Analysis Of Biological Samples Research Articles

Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3,5-Dinitrosalicylic Acid Assay.

Use of the 3,5-dinitrosalicylic acid reagent allows the simple, rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in characterization of enzyme reactions, as new reducing ends are generated when a polysaccharide substrate undergoes hydrolytic cleavage. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance versus sugar concentration.

Consecutive baicalin treatment relieves its accumulation in rats with intrahepatic cholestasis by increasing MRP2 expression

Baicalin, an important flavonoid isolated from Scutellaria baicalensis Georgi, is a Chinese herb widely used in clinical practice. We previously reported the in vivo accumulation of baicalin in rats with intrahepatic cholestasis (IHC) after a single dose. However, the effects of the long-term administration of baicalin on its pharmacokinetics are unknown. Thus, we investigated the disposition of baicalin in normal rats and those with IHC after single and multiple consecutive administrations. In addition, we further investigated the effect of baicalin on multidrug resistance protein 2 (MRP2) in vivo to explore the underlying mechanism. In our study, the liquid chromatography-mass spectrometry (LC-MS) method established to determine baicalin concentrations in rat blood was simple, specific, and with linearity (R2 = 0.9980) in the range of 1.01–506.00 μg/mL. The relative standard deviations (RSD) for intra-day and inter-day precision were not more than 10.55%, and the intra-day and inter-day accuracies were 94.94%–109.13%. The recovery rate and stability were in line with the requirements of the quantitative analysis of biological samples as stated in the Chinese Pharmacopoeia (2020 Edition). Compared with that in normal rats, the Cmax and t1/2 increased significantly in EE-induced rats with IHC, whereas the clearance (CL) decreased after a single administration of baicalin. However, the area under the curve decreased, CL increased, and the t1/2 was shortened after the continuous administration of baicalin in the IHC rat model compared with the single administration of baicalin, and the pharmacokinetic characteristics were similar to those in normal rats. Moreover, MRP2 expression increased in rats with IHC with the continuous administration of baicalin. Continuous baicalin intervention could effectively reduce its accumulation in rats with IHC, and the mechanism may be attributed to its enhancement of MRP2 expression.

Metasurface-enhanced infrared spectroscopy in multiwell format for real-time assaying of live cells.

Fourier transform infrared (FTIR) spectroscopy is a popular technique for the analysis of biological samples, yet its application in characterizing live cells is limited due to the strong attenuation of mid-IR light in water. Special thin flow cells and attenuated total reflection (ATR) FTIR spectroscopy have been used to mitigate this problem, but these techniques are difficult to integrate into a standard cell culture workflow. In this work, we demonstrate that the use of a plasmonic metasurface fabricated on planar substrates and the probing of cellular IR spectra through metasurface-enhanced infrared spectroscopy (MEIRS) can be an effective technique to characterize the IR spectra of live cells in a high-throughput manner. Cells are cultured on metasurfaces integrated with multiwell cell culture chambers and are probed from the bottom using an inverted FTIR micro-spectrometer. To demonstrate the use of MEIRS as a cellular assay, cellular adhesion on metasurfaces with different surface coatings and cellular response to the activation of the protease-activated receptor (PAR) signaling pathway were characterized through the changes in cellular IR spectra.

X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Biobanking as a Tool for Genomic Research: From Allele Frequencies to Cross-Ancestry Association Studies.

In recent years, great advances have been made in the field of collection, storage, and analysis of biological samples. Large collections of samples, biobanks, have been established in many countries. Biobanks typically collect large amounts of biological samples and associated clinical information; the largest collections include over a million samples. In this review, we summarize the main directions in which biobanks aid medical genetics and genomic research, from providing reference allele frequency information to allowing large-scale cross-ancestry meta-analyses. The largest biobanks greatly vary in the size of the collection, and the amount of available phenotype and genotype data. Nevertheless, all of them are extensively used in genomics, providing a rich resource for genome-wide association analysis, genetic epidemiology, and statistical research into the structure, function, and evolution of the human genome. Recently, multiple research efforts were based on trans-biobank data integration, which increases sample size and allows for the identification of robust genetic associations. We provide prominent examples of such data integration and discuss important caveats which have to be taken into account in trans-biobank research.

All-in-One Preparation Strategy Integrated in a Miniaturized Device for Fast Analyses of Biomarkers in Biofluids by Surface Enhanced Raman Scattering.

Complex and tedious sample preparation processes have greatly limited rapid analyses of biological samples. In this work, an all-in-one sample preparation strategy based on a miniaturized gas membrane separation/oven ring enrichment (GMS/ORE) device was developed for efficient surface enhanced Raman scattering (SERS) analyses of trace biomarkers in biofluid samples. This strategy integrating gasification separation, liquid trapping, derivatization SERS activation, and coffee-ring enrichment could highly promote the efficiency of sample preparation. Meanwhile, the edges of membranes modified by the hydrophobic-infusing slippery liquid-induced uniform "coffee-ring" effect could significantly improve the sensitivity and stability for SERS quantification. By adapting proper derivatization approaches to the miniaturized GMS/ORE pretreatment, the matrix effects in samples could be prominently eliminated, and clear SERS responses could be obtained for the selective analyses of target biomarkers. The miniaturized GMS/ORE device was practically applied for SERS analyses of trace biomarkers in biofluids, including hydrogen sulfide in saliva samples, creatinine in serum samples, and sarcosine, creatinine, and dimethyl disulfide in urine samples. Accurate quantification of all biomarkers was achieved with recoveries of 89.5%-120.0%, and the contents found by GMS/ORE-SERS matched well with those found by corresponding chromatographic methods with relative errors from -8.6% to 9.3%. The miniaturized GMS/ORE device with multiple parallel processing units could simultaneously treat eight samples in one run with a total analysis time of 40 min. Such an efficient all-in-one strategy integrated on a miniaturized device possesses great potential for fast on-site/point-of-care detection in analytical science and clinical medicine.

Sensitive Near-Infrared Fluorescent Determination of Hydrogen Peroxide and Glucose Using Glutathione-Coated Gold Nanoparticles

Near-infrared luminescence glutathione-coated gold nanoparticles (NGS-AuNPs) were synthesized and employed as fluorescent probes for the selective determination of hydrogen peroxide and glucose. This approach was based upon the coordination-induced fluorescence quenching of tris(2-carboxyethyl)phosphine (TCEP) with the NGS-AuNPs and its reduction of H2O2. The NGS-AuNPs possessed a highly monodispersed spherical morphology with a uniform diameter of 2.1 ± 0.5 nm and a maximal fluorescence emission wavelength of 810 nm. The results of the H2O2 determination demonstrate the excellent selectivity of this strategy with a limit of detection of 5.4 μM. This system was also employed for the selective determination of glucose which generated H2O2 by the catalytic oxidation of glucose oxidase with a limit of detection of 0.42 μM. This strategy exhibited excellent performance for the test of H2O2 and glucose in urine with recoveries from 93% to 103%. This methodology may allow the analysis of biological samples and clinical diagnosis.

Current Approaches for Image Fusion of Histological Data with Computed Tomography and Magnetic Resonance Imaging.

Classical analysis of biological samples requires the destruction of the tissue's integrity by cutting or grinding it down to thin slices for (Immuno)-histochemical staining and microscopic analysis. Despite high specificity, encoded in the stained 2D section of the whole tissue, the structural information, especially 3D information, is limited. Computed tomography (CT) or magnetic resonance imaging (MRI) scans performed prior to sectioning in combination with image registration algorithms provide an opportunity to regain access to morphological characteristics as well as to relate histological findings to the 3D structure of the local tissue environment. This review provides a summary of prevalent literature addressing the problem of multimodal coregistration of hard- and soft-tissue in microscopy and tomography. Grouped according to the complexity of the dimensions, including image-to-volume (2D ⟶ 3D), image-to-image (2D ⟶ 2D), and volume-to-volume (3D ⟶ 3D), selected currently applied approaches are investigated by comparing the method accuracy with respect to the limiting resolution of the tomography. Correlation of multimodal imaging could position itself as a useful tool allowing for precise histological diagnostic and allow the a priori planning of tissue extraction like biopsies.

Influence of measurement mode on the results of glioblastoma multiforme analysis with the FTIR microspectroscopy

Fourier Transform Infrared (FTIR) microspectroscopy is well known for its effectiveness in spectral and biochemical analyses of various materials. It enables to determine the sample biochemical composition by assigning detected frequencies, characteristic for functional groups of main biological macromolecules. In analysis of tissue sections one of two measurement modes, namely transmission and transflection, is usually applied. The first one has relatively straightforward geometry, hence it is considered to be more precise and accurate. However, IR-transparent media are very fragile and expensive. Transflection does not require expensive substrates, but is more prone to disruptive influence of Mie scattering as well as electric field standing wave effect.The excessive comparison of spectra’ characteristics, obtained via both measurement modes, was performed in this paper. By the means of Mann-Whitney non-parametrical U test and PCA, the comparison of results obtained with both modes and assessment of usefulness of IR spectra obtained with transmission and transflection modes to differentiate between healthy and GBM-affected tissue, were performed.The main objective of the presented research is to compare the results of FTIR analysis of unfixed biological samples performed with transflection and transmission mode. In frame of the study we demonstrated the discrepancies between results of biochemical analysis performed based on data obtained with transmission and transflection. Such observation suggests that caution should be taken in drawing conclusions from the results obtained with transflection geometry, as its more prone to disruptive effects. Despite that, IR spectra developed with both modes allowed to distinguish GBM area from healthy tissue, which proves their diagnostic potential. Especially, application of the ME-EMSC correction of spectra before PCA enhances the performance of both methods to distinguish the analysed tissue areas.

Accuracy of substance exposure history in patients attending emergency departments after substance misuse; a comparison with biological sample analysis

Context Acute toxicity caused by illicit substance use is a common reason for emergency department (ED) presentation. Knowledge of the substances involved is helpful for predicting and managing potential toxicity, but limited information is available about the accuracy of patient-reported substance exposure. This study assessed the accuracy of the history of exposure in those reporting use of a single substance by comparison with those identified by detailed toxicological analysis, focusing on synthetic cannabinoid receptor agonists (SCRA). Methods Adults (≥16 years) presenting between March 2015 and July 2021 to participating UK hospitals with toxicity after reporting use of a single illicit substance were included. Exposure details were documented from medical records and blood and/or urine samples analysed using high-resolution accurate mass liquid chromatography-mass spectrometry (HRAM LCMS). Sensitivity, specificity, and positive and negative predictive values of the exposure history were calculated by comparison with biological sample analysis (“gold standard”). Results Single substance exposure was reported for 474 (median age 33 years, IQR: 18 range 16–75, 80% males) patients. Analysis commonly identified multiple substances (Median 3, IQR 2–5). A history of exposure was documented for 121 of 151 patients where a SCRA or metabolite was detected on analysis (sensitivity 80.1%, 95% CI 72.9, 86.2%). Corresponding proportions were lower for 3,4-methylenedioxymethamphetamine (MDMA, 44/70, 62.9%., 95% CI 50.5%, 74.1%), heroin 41/108 (38.0% 95% CI 28.8–47.8%) and cocaine (22/56, 31.3%, 95% CI 20.9, 43.6%). Conclusions Multiple undeclared substances were detected analytically in most patients reporting single substance use. Clinicians should be alert to the potential presence and toxicity of unreported substances when managing patients presenting after substance misuse.

Generation of 13C-Labeled Inositol and Inositol Phosphates by Stable Isotope Labeling Cell Culture for Quantitative Metabolomics.

Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, U13C-inositol and U13C-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating U13C-inositol and U13C-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with U13C-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated U13Canalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, U13Canalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. U13CIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating U13Canalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Parallelized continuous flow dielectrophoretic separation ofDNA.

Numerous microfluidic separation applications have been shown in the past years providing a fast analysis of biological samples like DNA or proteins. Microfluidic separation based on dielectrophoresis (DEP), that is the migration of a polarizable object in an inhomogeneous electric field, provides numerous advantages. However, the main drawback of DEP separation devices is that they are not sufficient for large-scale sample purification due to the lack of high sample throughput. In this work, we present for the first time a microfluidic device with two parallelized dielectrophoretic separations of (biological) samples smaller than 1µm. The separation is carried out by means of insulator-based DEP, that is an insulating ridge reduced the flow through height and thus created a nanoslit at which the selective DEP forces occur. The device consists of a cross injector, two parallel operation regions and separate harvesting reservoirs where the samples are collected. Each DEP operation region contains an insulating ridge. We successfully demonstrate the separation of 100 and 40nm beads and 10 and 5kbp DNA with a separation purity of more than 80%. This states the proof-of-concept for up-scaling of dielectrophoretic separation by parallelization. As the present technique is virtually label-free, it offers a fast purification, for example in the production of gene vaccines.

DD-03 ANALYSING PORCINE ANTERIOR ABDOMINAL MIDLINE LAYERS USING RAMAN SPECTROSCOPY

Abstract Aim Evidence shows that incisional hernia (IH) formation may result from connective tissue alterations in a subgroup of individuals. Collagen signature typical of weaker tissues and healing wounds was reported by several groups. Such changes in the incisional scar may lead to IH formation. With the use of advanced imaging (Raman spectroscopy) we aim to investigate the composition and structure of the abdominal midline. We selected the porcine model to validate the use of Raman Spectroscopy for studying the anterior abdominal wall and calibrating it as a reference tool for human studies. Materials & Methods Raman spectroscopy, a technique widely used in physical sciences, is deployed to provide a chemical and structural fingerprint of the midline. Collagen produces specific Raman spectra, which allows its identification and analysis in biological samples. The porcine abdomen is similar in structure and composition to the human abdomen and is therefore suitable as reference tissue to validate Raman spectroscopy for human studies. Results Preliminary spectral data (linea alba, rectus sheet, muscle, skin, fat) showed differential collagen, lipid and muscle fibre content in corresponding layers of the porcine abdomen. Notably, characteristic collagen related Raman bands were confirmed. Conclusions The porcine abdominal wall is similar to that of humans. Hence using this model system, we can validate the use of Raman spectroscopy for the investigation of collagen in IH scars. A pilot trial has now been set up to study human scar collagen in patients undergoing IH repair and patient recruitment has begun.

Multimodal X-ray nano-spectromicroscopy analysis of chemically heterogeneous systems.

Understanding the nanoscale chemical speciation of heterogeneous systems in their native environment is critical for several disciplines such as life and environmental sciences, biogeochemistry, and materials science. Synchrotron-based X-ray spectromicroscopy tools are widely used to understand the chemistry and morphology of complex material systems owing to their high penetration depth and sensitivity. The multidimensional (4D+) structure of spectromicroscopy data poses visualization and data-reduction challenges. This paper reports the strategies for the visualization and analysis of spectromicroscopy data. We created a new graphical user interface and data analysis platform named XMIDAS (X-ray multimodal image data analysis software) to visualize spectromicroscopy data from both image and spectrum representations. The interactive data analysis toolkit combined conventional analysis methods with well-established machine learning classification algorithms (e.g. nonnegative matrix factorization) for data reduction. The data visualization and analysis methodologies were then defined and optimized using a model particle aggregate with known chemical composition. Nanoprobe-based X-ray fluorescence (nano-XRF) and X-ray absorption near edge structure (nano-XANES) spectromicroscopy techniques were used to probe elemental and chemical state information of the aggregate sample. We illustrated the complete chemical speciation methodology of the model particle by using XMIDAS. Next, we demonstrated the application of this approach in detecting and characterizing nanoparticles associated with alveolar macrophages. Our multimodal approach combining nano-XRF, nano-XANES, and differential phase-contrast imaging efficiently visualizes the chemistry of localized nanostructure with the morphology. We believe that the optimized data-reduction strategies and tool development will facilitate the analysis of complex biological and environmental samples using X-ray spectromicroscopy techniques.

Insight of development and validation of bio-analytical method in the reference of anti-diabetic drugs by using LC-MS/MS

Diabetes mellitus, which has high rates of disability and mortality, is one of the main causes of public health concerns worldwide. It currently poses serious medical and societal issues. Different bio-analytical techniques have been developed to identify the pharmaceutical formulation of the anti-diabetic drug. Due to the continual requirement to achieve improved sensitivity, accuracy, and speed of analysis in complex biological samples, the development of bio-analytical sample preparation techniques has grown more difficult over time (e.g., blood, serum, plasma, saliva, feces, and urine). This is a mini review, and its goal is to analyze how LC-MS/MS has been used to for the pharmaceutical formulation of the anti-diabetic drugs. The method was validated for linearity, accuracy, precision, specificity, selectivity, and stability.

Azure A embedded in carbon dots as NADH electrocatalyst: Development of a glutamate electrochemical biosensor

Carbon nanodots modified with azure A (AA-CDs) have been synthesized and applied as redox mediator of bioelectrocatalytic reactions. A deep characterization of AA-CDs nanomaterial has been carried out, proving the covalent attachment of azure A molecules into the carbon dots nanostructure. Disposable screen-printed carbon electrodes (SPCE) have been modified with AA-CDs, through the action of chitosan polymer (Chit-AA-CDs/SPCE). The Chit-AA-CDs/SPCE electrocatalytic activity towards the oxidation of NADH has been proved, obtaining excellent results regarding the low oxidation potential achieved (−0.15 V vs. Ag) and low detection and quantification limits (LOD and LOQ) for NADH, 16 and 53 µM, respectively. The developed electrochemical platform has been applied for the construction of a glutamate biosensor by immobilizing L-glutamic dehydrogenase (GLDH/Chit-AA-CDs/SPCE). The morphology of GLDH/Chit-AA-CDs/SPCE platform was analysed by AFM at each different step of the electrode modification process. The resulting biosensing platform is capable of detect NADH enzymatically generated by GLDH in the presence of glutamate and NAD+. Good analytical parameters were obtained for glutamate analysis using GLDH/Chit-AA-CDs/SPCE, as LOD and LOQ of 3.3 and 11 µM, respectively. The biosensor has been successfully applied to the analysis of food and biological samples.

Optimization of a GC-MS Injection-Port Derivatization Methodology to Enhance Metabolomics Analysis Throughput in Biological Samples.

Advances in metabolomics analysis and data treatment increase the knowledge of complex biological systems. One of the most used methodologies is gas chromatography-mass spectrometry (GC-MS) due to its robustness, high separation efficiency, and reliable peak identification through curated databases. However, methodologies are not standardized, and the derivatization steps in GC-MS can introduce experimental errors and take considerable time, exposing the samples to degradation. Here, we propose the injection-port derivatization (IPD) methodology to increase the throughput in plasma metabolomics analysis by GC-MS. The IPD method was evaluated and optimized for different families of metabolites (organic acids, amino acids, fatty acids, sugars, sugar phosphates, etc.) in terms of residence time, injection-port temperature, and sample/derivatization reagent ratio. Finally, the method's usefulness was validated in a study consisting of a cohort of obese patients with or without nonalcoholic steatohepatitis. Our results show a fast, reproducible, precise, and reliable method for the analysis of biological samples by GC-MS. Raw data are publicly available at MetaboLights with Study Identifier MTBLS5151.

Recent molecularly imprinted polymers applications in bioanalysis.

Molecular imprinted polymers (MIPs) as extraordinary compounds with unique features have presented a wide range of applications and benefits to researchers. In particular when used as a sorbent in sample preparation methods for the analysis of biological samples and complex matrices. Its application in the extraction of medicinal species has attracted much attention and a growing interest. This review focus on articles and research that deals with the application of MIPs in the analysis of components such as biomarkers, drugs, hormones, blockers and inhibitors, especially in biological matrices. The studies based on MIP applications in bioanalysis and the deployment of MIPs in high-throughput settings and optimization of extraction methods are presented. A review of more than 200 articles and research works clearly shows that the superiority of MIP techniques lies in high accuracy, reproducibility, sensitivity, speed and cost effectiveness which make them suitable for clinical usage. Furthermore, this review present MIP-based extraction techniques and MIP-biosensors which are categorized on their classes based on common properties of target components. Extraction methods, studied sample matrices, target analytes, analytical techniques and their results for each study are described. Investigations indicate satisfactory results using MIP-based bioanalysis. According to the increasing number of studies on method development over the last decade, the use of MIPs in bioanalysis is growing and will further expand the scope of MIP applications for less studied samples and analytes.

Sulphur-doped graphene based sensor for rapid and efficient gallic acid detection from food related samples

BackgroundNowadays, the food safety issue represents a hot topic. The emerging evidence over the past decade confirms that gallic acid (GA) plays a crucial role, with multiple pharmacological and therapeutic interventions in numerous health complications. MethodsSulphur-doped graphene material (exf-SGR) was obtained from graphite rod electrochemical exfoliation in the presence of inorganic salts mixture such as sodium thiosulfate and ammonium sulphate, Na2S2O3 / (NH4)2SO4 (0.2 M each), at low applied bias (6 V). The complete physico-chemical characterization of the as-prepared material was performed by means of TEM, SEM, STEM-EDS, XRD and XPS analysis. Furthermore, the electrochemical performances of bare glassy carbon (GCE) and modified (exf-SGR/GCE) electrodes for GA assay were evaluated. Significant findingsTEM/SEM images reveal the formation of large dimension graphene sheets having uniformly distributed spherical sulphur crystallites on the surface. XRD demonstrates that the material consists of few-layer S-doped graphenes. From XPS analysis the C/O ratio was determined to be 0.473 while C/S ratio equals 2.741, indicating a high heteroatom doping degree. The exf-SGR/GCE modified electrode shows a superior electrochemical behaviour over a broad GA detection range (0.1–100.0 µM), with a low detection limit (3.03 × 10−8 M). The electrode retains 96.83% of its initial response after 50 consecutive CV measurements indicating an excellent long-term stability and repeatability. The sensor possesses very good anti-interfering capabilities in complex matrix. The developed electrochemical sensor can provide a fast model for GA detection in food related and biological real sample analysis.