Human Blood Plasma Samples Research Articles

Towards Label-free detection of viral disease agents through their cell surface proteins: Rapid screening SARS-CoV-2 in biological specimens.

Current methods for the screening of viral infections in clinical settings, such as reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), are expensive, time-consuming, require trained personnel and sophisticated instruments. Therefore, novel sensors that can save time and cost are required specially in remote areas and developing countries that may lack the advanced scientific infrastructure for this task. In this work, we present a sensitive, and highly specific biosensing approach for the detection of harmful viruses that have cysteine residues within the structure of their cell surface proteins. We utilized new method for the rapid screening of SARS-CoV-2 virus in biological fluids through its S1 protein by surface enhanced Raman spectroscopy (SERS). The protein is captured from aqueous solutions and biological specimens using a target-specific extractor substrate. The structure of the purified protein is then modified to convert it into a bio-thiol by breaking the disulfide bonds and freeing up the sulfhydryl (SH) groups of the cysteine residues. The formed biothiol chemisorbs favourably onto a highly sensitive plasmonic sensor and probed by a handheld Raman device in few seconds. The new method was used to screen the S1 protein in aqueous medium, spiked human blood plasma, mucus, and saliva samples down to 150 fg/L. The label-free SERS biosensing method has strong potential for the fingerprint identification many viruses (e.g. the human immunodeficiency virus, the human polyomavirus, the human papilloma virus, the adeno associated viruses, the enteroviruses) through the cysteine residues of their capsid proteins. The new method can be applied at points of care (POC) in remote areas and developing countries lacking sophisticated scientific infrastructure.

Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates for multiplex determination of dopamine, norepinephrine, and epinephrine.

Aunique approach based on Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates is proposed for sensitive and rapid determination of the catecholamines (CA)dopamine, norepinephrine, and epinephrine. The ternary (CA)2Cu(4AAP)2 complexes were characterized by the appearance of new absorbance bands at 555, 600, and 500nm for dopamine, norepinephrine, and epinephrine, respectively. The new absorbance band matched with a broad surface plasmon resonance band of utilized silver nanoparticles: 450-600nm, and 633excitation wavelength. We observed enhancement factors up to 3.6·106 due to the additional resonant enhancement. The multiplexing capabilities of quantitative spectral unmixing for Raman spectra of a group of CAs, which differ by only either hydroxy or methyl group, at the fingerprint region were successfully demonstrated with the direct classic least squares model. The achieved nM limits of detection with only 1.5 mW laser power and analysis of spiked human blood plasma samples proved the possibility of the multiplex determination of the catecholamines at the level of reference concentrations in the blood of healthy people as well as promise for the future facilitation in the precision diagnosis of neuroendocrine tumors and neurodegenerative diseases.

Nonenzymatic glucose sensor design based on carbon fiber ultra-microelectrode: Controlled with a manual micro adjuster

Interest in designing and manufacturing glucose sensors based on metal oxide-modified microelectrodes is growing and leading to the increased research efforts to develop continuous glucose measurements. A non-enzymatic glucose sensor based on an ultra-microelectrode is presented. A carbon fiber microelectrode electrodeposited by nickel nanoparticles (NiCFME) and activated as a nonenzymatic glucose sensor. The modified carbon microfiber was attached to a micro adjuster to adjust the height of the electrode inside the solution, which improved the accuracy of the microelectrode performance. The microstructure and morphology of the electrodes and nanoparticles investigated using SEM, EDX and XRD. The electrocatalytic glucose oxidation behavior of the sensing NiCFME got analyzed by cyclic voltammetry and amperometric measurements in alkaline medium. Achieved results demonstrated that the fabricated sensor displays the sensitivity up to 8.5 μA μM−1 cm−2 with a low detection limit of 3.0 μM, LOQ of 10.0 μM, with a linearity range of 10.0 μM–150.0 μM and response time about 0.4 s for glucose detection. Designed sensor had an appropriate good stability and significant selectivity towards glucose. Finally, the proposed sensor was successfully applied in determination of glucose in human blood plasma samples. The results illustrated; the proposed design is a promising candidate for the development of nonenzymatic glucose sensors.

IDF21-0324 Clinical Validation of Nano-Interfaced Electrochemical Biosensor to Detect Methylglyoxal in subjects with T2DM

Background: Methylglyoxal (MG), a product of glycolysis, is involved in formation of advanced glycation end-products. It is associated with complications including neuropathy, kidney disease, retinopathy, and cardiovascular disease. Point of care devices to detect MG may have greater utility in screening of complications in diabetes. Aim: The present study was designed to determine the level of MG in human blood plasma of patients with type 2 diabetes mellitus using a newly developed electrochemical biosensor. The concordance of the MG values was compared with the commercially available ELISA kit. Method: We have designed and developed an electrochemical biosensor to detect MG using a surface-modified platinum electrode. This involved synthesis and characterization of ceria nano-interface material to be used to functionalize the working electrode. Electrochemical studies were carried out with optimized experimental parameters using the Glyoxalase-1 enzyme immobilized surface modified electrode. Subsequently, clinical studies were conducted to detect various concentrations of MG in human blood plasma samples and the results were validated against Immunotag Human Methylglyoxal ELISA kit (United states). We collected plasma samples from DM subjects (number of 175) and equal number of non-diabetic subjects to validate the MG sensor. Results: The developed biosensor for detection of MG exhibited a linear range of 1.0 to 7.5 micromolar with a sensitivity of 4.163 microampere per micromolar, a limit of detection of 0.21 micromolar, a limit of quantification of 0.70 micromolar and a response time less than 10 s towards the detection of MG. There was a 90 percent concordance of the MG measured by the sensor to that of MG measured by commercially available ELISA kit. Discussion: The developed biosensor showed a significant correlation with HbA1c (correlation coefficient (r) is equal to 0.674, p value is less than 0.05), fasting glucose (correlation coefficient (r) is equal to 0.780, p value is less than 0.05). To conclude, we have developed an electrochemical biosensor to detect MG. This MG detected by the sensor correlates with HbA1c, fasting glucose and can be explored to use as a point of care device to screen for complications of diabetes.

Infrared pulsed fiber laser-produced silver-109-nanoparticles for laser desorption/ionization mass spectrometry of amino acids.

Application of monoisotopic cationic 109Ag nanoparticles (109AgNPs) obtained by pulsed fiber laser (PFL) 2D galvo-scanner (GS) laser generated nanomaterial (LGN) for both high resolution laser desorption/ionization mass spectrometry and mass spectrometry imaging of amino acids is presented. Four amino acids, alanine, isoleucine, lysine, and phenylalanine were used as test compounds for quantification with matrix-assisted laser desorption/ionization mas (MALDI)-type mass spectrometer. Comparison of commonly made manual measurements with semiautomatic mass spectrometry imaging (MSI) was performed providing very interesting findings. Amino acids were directly tested in 1000 000-fold concentration change conditions ranging from 1mg/ml to 1ng/ml, which equates to 500 ng to 500 fg of amino acid per measurement spot. Methods were also tested on samples of human blood plasma for quantification of endogenous amino acids.

A Nuclear Magnetic Resonance Spectroscopy Method in Characterization of Blood Metabolomics for Alzheimer's Disease.

There is currently a crucial need for improved diagnostic techniques and targeted treatment methods for Alzheimer’s disease (AD), a disease which impacts millions of elderly individuals each year. Metabolomic analysis has been proposed as a potential methodology to better investigate and understand the progression of this disease. In this report, we present our AD metabolomics results measured with high resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) on human blood plasma samples obtained from AD and non-AD subjects. Our study centers on developments of AD and non-AD metabolomics differentiating models with procedures of quality assurance (QA) and quality control (QC) through pooled samples. Our findings suggest that analysis of blood plasma samples using HRMAS NMR has the potential to differentiate between diseased and healthy subjects, which has important clinical implications for future improvements in AD diagnosis methodologies.

Bio-enhanced polyrhodanine/graphene Oxide/Fe3O4 nanocomposite with kombucha solvent supernatant as ultra-sensitive biosensor for detection of doxorubicin hydrochloride in biological fluids

Cardiotoxicity and other side effects of current antineoplastic agents with narrow therapeutic windows, such as doxorubicin hydrochloride (DOX), have increased the need for advanced real-time monitoring approaches to control their dosage and minimize common side effects. In this study, we reinforced the biocompatible and electrosensitive polymeric structure of polyrhodanine with iron(II,III) oxide (Fe3O4) and coated graphene oxide (GO) with Fe3O4 (GO-Fe3O4) nanoparticles. The resulting hybrid polymeric platform was bio-enhanced by extraction of kombucha solvent supernatant and used as a highly sensitive biosensor with enhanced electroactive CN+ functional groups for real-time detection of DOX in the human blood plasma sample. The developed platform was well characterized by performing different analyses, and its applicability was evaluated using an electrochemical approach. The results showed the superior sensitivity (167.62 μA μM−1 cm−2), low detection limit (DL, 0.008 μM), and quantification limit (QL, 0.056 μM) of the proposed biosensor. The biosensor detected DOX in the biological fluid with optimal accuracy (>99%), highlighting the ideal performance of the fabricated polymeric biosensor.

Simultaneous electrochemical detection of Cd and Pb in aquatic samples via coupled graphene with brominated white polyaniline flakes

Severe toxicity of heavy metal ions and their adverse effects on the ecosystem and health of living species raised a requirement to develop practical detection configurations capable of simultaneous and differentiable detection of toxic metal ions in aquatic samples. In this regard, we have fabricated a high-performance sensor configuration based on the integrated reduced graphene oxide (rGO) flakes with brominated white polyaniline (PANi) flakes toward the prompt and accurate detection of Pb (II) and Cd (II) in biological and non-biological specimens. The as-developed configuration simultaneously identified both Pb (II) and Cd (II) through a differentiable manner with a limit of detection (LOD)/quantification limit (QL) of 7.3 nM/85 nM and 6.5 nM/92 nM, respectively. The sensor also showed superior sensitivity of about 4547.77 and 3914.01 µA.µM−1.cm−2 toward Pb (II) and Cd (II), respectively, along with a favorable recovery rate in actual human blood plasma and wastewater samples. The as-developed complex proved to be an ideal platform for simultaneous detection/recovery of heavy metal ions in various aquatic samples.

Highly selective and sensitive detection of glutathione over cysteine and homocysteine with a turn-on fluorescent biosensor based on cysteamine-stabilized CdTe quantum dots

In this work, cysteamine (CA) stabilized CdTe quantum dots (QDs) (CA-CdTe QDs) and sodium citrate stabilized gold nanoparticles (AuNPs) were prepared. Because of the strong electrostatic interaction and spectral overlap of emission spectrum of CA-CdTe QDs and absorption spectrum of AuNPs, a highly effective fluorescence resonance energy transfer (FRET) system was formed and the fluorescence of CA-CdTe QDs was strongly quenched. The synthesized CA-CdTe and AuNPs were self-assembled to large clusters due to the electrostatic attraction and the fluorescence of CA-CdTe was sharply quenched as a result of FRET. Under the optimum pH of 5.5, the positive GSH could assemble with negative AuNPs through electrostatic interaction and destroy the FRET system of CA-CdTe and AuNPs, due to the red shift of absorption wavelength of AuNPs caused by aggregation. The fluorescence of CA-CdTe recovered, and the recovered fluorescence efficiency shows a linear function against the GSH concentrations from 6.7 nM to 0.40 μM, with a detecting limit of 3.3 nM. The quenched emission of CA-CdTe could be recovered attributed to the aggregation of AuNPs by GSH. Under optimal conditions, the sensing system was successfully applied in the detection of GSH in real human blood plasma samples with a recovery of 99.5–102.3%, showing a promising future for the highly sensitive and selective GSH detection in the human blood plasma samples.

Organobase assisted synthesis of Co(OH)2 nanosheets enriched with oxygen vacancies for nonenzymatic glucose sensing at physiological pH

Herein, a two-dimensional (2D) Co(OH)2 nanosheet (NS) was prepared by reacting Co(II) acetate with 2-ethylimidazole and trimethylamine at room temperature. The as-prepared Co(OH)2 NS-modified screen-printed electrode enabled glucose sensing in human blood plasma samples (HBP) diluted with phosphate buffer saline (PBS, pH 7.4) and NaOH (0.1 M, pH 13.0) solutions. The electrocatalytic activity of Co(OH)2 NS for glucose oxidation in a wide pH range can be ascribed to the enriched oxygen vacancies with a high surface area of the porous NS, which enabled the facile formation of CoII/CoIII redox pair. The oxidation current response as a function of glucose concentration displayed two straight lines in the ranges 55.12–117.50 μM and 180–12055 μM in PBS diluted HBP, and 62.80–180 μM and 305–12055 μM in NaOH diluted HBP with the sensitivity of 430 and 98.18 μA/cm2/mM and the limit of detection of 0.082 and 2.2 μM, in PBS and NaOH diluted HBP solutions, respectively. The sensor also exhibited good long-term stability, repeatability, and interference-free signals. The good analytical performance of the proposed sensor demonstrates the suitability of Co(OH)2 NS as a material to fabricate disposable systems for the direct detection of glucose in human body fluids at physiological pH.

A facile method for the fabrication of hierarchically structured Ni2CoS4 nanopetals on carbon nanofibers to enhance non-enzymatic glucose oxidation.

Unique Ni2CoS4-carbon nanofiber (CNF) composite nanostructures were fabricated using a simple electrospinning-assisted hydrothermal route and used for the rapid and accurate electrochemical oxidation of glucose in real samples at the trace level. Electrochemical impedance spectroscopy and cyclic voltammetry of unmodified and modified electrodes revealed low charge-transfer resistance and the excellent electrocatalytic sensing of glucose when using the Ni2CoS4-CNF at a low potential due to the combined benefits of the highly conductive Ni2Co2S4 anchored to the large surface area of the CNFs. Amperometric analysis of the fabricated sensor has shown an extremely low limit of detection (0.25nM) and a large linear range (5-70nM) for glucose at a working potential of 0.54V (vs. Hg/HgO). The practicability of the Ni2CoS4-CNF for use in glucose determinationwas tested withl human saliva, blood plasma, and fruit juice samples. The Ni2CoS4-CNF/GCE showed acceptable recovery values for human saliva (99.1-100.8%), blood plasma (98.6-101.5%), and fruit juice (95.1-105.7%) samples. The proposed sensor also exhibited outstanding electroanalytical characteristics for glucose oxidation in these samples, including reusability, repeatability, and interference resistance, even in the presence of other biological substances and organic and inorganic metal ions.

Terahertz spectroscopy of diabetic and non-diabetic human blood plasma pellets.

.Significance: The creation of fundamentally new approaches to storing various biomaterial and estimation parameters, without irreversible loss of any biomaterial, is a pressing challenge in clinical practice. We present a technology for studying samples of diabetic and non-diabetic human blood plasma in the terahertz (THz) frequency range.Aim: The main idea of our study is to propose a method for diagnosis and storing the samples of diabetic and non-diabetic human blood plasma and to study these samples in the THz frequency range.Approach: Venous blood from patients with type 2 diabetes mellitus and conditionally healthy participants was collected. To limit the impact of water in the THz spectra, lyophilization of liquid samples and their pressing into a pellet were performed. These pellets were analyzed using THz time-domain spectroscopy. The differentiation between the THz spectral data was conducted using multivariate statistics to classify non-diabetic and diabetic groups’ spectra.Results: We present the density-normalized absorption and refractive index for diabetic and non-diabetic pellets in the range 0.2 to 1.4 THz. Over the entire THz frequency range, the normalized index of refraction of diabetes pellets exceeds this indicator of non-diabetic pellet on average by 9% to 12%. The non-diabetic and diabetic groups of the THz spectra are spatially separated in the principal component space.Conclusion: We illustrate the potential ability in clinical medicine to construct a predictive rule by supervised learning algorithms after collecting enough experimental data.

2D metal azolate framework as nanozyme for amperometric detection of glucose at physiological pH and alkaline medium.

Thesynthesis of Co-based two-dimensional (2D) metal azolate framework nanosheets (MAF-5-CoII NS) is described usinga simple hydrothermal method. The product was isostructural to MAF-5 (Zn). The as-prepared MAF-5-CoII NS exhibited high surface area (1155m2/g), purity, and crystallinity. The MAF-5-CoII NS-modified screen-printed electrode (MAF-5-CoII NS/SPE) was used for nonenzymatic detection of glucose in diluted human blood plasma (BP) samples with phosphate buffer saline (PBS, pH7.4) and NaOH (0.1M, pH13.0) solutions. The MAF-5-CoII NS nanozyme displayed good redox activity in both neutral and alkaline media with the formation of CoII/CoIII redox pair, which induced the catalytic oxidation of glucose. Under the optimized detection potential, the sensor presented a chronoamperometric current response for the oxidation of glucose with two wide concentration ranges in PBS-diluted (62.80 to 180μM and 305 to 8055μM) and NaOH-diluted (58.90 to 117.6μM and 180 to 10,055μM) BP samples, which were within the limit of blood glucose levels of diabetic patients before (4.4-7.2mM) and after (10mM) meals (recommended by the American Diabetes Association). The sensor has a limit of detection of ca. 0.25 and 0.05μM, respectively, and maximum sensitivity of ca. 36.55 and 1361.65mA/cm2/mM, respectively, in PBS- and NaOH-diluted BP samples. The sensor also displayed excellent stability in the neutral and alkaline media due to the existence of hydrophobic linkers (2-ethyl imidazole) in the MAF-5-CoII NS, good repeatability and reproducibility, and interference-free signals. Thus, MAF-5-CoII NS is a promising nanozyme for the development of the disposable type of sensor for glucose detection in human body fluids. Graphical abstract.

Identification of 2-(diethylamino)ethylthiol dipeptide (Cys-Pro) adduct as biomarker of nerve agents VR and CVX in human plasma using liquid chromatography-high-resolution tandem mass spectrometry.

Organophosphorus nerve agents pose a significant threat to human health. The most toxic compounds in this class include V-type poisonous substances such as VX, CVX, and VR. Although all stockpiles of this type of substance are subject to destruction under the Chemical Weapons Convention (CWC), there is still a risk that they could be used for criminal and terrorist purposes. The latter determines the relevance of studies aimed at identification of biomarkers that may indicate the exposure of these group substances to the organism. A liquid chromatography mass spectrometry/high-resolution mass spectrometry (LC-MS/HR MS) method for determination of trace amounts of nerve agents such as VR and CVX in human plasma was proposed. The method is based on enzymatic plasma hydrolysis with the use of pronase to form a stable adduct of 2-(diethylamino)ethylthiol with dipeptide cysteine-proline (DEAET-CP) with its subsequent determination by LC-MS/HR MS. Synthesis of DEAET-CP as reference compound was conducted using non-toxic precursors. Sample preparation of human blood plasma samples exposed to VR was carried out with the use of solid-phase extraction (SPE). Liquid chromatography (LC) separation on the reversed-phase column and mass spectrometric detection (selection of optimal transitions and detection modes) were performed. The achieved limit of detection (LOD) of VR (in the form of DEAET-CP) in human blood plasma was 0.05ngmL-1. The proposed approach was developed using plasma samples exposed to VR and CVX obtained in the frame of the Fifth Official Biomedical Test of the Organization for the Prohibition of Chemical Weapons (OPCW) and showed good specificity of detection.

Developing an integrated microfluidic and miniaturized electrochemical biosensor for point of care determination of glucose in human plasma samples

A cost-effective, point of care (POC) device based on highly oriented CNT arrays was developed as an electrochemical assay for real-time and sensitive detection of glucose in complex samples. A low-cost, microcontroller-based potentiostat consisting of Arduino Due and LMP9100-EVM was developed to perform electrochemical measurements such as cyclic voltammetry (CV) and amperometry. A syringe pump based on open-source electronics was designed to direct the flow through a microfluidic chip. Vertically aligned carbon nanotube (VACNT) sensor arrays, in combination with the miniature potentiostat and the syringe pumps, were utilized as a POC device for the rapid and accurate detection of glucose. The structure and morphology of samples were characterized by field emission scanning electron microscopy (FESEM) and attenuated total reflectance Fourier transform infrared spectrometry (ATR-FTIR). CV as well as electrochemical impedance spectroscopy (EIS) was performed to investigate the electrochemical behavior of the electrode with respect to different diffusion regimes. The mediator-less biosensor had a limit of detection of 23μM and sensitivity of 1462μAmM-1cm-2 and 1050μAmM-1cm-2 at the linear range of 1.2-7.8mM and 7.8-11.2mM, respectively. The presence of other biological compounds such as uric acid (UA) and ascorbic acid (AA) did not interfere with the detection of glucose. Finally, the designed POC device was successfully applied for the determination of glucose in human blood plasma samples.

Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics.

Proteomic tools are especially useful when it comes to investigating complex samples such as human blood plasma, in which protein quantities can span across up to ten orders of magnitude. Ultra definition mass spectrometry, in combination with two-dimensional liquid chromatography, provides better coverage of complex proteomes and allows for better control of collision energy, keeping the fragmentation benefits of high collision energy associated with drift time measurements from ion mobility separation. Here, we present a protocol to assist in the identification of proteins in human blood plasma and other similar samples with a large dynamic range.

A Voltammetric Sensor Based on a 3,4,9,10-Perylenetetracarboxylic Acid Composite for the Recognition and Determination of Tyrosine Enantiomers

An enantioselective voltammetric sensor based on a glassy carbon electrode modified with a composite of a polyelectrolyte complex of chitosan, Carboblack C graphitized thermal carbon black, and 3,4,9,10-perylenetetracarboxylic acid is developed for the recognition and determination of tyrosine enantiomers. The enantioselectivity of the sensor is due to the formation of self-organizing chiral nanoclusters of 3,4,9,10-perylenetetracarboxylic acid. The electrochemical and analytical characteristics of the sensor are studied. The proposed sensor is used to recognize and determine L- and D-enantiomers of tyrosine in samples of urine and human blood plasma, as well as in a mixture. To increase the probability of the recognition of tyrosine enantiomers in determining them in a mixture, the chemometric method of projection to latent structures is used. It is shown that the proposed sensor ensures the determination of the ratio of L- and D-enantiomers of tyrosine in a mixture with a high probability and a relative error of less than 8%.

A Disposable Amperometric Immunosensor for the Monitoring of Granzyme B in Blood Plasma Samples of Cancer Patients

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which participates in inducing apoptosis of target cells for NK cells and cytotoxic CD8+ lymphocytes [1]. GzmB is the major effector of the CTLs and NK cells in its killing attack on cancer cells, although the level of GzmB expression and its cytotoxic potential decrease significantly in the presence of cancer. This decrement is due to a ‘pro-cancer’ environment, where the cancer cells secrete soluble mediators that could inhibit and thus contribute to the decreased GzmB by CD8+ T cells and a reduction in their ability to cause apoptosis of the cancer cells. Once GzmB is secreted from cytotoxic cells, it can be discovered in two different host compartments, namely inside the target cell or extracellularly [2]. Due to this, the change of GzmB level in blood plasma may serve as indication of the patient’s cancer cure progress. Hence, we developed a disposable amperometric GzmB sensor for the blood plasma samples.In developing a robust electrochemical sensing platform, an efficient device is required. To enhance the electrochemical response, catalytic nanoparticles can be used as the electrode material, particularly nanocomposites composed of functionalized conducting polymers. They have been well-thought-of as the candidate of electrode substrate materials for the electrochemical sensing platform due to their ability to augment the electrical conductivity, electron-transfer rate, mechanical strength, surface area, and binding affinity to a specific molecule [3]. Therefore, 3’-(2-aminopyrimidyl)-2,2’:5’,2’’-terthiophene (PATT) bearing an amine group was electro-polymerized onto gold nanoparticles (AuNPs) modified electrode. Then, the stabilized immobilization of antibody on the SPCE/AuNPs/pPATT layer was accomplished through the formation of an amide bond between amine groups of the polymer and carboxylic acid groups of the antibody.In this study, a disposable amperometric immunosensor composed of a sensing probe and a bioconjugate was developed for the detection of GzmB. The sensing probe was fabricated by immobilizing the GzmB monoclonal antibody (Ab1) on the polyPATT/AuNPs layer. The bioconjugate particle was synthesized by self-assembling the monomer mixture of 2,2:5,2-terthiophene-3-(p-benzoic acid) (TBA) and PATT onto AuNPs, then covalently bonding brilliant cresyl blue (BCB) on pTBA and GzmB polyclonal antibody (Ab2) on pPATT layer, respectively. The sensing layers were characterized using the surface analysis, cyclic voltammetry, impedance spectroscopy, and X-ray photoelectron spectroscopy.A redox peak of the bioconjugate (Ab2-SAM-BCB) in the presence of GzmB in the sample was observed at -0.35/-0.37 V, corresponding to the redox reaction of the redox indicator BCB. Optimization of experimental parameters were carried out in terms of temperature (35°C), pH (7.4), the concentration of antibody (300 μg/ml), applied potential (-0.38 (reduction)), and binding time (20 min). At the optimized conditions, the developed immunosensor showed a dynamic range from 3.0 to 50.0 pg/ml and from 50.0 to 1000.0 pg/ml in two slops, with a detection limit of 1.75 ± 0.14 pg/ml. Interference effect, stability, and reproducibility were also evaluated. Reliability of the proposed immunosensor was evaluated through comparison of the results among GzmB concentration of lung cancer patients before and after medicine treatment, and healthy volunteer using human blood plasma samples, where the low level of GzmB was present in the blood plasma of lung cancer patients before medicine treatment (10.50 ± 1.02 pg/ml), which slightly increased level in patients after medicine treatment (15.85 ± 1.99 pg/ml), and the significantly higher level was present in the normal healthy individual (39.62 ± 0.79 pg/ml). Conclusively, the progress of cancer before and after the cure of the patients has been evaluated through monitoring GzmB concentration in the blood plasma.[1] Kurschus, F.C., Jenne, D.E., 2010. Immuno. Rev. 235, 159-171.[2] Boivin, W.A., Cooper, D.M., Granville, D.J., 2009. Lab. Invest. 89, 1195-1220.[3] Naveen, M.H., Gurudatt, N.G., Shim, Y.-B., 2017. Appl. Mater. Today 9, 419–433.

Trial Results for ELISA Test Kits for HBsAg Subtype and Hepatitis B Virus Genotype Identification in Human Blood Plasma

For personalized treatment of chronic HBV-infected patients, it is necessary to identify hepatitis B virus (HBV) surface antigen (HBsAg) subtypes and HBV genotypes circulating among infected individuals. To this end, a laboratory version of the test kit for HBsAg subtype and HBV genotype identification in human blood serum/plasma using enzyme-linked immunosorbent assay (ELISA) and a custom monoclonal antibody (MAb) panel has been developed. With this kit version, it appears possible to identify the main HBsAg subtypes (ayw2, ayw3, adw2, and adrq+) and HBV genotypes (A, C, and D) circulation in Russian Federation. The aim of the work was to test the laboratory-developed ELISA kits for HBsAg subtyping and HBV genotyping in human HBsAg-positive blood plasma samples. A total of 146 HBsAg-positive plasma samples were tested. For 108 samples with detectable viral DNA, the results of HBsAg subtyping and HBV genotyping using the suggested MAb panel were compared with the results of genomic DNA sequencing for HBV isolates. For 38 HBsAg-positive samples in which HBV DNA could not be detected, ELISA, using the highly specific MAb panel kindly provided by P. Swenson, PhD, was employed as a reference technique. The study demonstrated that for the group of HBsAg-positive samples with detectable HBV DNA, the coincidence between the results obtained using the suggested MAb panel and the results of HBV DNA genome sequence analysis was 85% for HBsAg subtyping and 98% for HBV genotyping. For the group of HBsAg-positive samples with undetectable HBV DNA, the coincidence between the two techniques (the one using the suggested MAb panel and one using Swenson’s MAb panel) was 95% in the case of HBsAg subtyping and 100% for HBV genotyping. The reported results allow recommending the suggested laboratory ELISA test kit for HBsAg subtype and HBV genotype identification in human blood plasma as an alternative or a complementary technique to genomic analysis in order to study HBV characteristics especially in those cases when HBV DNA isolation appears impossible.

Human biomonitoring of per- and polyfluoroalkyl substances in German blood plasma samples from 1982 to 2019

The findings of per- and polyfluoroalkyl substances (PFAS) in humans and the environment all over the world have raised concerns and public awareness for this group of man-made chemicals. In the last three decades, this led to different regulatory restrictions for specific PFAS as well as shifts in the production and usage of these substances. In this study, we analyzed the PFAS levels of 100 human blood plasma samples collected from 2009 to 2019 for the German Environmental Specimen Bank (ESB) to further elucidate the time course of exposure towards this substance group as shown by Schröter-Kermani et al., (2013) with samples from 1982 to 2010. A spectrum of 37 PFAS, including perfluorocarboxylic (PFCA) and –sulfonic acids (PFSA) as well as potential precursors and substitutes like ADONA, GenX or F-53B was analyzed by UHPLC coupled with high-resolution mass spectrometry. Validation was successful for 33 of the substances.The two legacy substances perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) were detected in every sample of the 2009–2019 dataset and showed the highest concentrations with ranges of 0.27–14.0 ng/mL and 1.21–14.1 ng/mL, respectively. A significant portion of total PFOS analytes was present as branched isomers (mean: 34 ± 7%). High detection frequencies of 95% and 82% were also found for perfluorohexane sulfonic acid (PFHxS) and perfluorononanoic acid (PFNA), respectively, but in lower concentrations (PFHxS: <LOQ – 4.62 ng/mL; PFNA: <LOQ – 3.66 ng/mL) than PFOA and PFOS. Besides other PFCA and PFSA only 8:2 fluorotelomer sulfonic acid (8:2 FtS) and N-methyl perfluorooctane sulfonamidoacetic acid were detected in very few samples. In combination with the previous results from 1982 to 2010, declining temporal trends were observed for all PFAS (PFOA, PFNA, PFHxS, and PFOS) frequently detected in the ESB samples. The results of this study indicate a decrease in human exposure to known PFAS in Germany over the last three decades and emphasize the importance of long-term human biomonitoring studies for investigating the effects of chemical regulation.