A. Ozcan riconosce con gratitudine il sostegno del Premio CARRIERA NSF, l&#39;Investigatore ONR Premio Giovani 2009 e Innovator Award nuovo direttore NIH DP2OD006427 presso l&#39;Ufficio del Direttore, NIH. Gli autori riconoscono anche il sostegno della Bill &amp; Melinda Gates Foundation, Fondazione Vodafone Americhe, e NSF programma Bish (sotto Awards # 0754880 e 0930501).

<ol>
	<li>Coskun, A. F., Su, T., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide+field-of-view+lens-free+fluorescent+imaging+on+a+chip.">Wide field-of-view lens-free fluorescent imaging on a chip.</a> Lab Chip. 10, 824-824 (2010).</li><li>Coskun, A. F., Sencan, I., Su, T., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensless+wide-field+fluorescent+imaging+on+a+chip+using+compressive+decoding+of+sparse+objects.">Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects.</a> Opt. Express. 18, 10510-10523 (2010).</li><li>Coskun, A. F., Sencan, I., Su, T., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+Fluorescent+On-Chip+Imaging+of+Transgenic+Caenorhabditis+elegans+Over+an+Ultra-Wide+Field-of-View.">Lensfree Fluorescent On-Chip Imaging of Transgenic Caenorhabditis elegans Over an Ultra-Wide Field-of-View.</a> PLoS ONE. 6, e15955-e15955 (2011).</li><li>Coskun, A. F., Sencan, I., Su, T., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+lensless+fluorescent+microscopy+using+a+tapered+fiber-optic+faceplate+on+a+chip.">Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.</a> Analyst. , (2011).</li><li>Seo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Lens-Free+Blood+Analysis+on+a+Chip.">High-Throughput Lens-Free Blood Analysis on a Chip.</a> Analytical Chemistry. 82, 4621-4627 (2010).</li><li>Mudanyali, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact,+light-weight+and+cost-effective+microscope+based+on+lensless+incoherent+holography+for+telemedicine+applications.">Compact, light-weight and cost-effective microscope based on lensless incoherent holography for telemedicine applications.</a> Lab Chip. 10, 1417-1417 (2010).</li><li>Tseng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+microscopy+on+a+cellphone.">Lensfree microscopy on a cellphone.</a> Lab Chip. 10, 1787-1787 (2010).</li><li>Lucy, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+iterative+technique+for+the+rectification+of+observed+distributions.">An iterative technique for the rectification of observed distributions.</a> The Astronomical Journal. 79, 745-745 (1974).</li><li>Richardson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian-Based+Iterative+Method+of+Image+Restoration.">Bayesian-Based Iterative Method of Image Restoration.</a> J. Opt. Soc. Am. 62, 55-59 (1972).</li><li>Biggs, D. S. C., Andrews, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+iterative+image+restoration+algorithms.">Acceleration of iterative image restoration algorithms.</a> Appl. Opt. 36, 1766-1775 (1997).</li><li>Candes, E., Wakin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+To+Compressive+Sampling.">An Introduction To Compressive Sampling.</a> Signal Processing Magazine, IEEE. 25, 21-30 (2008).</li><li>Kim, S., Koh, K., Lustig, M., Boyd, S., Gorinevsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Interior-Point+Method+for+Large-Scale+L1-Regularized+Least+Squares.">An Interior-Point Method for Large-Scale L1-Regularized Least Squares.</a> Selected Topics in Signal Processing, IEEE. 1, 606-617 (2007).</li><li>Candes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+restricted+isometry+property+and+its+implications+for+compressed+sensing.">The restricted isometry property and its implications for compressed sensing.</a> Comptes Rendus Mathematique. 346, 589-592 (2008).</li><li>Baraniuk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compressive+Sensing+[Lecture+Notes].">Compressive Sensing [Lecture Notes].</a> Signal Processing Magazine, IEEE. 24, 118-121 (2007).</li><li>Romberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+via+Compressive+Sampling.">Imaging via Compressive Sampling.</a> Signal Processing Magazine, IEEE. 25, 14-20 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo dimostrato una piattaforma on-chip microscopia a fluorescenza che può raggiungere ad esempio, &lt;4 _m risoluzione spaziale oltre ad esempio,&gt; 0,6-8 cm 2 di campo di vista, senza l&#39;uso di lenti, meccanico-scansione o film sottile interferenze filtri. In questa tecnica, con l&#39;utilizzo di una fibra ottica frontalino o un cono, l&#39;emissione fluorescente dagli oggetti viene raccolta con un 2D-array di cavi in ​​fibra ottica prima di essere consegnato ad un sensore opto-elettronico-array come un CCD / chip CMOS. Queste immagini acquisite lensfree vengono poi rapidamente elaborati per produrre &lt;risoluzione 4 _m oltre&gt; 0,6-8 cm 2 di campo di vista utilizzando un campionamento di compressione / rilevamento basato algoritmo di decodifica. Tale piattaforma compatta e grande campo di imaging fluorescente potrebbe essere molto utile per high-throughput citometria, rare cellule di ricerca così come per microarray-analisi.

<table border="1"><tbody><tr><th> Nome del materiale </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Charge-coupled device (CCD) </td><td> KODAK </td><td> KAF-8300 </td></tr><tr><td> Charge-coupled device (CCD) </td><td> KODAK </td><td> KAF-11002 </td></tr><tr><td> Charge-coupled device (CCD) </td><td> KODAK </td><td> KAF-39000 </td></tr><tr><td> Complementary Metal-Oxide-Semiconductor (CMOS) </td><td> Micron </td><td> MT9T031C12STCD </td></tr><tr><td> LED ad alta potenza sorgente luminosa </td><td> Thorlabs </td><td> M455L2-C2 </td></tr><tr><td> Alta conducente LED </td><td> Thorlabs </td><td> LEDD1B </td></tr><tr><td> Fibra accoppiata sorgente luminosa a LED </td><td> Mightex </td><td> FCS-0625-000 </td></tr><tr><td> Vuoto Penna </td><td> Edmund Optics </td><td> NT57-636 </td></tr><tr><td> 2, 4, 10 micron Fluospheres </td><td> Invitrogen </td><td> F-8826, F-8859, F-8836 </td></tr><tr><td> RBS lisi tampone 1X </td><td> eBioscience </td><td> 00-4333 </td></tr><tr><td> SYTO 16 etichettatura dei reagenti </td><td> Invitrogen </td><td> S7578 </td></tr><tr><td> Fibra ottica frontale </td><td> Edmund Optics </td><td> NT55-142 </td></tr><tr><td> Cono in fibra ottica </td><td> Edmund Optics </td><td> NT55-134 </td></tr><tr><td> Prismi </td><td> Edmund Optics </td><td> NT47-626, NT45-403 </td></tr><tr><td> Filtri </td><td> Edmund Optics </td><td> NT39-417 </td></tr><tr><td> PDMS Elastomeri </td><td> Dow Corning </td><td> Slygard 184 </td></tr></tbody></table>

lensless fluorescent microscopy on a chip

On-chip di imaging senza lenti, in generale, mira a sostituire ingombranti microscopi ottici con lenti a base di più semplice e più compatto design, soprattutto per le applicazioni high-throughput screening. Questa piattaforma tecnologia emergente ha il potenziale per eliminare la necessità di componenti ottici ingombranti e / o costose attraverso l&#39;aiuto di nuove teorie e gli algoritmi di ricostruzione digitale. Sulla stessa linea, qui si dimostrano una modalità on-chip microscopia a fluorescenza che può raggiungere ad esempio, &lt;4 _m risoluzione spaziale su un ultra-ampio campo di vista (FOV) di&gt; 0,6-8 cm 2 senza l&#39;uso di lenti , meccanico-scansione o film sottile basati filtri interferenziali. In questa tecnica, eccitazione fluorescente è raggiunto attraverso una interfaccia prisma o emisferica di vetro illuminato da una sorgente incoerente. Dopo aver interagito con il volume intero oggetto, questa luce di eccitazione è respinta da total-interno-riflessione (TIR), processo che si sta verificando nella parte inferiore del chip campione di micro-fluidico. L&#39;emissione di fluorescenza dagli oggetti eccitato è poi raccolto da una fibra ottica frontalino o una candela e viene consegnato a una matrice di sensori optoelettronici come un accoppiamento di carica-device (CCD). Utilizzando un algoritmo di compressione-campionamento basato decodifica, l&#39;acquisito lensfree prime immagini fluorescenti del campione può essere rapidamente trattati per produrre ad esempio, &lt;4 _m risoluzione su un campo visivo di&gt; 0,6-8 cm 2. Inoltre, impilati verticalmente micro-canali separati da esempio, 50-100 micron può essere ripreso con successo utilizzando le stesse lensfree on-chip piattaforma microscopia, che aumenta ulteriormente il throughput complessivo di questa modalità. Questa compatta on-chip piattaforma di imaging fluorescente, con un decoder rapida compressione dietro di esso, potrebbe essere piuttosto utile per high-throughput citometria, rare cellule di ricerca e di analisi dei microarray.

Watch this Scientific Journal Video about Lensless Fluorescent Microscopy on a Chip at JoVE.com

Lensless Fluorescent Microscopy on a Chip

Un senza lenti on-chip piattaforma microscopia a fluorescenza è dimostrato in grado di oggetti immagini fluorescenti su un ultra-ampio campo di vista di esempio,&gt; 0,6-8 cm2 con &lt;4 _m risoluzione utilizzando un campionamento di compressione basata algoritmo di decodifica. Tale compattezza e grande campo fluorescente on-chip modalità di imaging potrebbe essere prezioso per high-throughput citometria, rare cellule di ricerca e di analisi dei microarray.

Senza lenti microscopia a fluorescenza su un chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

lensless-fluorescent-microscopy-on-a-chip

Nanyang Technological University

Research

JoVE Journal

Bioengineering

17.5K Views.  University of California, Los Angeles. Un senza lenti on-chip piattaforma microscopia a fluorescenza è dimostrato in grado di oggetti immagini fluorescenti su un ultra-ampio campo di vista di esempio,> 0,6-8 cm2 con 

Video: Lensless Fluorescent Microscopy on a Chip

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Lensfree On-chip Tomographic Microscopia Utilizzando Multi-angolo di illuminazione e Pixel Super-risoluzione

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Ricerca

Bioingegneria

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

On-Chip Fenotipizzazione Endothelial infiammatoria

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Ampio campo di microscopia a fluorescenza e citometria di flusso Imaging fluorescente su un telefono cellulare

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Procedura per lo sviluppo di multi-profondità circolare a sezione trasversale Endothelialized microcanali-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Test di suscettibilità agli antibiotici indotti dallo stress su un chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Un nuovo metodo per la localizzazione Reporter perline fluorescenti vicino alla superficie coltura cellulare per la trazione microscopia a forza

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip: Uno studio privo di etichette delle interazioni proteine-fosfoinositide

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Film secco basati su Photoresist biosensore elettrochimico Microfluidic Platform: Fabbricazione di dispositivi, saggio su chip preparazione e funzionamento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabbricazione e validazione di un sistema di organo-on-chip con elettrodi integrati direttamente quantificare transendoteliale resistenza elettrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observed&#160;in vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.

Hypoxia is a hallmark of tumor microenvironment and plays a crucial role in cancer progression. This article describes the fabrication process of a hypoxic cancer-on-a-chip based on 3D cell-printing technology to recapitulate a hypoxia-related pathology of cancer.

Cancro ipossia stampato in cellule 3D su chip per ricapitolare la progressione patologica del cancro solido

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, ...