Improving Sample Preparation Research Articles

Improving sample preparation by biochar-coated sampling tubes: proof-of-concept extraction of sex hormones from real waters

This work showcases a novel application of biochar for analytical sample treatment. The carbonaceous material, obtained by pyrolysis of orange peel waste (550°C) without any post-synthesis treatment or functionalization, was thoroughly characterized and easily immobilized on the inner wall of sampling tubes in order to perform a sort of “in-vial” solid-phase extraction (SPE). The as-obtained device was tested for the extraction of seven sexual steroids, as probe water pollutants, from tap, lake, river water and wastewater treatment plant effluent samples spiked with 0.2-5 µg L−1 of each compound. The sorption kinetics profiles showed quantitative uptake from the sample (25 mL) in 20 min contact time, followed by complete elution in pure ethanol (2 mL, 15 min), thanks to the proper balance between sorption affinity and ease of elution. Under the selected conditions, recovery was in the range 60-123 %, with good inter-day precision (RSD 10-18 %, n=3). As evidence of the excellent reproducibility, an overall RSD below 15 % was observed from inter-day inter-batch recovery tests on three individually prepared sampling tubes. The procedure, carried out on a roller mixer, allows 10 samples to be extracted simultaneously, improving the sample throughput. Moreover, reusability tests showed that the same device maintains its efficiency for 10 consecutive sorption/desorption cycles. The greenness assessment, carried out by two dedicated software, further supported the sustainability of this biochar-based sample preparation as an alternative SPE.

A Comprehensive Review of Pesticide Residues in Peppers

Pesticides are chemicals that are used to control pests such as insects, fungi, and weeds. Pesticide residues can remain on crops after application. Peppers are popular and versatile foods that are valued for their flavor, nutrition, and medicinal properties. The consumption of raw or fresh peppers (bell and chili) can have important health benefits due to their high levels of vitamins, minerals, and antioxidants. Therefore, it is crucial to consider factors such as pesticide use and preparation methods to fully realize these benefits. Ensuring that the levels of pesticide residues in peppers are not harmful to human health requires rigorous and continuous monitoring. Several analytical methods, such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), infrared spectroscopy (IR), ultraviolet-visible spectroscopy (UV-Vis), and nuclear magnetic resonance spectroscopy (NMR), can detect and quantify pesticide residues in peppers. The choice of analytical method depends on the specific pesticide, that is being tested for and the type of sample being analyzed. The sample preparation method usually involves several processes. This includes extraction, which is used to separate the pesticides from the pepper matrix, and cleanup, which removes any interfering substances that could affect the accuracy of the analysis. Regulatory agencies or food safety organizations typically monitor pesticide residues in peppers by stipulating maximum residue limits (MRLs). Herein, we discuss various sample preparation, cleanup, and analytical techniques, as well as the dissipation patterns and application of monitoring strategies for analyzing pesticides in peppers to help safeguard against potential human health risks. From the authors' perspective, several challenges and limitations exist in the analytical approach to monitoring pesticide residues in peppers. These include the complexity of the matrix, the limited sensitivity of some analytical methods, cost and time, a lack of standard methods, and limited sample size. Furthermore, developing new analytical methods, using machine learning and artificial intelligence, promoting sustainable and organic growing practices, improving sample preparation methods, and increasing standardization could assist efficiently in analyzing pesticide residues in peppers.

Lateral flow test engineering and lessons learned from COVID-19

The acceptability and feasibility of large-scale testing with lateral flow tests (LFTs) for clinical and public health purposes has been demonstrated during the COVID-19 pandemic. LFTs can detect analytes in a variety of samples, providing a rapid read-out, which allows self-testing and decentralized diagnosis. In this Review, we examine the changing LFT landscape with a focus on lessons learned from COVID-19. We discuss the implications of LFTs for decentralized testing of infectious diseases, including diseases of epidemic potential, the ‘silent pandemic’ of antimicrobial resistance, and other acute and chronic infections. Bioengineering approaches will play a key part in increasing the sensitivity and specificity of LFTs, improving sample preparation, incorporating nucleic acid amplification and detection, and enabling multiplexing, digital connection and green manufacturing, with the aim of creating the next generation of high-accuracy, easy-to-use, affordable and digitally connected LFTs. We conclude with recommendations, including the building of a global network of LFT research and development hubs to facilitate and strengthen future diagnostic resilience. The feasibility of large-scale testing with lateral flow tests has been demonstrated in the COVID-19 pandemic. This Review examines lessons learned from the COVID-19 pandemic to inform the design and bioengineering of next-generation lateral flow tests to strengthen future diagnostic resilience.

Implementing and evaluating far-field 3D X-ray diffraction at the I12 JEEP beamline, Diamond Light Source.

Three-dimensional X-ray diffraction (3DXRD) is shown to be feasible at the I12 Joint Engineering, Environmental and Processing (JEEP) beamline of Diamond Light Source. As a demonstration, a microstructually simple low-carbon ferritic steel was studied in a highly textured and annealed state. A processing pipeline suited to this beamline was created, using software already established in the 3DXRD user community, enabling grain centre-of-mass positions, orientations and strain tensor elements to be determined. Orientations, with texture measurements independently validated from electron backscatter diffraction (EBSD) data, possessed a ∼0.1° uncertainty, comparable with other 3DXRD instruments. The spatial resolution was limited by the far-field detector pixel size; the average of the grain centre of mass position errors was determined as ±∼80 µm. An average per-grain error of ∼1 × 10-3 for the elastic strains was also measured; this could be reduced in future experiments by improving sample preparation, geometry calibration, data collection and analysis techniques. Application of 3DXRD onto I12 shows great potential, where its implementation is highly desirable due to the flexible, open architecture of the beamline. User-owned or designed sample environments can be used, thus 3DXRD could be applied to previously unexplored scientific areas.

Embryonic Toxicity Test of Medical Devices for Human Assisted Reproductive Technology

To introduce the test methods of embryo toxicity applied to medical devices for human assisted reproductive technology (ARTMD), and provide the evaluation reference. The embryo toxicity test methods of ARTMD were summarized, and the key procedures and challenges in their safety evaluation were also discussed. Establishing sensitive and stable test system is important to guarantee the safety and efficacy of ARTMD. It remains development opportunities in improving sample preparation, extending test technology and expending evaluation method.

Utilizing laser scribing for graphene ablation

We present the investigation of graphene ablation by laser scribing from a LightScribe DVD. In laser scribed graphene (LSG) samples, laser scribed holes (LSHs) were observed as dark features filled with residuals from copper etching. The LSHs tend to form a series of discontinuous lines aligning to laser scribed traces on the disk. The calculated fluence of laser scribing was about 93 and 124 mJ/cm2 for a pulse train and a single pulse, respectively. These fluences are in the range of fluence threshold of graphene ablation by femtosecond laser and are much higher than that of cw laser. From characterizations and electrical measurements, evidence of extended defects in graphene from LSHs in LSG was observed. From testing of graphene-based gas sensors, we can see that the response of LSG to ethanol is almost 100 times higher than that of graphene. This work provides a low-cost, facile method of graphene ablation by utilizing laser scribing. By improving sample preparation, our method could be applied to patterning graphene into microstrips, which have applications in novel devices such as THz sources and gas sensors.

Agile fusion method for the determination of Pu isotopes in diverse sediments

Atmospheric nuclear testing has occurred worldwide resulting in trace amounts of plutonium (Pu) isotopes in soils and sediments across the globe. Soils and sediments can vary greatly in both physical and chemical properties within close geographic regions presenting a major challenge for sample preparation in the analysis of fallout radionuclides. It is vital to have a rapid, agile method for the complete digestion of samples containing refractory Pu particles to address this challenge. A radioanalytical procedure for the analysis of Pu isotopes in soils/sediments has been developed improving sample preparation using fusion. This is done by eliminating the need for pre-dissolution combustion to remove organic material and providing greater application to a diverse range of samples including those of metal-bearing mineralogy. The integration of borate and peroxide flux materials creates a sample dissolution procedure that is greater than the sum of its parts. This method is applicable to the analysis of 239/240Pu in soil/sediment samples with a sample mass of 3 g, ranging from 0.05 to 5000 mBq/g achieving 242Pu tracer recoveries between 50 and 94%, with a trace-level detection limit of 0.04 mBq/g and measurement uncertainties down to 19%; adjustable to measurement priorities with variation in count time, background counts, chemical recovery and total activity of the sample. This sample preparation technique can be utilized with complementary analysis of alpha spectrometry or ICP-MS. Achieving these parameters provides a highly adaptable standard method for 239/240Pu in soil/sediment analysis applicable to samples from radioecological research at trace environmental levels to emergency response scenarios with 48-h turn-around producing highly repeatable, succinct and high-quality analysis.

A Universal and High-Throughput Proteomics Sample Preparation Platform.

Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ∼300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ∼4,000 proteins for each sample in a single high-performance liquid chromatography (HPLC)-tandem mass spectrometry injection with only 100-10K cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with two heavy labeled internal standard peptides spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.

Improved Sample Preparation and Optimized Solvent Extraction for Quantitation of Carotenoids.

Accurate, rapid quantitation of key antioxidants such as carotenoids is important for assessment of food quality. Carotenoids are lipid-soluble pigments that are susceptible to oxidation due to their highly conjugated carbon-carbon double bonds. Therefore, the present work focuses on improving sample preparation to facilitate rapid analysis of carotenoids. The method involves optimized carotenoid extraction followed by direct HPLC analysis without further concentration and redissolution. For extraction, we tested the effect of blending time (1, 3 and 5min) and 12 different solvent combinations for carotenoid extraction from cantaloupe (Cucumis melo var. cantalupensis) and oranges (Citrus sinensis), two popular fruits that are high in carotenoids. The identification of carotenoids was performed by LC-APCI-QTOF-HR-MS in positive-ionization mode. In melon, 1min blending time gave significantly higher β-carotene content with CHCl3: Ace (1:1) solvent. The optimized method was validated with tomato, watermelon, oranges, grapefruit, melon varieties and commercial products such as fruit juices. Among the different melon varieties, Western Shipper had significantly higher β-carotene (25.1 ± 0.4µg/g) contents. In oranges, β-carotene and (all-E)-lycopene contents were 4.4 ± 0.1and 3.8 ± 0.1µg/g, respectively. The optimized method has fewer unit operations and is reproducible for the quantitation of carotenoids and their isomers. This is the first report on the identification of ζ-carotene isomers, and lycopene isomers from cantaloupe varieties and lycopene from oranges. Graphical Abstract.

Improving sample preparation to investigate lignin intensity of xylem at the cellular level by confocal Raman microspectroscopy of Populus tomentosa

Confocal Raman microspectroscopy (CRM) is an important tool for analyzing the compositional distribution of cell walls in situ. In this study, we improved the sample preparation method using paraffin-embedded sections combined with hexane dewaxing to obtain high resolution Raman images. We determined that the cell wall components of fiber cells were different from those of ray cells and vessel cells in the xylem of Populus tomentosa. Acetyl bromide and CRM methods produced similar trends when the difference in lignin intensity in the xylem region was compared between transgenic PtrLac4 and wild-type P. tomentosa. However, CRM proved more useful to analyze the lignin distribution in each cell type and distinguished the detailed difference in lignin intensity at the cellular level. Thus, CRM proved to be a useful in situ method to rapidly analyze the spatial variation of lignin content in the xylem of woody plants.

Abstract 6197: Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors

Abstract Single cell multiomic (scM) methods are powerful tools to characterize patient-specific tumor-immune microenvironment, but current methods of sample handling and preparation can introduce bias. Obtaining non-biased results is particularly important in studies to identify new biomarkers for patient stratification of responders versus non-responders for immunotherapies in oncology. This problem is becoming acute as the number of immune-therapy modalities and combinations in clinical trials and in medical practice continue to expand. Current methodologies used to analyze patient specimens in routine pathology are not adequate to address heterogeneity and complexity of immune cell ecosystem of individuals. SCM analysis of immune cells from tumor microenvironment using technologies that allow high parameter analysis of genes and proteins will allow a comprehensive atlas of immune cells in the tumor tissue, however the quality of single cell sample preparation is critical. Here, we report evaluation of several available solid tumor dissociation methods, including mechanical dissociation by scalpel, BD™ Medimachine System, Miltenyi gentleMACS™ dissociator. Mechanical dissociation was followed by enzymatic dissociation using BD Horizon™ Dri Tumor & Tissue Dissociation Reagent or, in the case of gentleMACS™, the Miltenyi Tumor Dissociation Kit. Metrics evaluated were cell recovery, viability, and proportion of various cell lineage populations using flow cytometry; and expression of 399 mRNA transcripts and 31 proteins using single cell multiomics (BD Rhapsody™, BD Rhapsody™ Immune Response Targeted Panel, and BD™ AbSeq). Tumor tissues were from a mouse a pancreatic (PDAC) tumor model and humanized Patient-Derived Xenograft (PDX; from Onco-hu® mice) models of colon and lung cancer. Method dependent differences in viable cell yield and proportions of immune vs. tumor cells were observed. SCM analysis of FACS sorted CD45+ tumor-infiltrating lymphocytes (TILs) showed differences in gene and protein expression profiles, particularly for expression of rare vs. abundant genes depending on the dissociation method. Our data identify protocols to interrogate tumor immune microenvironment starting from tumor tissue specimen through scM all the way to informatics analysis with consistent results. Similar studies and further optimization are recommended for other tumor types. Consistent adoption of standardized protocols will enable robust findings in research and clinical trials, with the ultimate goal of improved therapies, biomarkers and outcomes. For Research Use Only. Not for use in diagnostic or therapeutic procedures. GentleMACS™ is trademark of Miltenyi Biotec. Onco-hu® is a registered trademark of The Jackson Laboratory. BD, the BD Logo, Horizon, Medimachine, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Aaron J. Middlebrook, Wei Huang, Margaret Nakamoto, Xiaoshan Shi, Evelyn Lo, Patrick Neuhoefer, Scott Bornheimer, Smita Ghanekar. Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6197.

Reducing variability in the measurement of gluten contamination in oats, oilseeds, and pulses by improving sample preparation

Reducing variability in the measurement of gluten contamination in oats, oilseeds, and pulses by improving sample preparation

Effect of sample preparation procedure for the determination of As, Sb and Se in fruit juices by HG-ICP-OES

Various sample preparation procedures for the simultaneous determination of As, Sb and Se in fruit juices by hydride generation inductively coupled plasma optical emission spectrometry (HG-ICP-OES) were examined. Applicability of total wet digestion with HNO3/H2O2, partial decomposition (solubilisation in aqua regia), 1:1 dilution with 2% (v/v) HNO3 and direct analysis were evaluated. Hydrides were generated in the reaction of an acidified sample with NaBH4 after pre-reduction with KI–ascorbic acid for total As and Sb, and boiling with HCl for total Se. Best results, i.e. limits of detection (LODs) of 0.51–0.73ngmL−1, precision (RSD) within 1.7–3.6% and recoveries for spiked samples between 101% and 106% were found using aqua regia treatment. This procedure simplifying and improving sample preparation step prior to As, Sb and Se measurements in fruit juices by HG-ICP-OES, thus could be adequate for the routine analysis in terms of the quality control of these drinks.

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.

Nanotechnologies for pathogen detection: Future alternatives?

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies. Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.

Preliminary study on two-dimensional gel electrophoresis of human pancreatic juice for proteome analysis

Objective To establish an optimal method of two-dimensional gel electrophoresis (2-DE) for proteomic analysis of human pancreatic juice (pj) on the basis of former work and explore the feasibility of differential proteomic analysis of human pancreatic juice by the use of mixed pancreatic juice samples. Methods Pj samples from 5 pancreatic cancer patients (two cases were pathologically diagnosed moderately differentiated adenocarcinoma and intraduetal papilloma canceration respectively,three cases were diagnosed by inageology and clinical manifestations) and 6 chronic pancreatitis pa-tients were included in this study. Pj samples were collected by naso-pancreatic drainage during endo-scopic retrograde cholangiopancreatography. 2-DE was conducted on one pj sample of chronic panerea-titis by improving sample preparation techniques. Compositions of rehydration solutions and methods of isoelectric focusing and 2-DE maps were compared with those got from former 2-DE method. 2-DE method and 2-DE maps of single pj sample and mixed pj samples of the same pancreatic disease were also compared by the optimized method of 2-DE. Results With the improved 2-DE method and when 200 μg protein of pancreatic juice was loaded, about 280 protein spots could be successfully separated with better differentiation rate as compared with the former method. There was higher similarity (>75%)between the 2-DE maps of single pj sample and mixed pj samples of the same pancreatic disease. Conclusion Optimized method of 2-DE has reliable effect and the comparison of 2-DE maps of mixed pj samples of different pancreatic diseases would become the basis of differential proteomic analysis of human pancreatic juice. Key words: Human; Pancreatic juiee; Proteomics; Two-dimensional gel electrophoresis

Improving sample preparation of gold-containing ores of the second and third groups for assay analysis

A procedure is proposed for the sample preparation of gold-containing ores of the second and third groups for assay analysis; it allows for an improvement in the accuracy of the results of analysis.

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Comparison of Precision XTEM Specimen Preparation Techniques for Semiconductor Failure Analysis

Abstract The demand for TEM analysis in semiconductor failure analysis is rising sharply due to the shrinking size of devices. A well-prepared sample is a necessity for getting meaningful results. In the past decades, a significant amount of effort has been invested in improving sample preparation techniques for TEM specimens, especially precision cross-sectioning techniques. The most common methods of preparation are mechanical dimpling & ion milling, focused ion beam milling (FIBXTEM), and wedge mechanical polishing. Each precision XTEM technique has important advantages and limitations that must be considered for each sample. The concept for both dimpling & ion milling and wedge specimen preparation techniques is similar. Both techniques utilize mechanical polishing to remove the majority of the unwanted material, followed by ion milling to assist in final polishing or cleaning. Dimpling & ion milling produces the highest quality samples and is a relatively easy technique to master.