Metal free graphene oxide (GO) nanosheets and pristine-single wall carbon nanotubes (p-SWCNTs) biocompatibility investigation: a comparative study in different human cell lines

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests

Measurement Technologies for up- and Downstream Bioprocessing

This book is devoted to new developments in measurement technologies for upstream and downstream bioprocessing. The recent advances in biotechnology and bioprocessing have generated a number of new biological products that require more qualified analytical technologies for diverse process analytical needs. These includes especially fast and sensitive measurement technology that, early in the process train, can inform on critical process parameters related to process economy and product quality and that can facilitate ambitions of designing efficient integrated end-to-end bioprocesses. This book covers these topics as well as analytical monitoring methods based either on real-time or in-line sensor technology, on simple and compact bioanalytical devices, or on the use of advanced data prediction methods

Characterization of the flavin–protein interaction in L ‐Lactate oxidase and old yellow enzyme by resonance inverse Raman spectroscopy

Resonance inverse Raman spectroscopy was used to examine the interactions between the flavin and surounding protein in L ‐lactate oxidase (from Mycobacterium smegmatis ) and Old Yellow Enzyme (from brewer's bottom yeast). Spectra were taken of the enzymes both free and bound to various ligands. For L ‐lactate oxidase, the ligands consisted of substrate analogs (acetate, propionate) and inorganic anions (phosphate, sulfate and nitrate). For the inorganic anions, the expected attenuation with detuning was not observed for several bands which are associated with flavin rings II and III. This effect appears to be due to a disruption of ring stacking between the uracil–pyrazine end of the flavin moiety and an aromatic amino acid. A shift in the 1232 cm −1 band and changes in the bandwidths of bands in the 1450–1600 cm −1 region indicate minor reorganization of the hydrogen bonding structure around the isoalloxazine on ligand binding. For Old Yellow Enzyme, the ligand was chloride. Chloride caused a slight change to the Raman spectrum, indicative of decreased hydrogen bonding to the flavin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91123/1/1250180403_ftp.pd

The Role of HOSCN in the Oxidation of Proteins and Cellular Damage in Atherosclerosis

Myeloperoxidase forms the reactive oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). While HOCl is an extremely reactive oxidant that causes extensive damage to all manner of biomolecules, HOSCN reacts preferentially with protein thiols. This targeting of thiols can lead to protein inactivation and inhibition, and has led to HOSCN being implicated in the alteration of numerous cellular redox pathways. This Thesis compares the ability of HOCl and HOSCN to form reversibly oxidised cysteine products in murine J774A.1 macrophages, examining changes to the protein thiol levels and the manner of oxidised cysteine products formed in the cells upon oxidation, with changes to the cellular chemistry observed via WB analysis, FTIR and Raman microscopy. Further studies employ real-time analysis into the functional changes in metabolism of J774A.1 cells after HOSCN treatment. Results show that HOSCN is able to affect macrophage glucose metabolism via the oxidation of glycolytic proteins. The oxidation of glycolytic proteins causes a reduction in the glycolytic end product, pyruvate, and extend to mitochondrial oxidative phosphorylation, mitochondrial permeability transition pore function and ATP production. In summary, the studies of this Thesis may be significant in understanding the inflammatory process of atherosclerosis, especially in cigarette smokers, who have elevated levels of thiocyanate, the parent ion of HOSCN in their plasma

Identification and Quantitative Determination of Lactate Using Optical Spectroscopy—Towards a Noninvasive Tool for Early Recognition of Sepsis

Uninterrupted monitoring of serum lactate levels is a prerequisite in the critical care of patients prone to sepsis, cardiogenic shock, cardiac arrest, or severe lung disease. Yet there exists no device to continuously measure blood lactate in clinical practice. Optical spectroscopy together with multivariate analysis is proposed as a viable noninvasive tool for estimation of lactate in blood. As an initial step towards this goal, we inspected the plausibility of predicting the concentration of sodium lactate (NaLac) from the UV/visible, near-infrared (NIR), and mid-infrared (MIR) spectra of 37 isotonic phosphate-buffered saline (PBS) samples containing NaLac ranging from 0 to 20 mmol/L. UV/visible (300–800 nm) and NIR (800–2600 nm) spectra of PBS samples were collected using the PerkinElmer Lambda 1050 dual-beam spectrophotometer, while MIR (4000–500 cm−1) spectra were collected using the Spectrum two FTIR spectrometer. Absorption bands in the spectra of all three regions were identified and functional groups were assigned. The concentration of lactate in samples was predicted using the Partial Least-Squares (PLS) regression analysis and leave-one-out cross-validation. The regression analysis showed a correlation coefficient (R2) of 0.926, 0.977, and 0.992 for UV/visible, NIR, and MIR spectra, respectively, between the predicted and reference samples. The RMSECV of UV/visible, NIR, and MIR spectra was 1.59, 0.89, and 0.49 mmol/L, respectively. The results indicate that optical spectroscopy together with multivariate models can achieve a superior technique in assessing lactate concentrations

ELECTROCHEMICAL CHIRAL BIOSENSORS

Recognition of chiral molecules in biological assemblies has been a subject of extensive research. The aim of this work was to fabricate and characterise biocompatible composite materials suitable for chiral recognition. Collagen, the most abundant chiral, extracellular protein, was chosen as a possible matrix. The chiral recognition properties were evaluated by a comparative study in collagen, collagen incorporated in tetramethyl orthosilicate (TMOS) and TMOS. In electrochemical studies, ferrocene was incorporated to facilitate electron transfer. The recognition characteristics of two chiral enzymes, L-lactate oxidase and D-glucose oxidase were tested using circular dichroism (CD), Fourier Transform Infra-Red (FTIR) spectroscopy and electrochemical methods. A surprising result revealed an inversion of chiral selectivity. The effect of various parameters such as immobilisation, temperature, chemical modification, solvent systems, on enantioselectivity is well known. Stereoinversions caused by the ‘sergeants and soldiers’ effect in gel-forming p-conjugated molecules caused by co-assembly has been reported by several groups. The inversion of stereoselectivity observed in this study is probably due to a combination of the microenvironment and electrostatic interactions of the enzyme, mediator and substrate with the chiral collagen matrix. The results may have important implications for biosensing, asymmetric syntheses and understanding the nature of chiral interactions in biological systems

Sensors for foetal hypoxia and metabolic acidosis: a review

This article reviews existing clinical practices and sensor research undertaken to monitor fetal well-being during labour. Current clinical practices that include fetal heart rate monitoring and fetal scalp blood sampling are shown to be either inadequate or time-consuming. Monitoring of lactate in blood is identified as a potential alternative for intrapartum fetal monitoring due to its ability to distinguish between different types of acidosis. A literature review from a medical and technical perspective is presented to identify the current advancements in the field of lactate sensors for this application. It is concluded that a less invasive and a more continuous monitoring device is required to fulfill the clinical needs of intrapartum fetal monitoring. Potential specifications for such a system are also presented in this paper

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes