Immobilization of Antibodies on Magnetic Carbonaceous Microspheres for Selective Enrichment of Lysine‐acetylated Proteins and Peptides

Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody‐immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α‐casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody‐immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.     

Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180 °C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180 °C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80 °C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180 °C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80 °C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (−)-catechin, (+)-epicatechin, and (−)-epicatechin.     

A New Colorimetric Platform for Protein Detection Based on Recognition‐Induced Cascade of Polymeric Nanoparticles Disassembly

Despite great progress, it is still of high interest to explore new homogeneous assays for simple, visual, and selective protein detection. Herein, one new colorimetric sensor has been developed for visual detection of protein by using polymeric micelles as a sensing scaffold and the molecular recognition between protein and the ligand on the surface of the polymeric micelles as the driving force to trigger the readout of the detection signal. The polymeric micelles formed via the self‐assembly of the amphiphilic block polymer biotin‐labeled poly(ethylene glycol)‐block‐poly(3‐acryl aminophenylboronic acid) are endowed with colorful feature by incorporation of alizarin red S (ARS) into the hydrophobic core. Based on the response to streptavidin recognition, these micelles are further disintegrated through the competitive binding of α‐cyclodextrin with boronic acid for disassociation of ARS, which achieves orange–yellow to pink–purple transition in 2 h. This work will open the way to develop one new mix‐and‐measure, visual, and homogeneous assay.     

Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D₂O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r² = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Computer Assisted Spectrophotometric Analysis of Three Component Mixtures (Caffeine Mixtures and Codeine Mixtures)

Abstract

Two mixtures (caffeine mixture (I) and codeine mixture (II)) of 3-components, containing acetaminophen and salicylamide as majors, have been assayed using different computer-assisted spectrophotometric methods. These methods are the Unique Absorbance Method (UAM), the Unique Orthogonal Function Method (UOFM), and Unique Fourier Function Method (UFFM), and also their correspondences under the least squares approach. The parameters have been optimized for the accurate determination of the minor component caffeine in mixture (I). In mixture (II), however. codeine has been extracted - in two steps-prior to the correction of interferences from the major components acetaminophen and salicylamide.     

Synthesis of Novel Bisphosphonate Inhibitors of Phosphoglycerate Kinase (3-PGK) (E.C.2.7.2.3)

Abstract

Novel, aromatic bisphosphonates have been synthesised as non-systematic analogues of 1,3-bisphosphoglyceric acid (1,3-BPG). These incorporate non-scissile α-halo and α-methylene phosphonates and have submicromolar K _i values for 3 -PGK.     

Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach

Skin sensitisation is a key endpoint under REACH as it is costly and its assessment currently has a high dependency on animal testing. In order to reduce both the cost and the numbers of animals tested, it is likely that (quantitative) structure–activity relationships ((Q)SAR) and read-across methods will be utilised as part of intelligent testing strategies. The majority of skin sensitisers elicit their effect via covalent bond formation with skin proteins. These reactions have been understood in terms of well defined nucleophilic–electrophilic reaction chemistry. Thus, a first step in (Q)SAR analysis is the assignment of a chemical's potential mechanism of action enabling it to be placed in an appropriate reactivity domain. The aim of this study was to design a series of SMARTS patterns capable of defining these reactivity domains. This was carried out using a large database of local lymph node assay (LLNA) results that had had potential mechanisms of action assigned to them using expert knowledge. A simple algorithm was written enabling the SMARTS patterns to be used to screen a database of SMILES strings. The SMARTS patterns were then evaluated using a second, smaller, test set of LLNA results which had also had potential mechanisms of action assigned by experts. The results showed that the SMARTS patterns provided an excellent method of identifying potential electrophilic mechanisms. The findings are supported, in part, by molecular orbital calculations which confirm assignment of reactive mechanism of action. The ability to define a chemical's potential reaction mechanism is likely to be of significant benefit to regulators and risk assessors as it enables category formation and subsequent read-across to be performed.     

Design of Sensitive Biocompatible Quantum‐Dots Embedded in Mesoporous Silica Microspheres for the Quantitative Immunoassay of Human Immunodeficiency Virus‐1 Antibodies

A novel sandwich‐type electrochemiluminescence (ECL) immunosensor was developed to enable the sensitive detection of HIV‐1 antibodies. This system incorporated mesoporous silica (mSiO₂) complexed with quantum dots (QDs) and nano‐gold particles, which were assembled to enhance signal detection. Magnetic beads were used by immobilizing the secondary anti‐IgG antibody. This was first employed to capture HIV‐1 antibody (Ab) to form a Fe₃O₄/anti‐IgG/Ab complex. A high loading and signal‐enhanced nanocomposite (hereafter referred to as Au‐mSiO₂‐CdTe) was used as a HIV‐1 antigen label. The Au‐mSiO₂‐CdTe nanocomposite was conjugated with the Fe₃O₄/anti‐IgG/Ab complex to form an immunocomplex (hereafter referred to as Fe₃O₄/anti‐IgG/Ab/HIV‐1/CdTe‐mSiO₂‐Au). This complex could be further separated by an external magnetic field to produce ECL signals. Due to the large specific surface area and pore volume of mSiO₂, the loading of the CdTe QDs was markedly increased. Thus, the loaded QDs released a powerful chemiluminescent signal with a concordantly increased sensitivity of the immunosensor. The immunosensor was highly sensitive, and displayed a linear range of responses for HIV‐1 antibody across a dilution range of 1 : 1500 through 1 : 50 with the detection limit of 1 : 4500. The immunoassay can be a promising candidate in early diagnosis of HIV infection.     

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5 × 10⁻¹³ M and 4.8 × 10⁻¹⁰ M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75 °C while the scFv melts at ∼65 °C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1 μg L⁻¹. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.