Questo lavoro è stato sostenuto dalla Science Foundation Israele (concessione n ° 1118-1108 e concessione n ° 1146/12) e il Minna Kroll Memorial Fund Research. ES riconosce con gratitudine il sostegno finanziario del Russell Berrie Nanotechnology Institute.

1. Preparazione di Oxidized poroso SiO 2 <ol><li> Etch wafer di silicio (singolo lato lucido in &lt;100&gt; viso e pesantemente drogato, di tipo p, 0.0008 Ω · cm) in una soluzione 03:01 (v / v) di HF acquosa ed etanolo assoluto per 30 secondi a una corrente costante densità di 385 mA / cm 2. Si prega di notare che la HF è un liquido altamente corrosivo e deve essere maneggiato con estrema cura. </li><li> Risciacquare la superficie del risultante Si (PSI) pellicola porosa con etanolo assoluto più volte; asciugare i film sotto un gas di azoto secco. </li><li> Ossidare i campioni PSi appena incise in un forno tubolare a 800 ° C per 1 ora in aria ambiente (posizionare il campione nel forno a temperatura ambiente, riscaldare il forno a 800 ° C, lasciare in forno per 1 ora, spegnere il forno e togliere i campioni dal forno solo a temperatura ambiente). </li></ol> 2. Biofunzionalizzazione di PSIO 2 Ponteggi <ol><li> Incubare un PSi ossidato (PSiO 2) campione per 1 ora in mercaptopropil (95% (MPTS) soluzione trimetossisilano-3) (108 mM in toluene). </li><li> Risciacquare il PSIO 2 campione silano-trattato con toluene, metanolo e acetone; secco sotto un gas di azoto secco. </li><li> Incubare la PSIO 2 campione silano modificato per 30 min in 1 ml di 100 mM di soluzione biotina PEO-iodoacetyl. </li><li> Sciacquare il PSIO 2 campione di biotina-trattati con 0,1 M tampone fosfato soluzione (PBS) diverse volte. </li><li> Incubare la PSIO 2 campione biotina-modificato per 30 min in 1 ml di soluzione di streptavidina ml 100 pg / (SA). </li><li> Sciacquare il PSIO 2 del campione SA-trattato con soluzione 0,1 M PBS diverse volte. </li><li> Incubare la SA-modificato PSIO 2 campione risultante per 30 minuti in 1 ml di 100 pg / ml biotinilato E. soluzione di anticorpo monoclonale coli (Immunoglobulina G, IgG) o con IgG 100 pg / ml biotinilato-coniglio (come modello per un anticorpo monoclonale). </li></ol> 3. Etichettatura fluorescente e microscopia a fluorescenza <ol><li> Incubare le superfici IgG modificati con 15 ug / ml di fluoresceina (FITC) codificata anti IgG di coniglio per 40 min e con 15 ug / ml di fluoresceina (FITC) codificata IgG anti-topo come controllo. </li><li> Sciacquare i campioni modificati con 0,1 M PBS soluzione più volte. </li><li> Esaminare i campioni al microscopio a fluorescenza. </li></ol> 4. Cultura Batteri <ol><li> Coltivare E. batteri coli K12 in una provetta da 10 ml con 5 ml di Luria Bertani (LB) medio (tessitura media in 1 L di acqua deionizzata: 5 g di NaCl, 5 g di estratto di lievito, e 10 g di triptone). Incubare i batteri durante la notte agitazione a 37 ° C. </li><li> Controllare la concentrazione di batteri dalla lettura della densità ottica (DO) a una lunghezza d&#39;onda di 600 nm. Dopo la crescita durante la notte in mezzo LB, leggere la OD 600 utilizzando uno spettrofotometro per determinare la concentrazione batterica. Il numero di cellule è terribilectly proporzionale ai OD 600 misure (1 OD 600 = 10 8 cellule / ml). </li></ol> 5. Batteri Sensing <ol><li> Posizionare il PSIO IgG modificato 2 e ordinata PSIO 2 (come controllo) campioni in una cella di flusso plexiglas su misura. Fissare la cella di flusso per garantire che la riflettività campione viene misurato nella stessa posizione durante tutte le misurazioni. </li><li> Incubare i campioni con E. sospensione coli K12 (10 4 cellule / ml) per 30 min a temperatura ambiente. Quindi rimuovere la sospensione batteri svuotando la cella con 0,85% w / v soluzione salina per 30 minuti. </li><li> Monitorare i cambiamenti nei dati di riflettività in tutto l&#39;esperimento. Tutte le misurazioni ottiche devono essere effettuate in un acquoso circostante. Spettri debbono essere raccolti utilizzando uno spettrometro CCD e analizzato applicando trasformata veloce di Fourier (FFT), come precedentemente descritto 25,26. </li><li> Confermare la presenza dei batteri sullasuperficie biosensore, dall&#39;osservazione dei campioni sotto un microscopio ottico verticale subito dopo l&#39;esperimento biosensori. </li></ol>

<ol>
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Gli autori dichiarano interessi finanziari concorrenti.

Un immunosensor ottico privo di etichetta, basato su un PSIO 2 nanostruttura (un film sottile Fabry-Perot) è fabbricato, e il suo potenziale applicabilità come un biosensore per la rilevazione batteri è confermato. Modifiche e risoluzione dei problemi Uno dei principali problemi durante la progettazione di immunosensor è la suscettibilità di anticorpi a subire cambiamenti di conformazione indesiderati durante la deposizione e patterning sul substrato...

Precoce ed accurata identificazione di batteri patogeni è estremamente importante per la sicurezza degli alimenti e l&#39;acqua, il monitoraggio ambientale e diagnostica 1 point-of-care. Come le tecniche di microbiologia tradizionali sono molto tempo, laborioso, e non hanno la capacità di rilevare i microrganismi in &quot;real-time&quot; o al di fuori dell&#39;ambiente di laboratorio, biosensori stanno evolvendo per rispondere a queste sfide 2-5. Negli ultimi anni, poroso Si (PSI) è emerso come una piattaforma promettente per la progettazione di sensori e biosensori 6-20. Negli ultimi dieci anni sono stati pubblicati numerosi studi in materia di sensori e biosensori ottici basati psi 21,22. Lo strato nanostrutturati PSi è tipicamente fabbricata elettrochimica anodica incisione da un singolo cristallo di wafer di silicio. I nanomateriali PSi risultanti presentano molte caratteristiche vantaggiose, come la grande superficie e volume libero, poro misure che possono essere controllati e sintonizzabile optiproprietà cal 10,16. Le proprietà ottiche dello strato Psi, quali fotoluminescenza 8,11 e luce bianca a base di riflettanza-interferometria 7,19, sono fortemente influenzati dalle condizioni ambientali. Cattura di giudizi analiti molecole / destinazione entro i risultati strato poroso in una variazione dell&#39;indice di rifrazione medio del film, osservato come una modulazione nello spettro di fotoluminescenza o come uno spostamento della lunghezza d&#39;onda nello spettro di riflettività 10. Anche se la grande innovazione nella tecnologia PSi biosensore ottico, ci sono solo pochi rapporti sulle piattaforme basate PSI-per i batteri rilevamento 6,8,20,23-29. Inoltre, la maggior parte di questi studi proof-of-concept hanno dimostrato di rilevamento &quot;indiretta&quot; dei batteri. Così, generalmente prima lisi delle cellule è necessario per estrarre i frammenti di proteine ​​/ DNA mirati, caratteristici per i batteri studiati 29. Il nostro approccio è quello di acquisire direttamente i batteri bersagliocellule su il biosensore psi. Pertanto, gli anticorpi monoclonali, che sono specifici per i batteri bersaglio, sono immobilizzati sulla superficie porosa. Il legame di cellule di batteri, tramite interazioni anticorpo-antigene, sulla superficie del biosensore indurre variazioni di ampiezza (intensità) dello spettro di riflettività 24-26. In questo lavoro, si segnala la costruzione di un biosensore basato PSI-ottica e dimostrare la sua applicazione come una piattaforma di biosensori privo di etichetta per la ricerca di Escherichia coli (E. coli), batteri K12 (utilizzati come modello microrganismo). L&#39;monitorato segnale ottico è la luce riflessa dalla nanostruttura PSi causa di Fabry-Perot interferenze film sottile (Figura 1A). Cambiamenti nella luce ampiezza / intensità sono correlati alla specifica immobilizzazione delle cellule dei batteri bersaglio sulla superficie biosensore, consente il rilevamento rapido e quantificazione dei batteri.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

<html> Un biosensore ottico privo di etichetta basato su un poroso nanostrutturato Si è progettato per acquisizione rapida e rilevamento di batteri Escherichia coli K12, come modello microrganismo. Il biosensore deduce legame diretto delle cellule dei batteri bersaglio sulla sua superficie, mentre non è necessario alcun pretrattamento (ad esempio mediante lisi cellulare) del campione studiato. Un film sottile mesoporosi Si è utilizzato come elemento trasduttore ottico del biosensore. Sotto illuminazione a luce bianca, lo strato poroso mostra ben risolti, i modelli frange Fabry-Perot nel suo spettro di riflessione. L&#39;applicazione di una trasformata veloce di Fourier (FFT) per i risultati dei dati di riflettività in un unico picco. Variazioni dell&#39;intensità del picco FFT sono monitorati. Così, batteri bersaglio catturare sulla superficie biosensore, attraverso interazioni anticorpo-antigene, induce cambiamenti misurabili nell&#39;intensità dei picchi FFT, consentendo un osservazione &#39;tempo reale&#39; di attaccamento batteri. nt &quot;&gt; Il film di Si mesoporosi, fabbricato da un processo di anodizzazione elettrochimica, è coniugato con anticorpi monoclonali, specifici per i batteri bersaglio. L&#39;immobilizzazione, immunoactivity e specificità degli anticorpi sono confermati da esperimenti di marcatura fluorescente. Dopo il biosensore è esposto al batteri bersaglio, le cellule sono direttamente catturati sulla superficie porosa Si anticorpi modificati. Questi eventi di cattura specifici producono variazioni di intensità del film sottile spettro interferenza ottico del biosensore. Abbiamo dimostrato che questi biosensori possono rilevare relativamente basse concentrazioni batteriche (rilevamento limite di 10 4 cellule / ml) in meno di un&#39;ora.

PSi ossidato (2) PSIO film sono preparati come descritto nella sezione Text protocollo. Figura 1B mostra una ad alta risoluzione al microscopio elettronico a scansione della pellicola PSi risultante dopo ossidazione termica. Lo strato PSIO 2 è caratterizzata da pori cilindrici ben definiti con un diametro nell&#39;intervallo di 30-80 nm. L&#39;anticorpo monoclonale (IgG) molecole si innestano sulle PSIO 2 superfici utilizzando una tecnologia...

Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Viene introdotto un biosensore ottico privo di etichetta per il rilevamento di batteri rapidi. Il biosensore è basato su poroso nanostrutturato Si, che è stato progettato per catturare direttamente le cellule dei batteri bersaglio sulla sua superficie. Usiamo anticorpi monoclonali, immobilizzati sul trasduttore poroso, come le sonde di cattura. I nostri studi dimostrano l&#39;applicabilità di questi biosensori per il rilevamento di concentrazioni batteriche basse in pochi minuti, senza trasformazione campione prima (come lisi cellulare).

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

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

optical-detection-of-e-coli-bacteria-by-mesoporous-silicon-biosensors

Research

JoVE Journal

Bioengineering

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

16.8K Views.  Technion - Israel Institute of Technology.  Viene introdotto un biosensore ottico privo di etichetta per il rilevamento di batteri rapidi. Il biosensore è basato su poroso nanostrutturato Si, che è stato progettato per catturare direttamente le cellule dei batteri bersaglio sulla sua superficie. Usiamo anticorpi monoclonali, immobilizzati sul trasduttore poroso, come le sonde di cattura. I nostri studi dimostrano l'applicabilità di questi biosensori per il rilevamento di concentrazioni batteriche basse in p...

Video: Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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. ...

Ricerca

Bioingegneria

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

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

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

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

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

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

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

Collegamento sonde biologiche ai biosensori ottici che utilizza silice agenti di accoppiamento silanici

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Analizzando internalizzazione cellulare di nanoparticelle e batteri da Multi-spettrale Citometria a Flusso Imaging

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensori per il rilevamento di antibiotici batteri di stafilococco resistenti

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

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

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

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

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

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

Nanomanipulation of Single RNA Molecules by Optical Tweezers

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

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

Nanomanipolazione di Single RNA Molecole da pinzette ottiche

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

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 ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.