Biological Sample Preparation Research Articles

Comparing Free Radicals in Sunscreen-Treated Pig Skin by Using Electron Paramagnetic Resonance Spectroscopy

Melanoma, a type of cancer that develops in melanocytes, is usually caused by direct exposure of skin to ultraviolet (UV) radiation resulting in cellular damage. In this study, a procedure to determine the effects of various commercial sunscreens with SPF values ranging from 15 to 100 was developed using pig skin to mimic human skin. These sunscreens contain inorganic filters, such as zinc oxide and titanium dioxide; active organic ingredients, such as octocrylene and oxybenzone; or both. As a model for human skin, pig skin was analyzed before and after UV exposure, and the presence of free radicals was measured using electron paramagnetic resonance (EPR) spectroscopy. Using this method, students were able to quantify radical formation following irradiation and use this as a basis to compare the efficacies of sunscreens against UVA. This experiment allowed undergraduate students to characterize a complex chemical process (light-induced radical formation) and relate it to something they experience every day (sun damage). Interestingly, students found higher levels of postillumination radical formation in sunscreen-treated samples, perhaps indicating sunscreen-induced stabilization of these species. Student outcomes included learning how to collect and interpret EPR data, statistical analysis of these data, and the preparation of reproducible biological samples. Students also consulted literature sources to properly display their measurements.

Recent Trends in Solid-Phase Extraction for Environmental, Food and Biological Sample Preparation

Recent Trends in Solid-Phase Extraction for Environmental, Food and Biological Sample Preparation

Direct genotyping from whole blood using alkaline polyethylene glycol

In order to simplify biological sample preparation to meet the demand of rapid genotyping, we improve alkaline polyethylene glycol (APEG) based on Chomczynski's procedure for the PCR-based lateral flow genotyping system, which enables the rapid and efficient direct genotyping from whole blood without DNA purification. The improved APEG has a high tolerance for extreme storage conditions. Testing whole blood with an abnormal hematological index indicates that APEG efficiency would not be influenced by pathological factors. Compared with sequencing, the accuracy of this genotyping system was 100% on testing with 200 clinical samples.

An LC-MS/MS Method to Measure S-Methyl-l-Cysteine and S-Methyl-l-Cysteine Sulfoxide in Human Specimens Using Isotope Labelled Internal Standards.

This is the first report describing an analytical method for quantitative analysis of two naturally occurring sulphur compounds, S-methyl-l-cysteine (SMC) and S-methyl-l-cysteine sulfoxide (SMCSO), in human body fluids using isotope-labelled internal standards and liquid chromatography-mass spectrometry (LC-MS)/MS techniques. This method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. It offers significant advantages including simple and fast preparation of human biological samples. The limits of detection of SMC were 0.08 µM for urine and 0.04 µM for plasma. The limits of detection of SMCSO were 0.03 µM for urine and 0.02 µM for plasma. The calibration curves of all matrices showed linearity with correlation coefficients r2 > 0.9987. The intra and inter day precisions in three levels of known concentrations were >10% and >20%, respectively. The quantification accuracy was 98.28 ± 5.66%. The proposed method would be beneficial for the rapid and accurate determination of the SMC and SMCSO in human plasma and urine samples using by isotope labelled internal standards.

The evaluation of short- and long-term stability studies for brimonidine in aqueous humor by DPV/BDDE method-possible application for direct assay in native samples.

A novel voltammetric method was developed for brimonidine (BRIM) determination in deproteinized aqueous humor, simplifying preparation of biological samples for analysis for stability studies. The differential pulse voltammetric (DPV) method using boron doped diamond electrode (BDDE), based on characteristic oxidation peaks, was proposed and successfully applied. The linearity range was within 5.0 × 10-6 to 5.0 × 10-5M of brimonidine, and limit of detection and limit of quantitation were 1.94 × 10-6M and 6.46 × 10-6M, respectively. Intra-day and inter-day precision and accuracy were evaluated and all results were in accordance with validation ICH guidelines. The best short-term stability study results were obtained for a concentration level of 3.0 × 10-5M expressed by deviation of + 1.86% between initial and post storage concentrations. A long-term stability study was performed for two concentrations of 3.0 × 10-5M and 5.0 × 10-5M and resulted in deviations of + 1.63% and + 3.56%, respectively. A freeze and thaw stability study indicated that samples might be frozen only once. The enhancement of DPV/BDDE method sensitivity gained by modification, for the analysis of immeasurable BRIM quantities in native, untreated aqueous humor, was reached for quantities of 6 or 12nmol/0.1mL aqueous humor with acceptable accuracy (up to + 7.5%). The nature of the process-the irreversible one electron oxidation voltammetric peak of BRIM-limited the sensitivity. Only electrochemical pre-treatment of the BDD electrode before each measurement significantly speeded up the whole procedure. The advantages of the proposed method are simplicity, short-time performance, and good specificity/selectivity, as well as satisfactory accuracy, and no chemical modification of BDDE was necessary.

A rapid method of whole cell sample preparation for scanning electron microscopy using neodymium chloride

The quality of electron microscopy (EM) visualization of biological objects is constantly improving, primarily with the usage of more complex technologies, such as serial block-face scanning electron microscopy (SEM), focused ion beam scanning electron microscopy, and array tomography. Here we suggest a new rapid method of whole cell sample preparation for scanning EM using neodymium chloride treatment followed by staining with lead acetate. This variant of sample preparation does not require separate fixation, complete dehydration, and metal sputtering. By means of SEM in the back-scattered electron mode, in the neodymium-treated preparations, we visualized various morphological structures in human cells (nuclei with nucleoli, cytoskeleton, mitochondria) and microbial cells (Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli, and Candida albicans) preserving their species-specific shape and size. Thus, the suggested method provides additional information combining capabilities of SEM in visualizing cellular surface and transmission EM in detecting intracellular structures. Moreover, biological sample preparation with neodymium and lead is fast, informative, and cost-saving indicating a potential for its practical use for environmental SEM, and can be effectively combined with optical microscopy.

Solvent front position extraction procedure for preparation of biological samples with coccidiostats for liquid chromatography-tandem mass spectrometry determination

Summary Coccidiostats are a group of drugs used for the treatment of coccidiosis. This disease is common among animals for slaughter. Residues of the mentioned drugs can be potentially harmful for ...

Divide and conquer: A perspective on biochips for single-cell and rare-molecule analysis by next-generation sequencing

Recent advances in biochip technologies that connect next-generation sequencing (NGS) to real-world problems have facilitated breakthroughs in science and medicine. Because biochip technologies are themselves used in sequencing technologies, the main strengths of biochips lie in their scalability and throughput. Through the advantages of biochips, NGS has facilitated groundbreaking scientific discoveries and technical breakthroughs in medicine. However, all current NGS platforms require nucleic acids to be prepared in a certain range of concentrations, making it difficult to analyze biological systems of interest. In particular, many of the most interesting questions in biology and medicine, including single-cell and rare-molecule analysis, require strategic preparation of biological samples in order to be answered. Answering these questions is important because each cell is different and exists in a complex biological system. Therefore, biochip platforms for single-cell or rare-molecule analyses by NGS, which allow convenient preparation of nucleic acids from biological systems, have been developed. Utilizing the advantages of miniaturizing reaction volumes of biological samples, biochip technologies have been applied to diverse fields, from single-cell analysis to liquid biopsy. From this perspective, here, we first review current state-of-the-art biochip technologies, divided into two broad categories: microfluidic- and micromanipulation-based methods. Then, we provide insights into how future biochip systems will aid some of the most important biological and medical applications that require NGS. Based on current and future biochip technologies, we envision that NGS will come ever closer to solving more real-world scientific and medical problems.

Validation of a new magnetic bead-based biological sample preparation kits for urinary cortisol and comparison with a home method used routinely in the lab

In this study, a fully automated system was developed for the determination of more than 300 different pesticides from various food commodities. The samples were extracted with acetonitrile prior to the injection into the two-dimensional LC-system. No manual clean-up was needed. The separation of analytes and matrix compounds was carried out by a YMC-Pack Diol (2.1 mm × 100 mm; 5 μm; 120 Å) HILIC column in the first dimension. All analytes eluted within one small fraction at the beginning of the run. With a packed loop interface this fraction was transferred to the analytical reversed phase separation performed on an Agilent Poroshell 120 EC-C18 (2.1 mm × 100 mm; 2.7 μm; 120 Å). Some very polar compounds with a stronger retention on the HILIC column were measured directly. The method was validated for over 300 pesticides in cucumber, lemon, wheat flour, rocket, and black tea. For the large majority of the analytes, the recovery was between 70% and 120% and the relative standard deviation was clearly under 20%. The limits of detection for nearly all the compounds were at least at 0.01 mg/kg. For over 50% of the analytes, good sensitivity was observed even at 0.001 mg/kg. In spite of the injection of a pure sample extract, the method showed robust results even with dirty matrices like hops and tea.

Evaluation of a new magnetic bead-based biological sample preparation kits for vitamin D analysis by LC-MS/MS

Evaluation of a new magnetic bead-based biological sample preparation kits for vitamin D analysis by LC-MS/MS

Evaluation of a new magnetic bead-based biological sample preparation kits for a panel of 11 steroids determined by LC-MS/MS

Evaluation of a new magnetic bead-based biological sample preparation kits for a panel of 11 steroids determined by LC-MS/MS

Quantitative STEM imaging of electron beam induced mass loss of epoxy resin sections

In sample preparation of biological samples for electron microscopy, many types of embedding media are widely used. Unfortunately, none of them is perfectly resistant to beam induced damage. The article is focused on mass loss measuring of pure epoxy resin EMbed 812 that replaced Epon – the most widely used embedding resin for biological electron microscopy, in a form of ultrathin sections with thicknesses ranging from 30 to 100 nm. The STEM imaging was performed in a quantitative way which allowed us to estimate the mass loss directly up to the total dose of 3000 e−/nm2. For data acquisition we used SEM equipped with a commercial STEM detector working at a relatively low acceleration voltage of 30 kV. In this study we estimated the influence of various factors which can affect the endurance of the epoxy resin EMbed 812 ultrathin sections under an electron beam, such as the sample aging, differences between storing the samples in forms of ultrathin sections and whole blocks, ultrathin sections thicknesses, temperature of the sample, probe current, and one or two-sided carbon coating of ultrathin sections. The aim of this work is to investigate beam induced mass loss at electron energies of SEM and find out how to reduce the mass loss.

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Assessing tritium internalisation in zebrafish early life stages: Importance of rapid isotopic exchange

Tritium (3H) is mainly released into the environment in the form of tritiated water (HTO) by nuclear power plants and nuclear fuel reprocessing plants. To better understand how organisms may be affected by contamination to 3H it is essential to link observed effects to a correct estimation of absorbed dose rates. Due to quick isotopic exchanges between 3H and hydrogen, 3H measurement is difficult in small organisms such as zebrafish embryo, a model in ecotoxicological assay. This work aimed to optimise tritium measurement protocol to better characterise internalisation by early life stages of zebrafish. Zebrafish eggs were exposed at one HTO activity concentration of 1.22 × 105 Bq/mL. This activity was calculated to correspond to theoretical dose rates of 0.4 mGy/h, where some deleterious effects are expected on young fish. A protocol for the preparation of biological samples was adapted from the method classically used to segregate the different forms of tritium in organisms. To deal with very quick isotopic exchanges of 3H with hydrogen, the impacts of washing by non-tritiated water as well as the bias induced by absorbed tritium around organisms on the measured activity concentration were studied. We managed to develop protocols to perform total tritium and total organically bound tritium (OBT) activity concentrations measurements in zebrafish eggs and larvae. The measurement of these both forms allowed the calculation of tissue-free-water-tritium (TFWT). To better understand total tritium internalisation, a study of total tritium kinetics from 4 hpf (hour post-fertilization) to 168 hpf was performed. OBT and TFWT were also assessed to complete the total tritium internalisation kinetics. The internalisation is a rapid phenomenon reaching a steady-state within 24 h after the beginning of contamination for total tritium and TFWT, with concentration factors and TFWT/HTO close to unity. OBT formation seemed to be slower. It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf). To verify absorbed dose rates at key developmental stages (24 hpf eggs and 96 hpf larvae), they were calculated from total tritium activity concentrations after exposure at 1.22 × 105 and 1.22 × 106 Bq/mL, as these two activity concentrations were used to assess effects of tritium in another part of the study. Dose rates calculated from total tritium activity concentrations measured in 24 hpf eggs and 96 hpf larvae were consistent with the nominal ones, which validates the robustness of the protocol developed in the present study.

Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures

The surface-enhanced Raman spectroscopy (SERS)-based analysis of bacteria suffers from the lack of a standard SERS detection protocol (type of substrates, excitation frequencies, and sampling methodologies) that could be employed throughout laboratories to produce repeatable and valuable spectral information. In this work, we have examined several factors influencing the spectrum and signal enhancement during SERS studies conducted on both Gram-negative and Gram-positive bacterial species: Escherichia coli and Bacillus subtilis, respectively. These factors can be grouped into those which are related to the structure and types of plasmonic systems used during SERS measurements and those that are associated with the culturing conditions, types of culture media, and method of biological sample preparation.

Dimethyl 3,3′-dithiobispropionimidate (DTBP) as a cleavable disulfide-based polymer to encapsulate nucleic acids in biological sample preparation

Cleavable disulfide bonds are extremely valuable functional groups for numerous biological applications, including applications as antitumor treatments, gene delivery, and diagnosis. Herein, we demonstrate the use of dimethyl 3,3′-dithiobispropionimidate (DTBP) as a disulfide cleavable bond based cross-linker in nano-composites that encapsulate and release nucleic acids in sample preparation systems for disease diagnoses. We conjugated DTBP with nano-composites, such as amine group-modified diatomaceous earth (DA), which is a solid substrate, and a zinc oxide nanomultigonal shuttle (ZnO NMS) that disrupts cell membranes without chaotropic detergents and improved the quantity and purity of nucleic acids from various biological samples. After encapsulating nucleic acids, the cleavable disulfide bond of DTBP was used to release nucleic acids by breaking disulfide bonds using dithiothreitol (DTT), resulting in highly enhanced efficiency of downstream analyses without PCR inhibitors such as high salt concentrations and SDS. Therefore, nano-composites with cleavable disulfide linkages have potential for various biological applications, including disease diagnostics and therapies.

A microfluidic design for desalination and selective removal and addition of components in biosamples.

We present the development of a microfluidic device that is able to selectively and nondisturbingly remove or add components to liquid samples, which allows control and conditioning of the samples for biomedical tests. The device consists of a series of chambers for sample retention and a through channel. Because smaller particles diffuse faster, small particles in the sample such as salt ions rapidly escape the chamber by diffusion and are subsequently removed by a carrier flow in the channel, leaving macromolecules of interest in the "desalted" solution. Conversely, components lacking in the sample can be diffused in by reversing the concentration gradient between the flow and the sample chamber. The ability to control the ionic strength of a sample offers many advantages in biological sample preparation as most biofluids contain high salt contents, making them unsuitable for downstream molecular analyses without additional sample treatments which could cause sample loss, contamination, and cost increase. Making use of the nature of laminar flow in a microfluidic device and mass transport by diffusion, we have developed an analytical model to calculate concentration profiles for different particles. Excellent agreements were found between the theory and the experiment, making the results highly reliable and predictable. Since the device and the principle is applicable to a wide range of biological samples, it can be incorporated into the workflow of various applications for research and in vitro diagnosis such as ion exchange, DNA sequencing, immuno assay, vesicle, cell secretion analysis, etc.

3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3'-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and lab-made mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2'-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

Development and validation of HPLC/UV-procedures of secnidazole determination in blood and urine

Aim. The system of HPLC-analyzer MiLiChrome® A-02 is widely used in Ukrainian laboratories of forensic toxicology. The purpose is to apply the HPLC-analyzer system for secnidazole quantitative determination in biological liquids and carry out validation of the developed procedures.Methods. Sample preparation of blood and urine was carried out in three ways – 1) liquid-liquid extraction with organic solvents immiscible with water, 2) amphiphylic solvents extraction and salting-out with ammonium sulphate, 3) complex application of liquid-liquid extraction with organic solvents immiscible with water and amphiphylic solvents extraction with salting-out. Chromatographic conditions: column – AE2´75 mm, ProntoSIL 120-5-C18 AQ, 5 μm; temperature – 40°С; flow rate – 100 μl/min; Eluent A – 0.2 M LiClO4 – 0.005 M HClO4; Eluent B – acetonitrile; elution mode – linear gradient; detection – UV, 277 nm; volume of injection – 2 μl.Results. Validation of all developed procedures has been carried out by such parameters as specificity, recovery, linearity, accuracy and precision in the variant of the method of standard. The results of analysis have shown the absence of peaks with the retention time, which is coincident with the secnidazole retention time, on the chromatograms of blank-samples for all variants of procedures of analyte isolation. All procedures of sample preparation show the high efficiency of secnidazole isolation both for blood and urine (at the level of 90 %). All examined procedures are characterized by the acceptable parameters of linearity, within-run and between-run accuracy and precision.Conclusions. The set of HPLC-procedures of secnidazole quantitative determination in blood and urine has been developed. Validation of the developed procedures has been carried out; isopropanol application in the acid medium is optimal for biological liquids sample preparation