Il lavoro riportato nel presente documento è stato sostenuto da sovvenzioni da Auburn University AUDFS e USAF CRADA 07-277-60MDG-01. Le opinioni espresse in questo articolo sono quelle degli autori e non riflettono la politica o la posizione della United States Air Force, Dipartimento della Difesa, o il governo degli Stati Uniti ufficiale.

1. Ponendo le basi <ol><li> Ottenere tipo ceppo S. aureus ATCC 12600, S. aureus ATCC 27690 e di Bacillus subtilis ATCC 6051. Ceppi meticillino-resistenti di S. aureus - MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; Il fago litico 12600. </li><li> Ottenere PBP 2a anticorpi coniugati perle di lattice. </li><li> Preparare NZY media come descritto 21. </li><li> Ottenere Soluzione tampone fosfato (PBS). </li><li> Preparare acqua deionizzata come sottofase di Langmuir-Blodgett (LB) monostrato deposizione. </li></ol> 2. Batteriofago Propagazione e titolazione <ol><li> Incubare 5 ml di coltura durante la notte di S. aureus ATCC 12600 (3.6 x 10 8 unità formanti colonie (CFU) / ml) con 500 ml di fago (3 x 10 9 &lt;/ Sup&gt; PFU / ml) in 500 ml di mezzo NZY in 2 L Flack su agitatore-incubatore a 37 ° C durante la notte. </li><li> Centrifugare coltura a 4.424 xg per 10 min a 4 ° C. </li><li> Supernatante re-centrifugati a 11.325 xg per 10 min a 4 ° C. </li><li> Filtrare il surnatante attraverso un filtro Millipore 0,22 micron. </li><li> Centrifugare il filtrato a 75.395 xg per 1,5 ore. </li><li> Sciogliere pellet in 100 ml di acqua distillata. </li><li> Preparare diluizioni di 10 volte di sospensione fago in media NZY. </li><li> Piastra 1 ml di una notte (ON) la cultura di S. aureus ATCC 12600 su ciascuna delle due piastre con agar NZY per assicurarsi che tutta la superficie è coperta. Rimuovere l&#39;eccesso di cultura e di lasciare che la superficie asciutta per 30 min. </li><li> Individuare un campione (10 microlitri) di diluizione appropriata fago sulla superficie della piastra e incubare a 37 ° C per 18-24 ore. </li><li> Esaminare la formazione di placche e calcolare il titolo fago. Batteriofago titoli (T) viene stimato sulla base del numero di pLaques (N) formate per 10 microlitri di volume (v) di sospensione fago e il fattore di diluizione (F). Il titolo è stato calcolato con la formula:. T = N / (VXF) Ad esempio, per il numero di placche 75 alla diluizione 10 -7 e il volume 10 microlitri (10 x 10 -3 ml), il titolo è uguale 75 / (10 x 10 x 10 -3 -7) = 7,5 x 10 10 unità formanti placche (PFU) per ml di sospensione. </li></ol> 3. Phage Gold-immobilizzato <ol><li> Pezzi di quarzo oro rivestite Clean (60 mm 2) di incisione al plasma in argon per 10 minuti e poi sterilizzare per 6 ore sotto la luce UV in un armadio sterile. </li><li> Aggiungere 50 microlitri di 1 x 10 9 PFU / ml sospensione fago alla superficie d&#39;oro di ogni pezzo in una piastra di Petri sterile, e poi incubare una notte in una camera umida a temperatura ambiente. </li><li> Rimuovere e titolo per il restante sospensione fago. </li><li> Lavare pezzi con fago rilegato 5 volte con PBS per rimuovere non legatofago. </li></ol> 4. Test di Immobilizzato infettività Phage su una superficie asciutta <ol><li> Diffondere una cultura O / N di S. Aureus ATCC 12600 su una piastra di agar NZY e lasciare asciugare. </li><li> Posizionare un pezzo d&#39;oro con fago immobilizzato sul viso piatto verso il basso. </li><li> Osservare una zona di lisi dopo 12 ore di incubazione a 37 ° C che indica infettività. </li><li> Utilizzare pezzi coperti d&#39;oro senza fago in esperimenti di controllo. </li></ol> 5. Test di attività litica del fago libero e legato a liquido <ol><li> Aggiungi una cultura ON di S. aureus ATCC 12.600-10 ml di NZY in ogni dei quattro beute da 300 ml sidearm (6 x 10 6 UFC / ml in ciascun pallone). </li><li> Aggiungi In due palloni 2 x 10 6 PFU / ml di fago libero. </li><li> Usare gli altri due palloni come controlli. </li><li> Monitorare l&#39;andamento nel tempo di S. lisi cellulare aureus per 570 min a misura di densità ottica (OD 600) ad intervalli di 30 min per tutte le beute.</li><li> Effettuare una misurazione finale a 24 hr. </li><li> Ripetere i passaggi 5,1-5,5, ma utilizzare pezzi d&#39;oro con i fagi legati invece di fagi gratuiti e monete d&#39;oro senza fago in flaconi di controllo. </li></ol> 6. Perdita di fagi Bound durante l&#39;incubazione <ol><li> Aggiungere 50 ml di 1 x 10 9 PFU / ml fago sulla superficie del pezzo oro sterile in una piastra di Petri che è posto in una camera umida notte a temperatura ambiente. </li><li> Rimuovere la sospensione con non legato fago dalla superficie d&#39;oro e titolo. </li><li> Lavare il pezzo d&#39;oro con fago bound 5 volte con PBS e posto in 1 ml di soluzione NZY in 15 ml di tubo in agitatore-incubatore a 37 ° C per 8 ore. </li><li> Determinare il titolo del fago distaccato come descritto in 2. </li></ol> 7. Phage Spheroids Preparazione <ol><li> La combinazione di 400 ml di magazzino fago 12600 sospensione (10 11 PFU / ml) con un uguale volume di cloroformio spettrofotometrica di grado in 1 ml fialaa temperatura ambiente. </li><li> Delicatamente vortice della sospensione fago-cloroformio 5-6 volte (intervalli di 5 secondi), più di un minuto nella durata. </li><li> Consentito la sospensione di stabilizzare per 30 sec e poi pipetta della fase superiore contenente sferoidi è stato dispensato off per la preparazione monostrato. </li><li> Richiedere alcuni microlitri della sospensione sferoidi per la trasmissione e la microscopia elettronica a scansione. </li><li> Utilizzare un residuo sospensione sferoide per la produzione di biosensori. </li></ol> 8. Preparazione biosensore <ol><li> Pulire i sensori QCM-D di incisione al plasma in argon per 10 minuti PDC-32G Harrick pulitore plasma. </li><li> Risciacquare sensore con esano per rimuovere eventuali impurezze organiche. </li><li> Preparare e pulire un trogolo del saldo pellicola come descritto in 22. </li><li> Riempire LB attraverso una soluzione sottofase e pulirlo con una barriera depressione e stabilizzare la vasca a 20 ± 0,1 ° C </li><li> Distribuire 300 ml un&#39;aliquota del fago 12600 in acquosa suspension (10 11 PFU / ml) su LB sottofase. </li><li> Preparare fago monostrato sulla soluzione sottofase LB permettendo 300 microlitri aliquota di fago 12600 sospensione acquosa (10 11 PFU / ml) a correre giù una bacchetta di vetro bagnabile inclinato che è parzialmente immerso nella sottofase 22,23. </li><li> Stabilizzare il monostrato preparata per 10 minuti, e poi comprimerlo ad una velocità di 30 mm / min (45 cm 2 / min) fino ad una pressione costante di 19 N / m è raggiunto. </li><li> Eseguire una deposizione di film sulla verticale perpendicolare realizzata sensore QCM-D ad una velocità di 4,5 mm / min in successione immersione sensore in e fuori del monostrato sette volte. Microscopia elettronica di monostrati depositati fago prima esposizione a campioni batterici mostrato che gli strati erano continui, omogeneo e uniforme (dati non mostrati). </li><li> Ripetere i passaggi 4,5-4,8 con una sospensione sferoide fago che viene preparato in 3. </li><li> Utilizzare biosensori fago fabbricato per MRSA rilevazione, elettronemicroscopia, e ellissometria. </li></ol> 9. Test biosensore, la discriminazione di meticillino resistente e batteri di stafilococco sensibili <ol><li> In questo protocollo, biosensori con non modificato e modificato fago e sferoidi fagi sono preparati. Un microbalance cristallo di quarzo con quattro camere di flusso del sensore è utilizzato per monitorare legame dei batteri al fago immobilizzato sulla superficie del sensore QCM. PBP 2a anticorpi coniugati perle di lattice sono utilizzati per discriminare tra resistente alla meticillina (MRSA) e batteri di stafilococco sensibili. Tutte le misurazioni sono condotte nel modo di flusso (50 microlitri / min) a 5 MHz. </li><li> Stabilire una linea di base frequenza di risonanza del sensore QCM in acqua (~ 30 min). </li><li> Disegnare una sospensione di live meticillina sensibili stafilococco cellule batteriche in acqua (10 9 CFU / ml) attraverso la cella di prova. </li><li> Continuamente monitorare i cambiamenti nella frequenza di risonanza sensori e dissipazione di energia per la prima armonica, USIng il software Q-Soft. </li><li> Quando le variazioni della frequenza di risonanza sensori e dissipazione raggiunto livelli di saturazione, aggiungere la sospensione di PBP anticorpo coniugato perle di lattice (6,77 x10 10 perline / ml) per il flusso durante il monitoraggio continuo della frequenza e dissipazione. </li><li> Ripetere i passaggi 9,2-9,5 per stafilococco meticillino-resistenti cellule batteriche (MRSA). </li><li> Definire una variazione di frequenza attraverso la variazione di massa dovuta a batteri cogenti del Sauerbrey equazione 24,25 Af = Δm-xn / C, dove C è la sensibilità di massa costante (C = 17,7 ng x cm x -2 -1 Hz a 5 MHz ), m è la massa ed n è il numero armonico (n = 1). </li><li> Definire un cambiamento dissipazione energetica (Δ D) dalla registrazione il decadimento esponenziale di oscillazione (frequenza e ampiezza smorzamento, che ha permesso la quantificazione dell&#39;energia dissipata e immagazzinato durante un periodo di oscillazione,E dissipata ed E memorizzati, rispettivamente: Δ D = E dissipata / 2 πE memorizzato. Δ S è misurata in unità di dissipazione (DU), uno = 10 -6 unità relative DU). </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> J. Vis. Exp. (63), e3064 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

E &#39;noto che i fagi possono essere usati come sonde biosensore per patogeni batterici 28. Si dimostra in questo lavoro che fago insieme con anticorpi PBP 2a può essere utilizzata per risolvere il vecchio problema: ceppi resistenti e sensibili agli antibiotici discriminazione rapidi. Si è riscontrato però quelle normali fagi stafilococciche non modificate non sono adatti al rilevamento batteri con i dispositivi QCM, anche se si legano batteri. La coda fago è così lunga che ...

Ceppi meticillino-resistenti di Staphylococcus aureus sono stati suggeriti come fattore di infezioni essenziali e le epidemie nosocomiali 4-8. Modi comuni di riconoscimento di resistenza alla meticillina, come la diffusione oxacillina prova schermo agar disco, o brodo microdiluizione, affidano a condizioni di coltura su misura per migliorare l&#39;espressione di resistenza. Alterazioni comprendono l&#39;utilizzo di oxacillina, incubazione a 30 o 35 ° C invece di 37 ° C, e l&#39;inclusione di NaCl al mezzo di crescita. Inoltre, per una corretta rilevazione da questi tipi di tecniche, un lungo periodo di incubazione di 24 ore anziché 16 a 18 ore è necessaria. Tecniche rapide con appropriato livello (&gt; 96%) di sensibilità per l&#39;identificazione di resistenza alla meticillina comprendono tecniche microdiluizione automatici come il Vitek GPS-SA card, il sistema Rapid ATB stafilococco, e il sistema pannello Rapid Microscan che producono risultati dopo 3-11 ore 9-11. The Crystal MID sistema RSA è un metodo rapido basato sul riconoscimento della crescita di S. aureus in presenza di 2% di NaCl e 4 mg di oxacillina per litro con un sensore di fluorescenza sensibili all&#39;ossigeno. Sensibilità rivendicati variano tra il 91 e il 100% dopo 4 ore di incubazione di 12-14. Questi metodi fenotipici limita le precisioni dall&#39;impatto di ceppi prevalenti che esprimono resistenza eterogenea. Pertanto, i metodi più ampiamente accettato per il riconoscimento della resistenza alla meticillina è PCR o ibridazione DNA del gene mecA 15. Tuttavia questa tecnica richiede DNA purificato ed è estremamente sensibile a vari additivi (impurità), che includono cellule detriti 16. Inoltre, queste tecniche hanno bisogno di molto tempo per eseguire. Strategie per il riconoscimento del prodotto del gene mecA, proteina PBP 2a, potrebbero essere utilizzati per determinare la resistenza e possono essere più affidabile rispetto alle tecniche di prova standard 17. di Staphylococcus aureus compresi quelli aventi resistenza alla meticillina 1,2,18. In questo lavoro abbiamo proposto una nuova tecnica nel riconoscimento specifico e la rilevazione di MRSA, come ad esempio il riconoscimento di batteri con conformazione di MRSA in tempo reale. Per questo scopo specifico di una S. batteriofago aureus con un ampio spettro di ospiti (tra cui i ceppi di MRSA) in combinazione con anticorpi monoclonali contro la proteina (PBP 2a) sono stati utilizzati. PBP 2a è una proteina della parete cellulare ed è la causa di antibiotico resistività di MRSA. Tuttavia PBP 2a anticorpo non è specifico per S. aureus da altri batteri sono proteine ​​leganti antibiotici con similarità di sequenza di PBP 2a 19,20. Di conseguenza, in questo lavoro, S. aureus batteriofago e anticorpi contro proteine ​​PBP 2a sono stati utilizzati. Per essere in grado di sviluppare un biosensore per specificamente rilevare e identificare MRSA un dispositivo con un due stadi è stato utilizzato. Il passo iniziale utilizzato un S. aureus batteriofago monostrato come sonda sensore, mentre la seconda fase impiega anticorpi specifici PBP 2a. Pertanto, passo uno riconosceranno S. aureus batteri, come l&#39;altra saranno sensibili alla proteina antibiotico-binding. Quando i segnali ricevuti da due passi sono positivi, indica il rilevamento specifico di MRSA.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

Un batteriofago litico strutturalmente trasformato avere una vasta gamma di ospiti di ceppi di Staphylococcus aureus e di una proteina legante la penicillina (PBP 2a) anticorpi coniugati perle di lattice sono stati utilizzati per creare un biosensore progettato per la discriminazione di meticillino-resistente (MRSA) e sensibile (MSSA) S . specie aureus 1,2. I fagi litici sono state convertite in sferoidi fagiche dal contatto con all&#39;interfaccia acqua-cloroformio. Fago monostrati sferoidali sono stati spostati su una superficie biosensore da Langmuir-Blodgett (LB) tecnica 3. I biosensori creati sono stati esaminati da una microbilancia cristallo di quarzo con il monitoraggio di dissipazione (QCM-D) per valutare le interazioni batteri-fagi. Interazioni batterio-sferoidali portato alla frequenza di risonanza ridotta e un aumento della dissipazione energetica per entrambi i ceppi MRSA e MSSA. Dopo il legame batterico, questi sensori sono stati ulteriormente esposta alla penicillina-binding protein anticorpo lattice tallones. Sensori analizzati con MRSA hanno risposto a PBP 2a perline anticorpi, anche se i sensori controllati con MSSA hanno dato alcuna risposta. Questa distinzione sperimentale determina una discriminazione univoca tra resistente alla meticillina e S. sensibile aureus. Altrettanto legati e non legati batteriofagi sopprimere la crescita batterica sulle superfici e nelle sospensioni idriche. Una volta che i fagi litici vengono trasformati in sferoidi, conservano la loro forte attività litica e mostrano un&#39;elevata capacità di cattura batterica. Il fago e fago sferoidi possono essere utilizzati per il test e la sterilizzazione di microrganismi resistenti agli antibiotici. Altre applicazioni possono includere l&#39;uso in terapia batteriofago e superfici antimicrobiche.

Il fago dimostrato attività litica contro tutti i ceppi testati di S. aureus, compresi i ceppi di MRSA, come indicato dallo spot test fago. Dimensioni della placca in genere variavano da 5 a 15 mm. Nessuna attività è stata trovata contro altri test-culture (Tabella 1). Una crescita normale di S. aureus ATCC 12600 in terreno NZY on-agitatore incubatore a 37 ° C è mostrata nella Figura 1A (una curva etichettato da cerchi vuoti). Il numer...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Biosensori fagi litici e perline anticorpi sono in grado di discriminare tra resistente alla meticillina (MRSA) e batteri di stafilococco sensibili. I fagi sono stati immobilizzati con un metodo Langmuir-Blodgett su una superficie di un sensore microbilancia cristallo di quarzo e lavorato come larghe sonde stafilococco gamma. Perle di anticorpi riconoscono MRSA.

Biosensori per il rilevamento di antibiotici batteri di stafilococco resistenti

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

23.9K Views.  Auburn University. Biosensori fagi litici e perline anticorpi sono in grado di discriminare tra resistente alla meticillina (MRSA) e batteri di stafilococco sensibili. I fagi sono stati immobilizzati con un metodo Langmuir-Blodgett su una superficie di un sensore microbilancia cristallo di quarzo e lavorato come larghe sonde stafilococco gamma. Perle di anticorpi riconoscono MRSA.

Video: Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

Fabbricazione di Nanotubi di carbonio ad alta frequenza Nanoelectronic biosensore per la rilevazione in alta forza ionica Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Ricerca

Bioingegneria

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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 model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Rilevamento ottico di<em&gt; E. coli</em&gt; Batteri di mesoporosi Silicon Biosensors

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 ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Test di suscettibilità agli antibiotici indotti dallo stress su un chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificazione di batteri Polimeri repellente per dispositivi medici

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

L'uso di un'analisi di β-lactamase-based conduttimetriche biosensore per rilevare interazioni biomolecolari

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

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.

Sviluppo di un biosensore elettrochimico del DNA per rilevare un patogeni di origine alimentare

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

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Strategia di biopanning e analisi bioinformatica ad alta produttività basata su biosensore per la convalida globale delle interazioni farmaco-proteina

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Interfacce idrogel fotodegradabili per lo screening, la selezione e l'isolamento dei batteri

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

Pick any colony from the MRS agar plate containing electroporated L.reuteri cells and inoculate into a 96 well microplate containing filter sterilized MRS broth per well and an appropriate selection antibiotic. Incubate aerobically for 24 hours at 37 degrees Celsius without shaking. The next day, L.reuteri will precipitate out of the media in the stationary phase.Resuspend the culture by pipetting. Add 200 microliters of culture into a flat, clear, bottom 96 well plate. Transfer the plate to a plate reader and measure the optical density and fluorescence of M cherry two and other relevant reporters.Colony morphology noticeably changes in the presence of oxygen. Under low oxygen conditions colonies appear white, opaque, smooth and round. Under partial or leaky conditions, colonies appear undulate, and umbanet.Whereas under atmospheric oxygen levels colonies always appear low bait, round and dry. In wild Type L.rueteri, growth and reporter protein expression vary between colonies making accurate deductions difficult. Plasmid curing, and re-transformation can result in more stable expression but it is an uncommon and unique occurrence reliant on more stable mutant phenotypes spontaneously occurring.

Misurazione della proteina reporter fluorescente resistente agli acidi mCherry2

<meta charset="utf8"></head><body>Nanocompositi antibatterici di ossido di grafene/rame per batteri resistenti

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

<meta charset="utf8"></head><body>Autore in primo piano: Nanocompositi metallici per eliminare i batteri resistenti agli antibiotici

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Fabbricazione di nanocompositi antibatterici di ossido di grafene/rame

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...