Oxygen-Induced and pH-Induced Direct Current Artifacts on Invasive Platinum/Iridium Electrodes for Electrocorticography

Background: Spreading depolarization (SD) and the initial, still reversible phase of neuronal cytotoxic edema in the cerebral gray matter are two modalities of the same process. SD may thus serve as a real-time mechanistic biomarker for impending parenchyma damage in patients during neurocritical care. Using subdural platinum/iridium (Pt/Ir) electrodes, SD is observed as a large negative direct current (DC) shift. Besides SD, there are other causes of DC shifts that are not to be confused with SD. Here, we systematically analyzed DC artifacts in ventilated patients by observing changes in the fraction of inspired oxygen. For the same change in blood oxygenation, we found that negative and positive DC shifts can simultaneously occur at adjacent Pt/Ir electrodes. Methods: Nurses and intensivists typically increase blood oxygenation by increasing the fraction of inspired oxygen at the ventilator before performing manipulations on the patient. We retrospectively identified 20 such episodes in six patients via tissue partial pressure of oxygen (p(ti)O(2)) measurements with an intracortical O-2 sensor and analyzed the associated DC shifts. In vitro, we compared Pt/Ir with silver/silver chloride (Ag/AgCl) to assess DC responses to changes in pO(2), pH, or 5-min square voltage pulses and investigated the effect of electrode polarization on pO(2)-induced DC artifacts. Results: Hyperoxygenation episodes started from a p(ti)O(2) of 37 (30-40) mmHg (median and interquartile range) reaching 71 (50-97) mmHg. During a total of 20 episodes on each of six subdural Pt/Ir electrodes in six patients, we observed 95 predominantly negative responses in six patients, 25 predominantly positive responses in four patients, and no brain activity changes. Adjacent electrodes could show positive and negative responses simultaneously. In vitro, Pt/Ir in contrast with Ag/AgCl responded to changes in either pO(2) or pH with large DC shifts. In response to square voltage pulses, Pt/Ir falsely showed smaller DC shifts than Ag/AgCl, with the worst performance under anoxia. In response to pO(2) increase, Pt/Ir showed DC positivity when positively polarized and DC negativity when negatively polarized. Conclusions: The magnitude of pO(2)-induced subdural DC shifts by approximately 6 mV was similar to that of SDs, but they did not show a sequential onset at adjacent recording sites, could be either predominantly negative or positive in contrast with the always negative DC shifts of SD, and were not accompanied by brain activity depression. Opposing polarities of pO(2)-induced DC artifacts may result from differences in baseline electrode polarization or subdural p(ti)O(2) inhomogeneities relative to subdermal p(ti)O(2) at the quasi-reference

Vectorially Imprinted Hybrid Nanofilm for Acetylcholinesterase Recognition

Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium-terminated self-assembled monolayer (Prop-SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate-modified derivative of 3,4-propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop-SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10−6 to 0.4 × 10−6m. An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP-AChE binding is 4.2 × 10−7m indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer is the effective suppression of the cross-reactivity toward competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by i) the “shape-specific” MIP filter, ii) the propidium-SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid

Echtzeitdetektion von Punktmutationen mit DNA-Chips am Beispiel des SULT1A1*213-SNP

Der Identifizierung von Punktmutationen im menschlichen Genom kommt eine hohe Bedeutung zu. Die Entdeckung einer Vielzahl von SNPs ('single nucleotide polymorphisms'), also Mutationen einzelner Basen, die definitionsgemäß bei mehr als 1% der Bevölkerung auftreten, und die Erkenntnis, dass SNPs die Nebenwirkungen von Medikamenten determinieren können, führte zu der Vision einer 'personalisierten Medizin': Der Patient erhält nach einer Genotypisierung das für ihn verträglichste Medikament verschrieben. Notwendige Bedingung für das neue Paradigma ist eine schnelle und hochdurchsatzfähige DNA-Analytik. Da bis zu 3 Millionen von SNPs beim Menschen vermutet werden (www.snp.cshl.org), ist das etablierte Verfahren mittels PCR-Amplifikation, Restriktionsenzymverdau und Elektrophorese nicht praktikabel. Neben den zum Massenscreening geeigneten, spezialisierten MALDI- (z.B. Sequenom ®) und Primer-Extension-Verfahren (z.B. Orchid Biocomputer ®) wird insbesondere die DNA-Chip-Technologie als vielversprechende Methode für mittlere bis hohe Durchsätze und Vor-Ort-Anwendungen angesehen. Der Einsatz dieses Verfahrens zum SNP-Screening wird am Beispiel des SULT1A1*213-SNPs demonstriert. Das SULT1A1-Gen beim Menschen kodiert für eine cytosolische, thermostabile Phenol-Sulfotransferase (P-PST, EC 2.8.2.1), die in der Leber durch Sulfonierung von phenolischen Substraten Biosynthese und Entgiftungsfunktionen ausübt. Bisher wurden drei Punktmutationen in diesem Gen entdeckt (Raftogianis 1997). Die Variation *213Arginin nach *213Histidin, die bei ca. 37% der (kaukasischen) Bevölkerung auftritt, führt zu einem deutlich verschiedenen Phänotyp (geringere Aktivität, geringere Thermostabilität, Engelke 2000) und wird mit Übergewicht in Zusammenhang gebracht

Oxygen-Induced and pH-Induced Direct Current Artifacts on Invasive Platinum/Iridium Electrodes for Electrocorticography

Background!#!Spreading depolarization (SD) and the initial, still reversible phase of neuronal cytotoxic edema in the cerebral gray matter are two modalities of the same process. SD may thus serve as a real-time mechanistic biomarker for impending parenchyma damage in patients during neurocritical care. Using subdural platinum/iridium (Pt/Ir) electrodes, SD is observed as a large negative direct current (DC) shift. Besides SD, there are other causes of DC shifts that are not to be confused with SD. Here, we systematically analyzed DC artifacts in ventilated patients by observing changes in the fraction of inspired oxygen. For the same change in blood oxygenation, we found that negative and positive DC shifts can simultaneously occur at adjacent Pt/Ir electrodes.!##!Methods!#!Nurses and intensivists typically increase blood oxygenation by increasing the fraction of inspired oxygen at the ventilator before performing manipulations on the patient. We retrospectively identified 20 such episodes in six patients via tissue partial pressure of oxygen (p!##!Results!#!Hyperoxygenation episodes started from a p!##!Conclusions!#!The magnitude of p

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs

Peroxide-Dependent Analyte Conversion by the Heme Prosthetic Group, the Heme Peptide “Microperoxidase-11” and Cytochrome c on Chitosan Capped Gold Nanoparticles Modified Electrodes

In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c) in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11