This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1. Setup sperimentale e preparazione del campione <ol><li> Realizzare microtoroids utilizzando la litografia, incisione, e di fusione procedura descritta in precedenza 6. Realizzare microtoroids sulla cima di un wafer di silicio (chip) che tipicamente hanno un diametro maggiore di 80-100 micron, e un diametro minore di 2 micron. </li><li> Rilassatevi circa un metro di fibra ottica monomodale (125 rivestimento micron, 4,3 micron modo diametri campo) dalla bobina di fibra. </li><li> Al centro della parte srotolato della fibra ottica, spogliare un piccolo segmento (2,5 cm) del rivestimento polimerico attorno alla fibra ottica utilizzando spellafili. Nota: Questa è la porzione della fibra ottica che verrà utilizzato per evanescently luce coppia nel microtoroid. </li><li> Pulire la fibra spogliato con alcool isopropanolo e un panno senza strofinare. </li><li> Mantenere questa parte della fibra in posizione utilizzando un supporto di fibra in ganci magnetici. </li><li> Sottile la fibra spogliato a ~ 500 nm da Melting e tirando con una torcia idrogeno e due motori passo-passo che si muovono in direzioni opposte a 60 micron / min. Posizionare la fibra ottica all&#39;interno della porzione superiore della fiamma, che dovrebbe essere ~ altezza 10 mm. Arrestare tirando la fibra quando la trasmissione attraverso la fibra ferma fluttuante, che può essere controllato visivamente (guardando la luce che è dispersa lateralmente dal lampeggio fibra on e off) o collegando la fibra ad un fotodiodo che è attaccato ad un oscilloscopio . </li><li> Cleave una estremità della fibra ottica e inserirla in un adattatore fibra nuda. Inserire questa estremità della fibra all&#39;ingresso del fotoricevitore. </li><li> Fibra coppia l&#39;altra estremità della bobina fibra laser utilizzando un accoppiatore a fibre ottiche. </li><li> La fiche microtoroid sopra un portacampioni (acciaio inox, 37,8 mm x 6.4 mm x 3.2 mm) utilizzando resina epossidica o nastro biadesivo. </li><li> Montare il porta-campione in cima ad una fase di posizionamento che si compone di un 3 assi nano-posizionamento (nanocube) fase (vedere inventario) sulla cima di un micrometro a 3 assi. Eseguire tutti gli esperimenti su un tavolo ottico pneumaticamente isolata per ridurre al minimo le vibrazioni. </li><li> Posizione grossolana il chip campione utilizzando un micrometro a 3 assi. </li><li> Allineare il-microtoroid contenente chip di parallelamente alla fibra ottica utilizzando nano-posizionatore. Nota: allineare il microtoroid entro una distanza di una lunghezza d&#39;onda della luce di ingresso (~ 633 nm). Per la visualizzazione di questo processo utilizzare due colonne di imaging (tubo con lente obiettivo e telecamera, vedere inventario) posizionato sulla parte superiore e sul lato del chip. </li><li> Ottimizzazione della polarizzazione della luce laser diretta attraverso la fibra ottica utilizzando un controllore di polarizzazione in linea (vedere inventario) con una manopola per regolare la polarizzazione. Nota: polarizzazione ottimale si ottiene quando il dip misurata nella trasmissione della fibra ottica appare più stretto. Osserva questo tuffo su un oscilloscopio (vedere il punto 2.2 per maggiori dettagli). </li><li> Costruireuna camera del campione da epoxying vetrino coprioggetti sulla fase del campione con un vetrino per microscopio come distanziatore. Nota: Una recinzione in plexiglass che copre l&#39;intera installazione può essere utile per ridurre al minimo le correnti d&#39;aria. Una piccola apertura dovrebbe essere lasciato per consentire la soluzione da pompare in utilizzando tubi. </li><li> Equilibrare termicamente particelle sospensioni o soluzioni acquose singola molecola per ≥ 1 ora in un bagno d&#39;acqua RT (~ 500 ml). Nota: I campioni vengono diluiti alla concentrazione desiderata in provette da microcentrifuga utilizzando i buffer associati specificati dal costruttore, ad esempio, PBS o HEPES. Se non vengono rilevati eventi di legame, aumentare la concentrazione di sale del buffer. </li><li> Particella Vortex contenenti soluzioni (1 ml) per breve tempo per ~ 2 sec. </li><li> Iniettare soluzioni contenenti particelle nella camera campione a 1 ml / min usando una pompa a siringa da 1 ml. </li><li> Dopo la camera del campione ha riempito, spegnere la pompa a siringa. </li><li> Attendere 30 secondi prima di registrare i dati per ridurre al minimogli effetti delle vibrazioni meccaniche derivanti dal flusso di fluido che può influenzare la misura. </li></ol> Blocco 2. Frequenza <ol><li> Re-coppia toroide alla fibra ottica spostando il supporto del campione con il nanopositioner, perché l&#39;accoppiamento sarà disturbata a causa di iniezione di liquido. </li><li> Individuare la lunghezza d&#39;onda di risonanza del microtoroid attraverso la scansione laser ingresso computer controllato attraverso una varietà di lunghezze d&#39;onda. Eseguire questo passaggio inviando un segnale di forma d&#39;onda di tensione triangolare un elemento piezoelettrico all&#39;interno del controller laser che regola la lunghezza d&#39;onda del laser. Effettuare esperimenti usando la luce visibile (635 nm ± 2,5 nm) in quanto vi è un basso assorbimento di luce in acqua a questa lunghezza d&#39;onda. </li><li> Misurare la trasmissione della luce attraverso la fibra ottica collegando l&#39;uscita della fibra ottica in un fotoricevitore auto-equilibrata. Inserire l&#39;uscita del fotoricevitore in un oscilloscopio utilizzando un cavo BNC. Osservare on l&#39;oscilloscopio che alla lunghezza d&#39;onda di risonanza del microtoroid, trasmissione attraverso la fibra ottica gocce. </li><li> Attaccare l&#39;uscita del fotoricevitore all&#39;ingresso principale della frequenza bloccaggio regolatore a reazione (vedere inventario) tramite un cavo. </li><li> Eseguire il controller di feed back di blocco in modalità di blocco automatico con picco top di chiusura con una frequenza di dithering di 2 kHz e un&#39;ampiezza di lunghezza d&#39;onda di oscillazione del 19 fm. Empiricamente configurare le impostazioni proporzionale integrale derivati ​​nella finestra del software utilizzando Ziegler-Nichols regole di tuning 14. Nota: questi valori devono solo essere impostata una volta all&#39;inizio di tutti gli esperimenti. </li><li> Auto-bloccare la lunghezza d&#39;onda del laser alla lunghezza d&#39;onda di risonanza del microtoroid. Eseguire questo passaggio dopo aver riempito la camera del campione. Nota: Se lo spostamento della lunghezza d&#39;onda è troppo grande, quindi il regolatore di feedback perderà di blocco e passare automaticamente alla modalità di scansione al fine di individuare la posizione di risonanza lunghezza d&#39;onda. Questo occurs per lunghezza d&#39;onda si sposta più grande di circa un linewidth (almeno 600 fm per tutti i sistemi esaminati qui). </li><li> Registrare l&#39;uscita del regolatore a reazione a 20 kHz utilizzando una scheda di acquisizione dati a 24 bit. Esportare i dati come file di testo tramite software di acquisizione dati. </li></ol> 3. Elaborazione Dati e analisi <ol><li> Fourier trasformare i dati in MATLAB. </li><li> Passa-basso i dati utilizzando un filtro &quot;muro di mattoni&quot; con un cutoff a 1 kHz per rimuovere la frequenza di dithering imposto di 2 kHz (vedi codice supplementare sul file Schermata 1). </li><li> Notch computazionalmente filtrare i dati utilizzando una dimensione della finestra di 16 Hz. Nota: Questo viene fatto per rimuovere fonti note di rumore, in questo caso, 60 Hz rumore elettronico linea e le sue armoniche, nonché 100 Hz (proveniente dal driver laser) e sue armoniche (vedi codice supplementare file Schermata 1). </li><li> Fourier inversa trasformare i dati di nuovo nel dominio del tempo. </li><li> Filtro mediano i dati utilizzando una finestradimensioni di 1.001 campioni (vedi Codice supplementare sul file Schermata 2). </li><li> Individuare cambio di passo nella lunghezza d&#39;onda di risonanza utilizzando l&#39;algoritmo passo ricerca di Kerssemakers et al. 15. </li><li> Genera istogrammi di ampiezza di ciascuna fase di legame. </li><li> Calcola dimensione delle particelle con Eq. (1) (vedi Discussione). </li></ol>

<ol>
	<li>Lu, T., 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=High+sensitivity+nanoparticle+detection+using+optical+microcavities.">High sensitivity nanoparticle detection using optical microcavities.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 5976-5979 (2011).</li><li>Su, J., Goldberg, A. F. G., Stoltz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+single+nanoparticles+and+biological+molecules+using+microtoroid+optical+resonators.">Label-free detection of single nanoparticles and biological molecules using microtoroid optical resonators.</a> Light: Science and Applications. , (2016).</li><li>Knight, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single molecule biology. , (2009).</li><li>Jonsson, U., 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=Real-time+biospecific+interaction+analysis+using+surface+plasmon+resonance+and+a+sensor+chip+technology.">Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.</a> BioTechniques. 11, 620-627 (1991).</li><li>Vollmer, F., Arnold, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whispering-gallery-mode+biosensing:+label-free+detection+down+to+single+molecules.">Whispering-gallery-mode biosensing: label-free detection down to single molecules.</a> Nat. Methods. 5, 591-596 (2008).</li><li>Armani, D. K., Kippenberg, T. J., Spillane, S. M., Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-Q+toroid+microcavity+on+a+chip.">Ultra-high-Q toroid microcavity on a chip.</a> Nature. 421, 925-928 (2003).</li><li>Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+microcavities.">Optical microcavities.</a> Nature. 424, 839-846 (2003).</li><li>Arnold, S., Khoshsima, M., Teraoka, I., Holler, S., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shift+of+whispering-gallery+modes+in+microspheres+by+protein+adsorption.">Shift of whispering-gallery modes in microspheres by protein adsorption.</a> Opt. Lett. 28, 272-274 (2003).</li><li>Vollmer, F., Arnold, S., Keng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+virus+detection+from+the+reactive+shift+of+a+whispering-gallery+mode.">Single virus detection from the reactive shift of a whispering-gallery mode.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 20701-20704 (2008).</li><li>He, L., Ozdemir, S. K., Zhu, J., Kim, W., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+single+viruses+and+nanoparticles+using+whispering+gallery+microlasers.">Detecting single viruses and nanoparticles using whispering gallery microlasers.</a> Nat Nano. 6, 428-432 (2011).</li><li>Dantham, V. R., 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=Label-free+detection+of+single+protein+using+a+nanoplasmonic-photonic+hybrid+microcavity.">Label-free detection of single protein using a nanoplasmonic-photonic hybrid microcavity.</a> Nano Lett. 13, 3347-3351 (2013).</li><li>Baaske, M. D., Foreman, M. R., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+nucleic+acid+interactions+monitored+on+a+label-free+microcavity+biosensor+platform.">Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform.</a> Nat Nanotechnol. 9, 933-939 (2014).</li><li>Su, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Single+Exosome+Detection+Using+Frequency-Locked+Microtoroid+Optical+Resonators.">Label-Free Single Exosome Detection Using Frequency-Locked Microtoroid Optical Resonators.</a> ACS Photonics. (9), 1241-1245 (2015).</li><li>&#197;str&#246;m, K. J., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Feedback systems : an introduction for scientists and engineers. , (2008).</li><li>Kerssemakers, J. W., 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=Assembly+dynamics+of+microtubules+at+molecular+resolution.">Assembly dynamics of microtubules at molecular resolution.</a> Nature. 442, 709-712 (2006).</li><li>Su, T. -. T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Label-free detection of single biological molecules using microtoroid optical resonators. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

Come si lega una particella, la lunghezza d&#39;onda di risonanza (λ) del toroide aumenta. Se una particella non associa, la lunghezza d&#39;onda di risonanza diminuisce corrispondentemente (un evento step-down). Il diametro delle particelle (d) può essere determinata attraverso istogrammi di ampiezza di ogni passo di lunghezza d&#39;onda. L&#39;altezza di ogni gradino d&#39;onda varia a causa di variazioni dimensionali della particella legato e causa la posizione sulla microtoroid cui si lega la par...

Esperimenti rilevamento molecole singole sono utili per ridurre la quantità di analita usato in biosensori, per la diagnosi precoce della malattia, e per esaminare le proprietà fondamentali delle molecole 3. Tali esperimenti sono tipicamente eseguite utilizzando etichette, tuttavia, le etichette non sono sempre possibile ottenere per una particolare proteina, aumenta il costo, può perturbare gli eventi oggetto di studio, e può essere scomodo, particolarmente per tempo reale in loco esperimenti o point-of- Diagnostica di cura. Il gold standard attuale per biosensing label-free è risonanza plasmonica di superficie 4, ma i sistemi commerciali di risonanza plasmonica di superficie in genere hanno un tipico limite inferiore di rilevamento dell&#39;ordine di nM. Recentemente, risonatori ottici sono emersi come una tecnologia promettente per libero etichetta singola molecola biorilevazione 5. Risonatori ottica lavoro basato sul lungo termine (ns) confinamento della luce 6,7. La luce è evanescentlyaccoppiato in questi dispositivi tipicamente attraverso una fibra ottica. Quando la lunghezza d&#39;onda della luce che attraversa la fibra corrisponde alla lunghezza d&#39;onda di risonanza del risonatore, illuminare efficientemente accoppia il risonatore. Questa luce accoppiata riflette totalmente internamente la cavità del risonatore a generare un campo evanescente in prossimità della circonferenza del risonatore. Come particelle entrano campo evanescente e si legano al risuonatore, la lunghezza d&#39;onda di risonanza dei cambiamenti risonatore in proporzione al volume della particella 8. In termini di capacità di rilevamento, risonatori di microsfere sono stati in precedenza utilizzata per rilevare l&#39;influenza A solo particelle virali (100 nm) 9,10. Recentemente, plasmonically avanzata microsfere risonatori ottici sono stati utilizzati per rilevare siero albumina bovina singola molecole 11 e oligonucleotidi 8-mer 12, tuttavia questo approccio limita l&#39;area di cattura delle particelle di 0,3 micron 2 per device. Più grandi biosensori zona di cattura sono ideali per massimizzare la probabilità di rilevamento delle particelle. Le attuali tecnologie basate su soluzioni label-free biosensori con grandi (&gt; 100 micron 2) aree di acquisizione si sono limitati a rilevare particelle di polistirene ≥ 25 nm. Abbiamo sviluppato un sistema di biosensori label-free basato sulla tecnologia risonatore ottico noto come frequenza di blocco ottico Whispering Evanescent risonatore (FIORE) 13 (Figura 1), che è in grado di rilevare in tempo risolto di singole molecole in soluzione. FIORE utilizza la lunga durata fotone di risonatori ottici microtoroid combinati con frequenza di blocco di controllo di retroazione, il rilevamento equilibrato, e il filtraggio di calcolo per rilevare piccole particelle fino a singole molecole proteiche. L&#39;uso di aggancio in frequenza consente al sistema di monitorare sempre la risonanza spostamento del microtoroid come particelle si legano, senza la necessità di spazzare o la scansione della lunghezza d&#39;onda del laser sopragrandi intervalli. I principi di FLOWER possono essere utilizzati per migliorare le capacità di rilevazione delle altre tecniche, tra cui la valorizzazione plasmonica. In ciò che segue, vengono descritte le procedure per l&#39;esecuzione FIORE.

<table border="0" cellpadding="0" cellspacing="0" width="396">
	<tbody>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Tunable diode laser</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Laser controller</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300-LN</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Frequency locking feedback controller</td>
			<td class="xl63" style="width:99pt" width="132">Toptica Photonics</td>
			<td class="xl63" style="width:99pt" width="132">Digilock 110</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Auto-balanced photoreceiver</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">Model 2007</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">In-line polarization controller</td>
			<td class="xl63" style="width:99pt" width="132">General Photonics</td>
			<td class="xl63" style="width:99pt" width="132">PLC-003-S-90</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">24-bit data acquisition card</td>
			<td class="xl63" style="width:99pt" width="132">National Instruments</td>
			<td class="xl63" style="width:99pt" width="132">NI-PCI-4461</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">Recombinant human interleukin-2</td>
			<td class="xl63" style="width:99pt" width="132">Pierce Biotechnology</td>
			<td class="xl63" style="width:99pt" width="132">R201520</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">20 nm polystyrene beads</td>
			<td class="xl63" style="width:99pt" width="132">Thermo Scientific</td>
			<td class="xl63" style="width:99pt" width="132">3020A</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">NanoCube XYZ Piezo Stage</td>
			<td class="xl63" style="width:99pt" width="132">Physik Instrumente</td>
			<td class="xl63" style="width:99pt" width="132">P-611.3</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Optical table</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">VH3660W-OPT</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Objective lens for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl64">1-60228</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Imaging column (adaptor tube)</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl65">1-60228</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">High-Res CCD camera for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Edmund Industrial Optics</td>
			<td class="xl64">NT39244</td>
		</tr>
	</tbody>
</table>

label-free single molecule detection using microtoroid optical resonators

Rilevamento piccole concentrazioni di molecole fino al limite singola molecola ha un impatto su settori quali la diagnosi precoce della malattia, e studi fondamentali sul comportamento delle molecole. Tecniche di rilevazione di singola molecola comunemente utilizzano etichette come etichette fluorescenti o punti quantici, tuttavia, le etichette non sono sempre disponibili, aumentare i costi e la complessità, e possono perturbare gli eventi in fase di studio. Risonatori ottici sono emersi come un mezzo promettente per rilevare singole molecole senza l&#39;uso di etichette. Attualmente la più piccola particella rilevata da un sistema di risuonatore non plasmonically potenziato nudo ottica in soluzione è a 25 nm di polistirolo sfera 1. Abbiamo sviluppato una tecnica nota come frequenza di blocco ottico Whispering Evanescent risonatore (fiore) in grado di superare questo limite e ottenere la diagnosi singola molecola label-free in soluzione acquosa 2. La potenza del segnale scale con il volume delle particelle, il nostro lavoro rappresenta un&gt; 100x improvement nel rapporto segnale-rumore (SNR) rispetto allo stato attuale della tecnica. Qui le procedure dietro fiore sono presentati in uno sforzo per aumentare il suo utilizzo nel settore.

Eventi di legame delle particelle sono chiaramente visto come variazioni a gradino in lunghezza d&#39;onda di risonanza del microtoroid nel tempo (Figura 2A). Le altezze di questi passaggi sono indicati come un istogramma della figura 2B. Figure 2-4 mostrano tracce rappresentative del legame di esosomi (nanovesicles), 5 nm perline di silice, e single umane interleuchina-2 molecole, rispettivamente. Il fatto che gli eventi a gradino scala con granulometr...

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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.

Singola molecola di rilevamento senza etichetta di testo usando Microtoroid ottici Risonatori

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

label-free-single-molecule-detection-using-microtoroid-optical

Research

JoVE Journal

Bioengineering

9.1K Views.  California Institute of Technology. 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. 

Video: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

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

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

Multiplex singola molecola Forza Misure proteolisi con delle pinzette magnetiche

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

Ricerca

Bioingegneria

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Un Cre-Lox P approccio ricombinazione per la rilevazione di fusione cellulare In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

L&#39;uso di biosensori ottici privi di etichetta per rilevare modulazione dei canali del potassio da G-recettori accoppiati a proteine

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipolazione di Single RNA Molecole da pinzette ottiche

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

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.

Un Biochip elettrochimica Microfluidic-based per la libera-Label Ibridazione DNA Analysis

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

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una tecnica senza etichetta per l&#39;Imaging spazio-temporale delle secrezioni monocellulari

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Singola molecola microscopia a fluorescenza su Planar bilayers supportati

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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.

Multicolore fluorescenza Detection per Droplet microfluidica tramite fibre ottiche

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