Name: bacterial detection &amp; identification using electrochemical sensors
Uploaded: 2013-04-23T12:00:00
Description: Watch this Scientific Journal Video about Bacterial Detection & Identification Using Electrochemical Sensors at JoVE.com

Denne undersøgelse blev støttet af Cooperative aftale Award AI075565 (til DAH) fra National Institute of Allergy og smitsomme sygdomme samt af Wendy og Ken Ruby Fund for Excellence i Pediatric Urology Research. BMC er Judith og Robert Winston Chair i Pediatric Urology.

1.. Funktionalisering af elektrokemiske sensorer <ol><li> Forbered thiolerede opfangningsprobe ved en koncentration på 0,05 uM i 300 uM 1,6-hexandithiol (HDT), 10 mM Tris-HCI, pH 8,0, 0,3 M NaCl, 1 mM EDTA og inkuberes i mørke ved stuetemperatur i 10 min . Inkubation af thiolerede indfangningsproben med HDT sikrer, at thiolgruppe på opfangningsproben er reduceret, hvilket resulterer i mere ensartede resultater. </li><li> Påfør en strøm af nitrogen til bare guld 16 sensor-chip (r) i 5 sekunder for at fjerne fugt ...

<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. 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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. 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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>

Den elektrokemiske sensor assay beskrevet her muliggør hurtig påvisning af nukleinsyremål. Sensitivitet og specificitet til dels afhænge af den frie energi for target-probehybridisering, hvilket igen afhænger af længden og GC indhold opsamling og detektorprober. Vi udfører typisk hybridiseringstrin ved stuetemperatur (~ 20 ° C) 5, 6. Dog kan hybridiseringstrin (3.2 og 3.3) også udføres ved højere temperaturer i en hybridisering ovnen, hvis chippen er placeret i en overdækket kammer indeholdende fu...

Brug rRNA som en målmolekyle til bakteriel detektion og identifikation har en række fordele. Den overflod af rRNA i bakterieceller giver en følsomhed grænse så lav som 250 bakterier per milliliter uden behov for målamplifikation 1.. Bakteriel rRNA indeholder unikke arter-specifikke sekvenser, der er tilgængelige for hybridisering med DNA-prober. Følgelig kan et array af elektrokemiske sensorer anvendes til at identificere ukendte bakterier, hvor hver sensor er funktionaliseret med en anden arts-specifik opfangningsprobe. Positive Sensorer bør medtages for et syntetisk oligonukleotid mål om, at &quot;broer&quot; til opsamling og detektorprober at skabe et indre kalibrering signal. Elektrokemiske sensorer har en bred vifte af grundlæggende og translationel forskning applikationer. For eksempel som beskrevet assayet her er blevet anvendt til præcist at måle effekten af E. coli vækstfase på rrnA og præ-rRNA kopiantal, som er af stor interesse for forskere interesseret i bakteriel fysiologi 2.. Følsomheden af ​​den elektrokemiske sensor assayet bestemmes af signal-støj-forholdet. En række af signalforstærkning og støjreduktion metoder er blevet udforsket. Vi finder, at en forbedring af kemien i sensorens overflade er nøglen til at reducere ikke-specifik binding af detektorprobe og / eller HRP enzym. Navnlig har en blandet monolag af alkanedithiols og mercaptohexanol vist sig at reducere baggrund ved at dække elektroden overflade mere fuldstændigt samtidig bevare tilgængeligheden af indfangningsproben til target-hybridisering 3.. Disse overfladekemien behandlinger er særligt vigtige for analyser, der involverer komplekse biologiske prøver.

<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

Elektrokemiske sensorer er almindeligt brugt til hurtig og præcis måling af blodsukker og kan tilpasses til påvisning af en lang række analytter. Elektrokemiske sensorer virker ved at transducere et biologisk anerkendelse begivenhed til et nyttigt elektrisk signal. Signaltransduktion forekommer ved kobling af aktiviteten af ​​en redox-enzym til en amperometrisk elektrode. Sensor specificitet er enten en iboende egenskab af enzymet glucoseoxidase i tilfælde af en glukosesensor, eller et produkt af binding mellem enzymet og et antistof eller proben. Her beskriver vi en elektrokemisk sensor analysemetode til direkte detektere og identificere bakterier. I hvert tilfælde er proberne beskrevet her DNA-oligonukleotider. Denne metode er baseret på sandwich-hybridisering af opsamling og detektor sonder med target ribosomale RNA (rRNA). Opfangningsproben er forankret til sensoroverfladen, mens detektorproben er knyttet til horseradish peroxidase (HRP). Når et substrat, såsom 3,3 &#39;, 5,5&#39;-tetramethylbenzidin (TMB) sættes til en elektrode med capture-target-detektor komplekser bundet til sin overflade er substratet oxideres af HRP og reduceret med arbejdselektroden. Denne redox cyklus medfører shuttling af elektroner fra substratet fra elektroden til HRP, der producerer strøm i elektroden.

Vi beskriver en elektrokemisk analyse, der er struktureret på samme måde som en sandwich-ELISA. Som vist i figur 1, mål ribosomale RNA (rRNA) hybridisering med opsamling og detektor sonder er udviklet af et redox reaktion katalyseret af HRP konjugeret til anti-fluorescein-antistof-fragmenter, der binder til 3&#39;fluorescein binding på detektoren sonden. En vigtig del af analysesensitivitet er overfladen kemi guldelektroden. Vi har fundet, at en ternær monolag bestående af thiolerede indfangningsp...

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

Bacterial Detection & Identification Using Electrochemical Sensors

Vi beskriver en elektrokemisk sensor analysemetode for hurtig påvisning af bakterier og identifikation. Assayet involverer et sensorarray funktionaliseret med DNA oligonucleotid capture prober til ribosomale RNA (rRNA) artsspecifikke sekvenser. Sandwich hybridisering af mål-rRNA med indfangningsproben og en peberrodsperoxidase-bundet DNA-oligonukleotid detektorprobe frembringer en målelig amperometrisk strøm.

Bakteriel Detection og identifikation Brug elektrokemiske sensorer

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

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Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

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

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

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Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Hollow mikro-baserede sensor for Multiplexed Transdermal Elektrokemisk Sensing

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

Sensing af Barrier vævssprængning med en organisk Elektrokemisk Transistor

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Glutamine Flux Imaging Using Genetically Encoded Sensors

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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

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

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