Name: optical detection of e. coli bacteria by mesoporous silicon biosensors
Uploaded: 2013-11-20T17:00:00
Description: Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Dette arbejde blev støttet af Israel Science Foundation (tilskud nr. 1118-1108 og tilskud nr. 1146/12) og Minna Kroll Memorial Research Fund. ES taknemmelig økonomisk støtte fra Russell Berrie Nanoteknologi Institut.

1.. Fremstilling af oxideret porøs SiO2 <ol><li> Etch Si wafers (single side poleret på &lt;100&gt; ansigt og stærkt doteret P type, 0,0008 Ω · cm) i et 3:1 (v / v) opløsning af vandig HF og absolut ethanol i 30 sekunder ved en konstant strøm densitet på 385 mA / cm2. Bemærk venligst, at HF ​​er en stærkt ætsende væske, og det bør håndteres med største omhu. </li><li> Skyl overfladen af ​​den resulterende porøse Si (PSI) film med absolut ethanol adskillige gange, og tør f...

<ol>
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En etiket-fri optisk immunosensor, baseret på en PSiO 2 nanostruktur (en Fabry-Perot tynd film) er fremstillet, og dets potentielle anvendelighed som en biosensor for bakterier detektion er bekræftet. Ændringer og fejlfinding Et af de største problemer, når designe en immunosensor er modtageligheden af antistoffer til at undergå uønskede kropsbygning ændringer under afsætning og mønster på det faste substrat, hvilket kan føre til et fald i bio...

Tidlig og præcis identifikation af sygdomsfremkaldende bakterier er yderst vigtig for fødevare-og vandsikkerhed, miljøovervågning, og point-of-care diagnostik 1. Som traditionel mikrobiologi teknikker er tidskrævende, besværligt, og mangler evnen til at detektere mikroorganismer i &quot;real-time&quot; eller uden for laboratoriet miljø, er biosensorer udvikler sig til at imødekomme disse udfordringer 2-5. I de senere år har porøs Si (PSI) dukket op som et lovende platform for udformningen af sensorer og biosensorer 6-20. Gennem det seneste årti talrige undersøgelser vedrørende PSI-baserede optiske sensorer og biosensorer blev offentliggjort 21,22. Den nanostrukturerede PSi lag fremstilles typisk ved elektrokemisk anodisk ætsning fra en enkelt krystal Si wafer. De resulterende Psi nanomaterialer udviser mange fordelagtige egenskaber, såsom stor overflade og fri volumen, pore størrelser, der kan kontrolleres og tunable optical egenskaber 10,16. De optiske egenskaber af Psi lag, såsom fotoluminescens 8,11 og hvidt lys reflektans-baserede interferometry 7,19, er stærkt påvirket af miljømæssige forhold. Opsamling af gæstemolekyler / målanalytter i de porøse lag resulterer i en ændring i det gennemsnitlige brydningsindeks af filmen, observeres som en graduering i fotoluminescens spektrum eller som en bølgelængdeforskydning i reflektionsniveauet spektrum 10. Selv om langt innovation i PSi optisk biosensor teknologi, der er kun få rapporter om PSI-baserede platforme for bakterier afsløring 6,8,20,23-29. Desuden har de fleste af disse proof-of-concept studier har vist &quot;indirekte&quot; bakterier afsløring. Således er normalt, før lysis af celler kræves for at udtrække de målrettede protein / DNA-fragmenter, som er karakteristiske for de undersøgte bakterier 29. Vores tilgang er at direkte fange målbakterierneceller på PSI biosensor. Derfor er monoklonale antistoffer, som er specifikke til at målrette bakterier, immobiliseres på den porøse overflade. Binding af bakterieceller, via antistof-antigen-interaktioner på overfladen af biosensoren inducere ændringer i amplituden (intensiteten) af refleksionsevne spektrum 24-26. I dette arbejde, rapporterer vi om opførelsen af en optisk PSi-baserede biosensor og demonstrere dens anvendelse som en etiket-fri biosensorer platform til påvisning af Escherichia coli (E. coli) K12 bakterier (bruges som model mikroorganisme). Den overvågede optiske signal er reflekteret fra PSi nanostruktur grund Fabry-Perot tynd film interferens (figur 1A) lys. Ændringer i lyset amplitude / intensitet korreleret til specifik immobilisering af target bakteriecellerne på biosensoroverfladen, giver mulighed for hurtig påvisning og kvantificering af bakterierne.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

<html> En etiket-fri optisk biosensor baseret på en nanostruktureret porøs Si er designet til hurtig opsamling og påvisning af Escherichia coli K12 bakterier, som en model mikroorganisme. Biosensoren afhængig direkte binding af target bakteriecellerne på dens overflade, mens ingen forbehandling (f.eks cellelysis), i den undersøgte prøve er påkrævet. Et mesoporøst Si tynd film anvendes som optiske transducer element i biosensoren. Under hvidt lys belysning, viser det porøse lag vel løst Fabry-Perot fringe mønstre i sin refleksionsevne spektrum. Anvendelse af en hurtig Fourier transformation (FFT) til reflektivitet data resulterer i en enkelt top. Ændringer i intensiteten af ​​FFT peak overvåges. Således målbakterier fange på biosensoroverfladen gennem antistof-antigen interaktioner inducerer målbare ændringer i intensiteten af ​​FFT toppe, giver mulighed for en &quot;real time&quot; observation af bakterier vedhæftet fil. nt &quot;&gt; mesoporøse Si film, fremstillet ved en elektrokemisk anodiseringsproces, konjugeres med monoklonale antistoffer, der er specifikke for den pågældende bakterie. immobilisering, immunaktivitet og specificitet af antistofferne bekræftes ved fluorescerende mærkning eksperimenter. Når biosensoren er udsat for målbakterier cellerne direkte fanget på antistof-modificerede porøs Si overflade. Disse specifikke opfange begivenheder medfører intensitet ændringer i optisk interferens spektret er tynd film af biosensoren. Vi viser, at disse biosensorer kan påvise relativt lave bakteriekoncentrationer (afsløring grænse på 10 4 celler / ml) i mindre end en time.

Oxideret psi (PSiO 2) film fremstilles som beskrevet i protokol tekst afsnittet. Figur 1B viser en høj opløsning scanningselektronmikrografi af den resulterende PSi filmen efter termisk oxidation. Den PSiO 2 lag er karakteriseret ved veldefinerede cylindriske porer med en diameter i området 30-80 nm. Det monoklonale antistof (IgG)-molekyler podes på de PSiO 2 overflader ved anvendelse af en veletableret silanisering teknologi kombineret m...

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Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

En etiket-fri optisk biosensor for hurtige bakterier detektion introduceres. Biosensoren er baseret på en nanostruktureret porøs Si, som er designet til direkte at fange målet bakterieceller på dens overflade. Vi anvender monoklonale antistoffer immobiliseret på den porøse transducer, som bindingsprober. Vore undersøgelser viser anvendeligheden af ​​disse biosensorer til påvisning af lave bakterielle koncentrationer inden for få minutter uden forudgående forarbejdning prøve (såsom cellelysis).

Optisk påvisning af<em&gt; E. coli</em&gt; bakterier ved Mesoporøse Silicon biosensorer

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

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Bioengineering

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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