Biological Samples Research Articles

PCR amplification, sequencing, and in silico analysis of holandric genes TSPYand SRY in Nili Ravi buffalo

The present work has been designed to study the evolutionary perspectives of two important holandric genes, namely, testis-specific protein, Y-encoded (TSPY), and sex determining region, Y-encoded (SRY) in Nili Ravi buffalo. These Y-chromosomal genes are responsible for male sex development. The evolutionary aspects of these genes have not been studied in the Nili Ravi buffaloes from India. A study was carried out on the peripheral blood samples of three adult Nili Ravi bulls belonging to university farm. DNA was extracted using the phenol-chloroform isoamyl alcohol (PCI) technique and reported primer sequences were used to amplify the genes (partially) using a thermocycler. Three biological samples were analyzed using Sanger sequencing and were aligned using Clustal Omega. Separate phylogenetic trees for the genes were constructed using MEGA7 software. The selection pressure analysis and evolutionary divergence were individually studied for each of the genes. The current study reports the partial TSPY and SRY gene sequences of Indian Nili Ravi buffalo. Besides, the molecular evolution analysis indicates that the genes have experienced some purifying selection during the evolutionary process. Molecular characterization of these holandric genes (TSPY and SRY) in Nili Ravi will find applications in sex determination in Nili Ravi and other buffaloes.

Relationships between nitric oxide biomarkers and physiological outcomes following dietary nitrate supplementation

Dietary nitrate (NO3−) supplementation can increase nitric oxide (NO) bioavailability, reduce blood pressure (BP) and improve muscle contractile function in humans. Plasma nitrite concentration (plasma [NO2−]) is the most oft-used biomarker of NO bioavailability. However, it is unclear which of several NO biomarkers (NO3−, NO2−, S-nitrosothiols (RSNOs)) in plasma, whole blood (WB), red blood cells (RBC) and skeletal muscle correlate with the physiological effects of acute and chronic dietary NO3− supplementation. Using a randomized, double-blind, crossover design, 12 participants (9 males) consumed NO3−-rich beetroot juice (BR) (∼12.8 mmol NO3−) and NO3−-depleted placebo beetroot juice (PL) acutely and then chronically (for two weeks). Biological samples were collected, resting BP was assessed, and 10 maximal voluntary isometric contractions of the knee extensors were performed at 2.5–3.5 h following supplement ingestion on day 1 and day 14. Diastolic BP was significantly lower in BR (−2 ± 3 mmHg, P = 0.03) compared to PL following acute supplementation, while the absolute rate of torque development (RTD) was significantly greater in BR at 0–30 ms (39 ± 57 N m s−1, P = 0.03) and 0–50 ms (79 ± 99 N m s−1, P = 0.02) compared to PL following two weeks supplementation. Greater WB [RSNOs] rather than plasma [NO2−] was correlated with lower diastolic BP (r = −0.68, P = 0.02) in BR compared to PL following acute supplementation, while greater skeletal muscle [NO3−] was correlated with greater RTD at 0–30 ms (r = 0.64, P=0.03) in BR compared to PL following chronic supplementation. We conclude that [RSNOs] in blood, and [NO3−] in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the reduction of BP and the enhanced muscle contractile function following dietary NO3− ingestion in humans.

Eco-friendly synthesis of cluster-like Dy2Ce2O7 nanoparticles using orange juice and their application in electrochemical determination of isoniazid

In this study, cluster-like (CL) Dy2Ce2O7 nanoparticles were synthesized via an easy and rapid sonochemical pathway. X-ray diffraction (XRD), and field emission scanning electron microscopy (FE-SEM) were used for the characterization of nanoparticles. The construction of an electrochemical sensor was performed to determine isoniazid (IZN) based on ionic liquid (IL) and the cluster-like nanostructure of Dy2Ce2O7 modified carbon paste electrode (CL-Dy2Ce2O7/IL/CPE). Using the fabricated sensor, cyclic voltammetry (CV) along with differential pulse voltammetry (DPV) have been utilized to examine the catalytic activity of the modified electrode toward oxidation of IZN in 0.1 M phosphate buffer solution (PBS) with pH of 7.0. The large specific surface area and electrocatalytic properties of CL-Dy2Ce2O7 as well as the good electrical conductivity of ionic liquid greatly enhanced the current signal of the IZN at the CL-Dy2Ce2O7/IL/CPE. The process at the surface of the electrode was under the diffusion control mechanism and the diffusion coefficient for IZN was 1.08 × 10−5 cm2/s. Additionally, with a low detection limit of 9.0 nM, the peak current of IZN rose linearly with concentration in the range of 0.02–340.0 μM. Our findings demonstrate that the sensitive and selective detection of IZN in pharmaceutical and biological samples can be achieved using the CL-Dy2Ce2O7/IL/CPE.

Construction of selectively enhanced MOF-SERS sensor and highly sensitive detection of hCE1 in HepG2 cells

In this paper, a surface-enhanced Raman scattering (SERS) magnetic immunosensor for human carboxylesterase 1 (hCE1) assay was developed using a sandwich structure consisting of a magnetic support substrate, a SERS tag, and a target. The objective was to investigate the correlation between changes in hCE1 levels and liver injury. The utilization of Co-MOF-74@Au@methylene blue (MB) magnetic support substrate facilitated the expedient separation of biological samples while uniformly distributing the AuNPs, thereby enhancing the SERS intensity and improving the reproducibility. The introduction of Co-MOF-74 metal–organic frameworks (MOFs), endowed with its own selective SERS-enhancing properties, into the Co-MOF-74@Au@MB SERS tag further enhances the SERS sensing capability. The proposed SERS sensor exhibited good linearity over the concentration range of hCE1 from 10−2 to 102 μg·mL−1, with a correlation coefficient (R2) of 0.990 and a limit of detection (LOD) of 8.3 ng·mL−1. This demonstrates the potential of this strategy for the detection of trace proteins or enzymes in complex biological samples. In the meantime, the selective SERS enhancement mechanism of MOFs on Raman reporter molecules was preliminarily explored. This provides a new idea for the development of SERS substrates and SERS assays.

Magnetic dispersive solid-phase extraction of clarithromycin and azithromycin by Fe3O4@ionic salt g-C3N4 adsorbent and HPLC-UV

In this study, Fe3O4@ionic salt g-C3N4 was synthesized as a new adsorbent for magnetic dispersive solid phase extraction (MDSPE) of two antibiotics (clarithromycin and azithromycin) in biological samples followed by HPLC-UV. The new nanocomposite was characterized by Fourier-transform infrared (FT-IR), field emission-scanning electronic microscope (FE-SEM), transmission electron microscope (TEM), nitrogen adsorption–desorption analysis, X-ray powder diffraction (XRD), and vibrating sample magnetometer (VSM). Box-Behnken design (BBD) and response surface methodology (RSM) were used for the optimization and design of experiments. The method had a suitable linear range (1–300 µg/L), a high coefficient of determination (R2; 0.9988 and 0.9993), and a low detection limit (0.33 and 0.42 µg/L) for clarithromycin and azithromycin, respectively. The applicability of the method was evaluated by estimating the recovery of analytes from urine and blood serum samples. Recovery of more than 90.0 % and RSDs of less than 5.2 % show the acceptable accuracy and precision of the proposed method.

Prediction of inhibitor development in previously untreated and minimally treated children with severe and moderately-severe hemophilia A using a machine-learning network

BackgroundPrediction of inhibitor development in patients with hemophilia A (HA) remains a challenge. AimTo construct a predictive model for inhibitor development in HA using a network of clinical variables and biomarkers based on the individual similarity network. MethodsPreviously untreated and minimally treated children with severe/moderately-severe HA, participants of the HEMFIL Cohort Study, were followed-up until reaching 75 exposure days (ED) without inhibitor (INH-) or upon inhibitor development (INH+). Clinical data and biological samples were collected before the start of factor VIII (FVIII) replacement (T0). A predictive model (HemfilNET) was built to compare the networks and potential global topological differences between INH- and INH+ at T0, considering the network robustness. For validation, the "leave-one-out" cross-validation technique was employed. Accuracy, precision, recall, and F1-score were used as evaluation metrics for the machine-learning model. ResultsWe included 95 children with HA (CHA), of whom 31 (33%) developed inhibitors. The algorithm, featuring 37 variables, identified distinct patterns of networks at T0 for INH+ and INH-. The accuracy of the model was 74.2% for CHA INH+ and 98.4% for INH-. By focusing the analysis on CHA with high-risk F8 mutations for inhibitor development, the accuracy in identifying CHA INH+ increased to 82.1%. ConclusionOur machine-learning algorithm demonstrated an overall accuracy of 90.5% for predicting inhibitor development in CHA, which further improved when restricting the analysis to CHA with a high-risk F8 genotype. However, our model requires validation in other cohorts. Yet, missing data for some variables hindered more precise predictions.

Synthesis of polyethylene glycol-grafted magnetic nanoparticles for the fast and efficient extraction of anabolic substances from human urine

Anabolic steroids and β-agonists are commonly prohibited substances found in doping control studies; therefore, the determination of anabolic substances in biological samples is crucial. To analyze the anabolic compounds in urine, an adsorbent, polyethylene glycol (PEG)-grafted magnetic nanoparticle material (Fe3O4@SiO2-PEG), with low toxicity and strong biocompatibility was prepared in this investigation. Compared to those of Fe3O4 and Fe3O4@SiO2, the grafted PEG chains (approximately 5.4 wt.%) on the magnetic nanoparticles improved the extraction efficiencies by factors of 3.9–17.0 and 2.5–2.9, respectively, likely due to the electrostatic attraction and hydrogen bonding. To achieve maximum extraction efficiency, several extraction parameters were optimized, including the kind and volume of desorption solvent, pH, and the extraction and desorption time. The standard curves were linear within the range of 0.5–20 μg/L for methyltestosterone and trenbolone, and 0.02–5 μg/L for clenbuterol. The limits of detection for the three drugs were 0.01–0.12 μg/L. The limits of quantification were 0.02–0.40 μg/L. The levels of precision of the optimized method were assessed based on the respective intra- and inter-day and batch-to-batch relative standard deviations in the ranges of 3.2–5.2% (n = 5), 5.9–11.3% (n = 4), and 6.9–9.2% (n = 3). The Fe3O4@SiO2-PEG nanoparticles could exclude urine matrix interferences (matrix effect of 91.8–98.1%) and achieve satisfactory recoveries (75.5–116.1%), affording sensitive and accurate determination of trace anabolic substances in urine.

Polylactic acid micro/nanoplastic-induced hepatotoxicity: Investigating food and air sources via multi-omics

Micro/nanoplastics (MNPs) are detected in human liver, and pose significant risks to human health. Oral exposure to MNPs derived from non-biodegradable plastics can induce toxicity in mouse liver. Similarly, nasal exposure to non-biodegradable plastics can cause airway dysbiosis in mice. However, the hepatotoxicity induced by foodborne and airborne biodegradable MNPs remains poorly understood. Here we show the hepatotoxic effects of biodegradable polylactic acid (PLA) MNPs through multi-omics analysis of various biological samples from mice, including gut, fecal, nasal, lung, liver, and blood samples. Our results show that both foodborne and airborne PLA MNPs compromise liver function, disrupt serum antioxidant activity, and cause liver pathology. Specifically, foodborne MNPs lead to gut microbial dysbiosis, metabolic alterations in the gut and serum, and liver transcriptomic changes. Airborne MNPs affect nasal and lung microbiota, alter lung and serum metabolites, and disrupt liver transcriptomics. The gut Lachnospiraceae_NK4A136_group is a potential biomarker for foodborne PLA MNP exposure, while nasal unclassified_Muribaculaceae and lung Klebsiella are potential biomarkers for airborne PLA MNP exposure. The relevant results suggest that foodborne PLA MNPs could affect the “gut microbiota-gut-liver” axis and induce hepatoxicity, while airborne PLA MNPs could disrupt the “airway microbiota-lung-liver” axis and cause hepatoxicity. These findings have implications for diagnosing PLA MNPs-induced hepatotoxicity and managing biodegradable materials in the environment. Our current study could be a starting point for biodegradable MNPs-induced hepatotoxicity. More research is needed to verify and inhibit the pathways that are crucial to MNPs-induced hepatotoxicity.

A practical approach to semi-automated supported liquid extraction and measurement of 18 steroids in plasma by targeted liquid chromatography tandem mass spectrometry

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 μL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 20 steroids. The method can measure 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17β-estradiol and estriol). The workflow includes● SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method● Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching● Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

Improved Data Acquisition Settings on Q Exactive HF-X and Fusion Lumos Tribrid Orbitrap-Based Mass Spectrometers for Proteomic Analysis of Limited Samples.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Development and Validation of an LC-MS/MS Method for the Determination of Plasma and Red Blood Cell Omega Fatty Acids: A Useful Diagnostic Tool

Label-free electrochemical detection of glycated hemoglobin (HbA1c) and C-reactive protein (CRP) to predict the maturation of coronary heart disease due to diabetes

The pathophysiological link between diabetes and heightened propensity for the development of coronary heart disease (CHD) is well-established. Prevailing evidence confirms that small increases in low concentrations of high-sensitivity C reactive protein (hs-CRP) in the human body can determine the tendency of developing CHD. Additionally, glycated hemoglobin (HbA1c) is a well-recognized biomarker to evaluate diabetes progression. Given the positive correlation between diabetes and CHD, this research presents a notably unprecedented label-free electrochemical approach for the dual detection of %HbA1c regarding Total Hb and hs-CRP, facilitating early CHD prediction and cost-effective point-of-care diagnostics. Furthermore, a novel redox probe O-(4-Nitrophenylphosphoryl)choline (C11H17N2O6P) was used for the electrochemical detection of CRP, a method not documented in scientific literature before. The calibration curves demonstrate a limit of detection (LOD) of 5 mg/mL in PBS (pH 8) and 6 mg/mL in simulated blood (SB) for a linear range of 0–30 mg/mL of HbA1c. Conjointly, a LOD of 0.007 mg/mL and 0.008 mg/mL for measurement in PBS (pH 7.4) and SB are reported for a linear range of 0–0.05 mg/mL of CRP. The electrochemical systems presented could accurately quantify HbA1c and CRP in mixed samples, demonstrating reasonable specificity and practical applicability for complex biological samples.

When surface-enhanced Raman spectroscopy meets complex biofluids: A new representation strategy for reliable and comprehensive characterization

BackgroundSurface-enhanced Raman spectroscopy (SERS) has gained increasing importance in molecular detection due to its high specificity and sensitivity. Complex biofluids (e.g., cell lysates and serums) typically contain large numbers of different bio-molecules with various concentrations, making it extremely challenging to be reliably and comprehensively characterized via conventional single SERS spectra due to uncontrollable electromagnetic hot spots and irregular molecular motions. The traditional approach of directly reading out the single SERS spectra or calculating the average of multiple spectra is less likely to take advantage of the full information of complex biofluid systems. ResultsHerein, we propose to construct a spectral set with unordered multiple SERS spectra as a novel representation strategy to characterize full molecular information of complex biofluids. This new SERS representation not only contains details from each single spectra but captures the temporal/spatial distribution characteristics. To address the ordering-independent property of traditional chemometric methods (e.g., the Euclidean distance and the Pearson correlation coefficient), we introduce Wasserstein distance (WD) to quantitatively and comprehensively assess the quality of spectral sets on biofluids. WD performs its superiority for the quantitative assessment of the spectral sets. Additionally, WD benefits from its independence of the ordering of spectra in a spectral set, which is undesirable for traditional chemometric methods. With experiments on cell lysates and human serums, we successfully achieve the verification for the reproducibility between parallel samples, the uniformity at different positions in the same sample, the repeatability from multiple tests at one location of the same sample, and the cardinality effect of the spectral set. SERS spectral sets also manage to distinguish different classes of human serums and achieve higher accuracy than the traditional prostate-specific antigen in prostate cancer classification. SignificanceThe proposed SERS spectral set is a robust representation approach in accessing full information of biological samples compared to relying on a single or averaged spectra in terms of reproducibility, uniformity, repeatability, and cardinality effect. The application of WD further demonstrates the effectiveness and robustness of spectral sets in characterizing complex biofluid samples, which extends and consolidates the role of SERS.

First electrochemical nanosensor for ultrasensitive quantification of MET inhibitor, capmatinib based on carbon nanofiber networks incorporated with hybrid nanofiller nanogold-loaded porous acetylene black

In the current study, the first voltammetric method is constructed for the ultrasensitive quantification of capmatinib (CMT), a targeted therapy drug falls within the category of tyrosine kinase inhibitors (TKIs), using a modified carbon nanofibers paste electrode (CNFs-PE). Nanogold-loaded porous acetylene black (Au@PAB) as a conductive hybrid nanofiller incorporating carbon nanofiber networks (CNFs) was synthesized. The surface morphology of the synthesized Au@PAB and the ternary nanocomposite Au@PAB/CNFs was elucidated using various analytical techniques. The results show Au@PAB hybrid nanofiller incorporated CNFs has a critical property that increases electrocatalytic performance. The impact of scan rate, pH, and supporting electrolyte on the electrocatalytic response of CMT was explored on the Au@PAB/CNFs-PE nanosensor. Under optimum adsorptive stripping square-wave voltammetric (AdS-SWV) conditions, the developed probe displayed good sensitivity, selectivity, and a remarkable wide linear dynamic range of 1.0 × 10-8 – 2.0 × 10-5 M of CMT. The low limits of detection (LOD) and quantification (LOQ) were 4.6 nM and 15.4 nM of CMT, respectively. The features of easy operation and good selectivity make this nanosensor a talented candidate for CMT detection in complex samples, as demonstrated by its application in spiked biological samples with good recoveries.

Recent Advances in Molecular Imprinting for Proteins on Magnetic Microspheres.

The separation of proteins in biological samples plays an essential role in the development of disease detection, drug discovery, and biological analysis. Protein imprinted polymers (PIPs) serve as a tool to capture target proteins specifically and selectively from complex media for separation purposes. Whereas conventional molecularly imprinted polymer is time-consuming in terms of incubation studies and solvent removal, magnetic particles are introduced using their magnetic properties for sedimentation and separation, resulting in saving extraction and centrifugation steps. Magnetic protein imprinted polymers (MPIPs), which combine molecularly imprinting materials with magnetic properties, have emerged as a new area of research hotspot. This review provides an overview of MPIPs for proteins, including synthesis, preparation strategies, and applications. Moreover, it also looks forward to the future directions for research in this emerging field.

Electrochemical analysis of Ca2+ based on DNAzyme catalyzed degradation of DNA hydrogel

Calcium ion is a type of indispensable metal elements in biology, which participates in processes like maintaining the excitability of neuromuscular muscles. However, calcium content should be monitored in a safety range. Herein, a novel electrochemical method is developed for Ca2+ assay by monitoring electrochemical response after DNAzyme catalyzed DNA hydrogel degradation. Pure DNA hydrogel is first built with three-way junction scaffolds and linkers containing Ca2+-dependent DNAzyme sequences. In the presence of target Ca2+, the substrates in linkers are cleaved and DNA hydrogel can be degraded gradually. The encapsulated electrochemical species thus facilely interact with the electrode, leading to the increase of electrochemical responses. This electrochemical method for Ca2+ quantification is selective and sensitive, which also performs satisfactorily challenging biological samples like sweat and urine.

Efficient acid hydrolysis for compound-specific δ15N analysis of amino acids for determining trophic positions

Compound-specific isotope analysis of nitrogen in amino acids (CSIA-AA, δ15NAA) has gained increasing popularity for elucidating energy flow within food chains and determining the trophic positions of various organisms. However, there is a lack of research on the impact of hydrolysis conditions, such as HCl concentration and hydrolysis time, on δ15NAA analysis in biota samples. In this study, we investigated two HCl concentrations (6 M and 12 M) and four hydrolysis times (2 h, 6 h, 12 h, and 24 h) for hydrolyzing and derivatizing AAs in reference materials (Tuna) and biological samples of little egret (n = 4), night heron (n = 4), sharpbelly (n = 4) and Algae (n = 1) using the n-pivaloyl-iso-propyl (NPIP) ester approach. A Dowex cation exchange resin was used to purify amino acids before derivatization. We then determined δ15NAA values using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The results revealed no significant differences (p > 0.05) in δ15NAA values among samples treated with different HCl concentrations or hydrolysis times, particularly for δ15NGlx (range: 21.0–23.5‰) and δ15NPhe (range: 4.3–5.4‰) in Tuna (12 M). Trophic positions (TPs) calculated based on δ15NAA at 2 h (little egret: 2.9 ± 0.1, night heron: 2.8 ± 0.1, sharpbelly: 2.0 ± 0.1 and Algae: 1.3 ± 0.2) were consistent with those at 24 h (3.1 ± 0.1, 2.8 ± 0.1, 2.2 ± 0.1 and 1.1 ± 0.1, respectively), suggesting that a 2-h hydrolysis time and a 6 M HCl concentration are efficient pretreatment conditions for determining δ15NAA and estimating TP. Compared to the currently used hydrolysis conditions (24 h, 6 M), the proposed conditions (2 h, 6 M) accelerated the δ15NAA assay, making it faster, more convenient, and more efficient. Further research is needed to simplify the operational processes and reduce the time costs, enabling more efficient applications of CSIA-AA.

Increasing protein identifications in bottom-up proteomics of T. castaneum − Exploiting synergies of protein biochemistry and bioinformatics

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant.Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications.A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100 % increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant’s Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2 % must be considered for the data analysis.Our work with Tribolium castaneum larvae demonstrates that sometimes – depending on matrix and research question − more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Electrochemical Detection of Melphalan in Biological Fluids Using a g-C3N4@ND-COOH@MoSe2 Modified Electrode Complemented by Molecular Docking Studies with Cellular Tumor Antigen P53.

Melphalan (Mel) is a potent alkylating agent utilized in chemotherapy treatments for a diverse range of malignancies. The need for its accurate and timely detection in pharmaceutical preparations and biological samples is paramount to ensure optimized therapeutic efficacy and to monitor treatment progression. To address this critical need, our study introduced a cutting-edge electrochemical sensor. This device boasts a uniquely modified electrode crafted from graphitic carbon nitride (g-C3N4), decorated with activated nanodiamonds (ND-COOH) and molybdenum diselenide (MoSe2), and specifically designed to detect Mel with unparalleled precision. Our rigorous testing employed advanced techniques such as cyclic voltammetry and differential pulse voltammetry. The outcomes were promising; the sensor consistently exhibited a linear response in the range of 0.5 to 12.5 μM. Even more impressively, the detection threshold was as low as 0.03 μM, highlighting its sensitivity. To further enhance our understanding of Mel's biological interactions, we turned to molecular docking studies. These studies primarily focused on Mel's interaction dynamics with the cellular tumor antigen P53, revealing a binding affinity of -5.0 kcal/mol. A fascinating observation was made when Mel was covalently conjugated with nanodiamond-COOH (ND-COOH). This conjugation resulted in a binding affinity that surged to -10.9 kcal/mol, clearly underscoring our sensor's superior detection capabilities. This observation also reinforced the wisdom behind incorporating ND-COOH in our electrode design. In conclusion, our sensor not only stands out in terms of sensitivity but also excels in selectivity and accuracy. By bridging electrochemical sensing with computational insights, our study illuminates Mel's intricate behavior, driving advancements in sensor technology and potentially revolutionizing cancer therapeutic strategies.

Three-Dimensional Microfluidic Capillary Device for Rapid and Multiplexed Immunoassays in Whole Blood.

In this study, we demonstrate whole blood immunoassays using a microfluidic device optimized for conducting rapid and multiplexed fluorescence-linked immunoassays. The device is capable of handling whole blood samples without any preparatory treatment. The three-dimensional channels in poly(methyl methacrylate) are designed to passively load bodily fluids and, due to their linearly tapered profile, facilitate size-dependent immobilization of biofunctionalized particles. The channel geometry is optimized to allow for the unimpeded flow of cellular constituents such as red blood cells (RBCs). Additionally, to make the device easier to operate, the biofunctionalized particles are pretrapped in a first step, and the channel is dried under vacuum, after which it can be loaded with the biological sample. This novel approach and design eliminated the need for traditionally laborious steps such as filtering, incubation, and washing steps, thereby substantially simplifying the immunoassay procedures. Moreover, by leveraging the shallow device dimensions, we show that sample loading to read-out is possible within 5 min. Our results also show that the presence of RBCs does not compromise the sensitivity of the assays when compared to those performed in a pure buffer solution. This highlights the practical adaptability of the device for simple and rapid whole-blood assays. Lastly, we demonstrate the device's multiplexing capability by pretrapping particles of different sizes, each functionalized with a different antigen, thus enabling the performance of multiplexed on-chip whole-blood immunoassays, showcasing the device's versatility and effectiveness toward low-cost, simple, and multiplexed sensing of biomarkers and pathogens directly in whole blood.