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The aim of our work was to develop a low-cost, portable device for the fast and easy determination of total protein content by using PDMS-based lab-in-a-syringe technology with removal of 3D-printed channels. We proposed two designs with a one-step PDMS curing and a two-step PDMS-curing fabrication procedure. The one-step PDMS microdevices were found to be the best in the view of preparation, repeatability, and stability of the reagent. This design was then applied for the determination of total protein content in biomedical products using the Bradford assay.

Objectives: The objective of this study was to perform selenium yeast production and analyze the selenium and protein contained in selenium yeast obtained. Methods: Selenium yeast production was carried out by incubating the stationary phase culture of yeast with selenium solution for 24 hr, and yeast biomass was dried by Freeze-dryer to produce selenium yeast in powder form, then the selenium yeast was determined to the selenium content with atomic absorption spectrophotometer at a wavelength of 196 nm and protein content analyzing by the Bradford method at a wavelength of 595 nm using UV-Vis spectrophotometer. Results: Selenium yeast production reached 2.5 g of selenium yeast in powder form. The selenium and protein content of selenium yeast was 4258.0096 (±278.39) μg Se/g and 0.8505 (±0.0045) mg/mL, respectively. The method was easy, simple, and can prove that selenium yeast can still be produced and analyzed for the selenium and protein content. Conclusion: The present study demonstrated that production of selenium yeast in the form of dry powder could be carried out using the stationary phase culture of S. cerevisiae incubated with 30 μg/mL selenium solution, and the resulting selenium yeast can be analyzed for selenium content using atomic absorption spectrophotometer and protein content by Bradford method using UV-Vis spectrophotometer.

Developing accurate methods for the assessment of therapeutic protein release from polymer drug delivery systems (microcapsules, microspheres, nanoparticles, 3D-printed systems) is of paramount importance for new formulation development. The most straightforward method for protein release assessment is spectrophotometric analysis of the release medium surrounding the formulation. However, direct spectrophotometric analysis is inapplicable to formulations releasing interfering compounds (co-encapsulated drugs, additives) absorbing light in the same spectrum as proteins. Conventional protein release assays also require frequent release medium sampling and replacement, which reduces their accuracy. We propose a one-step method to assess protein release from core/shell microcapsules eliminating the need for sampling and allowing selective real-time protein quantitation in the release medium. To prevent spectral interferences, released protein is differentiated from interfering compounds by employing a colorimetric protein assay reagent, forming a colour complex selectively with the protein, as the release medium. To eliminate sampling, we employed a continuous flow closed loop set-up, where the release medium is constantly circulating between microcapsule-containing tank and spectrophotometer. A series of colorimetric protein assay reagents (bromocresol green, tetrabromophenol blue, eosin B, eosin Y, biuret) were evaluated in terms of their applicability as the release medium in described system. Only biuret reagent was found compatible with proposed method due to formation of color complex stable over extended period of time and low adsorption to microcapsules. Presented method allowed effective evaluation of albumin release from alginate-polyethersulfone microcapsules with accuracy equal to conventional ‘sample and separate’ technique. Albumin release followed first-order kinetics with plateau reached after 19 h.

Analysis and identification of seized doping-related products are important tasks for customs or forensic laboratories in order to prevent potentially dangerous and illegal compounds to go into circulation. At the Section of Forensic Chemistry in Copenhagen, we have a workflow consisting of four complimentary validated methods to identify common doping-related substances: liquid chromatography-ultraviolet (LC-UV), LC coupled with time of flight mass spectrometry (LC-TOF-MS), the colorimetric Bradford assay, and an immunoassay. The Bradford assay screens for peptide or proteins in the sample, and the immunoassay confirmed human chorionic gonadotropin (hCG). LC-UV was carried out with a C4 protein column for identification of peptides and proteins from a standard reference library, based on retention times and ratios between peak areas at 220, 254, and 280 nm. LC-TOF-MS was performed using a C18 column, and identification was based on comparison of the retention time and the accurate mass with those of reference standards. In 2019, we received 36 samples for peptide/protein analysis, all of which were tested using the LC-UV, LC-TOF-MS, and colorimetric method, and samples suspected of containing hCG were confirmed with an immunoassay. We found a total of 15 samples containing an illegal doping substance, 12 samples containing substances not prohibited by the Danish Doping List, and nine samples containing no peptides or proteins. In conclusion, the four complimentary methods constitute a suitable approach for identifying common peptide/protein doping substances in the day-to-day routine of a forensic laboratory, with limited sample preparation and interpretation of data.

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (

Lectins arecarbohydrate-bindingproteinsthat are highly specific for sugarmoietiesof other molecules. They perform recognition on the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Blood groups are inherited characters which give rise to antigen-antibody reaction. A total of 120 samples of local (Nigeria)Achatina achatinasnail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used for all the actual tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination tests, Protein Assay and Specific Sugar determinations. The molecular weight was assessed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. The respective haemagglutination tests on the crude, partially and affinity purified lectin showed on standardisation, preferential agglutination with Blood group A type. The Protein contents of the lectin was deduced to be as follows: The crude extract contains 13.5mg/dl, Dialysed precipitate – 5.7mg/dl, Dialysed supernatant – 5.0mg/dl and the Affinity purified Lectin – 0.422mg/dl. Galactose N-acetyl amine (Gal NAc) residue was determined to be its specific sugar. The SDS-PAGE analysis showed the molecular weight of the lectin to be 250 KDaltons. This research has therefore succeeded in the Purification, Characterisation and illustration of the lectinic properties of the local Nigeria snail -Achatina achatina.

Protein extraction is a preliminary step of protein purification which mainly focus on maximization of total protein yield. The heterogeneous properties cause diversification of protein; therefore, there is no absolute protocol in protein extraction. The ratio of buffer gives different protein concentrations in different types of mammalian tissues, and this condition leads to the study of optimization of buffer ratio to obtain a better total protein yield. The objectives of this study were to compare the total protein yield based on three different ratios of buffer used. The phosphate buffer saline (PBS), radioimmunoprecipitation assay (RIPA) buffer, and RIPA buffer with the addition of protease inhibitor (Pi) were used with the ratios of 1:1, 1:3, and 1:5. Fetal cotyledons removed from the placenta have undergone mechanical disruption, incubation, sonication, and centrifugation. The supernatant was retained and quantified with Bradford assay to determine the total protein yield based on the standard curve of bovine serum albumin (BSA). There was a statistically significant different between buffer ratio (p

The present study aims to investigate the effects of exposure of the seminal plasma and spermatozoa of the giant grouper Epinephelus lanceolatus to dry ice (−79 °C) during transport on their quality. In all, 15 amino acid compounds were determined. The quantification of total proteins were measured using the Bradford method, and amino acid concentration were measured using the HPLC method. The cryopreserved seminal plasma was transferred from a liquid nitrogen tank to a styrofoam box filled with dry ice. Total protein and amino acids were measured after 24, 48, and 72 h. For comparative purposes, total protein and fifteen compound of amino acid were also measured. Both parameters were also measured after the cryopreserved seminal plasma were immersed in liquid nitrogen after 24 and 48 h exposed to dry ice. The results showed that the exposure of seminal plasma to dry ice for 24, 48 and 72 h during transportation or immersion back into the liquid nitrogen after 24 and 48 h does not change the total protein levels either in seminal plasma or spermatozoa. However, the level of each amino acid compound in the seminal plasma had significantly decreased.

Abstract Characterization of selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates prepared in nanopowder form, their elemental composition, and particle size distribution were investigated with a microwave plasma optical emission spectrometry operating in a single-particle mode. This new analytical technique was used for the first time to examine the molecular interaction between selenium nanoparticles and human serum albumin regarding potential biomedical applications of selenium nanoparticles. Nanopowder sample was introduced to a helium plasma by pneumatic nebulization based on fluidized bed approach and measured with a time resolution of 20 ms. Both selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates were characterized by observation of synchronous signals from different particle components. Plots of the time correlation between Se and C signals for all particles in selenium nanoparticles and selenium nanoparticle–human serum albumin conjugates samples differed from each other in degree of correlation and synchronicity of recorded signals. The interaction between selenium nanoparticles and human serum albumin was confirmed using Bradford assay. For selenium nanoparticles synthesized using yeast cells, the percentage of bound protein was only of 4%, whereas for selenium nanoparticles synthesized using yeast extract as a stabilizing agent it was 16%. Graphic abstract

Commercial colorimetric protein assays have been widely used, but their performance for protein quantitation in thermally processed milk samples has not been investigated. This study evaluated four colorimetric assays for quantitation of soluble protein in nonfat milk samples that were subjected to different degrees of heat treatment. Copper ion-based Pierce™ bicinchoninic acid protein assay (BCA) and Pierce™ modified Lowry protein assay (modified Lowry) consistently registered higher protein levels than those determined by dye binding-based Pierce™ Coomassie Plus (Bradford) assay (Coomassie Plus) or Pierce™ 660-nm protein assay (660-nm). When milk samples were autoclaved or dry-heated at ≥ 121 °C for 10 min, reduction in soluble protein content was reflected in measurements of Coomassie Plus and 660-nm assays but was not indicated by BCA or modified Lowry assays. In samples dry-heated at 176 °C, while no protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie Plus or 660-nm, BCA, and modified Lowry indicated protein levels that were 215.7 and 79.4%, respectively, of those of unheated samples. Analysis of lactose samples showed that lactose may contribute to a 13.4 or 1.0% overestimation in BCA or modified Lowry measurement of protein content in unheated dry milk samples. This study demonstrated that quantitation of protein in thermally treated milk samples using copper ion-based BCA and modified Lowry assays could be significantly affected by lactose and, to a greater degree, other reducing substances formed during heating likely via the caramelization and Maillard reaction pathways. In contrast, dye binding-based Coomassie Plus and 660-nm assays were not affected and may be more accurate for measurement of protein content in thermally treated milk samples.

Objective: An injectable long acting In-Situ Forming Gel (ISFG) of human Growth Hormone (hGH) was prepared by using triblock PCL-­‐PEG-­‐PCL (Mw 1500-1500-1500). Ring-Opening Polymerization (ROP) of triblock using microwave was applied. Methods: The BCA protein assay Kit was used to determine the concentration of hGH in the in-vitro release medium. Finally, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) tests and Circular Dichroism (CD) spectrum were done to approve the stability of released hGH. The result of ROP demonstrated that the proportion of PCL to PEG accorded with the initial molar ratio of the monomers. The cross-section of the Surface Electron Microscopy (SEM) indicated the porous framework of the hydrogel could load the drug into its tridimensional matrixes structure. There is the low initial burst release of hGH from the supramolecular hydrogel. Results: The maximum in-vitro release of hGH was 71.2 % ± 1.5 that were due to hGH degrading after this time (21 days). The CD spectrum and SDS-PAGE results confirmed the stability of hGH during invitro release evaluation. Conclusion: The results suggest that the sustained-release formulation using PCL-PEG-PCL can be applied to control the release of hGH.

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter/min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ~30-fold. The complete western blot was applied to the detection of GAPDH and β-Tubulin from A431 cell lysate.

A microfluidic open circuit potential label-free protein assay was developed for the reagentless quantification of C-reactive protein (CRP), a model protein target, and further utilized to assess target-receptor binding kinetics. Generated sensors have very high baseline stabilities (

PDMS based multi-channel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,N'-diallyl L-tartardiamide) in an eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal-ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10-13 m2 ) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin and human serum albumin) based on their surface exposed histidine surface topography. A protein recovery of ∼95% (Bradford assay data) was achieved. The multi-monolith microchip can be reusable even after 3 protein adsorption-desorption cycles. This article is protected by copyright. All rights reserved.

Protein-based biological materials such as albumin, casein and collagen are found in various cultural heritage (CH) artefacts. This study focuses on the study of protein binders from easel paintings media. Proteins have complex structures which are difficult to identify with non-invasive spectroscopic methods (FT-IR, Raman, UV). Immunoassays such as ELISA determine the protein’s source of origin which is necessary for art objects. To increase the detection and identification of proteins by immunoassays, the efficiency of micro-extraction of proteins from heritage materials is a crucial step. Extractions mediated by cycles of orbital agitation and ultrasonic radiation give the possibility to extract proteins from easel painting sample. In this work, protein-based paint models coupled with silica nanoparticles were used for micro-extraction. Nanoparticles possess high surface-to-volume ratios that can attach bioactive molecules such as proteins and increase the total protein recovered from microsamples. Protein extracts were quantified with Bradford Assay in the presence of Coomassie blue. The protein recovery results were statistically computed, and the SPSS analysis shows significant (p 2 and above 1.6 times for MCM-41. The statistical data shows evidence that silica nanoparticles intensify the total protein recovered from paint microsamples. Finally, ELISA was realized on the protein extracts to verify and compare the immunodetection of protein from the paint models with and without the use of silica nanoparticles.

A low-cost and reliable analytical method based on the combination of a newly designed sticker-plastic sheet platform, digital image-based colorimetry and down scaled Kjeldahl digestion is proposed for the determination of protein content in food samples. The yellowish-brown colloidal products, obtained from the reaction between the ammonium–nitrogen after digestion and the working Nessler's reagent on the miniaturized sticker-plastic sheet platform, were captured for imaging with a smartphone camera. The operational parameters and reaction conditions were optimized. A down scaled Kjeldahl digestion procedure was performed using the newly designed digestion block. The parameters influencing the digestion efficiency, including the mass of the sample, volume of acid, mass of the catalyst and digestion time, were evaluated. Under the selected conditions, a linear calibration in the range of 5–60 mg L−1 ammonium–nitrogen was obtained with limits of detection and quantification of 2.8 and 7.6 mg L−1, respectively. The repeatability and reproducibility were 6.7% and 8.8%, respectively. The accuracy of the proposed method was evaluated by applying the developed procedure for milk powder and feeding stuff reference materials and comparing it with the conventional Kjeldahl method. The proposed method was successfully applied for the determination of protein contents in soy foods and protein-based foods. The results agreed well with those obtained from the conventional Kjeldahl method.

Protein quantification with high throughput and high sensitivity is essential in the early diagnosis and elucidation of molecular mechanisms for many diseases. Conventional approaches for protein assay often suffer from high costs, long analysis time, and insufficient sensitivity. The recently emerged nanoimpact electrochemistry (NIE), as a contrast, allows in situ detection of analytes one at a time with simplicity, fast response, high throughput, and the potential of reducing the detection limits down to the single entity level. Herein, we propose a NIE-enabled electrochemical immunoassay using silver nanoparticles (AgNPs) as labels for the detection of CYFRA21-1, a typical protein marker for lung carcinoma. This strategy is based on the measurement of the impact frequency and the charge intensity of the electrochemical oxidation of individual AgNPs before and after they are modified with anti-CYFRA21-1 and in turn immunocomplexed with CYFRA21-1. Both the frequency and intensity modes of single-nanoparticle electrochemistry correlate well with each other, resulting in a self-validated immunoassay that provides linear ranges of two orders of magnitude and a limit of detection of 0.1 ng/mL for CYFRA21-1 analysis. The proposed immunoassay also exhibits excellent specificity when challenged with other possible interfering proteins. In addition, the CYFRA21-1 content is validated by a conventional, well-known enzyme-linked immunosorbent assay and successfully quantified in a diluted healthy serum with a satisfactory recovery. Moreover, CYFRA21-1 detection in serum samples of lung cancer patients is successfully demonstrated, suggesting the feasibility of the NIE-based immunoassay in clinically relevant diagnosis. To the best of our knowledge, this is the first report to construct NIE-based electrochemical immunoassays for the specific detection of tumor protein biomarkers.

Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin®) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1–20 μg/mL (R2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab.

The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.

Upon intravenous administration of nanoparticles (NP) into the bloodstream, proteins bind rapidly on their surface resulting in a formation of a so-called ‘Protein Corona’. These proteins are strongly attached to the NP surface and provide a new biological identity which is crucial for the reaction at the nano-biointerface. The structure and composition of the protein corona is greatly determined by the physico-chemical properties of the NP and the characteristics of the biological environment. The overall objective of this study was to characterize the role of NP size/surface curvature and PEGylation on the formation of the protein corona. Therefore, we prepared NP in a size of 100 and 200 nm using the biodegradable polymers poly(DL-lactide-co-glycolide) (PLGA) and poly(DL-lactide-co-glycolide)-co-polyethylene glycol diblock (PLGA-PEG) and subsequently incubated them with fetal bovine serum (FBS) to induce the formation of a protein corona. After removal of unbound protein, we employed different analytical approaches to study the corona in detail. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to gain a first impression about amount and composition of the corona proteins. Identification was carried out after tryptic in-solution digestion and liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS). In addition, we successfully established the Bradford protein assay as a suitable colorimetric method to quantify total adsorbed protein amount after alkaline hydrolysis of PLGA based NP. Our results revealed that protein adsorption on PLGA- and PLGA-PEG-NP didn’t depend on NP size within the range of 100 and 200 nm. PEGylation led to a significant reduced amount of bound proteins. The depletion of proteins which are involved in immune response was remarkable and indicated a prolonged circulation time in body.

Keratin was extracted from different segments of disposable waste chicken feathers (CF) including the whole feathers, calamus/rachis (β-sheet) and barbs/barbules (α-helix), using sodium sulfide and l -cysteine. The yield of extracted keratin from sodium sulfide and l -cysteine was ˜88% and ˜66% respectively. The mass ratio of feathers to reducing agent was 1:20 and the reaction temperature was 40 °C for 6 h. Concentration of keratin extracted by each method was measured using the Bradford assay. The protein extracted from each feather section was characterised using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vibrational spectroscopy including FTIR and Raman, nuclear magnetic resonance, and thermogravimetry. These results confirmed the keratin structures after each extraction methods. The study showed that α-helix and s-sheet based keratin could be extracted from CF using sodium sulfide and l -cysteine with high yields. This is the first report of CF keratin extraction using l -cysteine.

Saxitoxin (STX) is one of the paralytic shellfish poisons (PSP) that endanger people's health. It is necessary to develop methods for the on-site rapid detection for STX in order to prevent safety accidents. An enzyme-linked immunosorbent assay (ELISA) is timesaving and effective, but it is not suitable for large-scale in-field tests due to the expensiveness of commercial ELISA kits and the bulkiness of a microtiter plate reader (MTPR). In this study, a portable smartphone-based colorimetric analyzer (SBCA) with a cost-effictive enhanced gold nanoparticle-based ELISA (EGNB-ELISA) was proposed for STX detection. In a bicinchoninic acid (BCA) protein assay (R2 = 0.9939) and a glucose assay (R2 = 0.9937), SBCA was shown to be in good agreement with MTPR. EGNB-ELISA had a 12.5-fold lower detection limit (0.4 ng/mL) and a lower detection range (1 - 50 ng/mL, Y = 0.4037X + 0.3564, R2 = 0.9797) than the classical ELISA. The recovery rate ranged over 89.1 - 112.2%. The whole detection system, combining both homemade SBCA and ENGB-ELISA, is expected to satisfy the needs of on-site STX sample tests to guarantee seafood safety.

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced.

Objective The study aims at evaluating mid-infrared human milk analyzer (HMA) accuracy and precision, in human milk (HM). Study design Röse-Gottlieb, high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), Kjeldahl and amino acid analysis (AA) were selected as references for total fat, lactose and total protein determination. Results No significant difference was observed in lactose content between HMA and HPAEC-PAD. Significant differences were observed in fat and protein content between HMA and reference methods. However, the difference in fat content was lower than 12%, and therefore within the variability declared by supplier. For protein determination, the BCA protein assay was selected. No significant differences were observed in total protein content measured by BCA assay, Kjeldahl and AA methods. Conclusions HMA was reliable for the quantification of total fat and lactose content, but not for total protein one. The latter was measured by BCA assay, which yielded comparable results to Kjeldahl and AA methods.

The objective of the present study was to evaluate different methodologies for peptide quantification in the supernatant of chitosan nanoparticles by removing the unliked polymer in the suspension. The ionic gelation method was used to prepare the chitosan nanoparticles encapsulating a 5.3 kDa peptide. Three different methodologies for the processment of the solutions were compared before subjecting the samples to the Bradford protocol or Qubit® kit for protein detection. For the quantification, it was necessary to create a standard peptide curve using different peptide concentrations. The suitable standard curve would be one in which the peptide was diluted in the empty chitosan supernatant (obtained after nanoparticles centrifugation) or in the filtrate of the empty chitosan supernatant. Our results indicated the safest methodology tested for peptide quantification in the supernatant chitosan nanoparticles was filtering the solution before subjecting the sample to the Bradford assay.

Epoxide hydrolases catalyze the hydrolysis of both exogenous and endogenous epoxides to the corresponding vicinal diols by adding water. Microsomal and soluble epoxide hydrolase are two main mammalian enzymes that have been intensely characterized. The purpose of this investigation was to develop and validate a proteomics assay allowing the simultaneous quantification of microsomal and soluble epoxide hydrolase in rats. Protein quantification was realized through targeted proteomics using liquid chromatography with tandem mass spectrometry for the determination of trypsin-specific surrogate peptides after digestion. Stable isotope-labeled peptides were used as the internal standards. The chromatography of the surrogate peptides was performed on an Agilent SB C18 column (100 mm × 4.6 mm, 1.8 µm) with gradient elution. Acetonitrile containing 0.1% formic acid and 0.1% formic acid aqueous solution were used as mobile phases. A multiple reaction monitoring method in a positive ionization mode was used for the simultaneous detection of the peptides. The method was validated concerning the specificity, linearity, within-day and between-day accuracy and precision, matrix effect, stability, and digestion efficiency. The developed assay was successfully used to quantify the protein levels of microsomal and soluble epoxide hydrolase in rat liver, kidney, and heart S9 samples.

Cactus seeds have unusual properties that resist protein extraction, e.g. a thick testa (seed coat) and a high amount of mucilage within the cells. This study was performed to determine which method (phenol extraction, TCA/acetone extraction, or phosphate-TCA/acetone extraction) is optimal for extracting protein from seeds of three species of Opuntia: Opuntia robusta, Opuntia phaeacantha, and Opuntia engelmannii. Extracted protein was analyzed using a Bradford assay and SDS-PAGE with silver staining. Overall results revealed that the TCA/acetone and phosphate-TCA/acetone methods were far more effective at extracting protein compared to the phenol extraction method. These protein extraction results can be applied to future studies of proteins important for Opuntia seed germination and conservation.

The Coomassie Blue dye binding assay is a sensitive assay for determining protein concentration in samples and is based on the method originally developed by Bradford. The assay is reproducible, straightforward and easy to perform. Furthermore, the dye is compatible with various biochemical reagents, e.g. detergent solutions, buffers, chaotropic salts, preservatives such as sodium azide, some acids such as HCI etc. The proteins dissolved or mixed in any of these reagents can be measured. The assay is based on the principle of an absorbance shift resulting from an acidic Coomassie Brilliant Blue G-250 solution. When the dye solution is added to a protein sample interaction of the dye’s sulfonic groups with the protein results in a color change from brown to blue. This change can be measured at 595, 650 and 470 nm corresponding to anionic, neutral and cationic equilibrium states respectively for the

Background and objectives Determination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes. Findings In case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay. Conclusions BSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions. Significance and novelty Findings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories.

The aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the invasive C. taxifolia and C. cylindracea and the autochthonous C. prolifera). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for C. prolifera and C. cylindracea, and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For C. taxifolia, instead, none of the tested protocols produced satisfactory results and further studies will be required.

Proteinuria is considered indicative of kidney damage that can be related to various adverse outcomes. Nowadays, there is a huge demand for routine urine screening methods to assess health risks in clinical setting without expensive procedures and long pretreatment of the sample. To address this issue, a polydopamine-based colorimetric assay to determine urinary albumin concentration in real samples is proposed here. The core of this approach relies on the established competitive adsorption of polydopamine film and human serum albumin onto the polystyrene surface of ELISA plates. Herein, we investigated the influence of temperature and the Tris-HCl buffer concentration on the polydopamine film growth. The absorbance of polydopamine film, after 24 h at 25 °C, decreases with the increase of HSA concentration, allowing the selective detection of HSA down to 0.036 ± 0.001 g L−1 in untreated urine. This simple and low-cost bioanalytical assay exhibited very good reproducibility, %CVmean = 2 in human urine, and was superior in terms of analytical performances to some standard methods available on the market, especially in comparison to the Bradford assay, for early screening and assessment of kidney damage.

This chapter explains how to perform a batch cultivation of Trichoderma reesei in bench top bioreactors, exemplarily using wheat straw as sole carbon source, and a selection of recommended, frequently used analyses to monitor the cultivation (intra- and extracellular as well), which are microscopic analysis, sodium hydroxide soluble protein, Bradford assay, and GC analysis.

This study describes the use of regenerated cellulose (RCA) membranes with molecular weight cut-off (MWCO) values of 10, 30, and 100 kDa, respectively, to separate phytosterols from orange juice for possible nutraceutical production. A desirable membrane separation rejects protein whilst transmitting phytosterols and other low molecular mass compounds such as sugars. The ultrafiltration was performed in a cross-flow membrane system with a total filtration area of 336 cm2. Total phytosterol analysis was carried out by using a Liebermann-Buchard-based method. Protein concentration was quantified by the Bradford method. The effects of three different membranes upon the rejection of total phytosterol content, proteins, sugar, and antioxidant activity were studied. Of the membranes tested, the 10-kDa membrane displayed the highest concentration of phytosterols in the permeate. The 30-kDa and 100-kDa membranes gave comparatively higher phytosterol rejection. The membrane surface roughness and corresponding pure water flux values varied as a function of MWCO such that RCA30 > RCA100 > RCA10. Membranes with rougher surfaces displayed higher fouling than those with smoother surfaces. Hydrophobicity and surface roughness both influenced filtration performance, by controlling the development of the protein-based foulant which modified membrane selectivity.

Publisher's version (útgefin grein), Immobilized enzymatic catalysts are widely used in the chemical and pharmaceutical industries. As Candida antarctica lipase B (CALB) is one of the more commonly used biocatalysts, we attempted to design an optimal lipase-catalytic system. In order to do that, we investigated the enantioselectivity and lipolytic activity of CALB immobilized on 12 different supports. Immobilization of lipase on IB-D152 allowed us to achieve hyperactivation (178%) in lipolytic activity tests. Moreover, the conversion in enantioselective esterification increased 43-fold, when proceeding with lipase-immobilized on IB-S861. The immobilized form exhibited a constant high catalytic activity in the temperature range of 25 to 55°C. Additionally, the lipase immobilized on IBD152 exhibited a higher lipolytic activity in the pH range of 6 to 9 compared with the native form. Interestingly, our investigations showed that IB-S500 and IB-S60S offered a possibility of application in catalysis in both organic and aqueous solvents. A significant link between the reaction media, the substrates, the supports and the lipase was confirmed. In our enzymatic investigations, highperformance liquid chromatography (HPLC) and the titrimetric method, as well as the Bradford method were employed., This work was supported by the National Science Centre Poland grant DEC-2013/09/N/NZ7/03557.

Background Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. Aim Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. Method We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. Result Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3–102.4%, 0.3–3.8 ​mg/mL, and 1.2–12.8 ​mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 ​± ​16.2 ​mg/mL and 2.1 ​± ​1.5 ​mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 ​> ​0.6). Conclusion Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples., Highlights • Thorough evaluation of colorimetric protein assays on paper-analytical device. • Bromocresol green and tetrabromophenol blue methods were found to be best suited for serum and urine samples, respectively. • The selected methods are comparable with spectrophotometric assay protocols for human serum and urine samples.

Aims:: We aim to evaluate the potential application of amine reactive fluorogenic reagents for estimating enzymatic proteolysis. Background:: Proteolytic enzymes play important roles in regulating many physiological processes in living organisms. Objectives:: Assessment of protein degradation by using reagents for protein assay techniques. Methods:: We have assayed samples at the start and after 30-60 minutes incubation with trypsin by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents and NanoOrange as non-amine reactive dye. Results:: All BSA prepared samples with trypsin have shown significantly higher fluorescence intensity (FI) versus controls (which reflects proteolysis) when assayed by Chromeo P503 (Py 1 pyrylium compound) and CBQCA (3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde) as amine reactive reagents. However, same samples assayed with NanoOrange as non-amine reactive reagent did not show any significant variation between samples containing trypsin and controls. Conclusion:: These results are confirming reliability of highly sensitive protein assays utilizing amine reactive fluorogenic reagents for general estimation of proteolysis.

A recombinant ricin vaccine from E. coli (RVEc™), was developed at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) and assessed in an FDA sponsored Phase 1a clinical trial. At the maximum dosage, two of the study participants developed physiological responses that were elevated to the level of severe adverse reactions. To stay within safe dosing guidelines, the FDA recommended that an assay be developed to accurately quantify the recombinant protein content in the vaccine. The RVEc™ vaccine Final Drug Product (FDP) contains the adjuvant Alhydrogel®, which by its colloidal nature interferes with most conventional protein assay methods. We decided to develop an assay measuring RVEc™ FDP using o-pthalaldehyde (OPA) reagent. The OPA reagent reacts to the primary amines and lysine side chains of proteins in the presence of a thiol under alkaline conditions with a quantifiable fluorescent signature, but does not react with Alhydrogel®. Protein content in the RVEc™ FDP can be determined by comparing the fluorescence of the test sample to the fluorescence of a standard curve of defined concentration. Each phase of the assay was tested to optimize and simplify the assay procedure. The accuracy, specificity, reproducibility, and stability of the assay were evaluated. Results indicated that the optimized and modified OPA assay was simple and able to quantify antigen concentration from a standard curve in the 25 µg/mL–600 µg/mL range. The assay accuracy and coefficient of variation (CV) was 95% and less than 8%, respectively, when determining the ricin protein content in the 200 µg/mL vialed RVEc™ FDP. The assay was simple to perform and used conventional laboratory equipment. This assay could be adapted to measure the protein content in the FDP of other vaccines, but with the proviso that each step of the assay would need to be optimized for each antigen.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid with the capacity to reduce the proinflammatory response of activated microglia that occurs during neuroinflammation. The uptake of DHA into microglia is essential for it to exert its neuroprotective effects. However, quantifying the uptake of DHA into microglia is complicated by the presence of endogenous DHA interfering with any quantification technique. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was therefore developed and validated in order to assess the microglial uptake of docosahexaenoic acid-d5 (DHA-d5) as a surrogate for DHA. Using a mobile phase consisting of 90 % (v/v) acetonitrile and 10 % (v/v) water containing 2 mM ammonium acetate, a flow rate of 0.3 mL/min, and MS/MS detection in the negative ionization mode, DHA-d5 was detected at m/z transitions of 332.1/228.3/234.2, with good linearity between chromatographic area under the curve (AUC) and DHA-d5 mass (R2 = 0.999) over the range of 0.0063-0.1 ng. The precision and accuracy values for the quality control samples (0.0063, 0.025, and 0.1 ng) were less than 9.3 % and 96.6-109.8 %, respectively, and a comparison of DHA-d5 AUC when prepared in PBS or in microglial cell lysate demonstrated no significant difference between quantification of these quality control samples. Utilizing this quantification approach (with preparation of DHA-d5 calibration standards in PBS), the uptake of DHA-d5 into BV-2 microglial cells over a 15 min period was assessed, following the spiking of DHA-d5 at 50 ng/mL. Following protein normalization using a BCA protein assay, a rapid and linear uptake of DHA-d5 into BV-2 cells was observed in the first 2 min, after which a plateau in uptake was observed, in line with that reported for DHA uptake in other cell types. This novel LC-MS/MS technique can now be exploited to unravel the processes involved in microglial uptake of DHA, insights that may be used to maximize the anti-inflammatory effects of DHA in neuroinflammation.

Wound dressings based on natural polymers are of considerable interest in the pharmaceutical industry owing to their improved performance in the human body when compared to synthetic polymers. Alginate, a polysaccharide from brown algae, is commonly studied as a wound dressing owing to its biocompatibility and biodegradability. To improve its therapeutic features and thereby increase wound healing, papain (a proteolytic enzyme from Carica papaya latex) was proposed to be incorporated. Papain is capable of promoting the debridement of devitalized or necrotic tissues. The development of dressing based on alginate and papain aggregates the healing properties of both materials. In addition, the adsorption on a support can stabilize the enzyme structure and permits its release in a controlled manner. The optimal conditions for immobilization were evaluated (initial concentration, temperature, and pH), and the amount immobilized was measured by Bradford assay. The enzyme activity stability over 28 days was measured. The release profile was determined using Franz cell. In vitro cytotoxicity assays were performed using fibroblasts and keratinocytes. Optimal immobilization conditions were identified in a neutral medium at a papain concentration of 20 mg/mL and temperature of 25 °C. The enzyme remained active after immobilization (80 % of its initial activity), and the matrix protected the enzyme from deactivation (70 % reduction on the matrix compared to 94 % in a buffer solution). Franz cell displayed a release profile of 64.1 % of the enzyme after 24 h. The biological assays indicated a bioactive material with proteolytic properties.

Background Many food recalls are related to the presence of undeclared allergens and microorganisms in food products. To reduce these occurrences, portable diagnostic assay kits are available to quantitate mycotoxins, to detect allergens and gluten in foods and on environmental surfaces, and for sanitation monitoring. Objective This article reviews diagnostic kits that can detect sources of contamination in food and ingredients as well as on surfaces and clean-in-place rinses. Method Mycotoxins and gluten were detected using lateral flow diagnostic assays. Sanitation monitoring of surfaces was completed using a chemiluminescent assay to detect adenosine 5′-triphosphate disodium salt hydrate (ATP) and another assay to detect protein. Results Gluten was detected at 10 ppm in spiked commodities and on wet and dry surfaces at 2.5 µg/100cm2. Deoxynivalenol was quantitated in dry distillers grains plus solubles and mean results were within two SDs of those determined by HPLC. The chemiluminescent assay had an LOD of 6 fmol of ATP and was able to detect a 1:10 000 dilution of orange juice from surfaces. The protein assay detected 5 µg of bovine serum albumin (BSA) directly applied to the sampler, 100 µg of BSA on surfaces, and detected 1:10 dilutions of Greek yogurt and raw beef from surfaces. Conclusions Portable diagnostic kits evaluated in this work provided accurate, rapid, and sensitive results for detection of mycotoxins, gluten, proteins, and ATP. These methods can be used in facilities with minimal training and provide results that are important to ensure food safety. Highlights Portable methods to detect gluten, mycotoxins, proteins, and ATP are presented.

This method for extracting protein from ground coral samples is based on the Bradford assay for the colorimetric detection and quantification of total protein (Bradford, 1976) and is compared to a known standard dilution of bovine serum albumin (BSA). Pierce Inc. and Bio-Rad have developed the reagents and standards necessary for completing the extraction. There are five parts to quantifying total soluble protein in ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) solubilize protein via cell disruption [detergent lysis and freeze-thaw lysis], 3) separate the dissolved protein from tissue and skeletal particles, 4) quantify the protein concentration via Bradford microassay procedure, and 5) standardize the protein concentration to ash-free dry weight (AFDW). This method was originally developed by Rowan McLachlan with the assistance of Jamie Price and Kerri Dobson and with the guidance of Dr. Noah Weisleder and Andréa Grottoli at The Ohio State University. This protocol was written by Rowan McLachlan and reviewed by all co-authors.

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated fromLeishmaniaparasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3(sodium carbonate), 1% CuSO4(copper sulfate), 2% KNaC4H4O6(potassium sodium tartrate), and 2 N Folin and Ciocalteu’s phenol. This modified protein assay was sensitive for quantifyingLeishmaniaproteins in a total crude extract or in a soluble fraction within the approximate range of 10–500 μg/ml (1–50 μg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

Background The combination of ketoconazole and dexamethasone acetate is one of the commonly used treatments in dermatology regiment for skin inflammation accompanied by fungal infections. This interaction of ketoconazole and dexamethasone may make the drug much effective, decrease the adverse reaction or toxic effects. At present, there was no information about the interaction of these two external drugs. Therefore, it was necessary to build a model to measure the levels and evaluate the interaction of ketoconazole and dexamethasone acetate in skin cells. Methods In our study, the determination methodology of ketoconazole and dexamethasone acetate in human keratinocyte (HaCaT cells) was established and the interaction of these two drugs in cells was explored. HaCaT cells were cultured in medium containing ketoconazole, then they were sequentially cultured with or without dexamethasone acetate treatment for another 1, 2, 4, 8 and 12 h. The samples were then harvested and the concentrations were quantified by enhanced BCA (the bicinchoninic acid) protein assay. Furthermore, the analytes in the cell suspension were also prepared and analyzed by LC-MS method. Results As a result, ketoconazole and dexamethasone acetate were detected at the concentration range of 0.02 to 5 μg/mL and 0.2 to 100 μg/mL, respectively. The RSD (relative standard deviation) and RE (relative error) of precision and accuracy of the two analytes in the cell suspension were all less than 15 %. The matrix effect values variations were all less than 15%. The results showed that there was no significant difference in the concentration of ketoconazole with or without dexamethasone acetate treatment within 12 h. Conclusions A simple, sensitive and rapid LC-MS method which could quantify these two analytes in cells simultaneously was developed for the first time. The results of the concentration changes meant that no interaction occurred when dexamethasone sequentially administrated for 12 h after ketoconazole treatment. This method was successfully applied to establish the cell pharmacokinetics methodology and preliminary studied the metabolism of drug-drug interaction of ketoconazole and dexamethasone acetate.

This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs; often referred to as protein contaminants) are present. The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit

Monodisperse porous silica microspheres were functionalized with the iminodiacetic acid/copper(II) complex and then evaluated as a group-specific peroxidase-mimicking nanozyme for colorimetric determination of histidine-tagged (His-tagged) proteins. The green fluorescent protein (GFP) was selected as a typical His-tagged protein. The specificity for GFP and the peroxidase-like activity for the selected substrate were obtained by immobilizing the complex on the porous microspheres. The modified microspheres were also evaluated as a group specific immobilized metal affinity chromatography (IMAC) sorbent for the purification of GFP from Escherichia coli extract. The peroxidase-like activity of the microspheres was inhibited by the GFP adsorbed onto the microspheres due to the interaction of His-tagged protein with the immobilized Cu(II) complex. Ortho-phenylenediamine is used as a substrate for the enzyme mimic. The photometric response (measured at 416 nm) is linear in the 9.0–92 μg·mL−1 GFP concentration range in E. coli lysate. The limit of detection is 6.9 μg·mL−1.

Accurate determination of residual protein content in hydroxypropyl chitin (HPCH), a temperature-sensitive chitin derivative developed recently, is of great importance for biomedical applications. Coomassie brilliant blue (Bradford) method, Lowry method and Bicinchoninic acid (BCA) method were investigated to estimate the content of residual protein in HPCH. It was found that both Bradford and Lowry methods are not suitable for the protein assay in the HPCH sample. The BCA method could be used to determine protein content in HPCH solutions at low concentrations with good accuracy and precision and reasonable limit of detection and limit of quantitation for medical applications.

Glomalin is a soil protein resembling heat shock protein (HSP) 60 and exerting high affinity to metals, causing retention of water in the environment and improving mechanical stability of soil. Currently, glomalin is determined in the soil or other samples by combination of autoclaving extraction and total protein determination typically by the Bradford method. In this paper, a piezoelectric biosensor was prepared to determine glomalin in a label-free measurement. The biosensor contained antibodies immobilized on quartz crystal microbalance (QCM), and the recognition layer was stabilized by iron oxide nanoparticles. The assay was tested on real soil samples and compared with the standard Bradford assay. Limit of detection of the assay was equal to 2.4 µg/g for a soil extract with a volume of 50 µl. The assay takes approximately half of an hour and was fully correlated to the Bradford assay. The biosensor had significant advantages than the other methods: it worked in a label-free mode and was fully applicable for practical samples.

The purpose of the study was to evaluate the surface substructure, inorganic and organic components present in partially demineralized tooth bone graft material in young and old, male and female subjects, which may vary due to formation of secondary dentin suggesting decrease in calcium content as we age. A total of 34 extracted teeth were collected, 15 belonging to young patients and, the remaining 15 belonging to old patients, and, divided into 4 subgroups and a 4 control group. All the samples were cleaned, washed, air dried and powdered and demineralized. However the control group having 4 samples each was not demineralized. The samples were subjected to Scanning electron microscope (SEM) and Energy dispersive X-ray spectroscopy (EDX) for surface substructure and inorganic component analysis, and Bradford Assay to assess the protein content. The SEM analysis revealed the presence of dentinal tubules, collagen and, dense crystals of hydroxyapatite in young subject samples, however in old subjects’ collagen fibers were not visible, and hydroxyapatite crystals randomly scattered in clusters. The EDS results showed statistical significance in the Calcium Phosphorus ratio between the groups. Bradford Assay, showed higher concentration of proteins in female subject samples irrespective of age. It can be concluded that inorganic analysis showed young subjects had more densely packed hydroxyapatite crystals and slightly higher Calcium Phosphorus ratio and, high protein concentration was seen in female subjects’ samples irrespective of age.

The present study aimed to formulate a novel strategy to prepare influenza A/H1N1 virosomes using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) as solubilizing agent and compared with that of known solubilizing agents like dipalmitoylphosphatidylcholine (DCPC), Dimyristoylphosphatidylcholine (DHPC), Octaethyleneglycol-mono-(n dodecyl) ether (OEG, C12E8), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (PG) and Octaethyleneglycol-mono-(n dodecyl)ether (OEG, C12E8). The study involves solubilization and reconstitution of virus membrane with DSPC. DSPC is nonionic detergent with short chain lecithin, which makes it a suitable solubilizing and reconstituting agent. DSPC efficiently dissolve virus membrane and it is approximately 99.9% dialyzable from the virosome formulation without affecting fusion and membrane binding properties of prepared influenza virosomes. The prepared virosomes were characterized using blend of characterization tools like TEM, SEM and Bradford assay. The TEM and SEM results for prepared virosomes confirms the size ranging between 60 and 95 nm which is two times smaller than the size obtained with DCPC as solubilizing agent. Bradford’s assay depicts the successful preparation of virosomes. Further the virosomes were used in encapsulation of herbal drug nanocurcumin, encapsulation efficiency and drug release profile was studied to evaluate its potential to be used in drug delivery.

Drugs are important xenobiotics in the environment. Their use increases with the growth of the human population, but also in agricultural primary production. Paracetamol (PAR) is a widely used analgesic and antipyretic and its production is still growing. Commonly available drug production technologies are being developed very intensively with nanotechnological modifications for their gradual and targeted release. Nanoparticles (ST/PAR) from starch were prepared: PAR (0, 1, 2, 3, 5 and 10 mg/L) was mixed with citric acid ester in a 1:8 v/v ratio for 30 min at 25 °C. By the centrifugation (16.000 g, 30 min) ST/PAR were obtained in the pellet. The effect of PAR was studied on Daphnia magna Straus (Cladocera, Crustacea). Adult females (70-400 mg) were used for self-evaluation. The EC50 was 3.749 mg/L after 48 h of PAR treatment. Total protein values determined by Lowry method were between 0.5-2.2 mg/mL and by Bradford method between 190-676 mg/L. Antioxidant activity values determined by CUPRAC method were between 4-15 μg/mL GAE and by ABTS method ranged between 40-103 μg/mL GAE. PAR values were between 9-40 μM. Subsequently, the biological activity of the prepared nanoparticles was tested.