Sandia è laboratorio multiprogram gestito da Sandia Corporation, una società Lockheed Martin, per gli Stati Uniti Detto Dipartimento dell&#39;Amministrazione nazionale per l&#39;energia di Sicurezza nucleare nell&#39;ambito del contratto DE-AC04-94AL85000. Gli autori riconoscono un finanziamento Laboratory Sandia National Laboratories &#39;Regia di Ricerca e Sviluppo (LDRD) del programma.

1. Microago Fabrication <ol><li> Utilizzando la modellazione tridimensionale del software Solidworks (Dassault Systemes SA, Velizy, Francia), la progettazione di un piramidale a forma di array di cava microneedle (Figura 1). 3-5 </li><li> Progettare una struttura di supporto per l&#39;array microneedle utilizzando software Magics RP 13 (Materialise NV, Leuven, Belgio). La struttura di supporto consente la resina di drenare dal dispositivo durante la fabbricazione e fornisce una base su cui sono costruiti i microaghi. Una struttura di supporto esempio è illustrato nella figura 1. </li><li> Il supporto dei file collegati e matrice microago vengono inseriti nella RP Perfactory software (envisionTEC GmbH, Gladbeck, Germania), che controlla il processo di fabbricazione. All&#39;interno di questo pacchetto software, scegliere il numero di array microago da fabbricati e determinare il posizionamento dei dispositivi sulla piastra di fabbricazione. </li><li> Eseguire calibrazione in modo ultravioletta a 180 mW per la rapida Perfactory prototyping sistema di produzione e verificare la deviazione di energia rientra in ± 2 mW. </li><li> Una volta che la fabbricazione, rimuovere gli array microago dalla piastra di base e sviluppare in isopropanolo per 15 minuti. Asciugare le matrici con aria compressa e curare i microaghi a temperatura ambiente per 50 secondi nel sistema Otoflash postindurimento (envisionTEC GmbH, Gladbeck, Germania) per garantire la completa polimerizzazione. </li><li> Convalida di fabbricazione microneedle tramite microscopia e verificare che ogni foro microneedle è cavo e senza ostacoli. Microaghi completamente fabbricati sono mostrati nella Figura 2. </li></ol> 2. Fabbricazione di pasta di carbonio array di elettrodi <ol><li> Utilizzare un modello di 60 W 6,75 CO 2 raster / vettoriale sistema laser (Universal Laser Systems, Inc., Scottsdale, AZ) per tagliare i fori ed esporre i sottostanti cavi di rame che collegano individualmente indirizzabili in un cavo piatto flessibile (21039-0249), che era ottenuta da una fonte commerciale (MoConnettore lex Corp., Lisle, IL) (Figura 3 (A e B)). Posizionare i cavi piatti flessibili, in una giga correttamente li allineare sulla piastra ablazione laser. Utilizzare un approccio rastering per creare cavità 500 micron di diametro nella porzione isolante del cavo flessibile. Patterns per l&#39;ablazione sono create in CorelDraw (Corel, Ottawa, Ontario) e inviati al sistema laser. </li><li> Pulire i modificati cavi piatti flessibili con un aerografo che l&#39;acetone spray a 40 psi. Terminare la pulizia loro da risciacquo con acqua deionizzata e isopropanolo. Verificare che sotto un microscopio che nessun film isolante rimane sopra le strisce di rame esposte. </li><li> Il passo successivo è quello di creare una cavità in possesso per il confezionamento delle paste di carbonio. Melinex nastro (0,002 &quot;spessore rivestito su un unico lato con adesivo acrilico sensibile alla pressione) è ablato con lo stesso schema delle strisce di elettrodo, orientate sulle strisce elettrodi asportate e compressi a 3000 psi per 2 minuti per assicurare un corretto. In questo case, il diametro della cavità è di 750 micron. </li><li> Un ulteriore strato di nastro Melinex (0,004 &quot;spessore rivestito su due lati con adesivo acrilico sensibile a pressione) viene successivamente ablato nello stesso modello singolo-adesivo e viene utilizzato dopo allineamento per legare gli array di microaghi per le matrici di elettrodi di carbonio pasta . </li></ol> 3. Sintesi di Paste di carbonio funzionali e Imballaggio di cavità elettrodi <ol><li> La pasta di carbonio sensibile glucosio è basata fuori di una ricetta precedente ed è ottenuta miscelando 10 mg di glucosio ossidasi e 2,2 mg di poli (etilenimmina) fino ad una miscela omogenea. 16 A questa miscela, 60 mg di rodio su carbone in polvere ( 5% di carico) è aggiunto. 40 mg di olio minerale viene aggiunta e successivamente miscelate. Le paste sono conservati a 4 ° C fino all&#39;uso, le paste sono utilizzati fino ad una settimana dopo la preparazione. </li><li> La pasta di carbonio sensibile pH è ottenuto miscelando olio minerale al 30% (w / w) e 70% (w / w) p grafiteowder. Confezione da incollare nella cavità elettrodo come descritto nella sezione 3.4. Fare soluzione di 10 mM veloce sale di diazonio Blu RR (4-benzoilammino-2 ,5-emi dimethoxybenzenediazonium cloruro (cloruro di zinco) sale) in 0,5 M di acido fosforico. 17, Mettere una goccia 20 pl di questa soluzione sull&#39;elettrodo pasta imballato per 30 minuti per chemisorb spontaneamente il Fast Blue sale di diazonio PR. Risciacquare con acqua deionizzata e conservare in tampone o acqua deionizzata quando non in uso. </li><li> La pasta lattato carbonio sensibile è basato su di una ricetta precedente ed è ottenuta miscelando 2,5 mg di rodio su polvere di carbone e 2,5 mg di lattato ossidasi, alternando 5 minuti di sonicazione e 5 minuti di vortex per cinque rotazioni 18. </li><li> Imballaggio delle paste modificate nel preparato cavo piatto flessibile viene eseguita applicando le paste rispettive cavità negli elettrodi. Utilizzando un pezzo sottile di plastica (per esempio, un bordo di una imbarcazione plastica peso) come una spatola e t paccosi incolla fino ad una superficie liscia si ottiene. Ripetere l&#39;operazione con una seconda barca pulita pesata, finché la pasta in eccesso viene rimosso. Lavare con acqua deionizzata. Uno schema che mostra ablazione laser per creare cavità, confezionamento di paste di carbonio, e integrazione microago (descritto nella sezione 2 e 3) è presentata in Figura 3. </li></ol> 4. Individuazione e calibrazione del sensore <ol><li> Lattato rilevamento avviene misurando la risposta crono del sensore a -0,15 V e registrando la corrente dopo 15 secondi in 0,1 M tampone fosfato (pH = 7,5). Figura 4 (a) contiene una schematica della reazione elettrocatalitico per il rilevamento di lattato . </li><li> Rilevamento del glucosio viene effettuato in modo analogo misurando la risposta crono del sensore a -0,05 V e registrando la corrente dopo 15 secondi in tampone fosfato 0,1 M (pH 7,0). Figura 4 (b) contiene una schematica della reazione per elettrocatalitico detection di glucosio. </li><li> pH è controllato eseguendo ciclici scansioni voltammetriche da -0,7 V a 0,8 V a 100 mV / s e registrare la posizione del picco potenziale ossidativo. Uno schema delle reazioni redox per il rilevamento pH viene mostrato nella Figura 5. </li><li> Le curve di calibrazione per il glucosio e sensori di lattato può essere creato con aggiunte successive del rispettivo analita; misurazione crono esegue dopo ogni aggiunta di analita, come descritto nelle sezioni 5.1 e 5.2. In alternativa, misure di potenziale fisso crono può essere effettuata sotto agitazione pur consentendo tempo sufficiente (circa 10-100 secondi) tra ogni aggiunta di analita per stabilizzazione della corrente. </li><li> curve di taratura pH può essere costruito misurando la posizione del potenziale di picco ossidativo su una serie di valori di pH noti 5-8 in incrementi di 1,0 unità di pH e registrare voltammograms ciclici come descritto nella sezione 5.3. </li></ol> 5. Rappresentante Resultati Quando si ottengono curve crono (ad esempio, per il rilevamento di glucosio o di rilevamento lattato) in soluzioni di riposo con il carbonio modificato incolla-riempite microaghi, la corrente diminuire immediatamente su richiesta del potenziale di rilevazione corrispondente. Si potrebbero decadere ad un valore di stato stazionario. Un risultato rappresentativo è mostrato nella figura 6; questo risultato è stato ottenuto da aggiunte 2 mM di lattato e registrazione al microago lattato. La soluzione deve essere agitata brevemente dopo ogni aggiunta lattato. La corrente dopo 15 secondi si erge su aumento della concentrazione di lattato, la reazione di corrente può essere quindi utilizzato per determinare la concentrazione di lattato in una soluzione sconosciuta. In alternativa, il monitoraggio continuo può essere utilizzato in una soluzione agitata (o in una soluzione scorrevole) come dimostrato per una soluzione con una concentrazione crescente di glucosio (Figura 5). Ancora, l&#39;aumento di corrente su crescente tegli concentrazione di glucosio può essere utilizzato per standardizzare la risposta di glucosio ad una soluzione sconosciuta. Tempo sufficiente deve essere consentita dopo ogni picco per permettere la soluzione di stabilizzazione. Voltammograms ciclico nella microago sensibile al pH in tampone fosfato 0,1 M sono mostrati in quattro diverse soluzioni pH da 5 a incrementi pH 8 a 1 unità in Figura 6. I cambiamenti ossidativi potenziale picco all&#39;aumentare del pH, questo fenomeno è utilizzato come indicatore del valore di pH. <img alt="Figura 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> Figura 1. Immagini del file STL della matrice microago creata in Solidworks (A) e dello schermo di stampa, che mostra la struttura di supporto (B). <img alt="Figura 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> Figura 2. Scansione micrografie elettroniche di matrice microago (A) e un microago unico entro questa matrice (B). Figura 3. Schema di appartamento assemblaggio cavo flessibile. I passaggi includono modificando il cavo piatto flessibile (A), i cerchi ablazione modellati (B), aggiungendo lo strato inizialmente ablata Melinex, che viene riempito con pasta di carbonio (C), così come l&#39;aggiunta del secondo strato ablato Melinex e l&#39;accoppiamento matrice microneedle (D). <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> Figura 4. Taratura di lattato-sensitive pasta con 15 scansioni al secondo crono a -0,15 V a 0,1 M tampone fosfato (pH = 7.5). Ogni aumento portata corrisponde ad un incremento 2 mM di lattato. <img alt="Figura 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> Figura 5. </sTrong Calibrazione&gt; pasta di glucosio-sensibile esegue scansioni crono a -0,05 V. Ciascun diminuzione nella risposta corrisponde ad una aggiunta 2 mM di glucosio. Il fosfato 0,1 M tampone (pH = 7,0) è stata agitata durante la calibrazione. Esterno Ag / AgCl e Pt elettrodi di riferimento e contatore sono stati utilizzati in questo studio. <img alt="Figura 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> Figura 6. Voltammogramma ciclica (CV) di carbonio sensibile al pH incolla in tampone fosfato 0,1 M pH sul 5-8 in incrementi di 1 unità di pH (pH = 8,0 ottanio, verde = pH 7,0, viola = pH 6,0, rosso pH = 5.0). Un curriculum quinto è stato utilizzato per l&#39;analisi rispetto a Ag / AgCl elettrodi di riferimento e Pt contatore a filo.

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Non ci sono conflitti di interesse dichiarati.

Molteplici aspetti del design di questo microago basato su sensori sono stati considerati prima di fabbricazione del dispositivo. Per utilizzare questo sensore per rilevare in tempo reale, il tempo di risposta del sensore deve essere bassa, in questo protocollo, ciascun sensore testato mostrato un tempo di risposta inferiore quindici secondi. Paste utilizzati nel presente protocollo sono stati scelti per la loro selettività nel vivo, in ambienti che contengono biomolecole elettroattivi che possono interferire ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Piatto cavo flessibile </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;tape Side lati </td><td> Melinex </td><td></td></tr><tr><td> 0.004 &quot;nastro biadesivo </td><td> Melinex </td><td></td></tr><tr><td> Lattato ossidasi </td><td> Sigma </td><td> L0638 </td></tr><tr><td> Glucosio ossidasi </td><td> Sigma </td><td> G7141 </td></tr><tr><td> Rodio su carbone </td><td> Sigma </td><td> 206164 </td></tr><tr><td> Polvere di grafite </td><td> Sigma </td><td> 385031000 </td></tr><tr><td> poli (etilenimmina) </td><td> Acros </td><td> 178570010 </td></tr><tr><td> Olio minerale </td><td> Sigma </td><td> M5904 </td></tr><tr><td> Glucosio </td><td> Sigma &lt;/ Td&gt; <td> G8270 </td></tr><tr><td> Lattato </td><td> Sigma </td><td> L1750 </td></tr><tr><td> Fast Blue RR sale </td><td> Sigma </td><td> F0500 </td></tr><tr><td> e-Shell 300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> e-Shell 200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCl elettrodo di riferimento </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> Pt filo </td><td> Basi </td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash postindurimento sistema </td><td> EnvisionTEC </td><td></td></tr><tr><td> Acido fosforico </td><td> Pescatore </td><td> A366-4 </td></tr><tr><td> 60W Modello 6,75 CO 2 raster / vettore sistema laser </td><td> Universal Laser Systems </td><td> PLS6.75 </td></tr><tr><td> CorelDraw </td><td> Corel </td><td></td></tr><tr><td> Solidworks </td><td> Dassault Systemes </td><td> 2009 </td></tr><tr><td> Magics RP13 </td><td> Materialise </td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

Lo sviluppo di un sistema di mini-invasiva di monitoraggio multiplex per l&#39;analisi rapida di molecole biologicamente rilevanti potrebbe offrire agli individui affetti da patologie croniche facile la valutazione del loro stato fisiologico immediato. Inoltre, potrebbe servire come strumento di ricerca per l&#39;analisi di complessi, multifattoriali condizioni mediche. Affinché tale sensore multianalyte da realizzare, deve essere minimamente invasiva, il campionamento di fluido interstiziale deve avvenire senza dolore o danni per l&#39;utente, e l&#39;analisi deve essere rapida e selettiva. Inizialmente sviluppato per il dolore senza somministrazione di farmaci, microaghi sono stati usati per fornire vaccini e agenti farmacologici (per esempio, insulina) attraverso la pelle. 1-2 Poiché questi dispositivi accedere allo spazio interstiziale, microaghi che sono integrati con i microelettrodi può essere utilizzato come transdermico sensori elettrochimici. Rilevamento selettivo di glucosio, glutammato, lattato, hacido perossido ydrogen, e ascorbico è stata dimostrata usando integrati microago elettrodi dispositivi con fibre di carbonio, paste carbonio modificati e platino rivestite microaghi polimero che servono come elementi di trasduzione. 3-7,8 Questa tecnologia dei sensori microneedle ha permesso un nuovo e raffinato approccio analitico per la rilevazione in situ e simultanea di più analiti. Multiplexing offre la possibilità di monitorare microambienti complessi, che sono altrimenti difficili da caratterizzare in modo rapido e minimamente invasiva. Ad esempio, questa tecnologia può essere utilizzata per il monitoraggio simultaneo dei livelli di extracellulari, glucosio, lattato e pH, 9 che sono importanti indicatori metabolici di stati di malattia 7,10-14 (ad esempio, cancro proliferazione) e esercizio indotta acidosi 15.

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Questo articolo descrive in dettaglio la costruzione di un multiplex microago basato su sensore. Il dispositivo viene sviluppato per il campionamento in situ e analisi elettrochimica di analiti multipli in maniera rapida e selettiva. Prevediamo medicina clinica e nella ricerca biomedica utilizza per questi microneedle basati su sensori.

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 ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

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Bioengineering

14.2K Views.  University of North Carolina and North Carolina State University. Questo articolo descrive in dettaglio la costruzione di un multiplex microago basato su sensore. Il dispositivo viene sviluppato per il campionamento in situ e analisi elettrochimica di analiti multipli in maniera rapida e selettiva. Prevediamo medicina clinica e nella ricerca biomedica utilizza per questi microneedle basati su sensori.

Video: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolecolare Detection impiega il sensore di immagine interferometrica riflettanza (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Ricerca

Bioingegneria

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 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Rilevamento e identificazione batterica Utilizzando Sensori elettrochimici

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

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

Rilevamento di Barriera tessuto Turbativa con una Transistor elettrochimica biologico

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

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 ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication dal solvente assistita doppio strato lipidico (SALB) Metodo

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Bioreattori fibra cava per<em&gt; In Vivo</em&gt; -come Mammiferi Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Capsule Estrazione di Plant-based per applicazioni di microincapsulazione

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

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

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

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 ...

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, ...