Questo studio è stato sostenuto da Cooperativa accordo Award AI075565 (a DAH) presso l&#39;Istituto Nazionale di allergie e malattie infettive e da Wendy e Ken Fondo Rubino per l&#39;Eccellenza in Pediatric Urology Research. BMC è la Giuditta e Robert Winston Cattedra di Urologia Pediatrica.

1. Funzionalizzazione di Sensori elettrochimici <ol><li> Preparare la sonda di cattura tiolato ad una concentrazione di 0,05 pM a 300 pM 1,6-hexanedithiol (HDT), 10 mM Tris-HCl, pH 8,0, 0,3 M NaCl, 1 mM EDTA e incubare al buio a temperatura ambiente per 10 min . Incubazione della sonda di cattura tiolato con HDT assicura che il gruppo tiolo sulla sonda di cattura è ridotto, con conseguente risultati più coerenti. </li><li> Applicare una corrente di azoto a nudo oro 16 chip di array di sensori (s) per 5 secondi per rimuovere l&#39;umidità e / o particolati. </li><li> Applicare 6 microlitri della HDT-tiolato mix sonda di cattura per l&#39;elettrodo di lavoro di tutti i 16 sensori della matrice di sensori e memorizzare il chip sensore (s) in una capsula di Petri coperta a 4 ° C durante la notte. Sonde di cattura tiolati legano direttamente l&#39;elettrodo d&#39;oro nudo e la HDT atti a prevenire addensamento delle sonde di cattura e di tenerli in una conformazione estesa che promuove l&#39;ibridazione con il target. </li><li> Glidopo giorno, lavare il chip sensore con H 2 O deionizzata per 2-3 secondi e secco sotto flusso di azoto per 5 sec. </li><li> Applicare 6 pl di 10 mM Tris-HCl, pH 8,0, 0,3 M NaCl, 1 mM EDTA, 1 mM 6-mercapto-1-esanolo (MCH) per l&#39;elettrodo di lavoro di tutti i sensori 16 e incubare per 50 min. Questa e tutte le successive fasi di incubazione chip del sensore vengono eseguiti in una capsula di Petri coperto a temperatura ambiente. MCH agisce come un agente bloccante, riempiendo tutti gli spazi in cui la sonda di cattura o tiolato HDT non è presente sulla superficie dell&#39;elettrodo. </li></ol> 2. Preparazione del campione <ol><li> Trasferire 1 ml di coltura batterica in fase logaritmica di crescita (OD 600 ≈ 0,1) in una provetta e centrifugare a 16.000 xg per 5 min. Rimuovere il surnatante cultura. Il pellet batterico può essere elaborata immediatamente o conservato a -80 ° C per un uso successivo. </li><li> Accuratamente risospendere il pellet batterico in 10 ml di 1 M di NaOH applicando la punta della pipetta al bottom della provetta e pipettando su e giù diverse volte. Incubare la sospensione a temperatura ambiente per 5 min. </li><li> Neutralizzare il lisato batterico aggiungendo 50 ml di 1 M di tampone fosfato, pH 7,2, contenente 2,5% di sieroalbumina bovina (BSA) e 0,25 mM di una sonda rivelatore fluoresceina modificata. Incubare il lisato neutralizzato per 10 min a temperatura ambiente. Sonde rivelatore fluoresceina modificati ibridano con rRNA batterici molecole bersaglio. </li></ol> 3. Sensore elettrochimico Assay <ol><li> Lavare il MCH dal chip sensore con H 2 O deionizzata per 2-3 secondi e secco sotto flusso di azoto per 5 sec. </li><li> Applicare 4 microlitri di lisato batterico neutralizzato per l&#39;elettrodo di lavoro di ciascuno dei 14 sensori e incubare per 15 min. Obiettivo-detector complessi sonda ibridano per sonde di cattura tiolati immobilizzati. </li><li> Applicare 4 microlitri di 1 nm ponte oligonucleotide in 1 M tampone fosfato, pH 7.2, contenente 2.5% BSA e 0.25 &amp;mu, M sonda rivelatore fluoresceina-modificato a 2 sensori di controllo positivo (utilizzati per la normalizzazione del segnale) e incubare per 15 min. </li><li> Lavare il chip sensore con H 2 O deionizzata per 2-3 secondi e secco sotto flusso di azoto per 5 sec. </li><li> Applicare 4 pl di 0,5 U / ml di anti-fluoresceina-HRP 1 M in tampone fosfato, pH 7,2, contenente 0,5% di caseina per l&#39;elettrodo di lavoro di tutti i sensori 16 e incubare per 15 min. L&#39;anti-fluoresceina-HRP si lega alle sonde rivelatore fluoresceina modificati immobilizzati. </li><li> Lavare il chip sensore con H 2 O deionizzata per 2-3 secondi e secco sotto flusso di azoto per 5 sec. </li><li> Applicare un film ben adesivo alla superficie del chip del sensore e del carico nel supporto chip del sensore. Pipettare 50 ml di substrato TMB su tutti i 16 sensori e chiudere il monte chip del sensore. </li><li> Ottenere amperometria e misure di voltammetria ciclica per tutti i 16 sensori che utilizzano la Helios Reader Chip. Corrente amperometrica è proporzionale al tasso di TMB reduction sulla superficie del sensore (vedi Figura 1). </li></ol>

<ol>
	<li>Wu, J., Campuzano, S., Halford, C., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+Surface+Monolayers+for+Ultrasensitive+(Zeptomole)+Amperometric+Detection+of+Nucleic+Acid+Hybridization+without+Signal+Amplification.">Ternary Surface Monolayers for Ultrasensitive (Zeptomole) Amperometric Detection of Nucleic Acid Hybridization without Signal Amplification.</a> Anal. Chem. 82, 8830-8837 (2010).</li><li>Halford, C., 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=Rapid+Antimicrobial+Susceptibility+Testing+by+Sensitive+Detection+of+Precursor+Ribosomal+RNA+Using+a+Novel+Electrochemical+Biosensing+Platform.">Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor Ribosomal RNA Using a Novel Electrochemical Biosensing Platform.</a> Antimicrob. Agents Chemother. 56, (2012).</li><li>Campuzano, S., 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=Ternary+monolayers+as+DNA+recognition+interfaces+for+direct+and+sensitive+electrochemical+detection+in+untreated+clinical+samples.">Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.</a> Biosens. Bioelectron. 26, 3577-3584 (2011).</li><li>Gau, V., 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=Electrochemical+molecular+analysis+without+nucleic+acid+amplification.">Electrochemical molecular analysis without nucleic acid amplification.</a> Methods. 37, 73-83 (2005).</li><li>Patel, M., 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=Target+Specific+Capture+Enhances+Sensitivity+of+Electrochemical+Detection+of+Bacterial+Pathogens.">Target Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens.</a> J. Clin. Microbiol. 49, 4293-4296 (2011).</li><li>Mastali, M., 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=Optimal+probe+length+and+target+location+for+electrochemical+detection+of+selected+uropathogens+at+ambient+temperature.">Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.</a> J. Clin. Microbiol. 46, 2707-2716 (2008).</li><li>Liao, J. C., 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=Use+of+electrochemical+DNA+biosensors+for+rapid+molecular+identification+of+uropathogens+in+clinical+urine+specimens.">Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.</a> J. Clin. Microbiol. 44, 561-570 (2006).</li><li>Liao, J. C., 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=Development+of+an+advanced+electrochemical+DNA+biosensor+for+bacterial+pathogen+detection.">Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection.</a> J. Mol. Diagn. 9, 158-168 (2007).</li><li>Pedrero, M., Campuzano, S., Pingarron, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroanalytical+sensors+and+devices+for+multiplexed+detection+of+foodborne+pathogen+microorganisms.">Electroanalytical sensors and devices for multiplexed detection of foodborne pathogen microorganisms.</a> Sensors (Basel). 9, 5503-5520 (2009).</li><li>Kuralay, F., Campuzano, S., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+disposable+nucleic+acid+biosensors+for+direct+bioelectronic+detection+in+raw+biological+samples.">Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.</a> Talanta. 85, 1330-1337 (2011).</li><li>Ecker, D. J., 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=Ibis+T5000:+a+universal+biosensor+approach+for+microbiology.">Ibis T5000: a universal biosensor approach for microbiology.</a> Nat. Rev. Microbiol. 6, 553-558 (2008).</li><li>Casalta, J. P., 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=Evaluation+of+the+LightCycler+SeptiFast+test+in+the+rapid+etiologic+diagnostic+of+infectious+endocarditis.">Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis.</a> Eur. J. Clin. Microbiol. Infect. Dis. 28, 569-573 (2009).</li></ol>

Tutti gli autori sono gli inventori di brevetti relativi ai metodi descritti. Uno di questi brevetti è stato concesso in licenza a Qvella Corporation. BMC e DAH hanno partecipazione in Qvella Corporation. VG è il Presidente della GeneFluidics, che è il produttore del chip matrice di sensori elettrochimici, montaggio di chip, e lettore di chip descritto in questo articolo.

Il test del sensore elettrochimico qui descritto consente una rapida individuazione di obiettivi di acidi nucleici. Sensibilità e specificità dipendono in parte l&#39;energia libera di ibridazione sonda-bersaglio, che a sua volta dipende dalla lunghezza e il contenuto di GC della cattura e sonde rivelatore. Noi di solito eseguire i passaggi di ibridazione a temperatura ambiente (~ 20 ° C) 5, 6. Tuttavia, la procedura di ibridazione (3.2 e 3.3) possono essere eseguite anche a temperature più elevate in un ...

Usando rRNA come molecola bersaglio per il rilevamento e l&#39;identificazione batterica ha una serie di vantaggi. L&#39;abbondanza di rRNA in cellule batteriche prevede un limite di sensibilità a partire da 250 batteri per ml senza la necessità di amplificazione del target 1. RRNA batterico contiene uniche sequenze specie-specifici che sono accessibili a ibridazione con sonde di DNA. Di conseguenza, un array di sensori elettrochimici può essere utilizzato per identificare batteri sconosciuti, dove ogni sensore è funzionalizzato con un diverso sonda di cattura specie-specifica. Sensori di controlli positivi devono essere inclusi per un target oligonucleotide sintetico che &quot;ponti&quot; la cattura e sonde rivelatore per creare un segnale di calibrazione interno. Sensori elettrochimici hanno una vasta gamma di applicazioni di ricerca di base e traslazionale. Per esempio, il saggio qui descritto è stato usato per misurare con precisione l&#39;effetto di E. coli fase di crescita su RRNA e numero di copie pre-rRNA, che è di grande interesse per i ricercatori interessati alla fisiologia dei batteri 2. La sensibilità del saggio sensore elettrochimico è determinato dal rapporto segnale-rumore. Una varietà di amplificazione del segnale e di metodi di riduzione del rumore sono stati esplorati. Troviamo che migliorando la chimica della superficie del sensore è fondamentale per ridurre il legame non specifico della sonda rivelatore e / o enzima HRP. In particolare, un monostrato misto di alkanedithiols e mercaptohexanol è stato trovato per ridurre sfondo coprendo la superficie dell&#39;elettrodo più completamente mantenendo accessibilità della sonda di cattura per bersaglio ibridazione 3. Questi trattamenti chimici superficiali sono particolarmente importanti per saggi con campioni biologici complessi.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>6-mercapto-1-hexanol (MCH)</td>
 <td>Sigma</td>
 <td>451088</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>1,6-hexanedithiol (HDT)</td>
 <td>Sigma</td>
 <td>H-12005</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>Thiolated capture probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Fluorescein-modified detector probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Bridging Oligonucleotide</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Fluorescein-HRP, Fab fragments</td>
 <td>Roche</td>
 <td>11 426 346 910</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Helios Chip Reader</td>
 <td>GeneFluidics</td>
 <td>GFR-2009</td>
 <td></td>
 </tr>
 <tr>
 <td>Sensor Chip Mount</td>
 <td>GeneFluidics</td>
 <td>GFR-003</td>
 <td></td>
 </tr>
 <tr>
 <td>Film well sticker</td>
 <td>GeneFluidics</td>
 <td></td>
 <td>Shipped with sensor chips</td>
 </tr>
 <tr>
 <td>Bare gold 16-sensor array chips </td>
 <td>GeneFluidics</td>
 <td>SC1000-16X-B </td>
 <td>Store in 100% N2 at room temperature</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin </td>
 <td>Sigma</td>
 <td>A7906 </td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>1M Phosphate Buffer, pH 7.2 </td>
 <td></td>
 <td></td>
 <td>0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2</td>
 </tr>
 <tr>
 <td>Blocker Casein in PBS </td>
 <td>Pierce</td>
 <td>37528 </td>
 <td>Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 &deg;C</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

bacterial detection &amp; identification using electrochemical sensors

Sensori elettrochimici sono ampiamente utilizzati per la misurazione rapida e accurata del glucosio nel sangue e possono essere adattati per il rilevamento di una grande varietà di analiti. Sensori elettrochimici operano trasducendo un evento di riconoscimento biologico in un segnale elettrico utile. Trasduzione del segnale avviene accoppiando l&#39;attività di un enzima redox a un elettrodo amperometrico. Sensore specificità è o una caratteristica inerente l&#39;enzima, glucosio ossidasi nel caso di un sensore di glucosio, o un prodotto di legame tra l&#39;enzima ed un anticorpo o sonda. Qui, descriviamo un metodo di saggio sensore elettrochimico per rilevare direttamente e identificare i batteri. In ogni caso, le sonde descritte qui sono oligonucleotidi di DNA. Questo metodo si basa sul panino ibridazione di catturare ed identificare sonde con bersaglio RNA ribosomiale (rRNA). La sonda di cattura è ancorata alla superficie del sensore, mentre la sonda rivelatore è legata horseradish perossidasi (HRP). Quando un substrato quale 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB) è aggiunto ad un elettrodo con complessi di cattura-target-rivelatore legati alla sua superficie, il substrato viene ossidato da HRP e ridotto l&#39;elettrodo di lavoro. Questo redox ciclo sono presenti spola di elettroni da parte del substrato dall&#39;elettrodo a HRP, producendo flusso di corrente nel dell&#39;elettrodo.

Descriviamo un saggio elettrochimico che è strutturato in modo simile a un sandwich ELISA. Come mostrato in Figura 1, l&#39;RNA bersaglio ribosomiale (rRNA) ibridazione con sonde di cattura e rivelatore è sviluppato da una reazione redox catalizzata dalla HRP coniugato frammenti anticorpali anti-fluoresceina che legano il collegamento 3 &#39;fluoresceina sulla sonda rivelatore. Una componente importante di sensibilità del test è la chimica di superficie dell&#39;elettrodo oro. Abbiamo trovato che un...

Watch this Scientific Journal Video about Bacterial Detection & Identification Using Electrochemical Sensors at JoVE.com

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Descriviamo un metodo di analisi sensore elettrochimico per la rilevazione rapida di batteri e di identificazione. Il saggio comporta una serie di sensori funzionalizzato con DNA oligonucleotide sonde di cattura per RNA ribosomiale (rRNA) sequenze specie-specifici. Sandwich ibridazione del target rRNA con la sonda di cattura e di una perossidasi-coniugati DNA sonda rivelatore oligonucleotide rafano produce una corrente amperometrico misurabile.

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 ...

bacterial-detection-identification-using-electrochemical-sensors

Research

JoVE Journal

Bioengineering

28.1K Views.  Veterans Affairs Greater Los Angeles Healthcare System. Descriviamo un metodo di analisi sensore elettrochimico per la rilevazione rapida di batteri e di identificazione. Il saggio comporta una serie di sensori funzionalizzato con DNA oligonucleotide sonde di cattura per RNA ribosomiale (rRNA) sequenze specie-specifici. Sandwich ibridazione del target rRNA con la sonda di cattura e di una perossidasi-coniugati DNA sonda rivelatore oligonucleotide rafano produce una corrente amperometrico misurabile.

Video: Bacterial Detection & Identification Using Electrochemical Sensors

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Volatili che caratterizzano batterica secondaria utilizzando Electrospray ionizzazione Spettrometria di Massa (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Ricerca

Bioingegneria

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Rilevazione e isolamento delle cellule del melanoma circolanti con flussimetria fotoacustico

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

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 ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

La glutammina Flux Imaging Utilizzando sensori geneticamente codificato

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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 ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

Volare Insetto rilevamento e classificazione con sensori Economico

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

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 ...

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 ...