Il presente lavoro è stato sostenuto dal programma IST dell&#39;Unione Europea (Progetto &quot;INTREPID - Una Realtà Virtuale intelligente multi-sensore di sistema indossabile per il trattamento Fobie &#39;&quot; - IST-2002-507464).

Il protocollo clinico per il trattamento del Disturbo d&#39;Ansia Generalizzato (GAD) è programmato simultaneamente all&#39;interno e all&#39;esterno dell&#39;ufficio del terapeuta. Programma di rilassamento è una combinazione di &quot;formazione progressiva rilassamento muscolare&quot; (PMRT), la procedura classica descritta da Jacobsen, che coinvolge tensione e il rilassamento gruppi muscolari vari, mentre prendendo atto delle sensazioni contrastanti, e &quot;guidate Imagery&quot; del programma. Nel nostro immaginario la formazione è sostituito dal segnale visivo di un ambiente rilassante virtuale. ALL&#39;INTERNO studio del terapeuta. I pazienti partecipare a 8 sessioni di allenamento con il terapeuta, distribuiti in quattro settimane (2 sedute a settimana). Ogni sessione inizia e si ferma con una valutazione clinica che dà la misura di ansia del paziente. Piano di sessioni in studio del terapeuta: <ol><li> Iniziale valutazione clinica dello stato del paziente; </li><li> Il paziente è collegato con biosensori che registrare il suo / suoi parametri fisiologici (conduttanza cutanea, frequenza cardiaca, la respirazione). Una misura di base di questi parametri è registrato per 3 minuti; </li><li> Il paziente indossa un casco dotato di display collegato a un pc e gestisce un joystick; </li><li> Il paziente inizia a esplorare l&#39;ambiente virtuale: una bellissima isola tropicale che si affaccia sull&#39;oceano. Il paziente, seguendo la narrazione registrata dal terapeuta, raggiunge l&#39;isola in barca e esplorarlo. Seguendo un sentiero che guida lui / lei attraverso l&#39;isola, il paziente arriva al punto di partenza, dove diversi pannelli indicano le indicazioni per le aree di destinazione diverse. In ciascuna di queste aree un esercizio rilassante viene fornito, durante questa formazione, seguendo le indicazioni fornite dalla voce-guida, il paziente cerca di rilassarsi se stesso / ae mentre alcuni elementi dell&#39;ambiente virtuale sono direttamente modificate dal suo / suoi parametri fisiologici registrati in tempo reale. Così, il paziente riceve un feedback immediato della sua / il suo livello di attivazione (come nelle tecniche di biofeedback tradizionali), ma con il valore aggiunto dato da un ambiente virtuale che lui / lei è immersivo esplorare. Le aree di destinazione sono tenuti a: <ul><li> Campfire: parametri fisiologici controllare l&#39;intensità del fuoco, in modo che la riduzione dei risultati attivazione fisiologica del paziente su una riduzione del fuoco fino a che non si spegne; </li><li> Onde: parametri fisiologici di controllo delle onde fino a quando il mare diventa completamente calma; </li><li> Cascata: la riduzione del risveglio fisiologico corrisponde ad una riduzione dell&#39;intensità del flusso finché l&#39;acqua smette di cadere. </li><li> Gazebo: diversamente dagli altri, questo esercizio è personalizzabile da parte del paziente, che può scegliere le parole o immagini relative a lui / lei vicende personali stressanti. Questa tecnica serve un programma di vaccinazione di stress, e il paziente può sperimentare la riduzione delle dimensioni-sbiadimento fluttuante della voce di scelta - a seconda del suo / la sua capacità di rilassarsi lui / lei; </li></ul></li><li> Una volta completata la sessione di realtà virtuale, parametri fisiologici sono registrati ancora una volta in condizioni di riposo; </li><li> Finale di valutazione dello stato clinico del paziente. </li></ol> UFFICIO TERAPISTA FUORI&#39;S: Al fine di migliorare l&#39;efficacia della formazione e per aumentare gli effetti di rilassamento, i pazienti pratica tecniche di rilassamento fuori ufficio terapeuta, sperimentando uno scenario Domiciliare. Ciò si realizza con la presentazione dello stesso ambiente virtuale sperimentato durante la terapia su un dispositivo mobile. Il dispositivo mobile è collegato via Bluetooth ad una serie di biosensori che comprende battito cardiaco e la conduttanza cutanea. Questo apparecchio è abbastanza piccolo e facile da usare per essere gestito autonomamente dal paziente (vedi fig. 1). Il paziente è richiesto di formare capacità di rilassamento, almeno una volta al giorno per tutta la durata del trattamento (un mese), seguendo il piano di trattamento fornito dal terapeuta. Rappresentante Risultati Poiché sia ​​il biofeedback e la realtà virtuale sono noti per essere efficace per indurre il rilassamento, il principale risultato atteso è che una combinazione di questi due metodi è più efficace nel ridurre l&#39;ansia nei pazienti affetti da disturbo d&#39;ansia generalizzato, che le due tecniche utilizzate singolarmente. <img src="/files/ftp_upload/1554/Fig1.jpg" alt="Figura 1" /> Figura 1: GSR / AR modulo biosensori.

<ol>
	<li>Arntz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+therapy+versus+applied+relaxation+as+treatment+of+generalized+anxiety+disorder.">Cognitive therapy versus applied relaxation as treatment of generalized anxiety disorder.</a> Behav Res Ther. 41 (6), 633-633 (2003).</li><li>Siev, J., Chambless, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+treatment+effects:+cognitive+therapy+and+relaxation+for+generalized+anxiety+and+panic+disorders.">Specificity of treatment effects: cognitive therapy and relaxation for generalized anxiety and panic disorders.</a> Journal of consulting and clinical psychology. 75 (4), 513-513 (2007).</li><li>Jacobsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progressive relaxation. , (1938).</li><li>Villani, D., Riva, F., Riva, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technologies+for+relaxation:+the+role+of+presence.">New technologies for relaxation: the role of presence.</a> International journal of Stress Management. 14 (3), 260-260 (2007).</li></ol>

The authors have nothing to disclose.

La nostra ipotesi è che la combinazione di realtà virtuale e biofeedback che modifica direttamente l&#39;ambiente virtuale, in confronto con la realtà virtuale da solo, si tradurrà in un relax migliore e più veloce apprendimento. Se è così, questo nuovo strumento creato per il trattamento di disturbi d&#39;ansia potrebbe essere applicato e testato anche in altre patologie legate ansia.

the use of biofeedback in clinical virtual reality: the intrepid project

Disturbo d&#39;ansia generalizzato (GAD) è un disturbo psichiatrico caratterizzato da una costante ansia e aspecifici che interferisce con le attività quotidiane. La sua elevata prevalenza nella popolazione generale e le gravi limitazioni che provoca, sottolineano la necessità di trovare nuove strategie efficaci per trattarla. Insieme ai trattamenti cognitivo-comportamentale, il rilassamento rappresenta un approccio utile per il trattamento del GAD, ma ha il limite che è difficile da imparare. INTREPID Il progetto si propone di implementare un nuovo strumento per trattare l&#39;ansia e disturbi legati per testare la sua efficacia clinica nel ridurre l&#39;ansia sintomi correlati. L&#39;innovazione di questo approccio è la combinazione di realtà virtuale e il biofeedback, in modo che la prima è direttamente modificato dal uscita del secondo. In questo modo, il paziente è a conoscenza delle reazioni proprio attraverso la modifica di alcune caratteristiche dell&#39;ambiente di realtà virtuale in tempo reale. Utilizzando esercizi mentali del paziente impara a controllare questi parametri fisiologici e utilizzando il feedback fornito dai ambiente virtuale è in grado di misurare il suo successo. L&#39;utilizzo supplementare di dispositivi portatili, come ad esempio PDA o smart-phone, permette al paziente di eseguire a casa, individualmente e autonomamente, gli stessi esercizi con esperienza in ufficio terapeuta. L&#39;obiettivo è quello di ancorare il protocollo appreso in un contesto di vita reale, in modo da migliorare la capacità dei pazienti &#39;a che fare con i loro sintomi. Il risultato atteso è una migliore e più veloce apprendimento di tecniche di rilassamento, e quindi una maggiore efficacia del trattamento rispetto ai tradizionali protocolli clinici.

Watch this Scientific Journal Video about The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project at JoVE.com

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Il Progetto su Virtual Reality intelligente multi-sensore di sistema per il trattamento di ansia disturbi legati all&#39;alimentazione (Intrepid) è finalizzato a sviluppare un multi-sensore di context-aware sistema di realtà virtuale per il trattamento di ansia disturbi correlati.

L&#39;uso del Biofeedback in Clinica Realtà Virtuale: Il Progetto INTREPID

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

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Research

JoVE Journal

Biology

15.0K Views.  Istituto Auxologico Italiano. Il Progetto su Virtual Reality intelligente multi-sensore di sistema per il trattamento di ansia disturbi legati all'alimentazione (Intrepid) è finalizzato a sviluppare un multi-sensore di context-aware sistema di realtà virtuale per il trattamento di ansia disturbi correlati.

Video: The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

L&#39;uso di Rotorod come un metodo per l&#39;analisi qualitativa del Camminare in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Ricerca

Biologia

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

Lo stress di valutazione psicofisiologica Utilizzando Biofeedback

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

L&#39;uso del kit di rilevamento a fluorescenza della proteasi per determinare l&#39;attività della proteasi

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

L&#39;uso di SC1 (Pluripotin) a supporto Mesc auto-rinnovamento in assenza di LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;uso di chemostati in Biologia microbica Sistemi

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Base-Probe Real-time PCR Approcci per la misurazione quantitativa dei microRNA

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

L&#39;utilizzo di biosensori enzimatica per quantificare endogena ATP o H<sub&gt; 2</sub&gt; O<sub&gt; 2</sub&gt; Nel rene

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...