Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation seen in commercially sourced hemoglobin.

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe3+, ferric) hemoglobin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemoglobin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain stabilizers and additives which contribute to CAgg formation.

Adsorption and Electropolymerization of p-Aminophenol Reduces Reproducibility of Electrochemical Immunoassays.

This paper investigates the electrochemical behavior of p-aminophenol (PAP) on commercially available carbon screen-printed electrodes (CSPEs) and gold screen-printed electrodes (GSPEs) at neutral and basic pHs for the development of inexpensive immunoassays. The electrochemical oxidative signal from PAP results from its adsorption to the electrode. The formation of self-assembled monolayers on gold electrodes prevented PAP adsorption but also reduced its oxidative current, confirming that adsorption increases signal production. On bare electrodes, PAP adsorption results in oxidative current variability depending on the electroactive surface area of the screen-printed electrode. This variability could not be remedied by cleaning and reusing the same GSPE. Decreasing the PAP concentration to 3.8 µM greatly improved the consistency of the measurements, suggesting that the adsorption of PAP is concentration-dependent. Multiple PAP oxidations on the same electrode caused polymerization, limiting PAP in continuous monitoring applications. Infrared and Raman spectroscopy allow the distinction between adsorbed PAP and electropolymerized PAP on the surface of a gold wafer. The results from this study suggest that the use of PAP production in immunoassays with SPEs must be fine-tuned, and electrodes must be cleaned or disposed of between measurements.

Trace Oxygen Affects Osmium Redox Polymer Synthesis for Wired Enzymatic Biosensors.

Electrochemical sensors that utilize enzymes are a sensitive, inexpensive means of detecting biologically relevant analytes. These sensors are categorized based on their construction and method of signal transport. Type I sensors consist of a crosslinked enzyme on an electrode surface and are potentially subject to interference from byproducts and other biological analytes. However, type II sensors help alleviate this problem with the addition of a redox polymer layer that assists in signal transduction, thus minimizing interferences. An osmium-loaded poly(vinylimidazole) polymer (Os-PVI) is commonly used with successful results, and when combined with an enzyme yields a type II sensor. Our initial attempts at the synthesis of this polymer resulted in an unexpected osmium precursor, which had fluorescent and redox properties that did not match with the desired Os-PVI polymer. Careful exclusion of oxygen during the Os complex precursor synthesis was necessary to avoid this unexpected oxygen containing Os-precursor, which had been seen previously in mass spectrometry studies. All precursors and osmium polymers were characterized with 1H NMR, fluorescence, mass spectrometry, and cyclic voltammetry to provide a better understanding of these compounds and assist in the building of new sensors.