Sandia ist Multiprogramm Labor von Sandia Corporation, ein Unternehmen Lockheed Martin, für die der Vereinigten Staaten, Department of National Nuclear Security Administration Energy unter Vertrag DE-AC04-94AL85000 betrieben. Die Autoren danken der Finanzierung von den Sandia National Laboratories &#39;Laboratory Directed Research &amp; Development (LDRD) Programm.

1. Microneedle Fabrication <ol><li> Mit dreidimensionalen Modellierung Software Solidworks (Dassault Systemes SA, Velizy, Frankreich), die Gestaltung eines pyramidal-förmige hohle Mikronadel Array (Abbildung 1). 5.3 </li><li> Entwerfen Sie eine Struktur zur Unterstützung der Mikronadelarrays mit Magics RP 13 Software (Materialise NV, Leuven, Belgien). Die Trägerstruktur kann das Harz aus der Vorrichtung während der Herstellung Drain und stellt eine Basis, auf der die Mikronadeln gebaut. Ein Beispiel Trägerstruktur in 1 gezeigt. </li><li> Die verknüpfte Unterstützung und Mikronadelarrays Dateien werden in den Perfactory RP-Software (envisionTEC GmbH, Gladbeck, Deutschland), die den Herstellungsprozess steuert hochgeladen. In diesem Software-Paket auswählen, die Anzahl der Mikronadel Arrays hergestellt werden, und die Platzierung von Vorrichtungen auf die Herstellung Platte. </li><li> Führen Kalibrierung im Ultraviolett-Modus bei 180 mW für den schnellen Perfactory prototyping Fertigungssystem und überprüfen Sie die Abweichung in der Energie liegt innerhalb von ± 2 mW. </li><li> Nach Fertigung abgeschlossen ist, entfernen Sie die Mikronadelarrays von der Grundplatte und in Isopropanol entwickeln für 15 Minuten. Trocknen Sie die Arrays mit Druckluft und heilen die Mikronadeln bei Raumtemperatur für 50 Sekunden in der Otoflash Nachhärtung System (envisionTEC GmbH, Gladbeck, Deutschland), um eine vollständige Polymerisation sicherzustellen. </li><li> Bestätigen Mikronadel Fertigung über Mikroskopie und überprüfen, ob jeder Mikronadel Bohrung hohl und nicht behindert wird. Voll hergestellt Mikronadeln sind in Abbildung 2 dargestellt. </li></ol> 2. Herstellung von Kohlenstoff-Paste Elektroden-Arrays <ol><li> Verwenden Sie eine 60-W-Modell 6,75 CO 2-Raster-/ Vektor-Laser-System (Universal Laser Systems, Inc., Scottsdale, AZ), um Löcher schneiden und legen das darunterliegende einzeln adressierbar Anschluss Kupferdrähte in einem Flachbandkabel (21039-0249), das war erhalten von einer kommerziellen Quelle (Molex-Connector Corp, Lisle, IL) (Abbildung 3 (A und B)). Legen Sie die flache flexible Kabel in einer Spannvorrichtung richtig auszurichten sie auf der Laser-Ablation Platte. Mit einem Ansatz Rasterung bis 500 Mikrometer Durchmesser Hohlräume in dem isolierenden Abschnitt des flexiblen Kabels zu schaffen. Patterns für die Ablation werden in CorelDraw erstellt (Corel, Ottawa, Ontario) und an das Lasersystem. </li><li> Reinigen Sie die modifizierte flache flexible Kabel mit einem Airbrush, dass Sprays Aceton bei 40 psi. Beenden der Reinigung sie durch Spülen mit entionisiertem Wasser und Isopropanol. Überprüfen unter einem Mikroskop, dass kein Isolierfilm über den freiliegenden Kupferstreifen bleibt. </li><li> Der nächste Schritt besteht darin, ein Haltehohlraum für die Verpackung von Kohlenstoff-Pasten zu schaffen. Melinex Bandes (0,002 &quot;Dicke auf einer einzigen Seite mit einem druckempfindlichen Acrylklebstoff beschichtet) mit dem gleichen Muster wie die Elektrodenstreifen, ausgerichtet in den abgetragenen Elektrodenstreifen und komprimiert bei 3000 psi für 2 Minuten, um eine sichere Verbindung zu gewährleisten abgetragen. In Dieses CAse ist der Hohlraum Durchmesser 750 um. </li><li> Eine zusätzliche Schicht Melinex Bandes (0,004 &quot;Dicke auf beiden Seiten mit druckempfindlichen Acrylkleber) wird anschließend in dem gleichen Muster wie dem einseitigen Klebeband abgetragen wird und nach der Ausrichtung zum Binden der Mikronadelarrays zu den Kohlenstoff-Paste Elektroden-Arrays . </li></ol> 3. Synthese von funktionellen Carbon-Pasten und Verpackung der Elektroden-Kavitäten <ol><li> Die Glucose-empfindliche Kohlepaste wird einer vorhergehenden Rezept und wird durch Mischen von 10 mg Glucoseoxidase und 2,2 mg Poly (ethylenimin), bis eine homogene Mischung erhalten wird. 16 Zu dieser Mischung wurden 60 mg Rhodium auf Kohlenstoff-Pulver ( 5% Beladung) zugegeben. 40 mg Mineralöl zugegeben und anschließend gemischt. Die Pasten werden bei 4 ° C bis zur Verwendung der Pasten sind bis zu einer Woche nach der Herstellung verwendet. </li><li> Die pH-empfindlichen C Paste durch Mischen von 30% (w / w) Mineralöl und 70% (w / w) p Graphit erhaltenowder. Packen Sie fügen in die Elektrode Hohlraum, wie in Abschnitt 3.4 beschrieben. Machen Lösung von 10 mM Fast Blue RR Diazoniumsalz (4-Benzoylamino-2 ,5-dimethoxybenzenediazonium Chlorid Hemi (Zinkchlorid) Salz) in 0,5 M Phosphorsäure. 17. Legen Sie eine 20 ul Tropfen dieser Lösung über das verpackte Paste-Elektrode für 30 Minuten, um spontan die Chemisorption Fast Blue PR Diazoniumsalz. Mit deionisiertem Wasser abspülen und zwischenspeichern oder entionisiertes Wasser bei Nichtgebrauch. </li><li> Die Laktat-sensitiven Kohlepaste wird von einem vorherigen Rezept und wird durch Mischen von 2,5 mg Rhodium auf Kohlenstoff-Pulver und 2,5 mg Lactat-Oxidase erhalten, im Wechsel zwischen 5 Minuten und 5 Minuten Beschallung von Vortexen für fünf Umdrehungen. 18 </li><li> Verpackung der modifizierten Pasten in den vorbereiteten flachen flexiblen Kabels wird durch Anlegen der jeweiligen Pasten in den Hohlräumen Elektrode erreicht. Durch eine dünne Stück Plastik (z. B. eine Kante aus einem Kunststoff wiegen Boot) als Packung Kelle und ter fügen, bis eine glatte Oberfläche erzielt wird. Wiederholen Sie mit einem zweiten sauberen Boot mit einem Gewicht von bis überschüssige Paste entfernt wird. Waschen mit destilliertem Wasser. Eine schematische Darstellung, Laserablation, um Hohlräume zu schaffen, Verpacken von Kohlenstoff-Pasten und Mikronadel Integration (beschrieben in Abschnitt 2 und 3) ist in Abbildung 3 dargestellt. </li></ol> 4. Erkennung und Kalibrierung des Sensors <ol><li> Lactate über die Messung der chronoamperometrischen Reaktion des Sensors auf -0,15 V und der Aufzeichnung des aktuellen nach 15 Sekunden in 0,1 erreicht M Phosphatpuffer (pH = 7,5). 4 (a) eine schematische Darstellung der elektrokatalytischen Reaktion zur Detektion von Laktat . </li><li> Glucose Nachweis wird in ähnlicher Weise durch Messen der chronoamperometrischen Reaktion des Sensors auf -0,05 V und der Aufzeichnung des aktuellen nach 15 Sekunden in 0,1 M Phosphatpuffer (pH 7,0) durchgeführt. 4 (b) eine schematische Darstellung der elektrokatalytischen Reaktion detection von Glukose. </li><li> pH-Wert wird durch Ausführen cyclovoltammetrische Scans von -0,7 V bis 0,8 V bei 100 mV / s und Aufzeichnen der Position des oxidativen Peakpotential überwacht. Ein Schema der Redox-Reaktionen für die pH-Detektion ist in 5 gezeigt. </li><li> Kalibrierkurven für Glukose und Laktat-Sensoren können durch aufeinanderfolgende Zugaben von der jeweiligen Analyten erstellt werden; chronoamperometrischen Messungen werden nach jeder Analyt Neben Auftritten als in den Abschnitten 5.1 und 5.2 beschrieben. Alternativ können feste Potential chronoamperometrischen Messungen unter Rühren aber ausreichend Zeit (~ 10-100 Sekunden) zwischen den einzelnen Analyten zusätzlich zur aktuellen Stabilisierung vorgenommen werden. </li><li> pH Eichkurven kann durch Messung der Position des oxidativen Peakpotential über eine Reihe von bekannten pH-Werten von 5 bis 8 in Schritten von 1,0 pH-Einheiten und Aufzeichnung Cyclovoltammogramme, wie in Abschnitt 5.3 beschrieben erstellt werden. </li></ol> 5. Vertreter ReErgebnisse Bei der Gewinnung chronoamperometrischen Kurven (z. B. für Glucose Nachweis oder Lactat-Detektion) in ruhenden Lösungen mit folgenden modifizierten Kohlenstoff-Mikronadeln gefüllt, wird der Strom sofort nach Anlegen des jeweiligen Detektionspotential verringern. Es wird schließlich zu einem stationären Wert verfallen. Ein repräsentatives Ergebnis ist in 6 gezeigt, das Ergebnis wurde aus 2 mM Zusätze von Lactat und Aufzeichnung der Lactat Mikronadel erhalten. Die Lösung muss kurz nach jeder Zugabe gerührt Laktat werden. Die aktuelle nach 15 Sekunden nach oben steigt die Konzentration von Laktat, die aktuelle Antwort kann dann verwendet, um die Konzentration von Lactat in einer unbekannten Lösung zu bestimmen. Alternativ kann eine kontinuierliche Überwachung in einer gerührten Lösung (oder in einer fließenden Lösung) als eine Lösung mit zunehmender Glucosekonzentration (5) gezeigt verwendet werden. Auch hier erhöht die Zunahme des Stroms, der bei ter Konzentration von Glukose verwendet werden, um die Glukose als Reaktion auf eine unbekannte Lösung zu standardisieren. Es muss genug Zeit nach jeder Spike gewährt werden, um damit die Lösung zu stabilisieren. Cyclovoltammogramme an der pH-empfindliche Mikronadel in 0,1 M Phosphatpuffer auf vier verschiedene Lösungen von pH 5 bis 8, 1 pH-Einheit Schritten in 6 gezeigt. Die oxidativen Peakpotential Schichten mit zunehmendem pH-Wert; dieses Phänomen wird als ein Indikator für die pH-Wert verwendet. <img alt="1" src="/files/ftp_upload/4067/4067fig1.jpg" /> 1. Bilder von der STL-Datei des Mikronadelarray in Solidworks (A) und der Druck Bildschirms, wobei die Trägerstruktur (B) zeigt erstellt. <img alt="2" src="/files/ftp_upload/4067/4067fig2.jpg" /> 2. Rasterelektronenmikroskopische Aufnahmen der Mikronadel-Anordnung (A) und einer Mikronadel in diesem Array (B). Abbildung 3. Schematische Darstellung des Flachbandkabels Montage. Die einzelnen Schritte das Modifizieren der flachen flexiblen Kabels (A), Abtragen der gemusterten Kreisen (B), indem die zunächst abgetragen Melinex Schicht, die mit Kohlenstoff-Paste (C) gefüllt ist, sowie das Hinzufügen der zweiten Schicht und abgetragenen Melinex Zusammensetzen der Mikronadelarrays (D). <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> . <img alt="Abbildung 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> Abbildung 4. Kalibrierung von Laktat-und Kleinschreibung Paste mit 15 Sekunden chronoamperometrischen Scans bei -0,15 V in 0,1 M Phosphatpuffer (pH = 7,5). Jede Erhöhung des Stroms entspricht einer 2 mM Zusatz von Laktat. <img alt="Abbildung 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> Abbildung 5. </sTrong&gt; Kalibrierung von Glukose-und Kleinschreibung Paste läuft chronoamperometrischen Scans bei -0,05 V. Jede Abnahme der Reaktion entspricht einer 2 mM Zugabe von Glucose. Die 0,1 M Phosphat-Pufferlösung (pH = 7,0) wurde während der Kalibrierung gerührt. Weitere Ag / AgCl-Referenz und Pt und Gegenelektroden wurden in dieser Studie verwendet. <img alt="6" src="/files/ftp_upload/4067/4067fig6.jpg" /> Abbildung 6. Cyclovoltammogramm (CV) der pH-sensitiven Kohlenstoff in 0,1 M Phosphatpuffer auf pH 5-8 fügen Sie in Schritten von 1 pH-Einheit (teal = pH 8,0, grün = pH 7,0, pH 6,0 = lila, rot = pH 5,0). Eine fünfte CV wurde für die Analyse gegen Ag / AgCl-Referenzelektrode und Pt-Draht Gegenelektroden verwendet.

<ol>
	<li>Henry, S., McAllister, D. V., Allen, M. G., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+microneedles:+a+novel+approach+to+transdermal+drug+delivery.">Microfabricated microneedles: a novel approach to transdermal drug delivery.</a> J. Pharm. Sci. 87, 922-925 (1998).</li><li>Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedles+for+transdermal+drug+delivery.">Microneedles for transdermal drug delivery.</a> Adv. Drug Deliv. Rev. 56, 581-587 (2004).</li><li>Miller, P. R., Gittard, S. D., Edwards, T. L., Lopez, D. M., Xiao, X., Wheeler, D. R., Monteiro-Riviere, N. A., Brozik, S. M., Polsky, R., Narayan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+carbon+fiber+electrodes+within+hollow+polymer+microneedles+for+transdermal+electrochemical+sensing.">Integrated carbon fiber electrodes within hollow polymer microneedles for transdermal electrochemical sensing.</a> Biomicrofluidics. 5, 013415-013415 (2011).</li><li>Windmiller, J. R., Zhou, N., Chuang, M. C., Valdés-Ramírez, G., Santhosh, P., Miller, P. R., Narayan, R., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array-based+carbon+paste+amperometric+sensors+and+biosensors.">Microneedle array-based carbon paste amperometric sensors and biosensors.</a> Analyst. 136, 1846-1851 (2011).</li><li>Windmiller, J. R., Vald&#233;s-Ram&#237;rez, G., Zhou, N., Zhou, M., Miller, P. R., Jin, C., Brozik, S. M., Polsky, R., Katz, E., Narayan, R., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicomponent+microneedle+array+biosensor+for+minimally-invasive+glutamate+monitoring.">Bicomponent microneedle array biosensor for minimally-invasive glutamate monitoring.</a> Electroanal. 23, 2302-2309 (2011).</li><li>Ricci, F., Moscone, D., Palleschi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+continuous+glucose+monitoringwith+microdalysis+technique:+The+example+of+GlucoDay.">Ex vivo continuous glucose monitoringwith microdalysis technique: The example of GlucoDay.</a> IEEE Sensors J. 8, 63-70 (2008).</li><li>Zimmermann, S., Fienbork, D., Flounders, A. W., Liepmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-device+enzyme+immobilization:+Wafer-level+fabrication+of+an+integrated+glucose.">In-device enzyme immobilization: Wafer-level fabrication of an integrated glucose.</a> Sens. Actuat. B. 99, 163-173 (2004).</li><li>Miller, P. R., Skoog, S. A., Edwards, T. L., Lopez, D. M., Wheeler, D. R., Arango, D. C., Xiao, X., Brozik, S. M., Wang, J., Polsky, R., Narayan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microneedle-based+biosensor+array+for+characterization+of+metabolic+acidosis.">Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.</a> Biomicrofluidics. 88, 739-742 (2012).</li><li>Miller, P. R., Skoog, S. A., Edwards, T. L., Lopez, D. M., Wheeler, D. R., Arango, D. C., Xiao, X., Brozik, S. M., Wang, J., Polsky, R., Narayan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microneedle-based+biosensor+array+for+characterization+of+metabolic+acidosis.">Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.</a> Talanta. 88, 739-742 (2012).</li><li>Rofstad, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment-induced+cancer+metastasis.">Microenvironment-induced cancer metastasis.</a> Int. J. Radiat. Biol. 76, 589-605 (2000).</li><li>Vander Heiden, M. G., Cantley, L. C., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Warburg+effect:+the+metabolic+requirements+of+cell+proliferation.">Understanding the Warburg effect: the metabolic requirements of cell proliferation.</a> Science. 324, 1029-1033 (2009).</li><li>Warburg, O., Wind, F., Negelein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+tumors+in+the+body.">The metabolism of tumors in the body.</a> J. Gen. Physiol. 8, 519-530 (1927).</li><li>Goode, J. A., Chadwick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tumour+Microenvironment:+Causes+and+Consequences+of+Hypoxia+and+Acidity.">The Tumour Microenvironment: Causes and Consequences of Hypoxia and Acidity.</a> Novartis Foundation Symposium 240. , (2008).</li><li>Cardone, R. A., Casavola, V., Reshkin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+disturbed+pH+dynamics+and+the+Na+/H++exchanger+in+metastasis.">The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis.</a> Nature Rev. Cancer. 5, 786-795 (2005).</li><li>Robergs, R. A., Ghiasvand, F., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+exercise-induced+metabolic+acidosis.">Biochemistry of exercise-induced metabolic acidosis.</a> Am. J. Phys. 287, R502-R516 (2004).</li><li>Wang, J., Liu, J., Chen, L., Lu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+membrane-free,+mediator-free+glucose+biosensor.">Highly selective membrane-free, mediator-free glucose biosensor.</a> Anal. Chem. 66, 3600-3603 (1994).</li><li>Makos, M. A., Omiatek, D. M., Ewing, A. G., Heien, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+voltammetric+carbon-fiber+microelectrode+pH+sensor.">Development and characterization of a voltammetric carbon-fiber microelectrode pH sensor.</a> Langmuir. 26, 10386-10391 (2010).</li><li>Wang, J., Chen, Q., Pedrero, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+biosensing+of+lactate+at+lactate+oxidase+containing+rhodium-dispersed+carbon+paste+electrodes.">Highly selective biosensing of lactate at lactate oxidase containing rhodium-dispersed carbon paste electrodes.</a> Anal. Chem. Acta. 304, 41-46 (1995).</li></ol>

Keine Interessenskonflikte erklärt.

Mehrere Aspekte der Gestaltung dieser Mikronadel-basierten Sensor wurden als vor der Herstellung der Vorrichtung. Um diesen Sensor für die Echtzeit-Detektion verwendet, muss die Reaktion des Sensors gering; in diesem Protokoll, zeigte jede getestete Sensor eine Reaktionszeit unter fünfzehn Sekunden. Pasten im Sinne dieses Protokolls wurden auch auf ihre Selektivität in In-vivo-Umgebungen, die elektrisch aktiven Biomoleküle, die mit Elektroden-Reaktion stören können enthalten gewählt. Neben der Kompositio...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Flexible Flachbandkabel </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;Side Klebeband </td><td> Melinex </td><td></td></tr><tr><td> 0.004 &quot;Doppelseitiges Klebeband </td><td> Melinex </td><td></td></tr><tr><td> Lactatoxidase </td><td> Sigma </td><td> L0638 </td></tr><tr><td> Glucose-Oxidase </td><td> Sigma </td><td> G7141 </td></tr><tr><td> Rhodium auf Kohle </td><td> Sigma </td><td> 206164 </td></tr><tr><td> Graphitpulver </td><td> Sigma </td><td> 385031000 </td></tr><tr><td> Poly (ethylenimin) </td><td> Acros </td><td> 178570010 </td></tr><tr><td> Mineralöl </td><td> Sigma </td><td> M5904 </td></tr><tr><td> Glucose </td><td> Sigma &lt;/ Td&gt; <td> G8270 </td></tr><tr><td> Laktat </td><td> Sigma </td><td> L1750 </td></tr><tr><td> Fast Blue RR Salz </td><td> Sigma </td><td> F0500 </td></tr><tr><td> e-Shell 300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> e-Shell 200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCl-Referenzelektrode </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> Pt-Draht </td><td> Basi </td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash Nachhärtung System </td><td> EnvisionTEC </td><td></td></tr><tr><td> Phosphorsäure </td><td> Fischer </td><td> A366-4 </td></tr><tr><td> Modell 60W 6,75 CO 2-Raster / Vektor-Laser-System </td><td> Unisal Laser Systems </td><td> PLS6.75 </td></tr><tr><td> CorelDraw </td><td> Corel </td><td></td></tr><tr><td> Solidworks </td><td> Dassault Systemes </td><td> 2009 </td></tr><tr><td> Magics RP13 </td><td> Materialise </td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

Die Entwicklung einer minimal-invasiven Multiplex-Monitoring-System für die schnelle Analyse von biologisch-relevante Moleküle bieten konnte Individuen mit chronischen Erkrankungen facile Bewertung ihrer unmittelbaren physiologischen Zustand. Darüber hinaus könnte es als Recherche-Tool für die Analyse von komplexen, multifaktoriellen Erkrankungen dienen. Um für einen solchen Multianalyt Sensor realisiert werden, muss es minimal-invasive, Probenahme von interstitiellen Flüssigkeit muss ohne Schmerzen oder Schäden für den Benutzer auftreten, und die Analyse muss schnell als auch selektiv. Zunächst für die schmerzfreie Drug Delivery entwickelt, Mikronadeln wurden zur Herstellung von Vakzinen und pharmakologische Mittel (zB Insulin) durch die Haut zu liefern. 1-2 Da diese Geräte in den interstitiellen Raum zuzugreifen, können Mikronadeln, die mit Mikroelektroden integriert sind als transdermales verwendet werden elektrochemischen Sensoren. Die selektive Detektion von Glucose, Glutamat, Lactat, hydrogen Peroxid und Ascorbinsäure wurde anhand von integrierten Mikronadel-Elektrodeneinrichtungen mit Kohlefasern, modifizierte Kohlenstoffprodukt Pasten, und Platin-beschichteten Polymer Mikronadeln, die als Wandlerelemente. 3-7,8 Diese Mikronadel Sensorik hat einen neuartigen und anspruchsvollen analytischen Ansatz zur in-situ und simultane Detektion von mehreren Analyten aktiviert. Multiplexing bietet die Möglichkeit der Überwachung komplexer Mikroumgebungen, die sonst nur schwer in einer schnellen und minimal-invasive Weise zu charakterisieren sind. Zum Beispiel kann diese Technik zur gleichzeitigen Überwachung der extrazelluläre Konzentration von könnte verwendet werden, Glukose, Laktat und pH-Wert 9 sind wichtige Indikatoren für metabolische Erkrankungen 7,10-14 (zB Krebs Proliferation) und belastungsinduzierten Azidose. 15

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Dieser Artikel beschreibt den Bau eines Multiplex-Mikronadel-basierten Sensor. Das Gerät wird für die in situ Probenahme und elektrochemischen Analyse von mehreren Analyten in einer schnellen und selektiven Weise entwickelt. Wir haben die Vision der klinischen Medizin und der biomedizinischen Forschung verwendet für diese Mikronadel-basierte Sensoren.

Hohle Mikronadel-basierten Sensor für Multiplex Transdermale elektrochemische Nachweis

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K Aufrufe.  University of North Carolina and North Carolina State University. Dieser Artikel beschreibt den Bau eines Multiplex-Mikronadel-basierten Sensor. Das Gerät wird für die in situ Probenahme und elektrochemischen Analyse von mehreren Analyten in einer schnellen und selektiven Weise entwickelt. Wir haben die Vision der klinischen Medizin und der biomedizinischen Forschung verwendet für diese Mikronadel-basierte Sensoren.

Serie: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolekulare Erkennung Einsatz der Interferometrische Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Forschung

Biotechnik

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexed Einzelmolekül-Kraftspektroskopie Proteolyse Messungen mit magnetischen Pinzette

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bakterielle Erkennung und Identifizierung mit elektrochemischen Sensoren

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

Erkundung der Barrier Gewebestörung mit einem organischen Transistor Elektro

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Einem mikrofluidischen Biochip-basierte Elektrochemische für Markierungsfreie DNA-Hybridisierungsanalyse

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembran Fabrication durch das Lösungsmittel-unterstützten Lipiddoppelschicht (SALB) Methode

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Hohlfaser-Bioreaktoren für<em&gt; In Vivo</em&gt; -ähnlichen Mammalian Gewebekultur

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Extraktion von Pflanzenbasis Kapseln für Mikroverkapselung Anwendungen

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Snap Chip für Cross-reactivity frei und Spotter Multiplex Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Entwicklung der eine elektrochemische DNA-Biosensor, einem Foodborne Krankheitserreger zu erkennen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...