Publikacja Sensory chemiczne i biosensory omawia podstawowe cechy, zalety i ograniczenia sensorów chemicznych i biosensorów oraz ich praktycznych zastosowań w klinicznej i procesowej kontroli analitycznej, w diagnostyce medycznej, w systemach kontroli bezpieczeństwa oraz w analizie środowiskowej. Przybliża zagadnienia związane z podstawami działania sensorów i biosensorów, materiałami i technologiami wykorzystywanymi do ich produkcji, a także zastosowaniem sensorów w praktyce. Pokazuje rozwój sensorów chemicznych i biosensorów indukowany pojawianiem się nowych materiałów i technologii oraz zapotrzebowaniem tego typu urządzenia zarówno analityce klinicznej, jak i przemysłowej. Autorami są doświadczeni pracownicy naukowo-badawczy z kilku ośrodków akademickich w Polsce, zajmujących się od wielu lat projektowaniem tego typu narzędzi analitycznych. * Fragment (epub) * Fragment (mobi)

Introduction: The relative resistance of children to severe course of the novel coronavirus infection remains unclear. We hypothesized that there might be a link between this phenomenon and observation from our previous studies concerning an inhibitory or cytotoxic effect of exhaled breath condensate (EBC) on endothelial cell cultures in children. Aim: Since we could not find any data on the similar effect caused by EBC in adults, the aim of our study was to evaluate and compare the biological activity of EBC in adults and children in an experimental in vitro model. Furthermore, in order to identify a putative agent responsible for these properties of EBC in children, we attempted to analyse the composition of selected EBC samples. Material and methods: The influence of EBC samples on metabolic activity of endothelial cell line C-166 was assessed using colorimetric tetrazolium salt reduction assay (MTT assay). Selected EBC samples were fractionated using size exclusion chromatography and subjected to mass spectrometry analysis. Results: Exhaled breath condensates in healthy children, but not in adults, revealed a cytotoxic effect on in vitro cell cultures. This effect was most significant in condensate fraction, which contained a prominent 4.8 kDa peak in the mass spectra. Conclusions: Breath condensates of healthy children contain the factor which reveals the inhibitory/cytotoxic effect on endothelial cell cultures. Although the physiological role of this agent remains unclear, its identification may potentially be useful in ongoing research on SARS-CoV-2/COVID-19.

PurposeIdentification of human insulin analogs' impurity with a mass shift +14Da in comparison to a parent protein.MethodsThe protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing.ResultsThe misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs.ConclusionsAlthough there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.

Isolation and identification of unknown impurities of recombinant insulin lispro (produced at IBA) formed during accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formulations of recombinant human insulin (Humulin S®; Lilly), recombinant insulin lispro (Humalog®; Lilly), recombinant insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recombinant insulin detemir (Levemir®; Novo Nordisk) and recombinant insulin glargine (Lantus®; Sanofi-Aventis) were analyzed.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

A monomer structure of a novel human insulin analog A22S-B3K-B31R (SK3R) has been characterized by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS) established in the same medium. The composition of the oligomer ensemble for neat insulins in water was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion coefficient Dix10-10m2s-1, whose value is a population averaged of individual coefficients for species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were established and compared to insulin glargine characterized by the same profile of action in diabetics. The oligomerization process of insulin during development of pharmaceutical formulation with routinely used excipients has been studied using translation diffusion coefficient Dix10-10m2s-1 established in water solution. These properties were compared with those of human insulin (HIS) which is a standard reference for novel recombinant insulins.

Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis.

A biosynthetic human insulin precursor displayed enhanced susceptibility to deamidation at one particular site. The present study was undertaken to monitor progress of precursor deamidation at successive manufacturing stages. MALDI-TOF/TOF MS in combination with controlled endoproteinase Glu-C and endoproteinase Asp-N proteolysis was used for rapid and unambiguous determination of deamidated residue within the investigated structure. Close inspection of isotopic distribution patterns of peptides resulting from enzymatic digestion enabled determination of distinct precursor forms occurring during the production process. Asn, Asp, isoAsp and succinimide derivatives of the amino acid at position 26 were unambiguously identified. These modifications are related to the leader peptide of a precursor encompassing amino acid sequence corresponding to that of superoxide dismutase [Cu-Zn] (SOD1 1, EC=1.15.1.1). Monitoring of precursor deamidation process at successive manufacturing stages revealed that the protein folding stage was sufficient for a prominent replacement of asparagine by aspartic and isoaspartic acid and the deamidated human insulin precursor constituted the main manufactured product. Conversion proceeded through a succinimide intermediate. Significant deamidation is associated with the presence of SNG motif and confirms results achieved previously on model peptides. Our findings highlight an essential role of the specific amino acid sequence on accelerated rate of protein deamidation. To our knowledge, this is the first time that such a dramatic change in the relative abundance of Asp and isoAsp resulting from protein deamidation process is reported.

Bioactive surface domains as well as low oxygen conditions were applied to investigate cellular developmental processes of human cord blood-derived stem cells and to direct their fate into desired neural lineages. Such microenvironmental cues should represent those found in vivo. For that purpose we have created miniaturized cell growth platforms with defined arrays of cell attractive biomaterials serving as functional domains. Applied technologies included a nano/micro-fabrication technique like microcontact printing and piezoelectric microspotting of biomolecules on plasma deposited cell repellent surface. Human Umbilical Cord Blood Neural Stem Cells (HUCB-NSC) were plated on biodomains at different cell densities and serum concentrations. HUCB-NSCs were shown to adhere and differentiate on microarray platforms in the protein type, serum concentration and cell density dependent manner. Receptor-mediated interactions with extracllular proteins promote neuronal differentiation, while non-specific adhesion to polyaminoacid molecules allows maintaining of stem cells immobilized to the surface in non-differentiated stage. “Smart” functional domains were created by immobilizing to the surface small signaling molecules (e.g wnt, shh, notch or jagged) together with ECM proteins. Stimulation of selected intracellular pathways by signaling molecules resulted in differentiation of HUCB-NSC to either neuronal or astroglial lineage. Miniaturization of such bioengineered active domains combined with appropriate stem cell model may allow their application of for the multiparameter bio-tests and can provide important information on the sensitivity of certain neural stem cell molecular pathways to the selected neurotoxins. Since HUCB-NSC line can be cultured at different developmental stages and was assigned for developmental toxicity testing, homogenous lineage related pluripotent cell population would be required. For that purpose iPs cells from HUCB-NSC are produced. The influence of low oxygen tension, similar to conditions found in neural stem cell niche in vivo, was tested for both: reprogramming and neural commitment of HUCB-NSC. Reprogramming efficiency of HUCB-NSC to pluripotency was shown to be stimulated upon hypoxia conditions, however its influence for neuronal differentiation was dependent on cell developmental stage. Acknowledgements Sponsored by grant from Polish Ministry of Scientific Research and Higher Education No 5978/B/P01/2010/38 and European Commission, JRC. LP2

This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.

This work presents the development of a novel construction of an integrated microelectrode array. The device was fabricated on a ceramic support, with the use of low temperature cofired ceramics technology. Model potassium-selective membranes were applied on the surface of PdAg/AgCl electrodes formed on the ceramic substrate. The obtained microsensors exhibited very good repeatability, reproducibility, and sensitivity. The array of microelectrodes covered with polymeric layers of various selectivities was applied as an electronic tongue to differentiate between various diet supplements.