Diese Studie wurde von Cooperative Agreement Auszeichnung AI075565 (bis DAH) von der National Institute of Allergy and Infectious Diseases und durch die Wendy und Ken Rubin Fund for Excellence in Pediatric Urology Forschung gefördert. BMC ist die Judith und Robert Winston Chair in Pediatric Urology.

1. Funktionalisierung von Elektrochemische Sensoren <ol><li> Vorbereiten des thiolierten Fangsonde mit einer Konzentration von 0,05 uM in 300 uM 1,6-Hexandithiol (HDT), 10 mM Tris-HCl, pH 8,0, 0,3 M NaCl, 1 mM EDTA und Inkubation im Dunkeln bei Raumtemperatur für 10 min . Inkubation der thiolierten Einfangsonde mit HDT sorgt dafür, dass die Thiolgruppe auf der Capture-Sonde reduziert wird, was zu mehr konsistente Ergebnisse. </li><li> Tragen Sie einen Stickstoffstrom auf blankem Gold 16 Sensorarraychips (e) für 5 Sek. Feuchtigkeit und / oder Partikel zu entfernen. </li><li> Übernehmen 6 ul der HDT-thiolierten Einfangsonde Mix zur Arbeitselektrode aller 16 Sensoren des Sensor-Arrays und speichern den Sensorchip (s) in einem überdachten Petrischale bei 4 ° C über Nacht. Thiolierte Fangsonden binden direkt auf den blanken Goldelektrode und der HDT wirkt, um Überspritzungen der Fangsonden verhindern und halten sie in einer gestreckten Konformation, die eine Hybridisierung mit dem Ziel fördert. </li><li> Diefolgenden Tag, waschen Sie den Sensor-Chip mit deionisiertem H 2 O für 2-3 sec und trocken unter einem Strom von Stickstoff für 5 sek. </li><li> Anwenden 6 ul 10 mM Tris-HCl, pH 8,0, 0,3 M NaCl, 1 mM EDTA, 1 mM 6-Mercapto-1-hexanol (MCH) an die Arbeitselektrode von allen Sensoren 16 und Inkubation für 50 min. Dieser und alle nachfolgenden Sensorchip Inkubationen werden in einem bedeckten Petrischale bei Raumtemperatur durchgeführt. MCH wirkt als Blockierungsmittel, Auffüllen der Lücken in dem thiolierten Fangsonde oder HDT nicht vorhanden ist auf der Elektrodenoberfläche. </li></ol> 2. Probenvorbereitung <ol><li> 1 ml der Bakterienkultur in der logarithmischen Wachstumsphase (OD 600 ≈ 0,1) zu einem Mikrozentrifugenröhrchens und Zentrifuge bei 16.000 xg für 5 min. Entfernen Sie den Kulturüberstand. Das bakterielle Pellet kann unmittelbar weiterverarbeitet oder bei -80 ° C für die spätere Verwendung. </li><li> Gründlich resuspendieren die bakterielle Pellet in 10 ul 1 M NaOH, indem die Pipettenspitze zu dem Bottom des Mikrozentrifugenröhrchens und Auf-und Abpipettieren mehrmals. Inkubieren der Suspension bei Raumtemperatur für 5 min. </li><li> Neutralisieren des bakteriellen Lysat durch Zugabe von 50 ul 1 M Phosphatpuffer, pH 7,2, enthaltend 2,5% Rinderserumalbumin (BSA) und 0,25 mM eines Fluorescein-modifizierten Detektorsonde. Inkubieren des neutralisierten Lysats für 10 min bei Raumtemperatur. Fluorescein-modifizierten Detektorsonden mit bakteriellen rRNA Zielmoleküle hybridisieren. </li></ol> 3. Elektrochemische Sensor Assay <ol><li> Waschen der MCH vom Sensorchip mit deionisiertem H 2 O für 2-3 Sekunden gewaschen und unter einem Stickstoffstrom für 5 Sekunden. </li><li> Übernehmen 4 ul neutralisiert Bakterienlysat zur Arbeitselektrode jeder 14 Sensoren und die Proben für 15 min. Ziel-Detektorsonde Komplexe immobilisiert thiolierten Fangsonden hybridisieren. </li><li> Übernehmen 4 ul 1 nM Überbrückungsoligonukleotids in 1 M Phosphatpuffer, pH 7,2, mit 2,5% BSA und 0,25 &amp;mu; M Fluorescein-modifizierten Detektorsonde bis 2 positive Kontrolle Sensoren (für Signal-Normalisierung) und Inkubation für 15 min. </li><li> Waschen des Sensorchips mit deionisiertem H 2 O für 2-3 Sekunden gewaschen und unter einem Stickstoffstrom für 5 Sekunden. </li><li> Übernehmen 4 ul von 0,5 U / ml anti-Fluorescein-HRP in 1 M Phosphatpuffer, pH 7,2, mit 0,5% Casein zur Arbeitselektrode aller 16 Sensoren und die Proben für 15 min. Die Anti-Fluorescein-HRP bindet an die immobilisierten fluoresceinmodifiziertem der Fühler. </li><li> Waschen des Sensorchips mit deionisiertem H 2 O für 2-3 Sekunden gewaschen und unter einem Stickstoffstrom für 5 Sekunden. </li><li> Tragen Sie einen Film auch Aufkleber auf der Oberfläche des Sensorchips und Last in den Sensor-Chip Halterung. Je 50 ul TMB Substrat, auf allen 16 Sensoren und schließen Sie den Sensor-Chip Halterung. </li><li> Erhalten Amperometrie und Cyclovoltammetrie Messungen für alle 16 Sensoren mit der Helios Chip Reader. Amperometric Strom ist proportional zur Geschwindigkeit der TMB reduction auf der Sensoroberfläche (siehe Abbildung 1). </li></ol>

<ol>
	<li>Wu, J., Campuzano, S., Halford, C., Haake, D. A., 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=Ternary+Surface+Monolayers+for+Ultrasensitive+(Zeptomole)+Amperometric+Detection+of+Nucleic+Acid+Hybridization+without+Signal+Amplification.">Ternary Surface Monolayers for Ultrasensitive (Zeptomole) Amperometric Detection of Nucleic Acid Hybridization without Signal Amplification.</a> Anal. Chem. 82, 8830-8837 (2010).</li><li>Halford, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Antimicrobial+Susceptibility+Testing+by+Sensitive+Detection+of+Precursor+Ribosomal+RNA+Using+a+Novel+Electrochemical+Biosensing+Platform.">Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor Ribosomal RNA Using a Novel Electrochemical Biosensing Platform.</a> Antimicrob. Agents Chemother. 56, (2012).</li><li>Campuzano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+monolayers+as+DNA+recognition+interfaces+for+direct+and+sensitive+electrochemical+detection+in+untreated+clinical+samples.">Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.</a> Biosens. Bioelectron. 26, 3577-3584 (2011).</li><li>Gau, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+molecular+analysis+without+nucleic+acid+amplification.">Electrochemical molecular analysis without nucleic acid amplification.</a> Methods. 37, 73-83 (2005).</li><li>Patel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Specific+Capture+Enhances+Sensitivity+of+Electrochemical+Detection+of+Bacterial+Pathogens.">Target Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens.</a> J. Clin. Microbiol. 49, 4293-4296 (2011).</li><li>Mastali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+probe+length+and+target+location+for+electrochemical+detection+of+selected+uropathogens+at+ambient+temperature.">Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.</a> J. Clin. Microbiol. 46, 2707-2716 (2008).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrochemical+DNA+biosensors+for+rapid+molecular+identification+of+uropathogens+in+clinical+urine+specimens.">Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.</a> J. Clin. Microbiol. 44, 561-570 (2006).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+advanced+electrochemical+DNA+biosensor+for+bacterial+pathogen+detection.">Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection.</a> J. Mol. Diagn. 9, 158-168 (2007).</li><li>Pedrero, M., Campuzano, S., Pingarron, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroanalytical+sensors+and+devices+for+multiplexed+detection+of+foodborne+pathogen+microorganisms.">Electroanalytical sensors and devices for multiplexed detection of foodborne pathogen microorganisms.</a> Sensors (Basel). 9, 5503-5520 (2009).</li><li>Kuralay, F., Campuzano, S., Haake, D. A., 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=Highly+sensitive+disposable+nucleic+acid+biosensors+for+direct+bioelectronic+detection+in+raw+biological+samples.">Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.</a> Talanta. 85, 1330-1337 (2011).</li><li>Ecker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ibis+T5000:+a+universal+biosensor+approach+for+microbiology.">Ibis T5000: a universal biosensor approach for microbiology.</a> Nat. Rev. Microbiol. 6, 553-558 (2008).</li><li>Casalta, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+LightCycler+SeptiFast+test+in+the+rapid+etiologic+diagnostic+of+infectious+endocarditis.">Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis.</a> Eur. J. Clin. Microbiol. Infect. Dis. 28, 569-573 (2009).</li></ol>

Alle Autoren sind Erfinder von Patenten relevant zu den beschriebenen Methoden. Eines dieser Patente hat Qvella Corporation lizenziert. BMC und DAH haben Kapitalanteil in Qvella Corporation. VG ist der Präsident der GeneFluidics, die der Hersteller der elektrochemischen Sensor-Array-Chip, Chip-Halterung und Chip-Leser in diesem Papier beschrieben.

Der elektrochemische Sensor beschriebenen Test hier ermöglicht die schnelle Detektion von Nukleinsäure-Targets. Sensitivität und Spezifität hängen teilweise von der freien Energie der Target-Sonden-Hybridisierung, die ihrerseits abhängig von der Länge und GC-Gehalt der Erfassung und der Fühler. Wir üblicherweise die Schritte Hybridisierung bei Raumtemperatur (~ 20 ° C) 5, 6. Jedoch können die Hybridisierung der Schritte (3.2 und 3.3) auch bei höheren Temperaturen in einem Wärmeschrank durchgefüh...

Mit rRNA als Zielmolekül für bakterielle Nachweis und zur Identifizierung hat eine Reihe von Vorteilen. Die Fülle der rRNA in bakteriellen Zellen sorgt für eine Nachweisgrenze von nur 250 Bakterien pro Milliliter, ohne die Notwendigkeit für Zielamplifikation 1. Bakterielle rRNA enthält eindeutige Spezies-spezifischen Sequenzen, die zugänglich Hybridisierung mit DNA-Sonden sind. Folglich kann eine Anordnung von elektrochemischen Sensoren verwendet werden, um unbekannte Bakterien, wobei jeder Sensor mit einer unterschiedlichen Spezies-spezifische Fangsonde funktionalisierte identifizieren. Positive Control-Sensoren sollten für ein synthetisches Oligonukleotid Ziel, dass &quot;Brücken&quot; die Gefangennahme und der Fühler eine interne Kalibrierung zu erzeugen einbezogen werden. Elektrochemische Sensoren haben eine breite Palette von Grundlagenforschung und translationale Forschung. Zum Beispiel beschriebene Test hier wurde verwendet, um präzise die Wirkung von E. coli Wachstumsphase auf RRnA und pre-rRNA Kopie Zahlen, die von großem Interesse für die Forscher in der bakteriellen Physiologie 2 interessiert ist. Die Empfindlichkeit des elektrochemischen Sensors Assay wird durch das Signal-Rausch-Verhältnis bestimmt. Eine Vielzahl von Signalverstärkung und Rauschunterdrückung Methoden erforscht worden. Wir finden, dass die Verbesserung der Chemie der Sensoroberfläche Schlüssel zur Verringerung der unspezifischen Bindung von Detektor-Sonde und / oder HRP-Enzym. Insbesondere wurde eine gemischte Monoschicht Alkandithiolen und Mercaptohexanol erwiesen Hintergrund durch Abdecken der Elektrodenoberfläche vollständiger Beibehaltung Zugänglichkeit der Einfang-Sonde für Zielhybridisierung 3 zu reduzieren. Diese Oberflächenchemie Behandlungen sind besonders wichtig für die Assays, die komplexen biologischen Proben.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>6-mercapto-1-hexanol (MCH)</td>
 <td>Sigma</td>
 <td>451088</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>1,6-hexanedithiol (HDT)</td>
 <td>Sigma</td>
 <td>H-12005</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>Thiolated capture probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Fluorescein-modified detector probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Bridging Oligonucleotide</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Fluorescein-HRP, Fab fragments</td>
 <td>Roche</td>
 <td>11 426 346 910</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Helios Chip Reader</td>
 <td>GeneFluidics</td>
 <td>GFR-2009</td>
 <td></td>
 </tr>
 <tr>
 <td>Sensor Chip Mount</td>
 <td>GeneFluidics</td>
 <td>GFR-003</td>
 <td></td>
 </tr>
 <tr>
 <td>Film well sticker</td>
 <td>GeneFluidics</td>
 <td></td>
 <td>Shipped with sensor chips</td>
 </tr>
 <tr>
 <td>Bare gold 16-sensor array chips </td>
 <td>GeneFluidics</td>
 <td>SC1000-16X-B </td>
 <td>Store in 100% N2 at room temperature</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin </td>
 <td>Sigma</td>
 <td>A7906 </td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>1M Phosphate Buffer, pH 7.2 </td>
 <td></td>
 <td></td>
 <td>0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2</td>
 </tr>
 <tr>
 <td>Blocker Casein in PBS </td>
 <td>Pierce</td>
 <td>37528 </td>
 <td>Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 &deg;C</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

bacterial detection &amp; identification using electrochemical sensors

Elektrochemische Sensoren sind weit verbreitet für die schnelle und genaue Messung des Blutzuckerspiegels verwendet werden und kann für den Nachweis einer Vielzahl von Analyten angepasst werden. Elektrochemische Sensoren arbeiten nach Transduktion einer biologischen Erkennung Veranstaltung in ein nützliches elektrisches Signal um. Die Signalübertragung erfolgt durch Koppeln der Aktivität eines Redox-Enzym zu einer amperometrischen Elektrode. Sensor Spezifität entweder eine inhärente Eigenschaft des Enzyms Glucoseoxidase bei einer Glucose-Sensor, oder ein Produkt der Verknüpfung zwischen dem Enzym und einem Antikörper oder der Sonde. Hier beschreiben wir einen elektrochemischen Sensor Assay-Verfahren direkt zu erfassen und zu identifizieren Bakterien. In jedem Fall sind die hier beschriebenen DNA-Sonden-Oligonukleotide. Diese Methode basiert auf Sandwich-Hybridisierung von capture und der Fühler mit Ziel ribosomalen RNA (rRNA) basiert. Die Fänger-Sonde an den Sensor Oberfläche verankert, während der Detektor-Sonde an h gebunden istorseradish Peroxidase (HRP). Wenn ein Substrat, wie 3,3 &#39;, 5,5&#39;-Tetramethylbenzidin (TMB) mit einer Elektrode mit capture-Ziel-Detektor Komplexe gebunden an dessen Oberfläche zugegeben wird, wird das Substrat durch HRP oxidiert und reduziert durch die Arbeitselektrode. Das Redox-Zyklus führt pendelt der Elektronen durch das Substrat von der Elektrode zum HRP, wodurch Strom in der Elektrode.

Wir beschreiben einen elektrochemischen Assay, ähnlich einer Sandwich-ELISA strukturiert. Wie in 1, Ziel ribosomalen RNA (rRNA) Hybridisierung mit Erfassung und der Fühler durch eine Redox-Reaktion katalysiert HRP konjugiert an Anti-Fluorescein-Antikörper-Fragmente, die an das 3&#39;-Fluorescein-Verknüpfung gebunden auf dem Detektor-Sonde entwickelt gezeigt. Ein wichtiger Bestandteil der Empfindlichkeit des Assays ist die Oberflächenchemie des Goldelektrode. Wir haben gefunden, dass eine ternäre M...

Watch this Scientific Journal Video about Bacterial Detection & Identification Using Electrochemical Sensors at JoVE.com

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Wir beschreiben einen elektrochemischen Sensor Testverfahren zum schnellen Nachweis von Bakterien und Identifikation. Der Test umfasst eine Sensoranordnung mit DNA-Oligonukleotid-Capture-Sonden für die ribosomale RNA (rRNA) artspezifische Sequenzen funktionalisiert. Sandwich-Hybridisierung der Ziel-rRNA mit dem Capture-Sonde und einem Meerrettichperoxidase-linked DNA-Oligonukleotid Detektorsonde erzeugt eine messbare amperometrischen Strom.

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 ...

bacterial-detection-identification-using-electrochemical-sensors

Research

JoVE Journal

Bioengineering

28.1K Aufrufe.  Veterans Affairs Greater Los Angeles Healthcare System. Wir beschreiben einen elektrochemischen Sensor Testverfahren zum schnellen Nachweis von Bakterien und Identifikation. Der Test umfasst eine Sensoranordnung mit DNA-Oligonukleotid-Capture-Sonden für die ribosomale RNA (rRNA) artspezifische Sequenzen funktionalisiert. Sandwich-Hybridisierung der Ziel-rRNA mit dem Capture-Sonde und einem Meerrettichperoxidase-linked DNA-Oligonukleotid Detektorsonde erzeugt eine messbare amperometrischen Strom.

Serie: Bacterial Detection & Identification Using Electrochemical Sensors

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Charakterisierung Bakterielle Flüchtige mit Secondary Elektrospray-Ionisation-Massenspektrometrie (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Forschung

Biotechnik

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

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 ...

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 ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Glutamin Flux Imaging Verwendung genetisch codierten Sensoren

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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 ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

Fliegen-Insekt Erkennung und Klassifizierung mit preiswerten Sensoren

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Markierungsfreie Einzelmoleküldetektion Mit Mikrotoroid optischen Resonatoren

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Mehrfarbenfluoreszenzdetektion für Droplet Mikrofluidik Verwendung von Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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, ...