Ringraziamo il Dr. Rob Sladek per l'uso del cercatore a getto d'inchiostro. Riconosciamo il supporto finale da istituti canadesi per Health Research (CIHR), scienza naturale e ingegneria ricerca Consiglio del Canada (NSERC), l'Istituto di ricerca di società cancri canadese e la Fondazione canadese per l'innovazione (CFI). D.J. grazie sostegno da un Canada Research Chair.

1. fabbricazione e deposito di scatto chip <ol><li>metodo di trasferimento singolo ( Figura 1a) <ol><li>Spot cabina soluzioni contenenti 400 µ g/mL anticorpi e glicerolo 20% in tampone fosfato salino (PBS) su una nitrocellulosa (o un bicchiere funzionalizzato) diapositiva di dosaggio con un getto di inchiostro microarray spotter 13 a un'umidità relativa del 60% (1.2 nL per ogni spot) con spaziatura di centro--centro 800 µm. Accertarsi che il vetrino sia fissato sul ponte spotter secondo un angolo (qui, nell'angolo inferiore sinistro è stato utilizzato).</li>
		<li>Incubare il vetrino di saggio macchiato a temperatura ambiente per 1 h con un'umidità relativa del 60%.</li>
		<li>Morsetto una guarnizione di modulo diapositiva a 16 scomparti sul vetrino di saggio di dividerlo in 16 pozzetti. Sciacquare il vetrino tre volte con l'aggiunta di 80 µ l di PBS contenente 0.1% Tween-20 (PBST) in ogni bene e agitazione a 450 giri/min con un agitatore, 5 min ogni volta.</li>
		<li>Aggiungere 80 µ l di bloccare soluzioni per ogni bene e agitare per 1 h a 450 giri/min.</li>
		<li>Rimuovere la guarnizione e far asciugare il vetrino di saggio con un flusso di azoto.</li>
		<li>Fissare un trasferimento sul ponte spotter e spingere nell'angolo inferiore sinistro della diapositiva contro angolo inferiore sinistro del ponte spotter. A getto d'inchiostro spot matrice di segni di allineamento (una soluzione di microsfere di polistirolo) con lo stesso layout che per i taxi.</li>
		<li>Asciugare i segni di allineamento. Capovolgere la diapositiva di trasferimento e rimetterlo sul ponte spotter, fissaggio contro l'angolo inferiore sinistro.</li>
		<li>Preparare il dAb spotting soluzioni contenenti 20 µ g/mL anticorpi, 20% glicerolo e BSA 1%.</li>
		<li>Utilizzando lo spotter &#39; s fotocamera, prendere una foto di un marchio di allineamento. Implementare questa immagine nel programma spotting come un fiducial e ottenere il sistema di riconoscimento di immagine del cercatore a getto d'inchiostro per identificare il marchio di sinistro più in alto. Usare la sua coordinata come il primo spot della matrice dAb. Spot 8 nL per goccia con una spaziatura da centro a centro di 800 µm.</li>
	</ol></li> <li>Metodo di trasferimento doppia ( Figura 1b) <ol><li>disporre un vetrino di trasferimento 1 sul ponte spotter inkjet contro l'angolo inferiore sinistro. Individuare soluzioni di cabine contenenti 400 µ g/mL anticorpi, glicerolo 20% e 1% BSA in PBS sulla diapositiva con un spotter di microarray a getto d'inchiostro ad un'umidità relativa del 60% (0,4 nL per ogni spot e ~ 200 µm di diametro). La spaziatura da centro a centro è 400 µm.</li>
		<li>a scatto il trasferimento con una nitrocellulosa rivestito (o un bicchiere funzionalizzato) dosaggio diapositive utilizzando un apparato di snap ( Figura 2) per trasferire le goccioline di cabina il vetrino di saggio.
		<ol><li>Disabilita tutti i sei pulsanti sul lato apparato snap di 90 gradi per tirare e bloccare tutti i tuffatori in posizione aperta. Inserire la diapositiva di trasferimento il portavetrini in suo recipiente con l'angolo tagliato rivolto verso il perno fisso pogo. Girare la prima serie di tre pulsanti per rilasciare i tuffatori con il perno d'oro pogo e assicurarsi che le diapositive vengono spinti contro i perni di allineamento.</li>
			<li>Inserire una diapositiva rivestite con nitrocellulosa in snap apparato capovolto udienza i 4 perni di pogo. Girare la seconda serie di tre pulsanti per rilasciare pressori con il perno d'argento e assicurarsi che le diapositive vengono spinti contro i perni di allineamento.</li>
			<li>Chiudere l'apparecchio snap posizionando il guscio superiore sulla porta diapositive utilizzando i pilastri di allineamento e fori per un posizionamento preciso.</li>
			<li>Inserire l'apparecchio snap chiuso nella sua gabbia e spingere la linguetta di chiusura completamente verso il basso per portare i microarrays faccia a faccia, esercitando la pressione appropriata. Tenere chiuso per 1 min.</li>
		</ol></li> <li>Separare le diapositive. Incubare il vetrino di saggio a temperatura ambiente per 1 h con un'umidità relativa del 60%. Lavare, bloccare e far asciugare il vetrino di saggio come descritto nei passaggi 1.1.3 - 1.1.5.</li>
		<li>Mettere un altro trasferimento slide sul ponte spotter inkjet e spingere contro l'angolo inferiore sinistro. Soluzioni punto dAb contenente 50 o 100 µ g/mL di anticorpi (Vedi tabella 1), glicerolo 20% e 1% BSA in PBS. Garantire che ogni luogo è 0,8 nL e la spaziatura da centro a centro tra spot è 400 µm.</li>
		<li>Memorizzare il dosaggio e lo scivolo di trasferimento. Guarnizione sia saggio e trasferimento i vetrini in un sacchetto ermetico contenente essiccante e messo in un congelatore a-20 ° C.</li>
	</ol></li></ol> 2. Multiplexed immunodosaggi con snap chips <ol><li>recuperare il vetrino di saggio dal freezer. Lasciare il sacchetto sigillato per 30 min fino a quando la diapositiva viene a temperatura ambiente.</li>
	<li>Preparare 7 punti serie campione soluzioni diluite di chiodare proteine in PBS contenente 0.05% Tween-20. 
	Nota: Qui, viene utilizzato un fattore di diluizione 5 volte. Le concentrazioni di partenza per ogni proteina sono elencate nella tabella 1.</li>
	<li>Preparare soluzioni campione come appropriato. Ad esempio, diluire il siero umano 4 volte in tampone PBST.</li>
	<li>Morsetto una guarnizione 16-vano sulla diapositiva test.</li>
	<li>Riempire una colonna di 8 pozzetti con le soluzioni di diluizione 7 proteina e una soluzione di tampone PBST privo di proteine di pipettaggio. Pipettare soluzioni campione in altri 8 pozzetti nella stessa diapositiva. 
	Nota: 80 soluzioni µ l possono essere in forma in ogni bene. Altre diapositive possono essere utilizzati per misurare campioni supplementari quando necessario.</li>
	<li>Incubare i campioni su un agitatore a 450 giri/min per 1 h a temperatura ambiente o per una notte a 4 ° C. lavare il vetrino tre volte con PBST sull'agitatore a 450 giri/min, 5 min ogni volta. Rimuovere la guarnizione e far asciugare il vetrino sotto un getto di gas azoto.</li>
	<li>Recuperare la diapositiva di trasferimento con i tocchi dal freezer. Tenere il sacchetto sigillato per 30 min a temperatura ambiente. Quindi, incubare il vetrino in una camera chiusa (ad esempio casella vuota suggerimenti) contenente microsfere di stabilizzazione di umidità 60% per 20 min per reidratazione.</li>
	<li>a scatto la diapositiva di trasferimento con il vetrino di saggio utilizzando un apparato di snap ( Figura 2) per trasferire le goccioline di dAb. Vedere sezione 1.2.2 per il funzionamento dell'apparato snap.</li>
	<li>Separare le diapositive. Incubare il vetrino di saggio in una camera chiusa contenente microsfere di stabilizzazione di umidità 60% per 1 h. morsetto il vetrino di saggio con una guarnizione di 16-vano e sciacquare il vetrino 4 volte con PBST su un agitatore a 450 giri/min, 5 min ogni volta.</li>
	<li>Pipetta 80 µ l di soluzioni contenenti 2,5 µ g/mL streptavidina fluoroforo in PBS in ciascun pozzetto. Incubare per 20 minuti su un agitatore a 450 giri/min.</li>
	<li>Sciacquare il vetrino 3 volte con PBST e una volta con acqua distillata sull'agitatore a 450 giri/min. Rimuovere la guarnizione e far asciugare il vetrino utilizzando gas azoto.</li>
</ol> 3. Far scorrere la scansione e analisi dei dati <ol><li>scansione il vetrino di saggio con uno scanner di microarray di fluorescenza usando il laser-nm-635.</li>
	<li>Estrarre l'intensità netta di ogni spot utilizzando un'analisi software (ad es. matrice-pro analizzatore) 16.</li>
	<li>Calcolare il limite di rilevabilità (LOD) di ogni proteina usando un software di statistiche e determinare le concentrazioni di proteina nei campioni. 
	Nota: Il LOD è definito come l'intercetta Y della norma cuRVE incrementato di tre volte la deviazione standard dei tre saggi indipendenti.</li>
</ol>

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McGill University ha presentato una domanda di brevetto su alcuni aspetti di questo lavoro con Huiyan Li e David Juncker come inventori.

In questo lavoro, abbiamo presentato una tecnologia di chip di snap che rende i test immunologici cross-reactivity-gratis multiplex ampiamente disponibili per i ricercatori con base messa a punto sperimentale. Diverso da microarrays dell'anticorpo esistente, nessun spotter microarray è necessaria per gli utenti finali. Sono dimostrati sia singola che doppia i metodi di trasferimento, e raddoppiare il transfer offre una precisione di allineamento superiore verso il basso per ~ 40 μm per macchie di 98%, con il più grand...

Un pannello di biomarcatori che comprende proteine multiple può fornire maggiore sensibilità e specificità rispetto al singolo biomarcatore nella diagnosi di malattie complesse come cancri1,2. L'analisi enzima-collegata dell'immunosorbente (ELISA) è stata la tecnologia di gold standard utilizzata nei laboratori clinici, raggiungimento di un limite di rilevazione a basso pg/mL nel plasma, ma limiti a una sola destinazione per dosaggio3,4,5. Microarrays dell'anticorpo sono stati sviluppati per accogliere migliaia di saggi miniaturizzati condotto in parallelo su un microscopio singola diapositiva6,7,8. Tuttavia, la capacità di multiplexing di questo metodo è limitata dalla reattività crociata reagente-driven, derivanti dall'applicazione di una miscela di dAbs, e diventa più problematico con un numero crescente di target9,10 , 11. Pla et al. hanno dichiarato che la vulnerabilità risultante di un'analisi multiplex panino bilance come 4N(N-1) dove N è il numero dei bersagli12.
Per attenuare la reattività crociata in microarrays dell'anticorpo, anticorpo colocalizzazione microarray (ACM) è stato sviluppato nel nostro laboratorio per sandwich multiplex assay12. Gli anticorpi di cattura (cabine) sono macchiati su un substrato con uno spotter di microarray. Dopo blocchi campioni vengono applicati sulla superficie, e quindi i singoli tocchi sono macchiati sulle stesse macchie con il complesso antigene-cabina. Tutti gli scenari di reattività crociata tra antigeni e anticorpi possono essere mitigati con ACM e limiti di rilevazione a pg/mL sono stati raggiunti. Tuttavia, il protocollo del test richiede preparazione e spotting i danti durante gli esperimenti usando un microarray in loco spotter con alta precisione per scopo di allineamento, che è costoso e richiede tempo, limitare l'ampia applicazione di questa tecnologia in altri laboratori. Un palmare ACM, denominato snap chip è stato sviluppato per cross-reactivity-gratis e privo di spotter multiplex panino immunoassays13,14,15. i taxi e i tocchi sono pre-macchiati su una diapositiva di dosaggio e un trasferimento rispettivamente nel formato microarray e memorizzati. Durante il dosaggio, le diapositive vengono recuperate e un microarray di tocchi sono trasferite collettivamente il vetrino di saggio semplicemente schioccando i due chip insieme. Un'apparecchiatura di snap è utilizzata per il trasferimento affidabile reagente. Diapositive di nitrocellulosa rivestita con una capacità di legame dell'anticorpo relativamente grandi sono stati usati come gli scivoli di dosaggio per assorbire le goccioline di liquide e facilitando così il trasferimento di reagente, tuttavia, le diapositive sono più costose di microarray e diapositive di vetro regolare scanner compatibile con diapositive non trasparenti sono necessari per l'acquisizione del segnale.
In questo lavoro, dimostriamo il protocollo di esecuzione di un immunodosaggio a sandwich multiplex con un chip di snap. Un apparato di romanzo snap è stato sviluppato per il trasferimento di reagente più conveniente e affidabile da microarray a DNA-microarray. Cosa importante, qui abbiamo stabilito il metodo di trasferimento di reagente su vetrini regolari con il chip di snap. 1024 punti correttamente trasferiti e allineati su una lastra di vetro, espandendo significativamente l'uso di questa tecnologia nella maggior parte dei laboratori.

<table>
	<tbody>
		<tr>
			<td>Phosphate buffered saline tablet</td>
			<td>Fisher Scientific</td>
			<td>5246501EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-conjugated Cy5</td>
			<td>Rockland</td>
			<td>s000-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>p1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer</td>
			<td>SurModics, Inc</td>
			<td>SG02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose coated slides</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>305116</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane coated slides</td>
			<td>Schott North America</td>
			<td>1064875</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Device</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-SD01</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkjet microarray spotter</td>
			<td>GeSiM</td>
			<td>Nanoplotter 2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide module gasket</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>204862</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Stabilization Beads</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-HU60</td>
			<td></td>
		</tr>
		<tr>
			<td>Array-Pro Analyzer software</td>
			<td>Media Cybernetics</td>
			<td>Version 4.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microarray scanner</td>
			<td>Agilent</td>
			<td>SureScan Microarray Scanner</td>
			<td></td>
		</tr>
		<tr>
			<td>Biostatistics software</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB10972</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin protein</td>
			<td>R&#38;D Systems</td>
			<td>1097-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF1097</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6a (see Table 1)</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6b (see Table 1)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays

Analisi multiplex della proteina ha mostrato sensibilità diagnostica superiore e precisione rispetto alle singole proteine. Microarrays dell'anticorpo consentono migliaia di test immunodiagnostici di micro-scala eseguite contemporaneamente su un singolo chip. Formato di dosaggio sandwich migliora la specificità dell'analisi rilevando ogni destinazione con due anticorpi, ma soffre di reattività crociata fra i reagenti, limitando così la loro capacità di multiplexing. Microarray di colocalizzazione dell'anticorpo (ACM) è stato sviluppato per la rilevazione di proteine "multiplex" cross-reactivity-gratis, ma richiede un costoso spotter in loco per la produzione di microarray durante le analisi. In questo lavoro, dimostriamo una tecnologia di chip di snap che trasferisce il reagente da microarray a DNA-microarray di schiocco semplicemente due chip insieme, che così nessun spotter è necessaria durante l'incubazione del campione e la successiva applicazione degli anticorpi di rilevamento (danti) su deposito di diapositive pre-macchiati, dissociando la preparazione del vetrino dall'esecuzione di test. Entrambi i metodi di trasferimento di singole e doppie sono presentati per ottenere l'allineamento tra i due microarray e la fabbricazione di diapositiva per entrambi i metodi sono descritti. I risultati indicano che &#60; 40 μm allineamento è stato realizzato con doppio trasferimento, raggiungendo una densità di matrice di 625 punti/cm2. Un immunoassay 50-plexed è stato condotto per dimostrare l'utilizzabilità del chip lo snap in analisi multiplex della proteina. Limiti di rilevazione delle 35 proteine sono nella gamma di pg/mL.

La procedura di analisi per singolo e doppio metodi di trasferimento è mostrato nella Figura 1. Nel singolo trasferimento, le cabine sono macchiate direttamente nella diapositiva saggio e i tocchi vengono trasferiti nella diapositiva di analisi al momento dell'impiego in un modello di specchio delle carrozze (Figura 1a). Un solo trasferimento procedura è necessaria, ma questo metodo soffre di disallineamento tra i due microarra...

Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Dimostriamo una tecnologia di chip di snap per l'esecuzione di test immunologici cross-reactivity-gratis multiplex panino semplicemente schioccando due diapositive. Un'apparecchiatura di snap è utilizzata per il trasferimento in modo affidabile i reagenti da microarray a DNA-microarray. Il chip di snap può essere utilizzato per eventuali reazioni biochimiche che richiedono colocalizzazione di reagenti diversi senza la contaminazione incrociata.

Chip a scatto per immunodosaggi panino multiplex Cross-reactivity e Spotter-free

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

snap-chip-for-cross-reactivity-free-spotter-free-multiplexed-sandwich

Research

JoVE Journal

Bioengineering

6.4K Views.  McGill University and Génome Québec Innovation Centre. Dimostriamo una tecnologia di chip di snap per l'esecuzione di test immunologici cross-reactivity-gratis multiplex panino semplicemente schioccando due diapositive. Un'apparecchiatura di snap è utilizzata per il trasferimento in modo affidabile i reagenti da microarray a DNA-microarray. Il chip di snap può essere utilizzato per eventuali reazioni biochimiche che richiedono colocalizzazione di reagenti diversi senza la contaminazione incrociata.

Video: Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Metodi di rilevamento a fluorescenza per piattaforme goccia microfluidica

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Ricerca

Bioingegneria

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

On-Chip Isotachophoresis per separazione degli ioni e purificazione di acidi nucleici

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

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

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

Hollow microago-based sensore per Multiplexed transdermico elettrochimica Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Un chip microfluidica per la Versatile analisi chimica di singole cellule

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip microfluidico per ICPMS introduzione del campione

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabbricazione di dispositivi microfluidici basati su carta tridimensionale per gli immunodosaggi

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Piattaforma microfluidica con multiplex di rilevamento elettronico per spaziale monitoraggio di particelle

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...