of blood by mass spectrometry after protein digestion. The proposed technique could be invaluable in point-of-care diagnostics, enabling rapid monitoring of metformin plasma concentration and major plasma proteins to help patients mitigate the risk of metformin-associated lactic acidosis. To reduce organic waste in clinical applications, micro-extraction is a simple and efficient protocol for sample preparation using minute volumes of blood samples.

Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of Vitamin E in plasma: the effect of sample preparation and data processing

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both “bind and elute” and “interference removal” modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ- tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to – 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels, as a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standard (SIL-IS) was used for the compensation. The significant differences between individual concentration levels and compounds were observed, even if the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. For the selected example of vitamin E, solid-phase extraction was least affected by matrix effects when used in interference removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

Rapid detection of SARS-CoV-2 with a mobile device based on pulse controlled amplification

In the past, vast research has been conducted on biosensors and point-of-care (PoC) diagnostics. Despite rapid advances especially during the SARS-CoV-2 pandemic in this research field a low-cost molecular biosensor exhibiting the user-friendliness of a rapid antigen test, and also the sensitivity and specificity of a PCR test, has not been developed yet. To this end we developed a novel microfluidics based and handheld PoC device, that facilitates viral detection at PCR sensitivity and specificity in less than 40 min, including 15 min sample preparation. This was attained by incorporation of pulse controlled amplification (PCA), a method which uses short electrical pulses to rapidly increase the temperature of a small fraction of the sample volume. In this work, we present a low-cost PCA device with a microfluidic consumable intended for the use in a decentralized or home-setting. We used finite element analysis (FEA) simulations to display the fundamental principle and highlight the critical parameter dependency of PCA, such as pulse length and resistor shape. Furthermore, we integrated a simple and fast workflow for sample preparation and evaluated the limit of detection (LoD) for SARS-CoV-2 viral RNA, which is 0.88 copies/μL (=44 copies/reaction), and thus, comparable to conventional RT-qPCR. Additionally, target specificity of the device was validated. Our device and PCA approach enables cost-effective, rapid and mobile molecular diagnostics while remaining highly sensitive and specific.

Tricks and tips for trips.

Finding out about sample preparation and transportation of structural biology samples in Acta Crystallographica F, Structural Biology Communications.

Development of UV Method for Simultaneous Estimation of Imipramine and Ferulic Acid in Bulk and Developed Freeze-Dried Mixed Micelles

Introduction:: A simple, precise, and sensitive UV spectrophotometric method was developed to estimate Imipramine and Ferulic acid in bulk and polymeric micelles formulation. Moreover, imipramine and ferulic acid showed maximum absorbance at 237 nm and 216 nm. Method:: The method was validated for linearity, accuracy, precision, robustness, and ruggedness. The detector response for the imipramine and ferulic acid was linear over the selected range of 2 to 12 μg/ml with a correlation coefficient of 0.996 and 0.997. The accuracy was 99.4 and 101.02%. The precision (RSD) among six sample preparations was 0.68% and 0.85%. The method was validated as per the ICH guidelines. A polymeric micelle formulation was developed containing Vitamin E TPGS and F-127 as a surfactant and block co-polymer using different solvents. The optimized formulation containing 40 mg of F-127 and 30 mg of TPGS yielded the desired attributes of the optimized formulation. Result:: The optimized formulation was subjected to freeze-drying and yielded nanoparticulate size and excellent flowability. In vitro, the release of both drugs from the polymeric micelles was evaluated using dissolution, and multi-fold enhancement in release behavior was demonstrated compared to pure drugs. Both drugs were simultaneously detected successfully with accuracy and precision in bulk form and during in vitro analysis Conclusion:: The developed method can be adopted in routine analysis of imipramine and ferulic acid in bulk, and it involves relatively low-cost solvents with no complex extraction techniques.

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.

Scanorama: integrating large and diverse single-cell transcriptomic datasets.

Merging diverse single-cell RNA sequencing (scRNA-seq) data from numerous experiments, laboratories and technologies can uncover important biological insights. Nonetheless, integrating scRNA-seq data encounters special challenges when the datasets are composed of diverse cell type compositions. Scanorama offers a robust solution for improving the quality and interpretation of heterogeneous scRNA-seq data by effectively merging information from diverse sources. Scanorama is designed to address the technical variation introduced by differences in sample preparation, sequencing depth and experimental batches that can confound the analysis of multiple scRNA-seq datasets. Here we provide a detailed protocol for using Scanorama within a Scanpy-based single-cell analysis workflow coupled with Google Colaboratory, a cloud-based free Jupyter notebook environment service. The protocol involves Scanorama integration, a process that typically spans 0.5-3 h. Scanorama integration requires a basic understanding of cellular biology, transcriptomic technologies and bioinformatics. Our protocol and new Scanorama-Colaboratory resource should make scRNA-seq integration more widely accessible to researchers.

A High-throughput, Turbulent-mixing, Condensation Aerosol Concentrator for Direct Aerosol Collection as a Liquid Suspension

Trace measurement of aerosol chemical composition in workplace atmospheres requires the development of high-throughput aerosol collectors that are compact, hand-portable, and can be operated using personal pumps. We describe the design and characterization of a compact, high flow, Turbulent-mixing Condensation Aerosol-in-Liquid Concentrator (TCALC) that allows direct collection of aerosols as liquid suspensions, for off-line chemical, biological, or microscopy analysis. The TCALC unit, measuring approximately 12 × 16 × 18 cm, operates at an aerosol sample flowrate of up to 10 L min-1, using rapid mixing of a hot flow saturated with water vapor and a cold aerosol sample flow, thereby promoting condensational growth of aerosol particles. We investigated the effect of operating parameters such as vapor temperature, growth tube wall temperature, and aerosol sample flowrate, along with the effect of particle diameter, inlet humidity, aerosol concentration, and operation time on TCALC performance. Nanoparticles with an initial aerodynamic diameter ≥ 25 nm could grow to droplet diameters > 1400 nm with an efficiency ≥ 80%. Good droplet growth efficiency was achieved for sampled aerosol relative humidity ≥ 9%. We measured complete aerosol collection for concentrations of ≤ 3×105 cm-3. The results showed good agreement between the particulate mass collected through the liquid collector and direct filter collection. The TCALC eliminates the need for sample preparation and filter digestion during chemical analysis, thereby increasing sample recovery and substantially improving the limit of detection and sensitivity of off-line trace analysis of collected liquid samples.

Development of a fit-for-purpose multi-marker panel for early diagnosis of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) suffers from lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including short gradient (<10 min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

A quantitative method for the analysis of total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma with liquid chromatography-tandem mass spectrometry assay.

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC-MS/MS was performed within a run time of 7.5min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1-50 and 0.05-100 mg/l, respectively) and quadratic for other analytes (0.1-50 mg/l, except for ampicillin: 0.1-20 mg/l; R2 > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.

Small data reliability analysis in concrete three-point bending tests: A Weibull mixture model approach based on Weibull fracture theory

Due to the high costs and time-consuming nature of experiments, data from concrete three-point bending tests are usually limited. Additionally, the performance of concrete is influenced by various factors such as mix composition and sample preparation methods, leading to significant variability in test results. This poses a major challenge for the reliability analysis of small data from repeated tests. In reliability analysis, statistical models are particularly important as they allow for the prediction of performance and failure probabilities based on existing data. However, traditional statistical methods require a large number of similar samples for data processing and may need specific adjustments due to the diversity of practical applications. To address these issues, this study proposes a mixture model that combines domain knowledge of concrete with Weibull fracture theory and uses the Clustering-Circular Block Bootstrap method for sample augmentation. By analyzing real data obtained from concrete three-point bending tests, we accurately estimated the failure probability of concrete. Additionally, the study uses Gaussian process regression with a spectral mixture kernel function to fit the threshold fracture strength of concrete and estimate failure probabilities. The results show that the proposed method can accurately fit small datasets, with a RMSE of 0.320 and a MAPE of 0.100. The nominal bending strength range at a 5% failure probability is 2.0 MPa to 6.5 MPa, depending on the sample height and pre-crack length. Statistical learning demonstrates that the proposed method excels in fitting small datasets, providing a solid framework for predicting concrete strength and reliability.

SPOT-RASTR-A cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface.

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.