Biz SAM formasyonu üzerinde yararlı tartışmalar için Melissa Grunlan kabul etmiş sayılırsınız. Bu araştırma, Robert A. Welch Vakfı (A-1585) tarafından kısmen desteklenmiştir.

Reaktiflerin hazırlanması BORU tamponu (pH 6,8 KOH ile ayarlanmış 80 mM BORU 1mm MgCl 2, 1mm EGTA) hazırlayın. -70 Ve mağaza kısım glukoz oksidaz, katalaz ve glikoz ° C ve taksol -20 ° C mevcut protokollere göre 1,2 Temsili miktar ve mağaza tubulin -70 ° C tedarikçi tarafından verilen talimatlara göre. Litografi kullanarak Desen Au Mikroelektronlar <ol><li> Bir katip veya gofret kesici kullanarak 1 cm 1.5 cm substratlar içine silikon gofret kesin. Kapak ve 5 dakika sonikasyon kadar aseton beher yerleştirerek substratlar temizleyin. Yüzeylerde kurumasına izin vermeden, taze aseton onları yıkayın, sonra hemen izopropanol ve kuru nitrojen içinde durulayın. </li><li> Au Mikroelektronlar temiz yüzeyler üzerinde istenen özellikleri boyutuna bağlı olarak, standart, pozitif tonu fotolitografi veya elektron demeti litografi kullanarak imal 3 desen geometri tasarlarken, kapsama sağlamak için her elektrot desen çapı 100 mikron altında tutmak PEG SAM (sonraki bölümde açıklandığı gibi). Yastık bağlayan bir elektrot (Şekil 1) ile 1 mm çizgi ile desen, 500 mm ile 300 konumlandırılmış temas pedleri (~ 300 mm 2) ekleyin . Akışını odasının montajı için desen etrafında yer kalmasına izin verecek yüzey ortasına yakın litografik kalıpları yerleştirin. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="Şekil 1" /> Şekil 1. Örnek geometri. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1390/1390fig1.jpg">buraya tıklayınız</a> . </li><li> Karşı katmanı desen kalkınma sonra, karşı temas pedi cımbız kullanarak (Şekil 1) substrat kenarına bir çizgi çizebilir. Metal birikimi üzerine, sıfırdan elektrot ve yüzey kenarı arasındaki elektriksel temas sağlayacaktır. Biriktirme haznesi, tercihen iki kaynaktan ile bir metal katmanı yatırmak için kullanılır. Elektrotlar vakum bozmadan, Au 6 nm takip Cr 2 nm birikimi (yapışma) tarafından oluşturulur. Odasının içine donatılmış bir kuvars kristali mikro depolanması sırasında filmlerin kalınlığı ölçmek için kullanılabilir. </li></ol> SAM hazırlayın <ol><li> Onceden ve iyi havalandırılan bir alanda fırın - 75 ° C 250 ml cam beher 20 mL toluen ekleyerek silanization çözüm hazırlayın. Daha sonra plastik bir pipet kullanarak toluen için 1 ml PEG-silan (yani% 5 v / v) ekleyin. Yukarı ve aşağı pipetleme 5 ila 10 kat ve daha sonra 30 saniye boyunca pipet ile karıştırarak PEG-silan iyi karıştırın. </li><li> PEG-silan toluen çözümü tamamen sular altında ve eşit olarak beher alt kısmında aralıklı emin olun desenli örneklerin beher desenli yüzü yukarı bakacak şekilde yerleştirin. Fırında behere koyun ve 18 ila 21 saat fırında 75 ° C. SAM oluşturan iken, counterelectrode hazırlamak (aşağıya bakınız) ve kalan adımlar için gerekli olan tüm ek parçaları bir araya toplamak. </li><li> Fırında 18-21 saat sonra, toluen, PEG-silan viskoz bir kalıntı desenli örnekler bırakarak buharlaşır gerekir. 20 mL toluen beher ekleyin ve 1-2 dakika örnekleri bekletin. Örnekleri kaptan çıkarın ve sonra toluen, izopropanol durulayın ve nitrojen ile nihayet kuru. Bu noktada, numuneler temiz bir görünüm ve SAM katmanı çıplak gözle görünmez olmalıdır. SAMs serin kuru hava şartlarında (25 ° C, orta nem) altında saklanan iki gün kadar kullanılabilen bu şekilde hazırladı. Yüzeyinde bulutlu veya düzensiz Mutfak dekorasyonunun puf noktalari kalıntı kaplama Başarısız SAM oluşumuna neden olur. </li></ol> Counterelectrode Üretiyor Mikroelektrot depolanması için kullanılan aynı birikimi odasının kurulumu, 22 x 50 mm No 1 ya da 0 no&#39;lu cam lamel üzerine Au 4 nm Cr 1 nm, para yatırma bir counterelectrode olun. Counterelectrode açık gri ya da pembe bir renk tonu ile neredeyse şeffaf olmalıdır. Temiz tutmak için bir Petri kabındaki kaplı yan counterelectrode saklayın. MTS içine polimerize tubulin <ol><li> Çözülme tubulin ve rodamin tubulin etiketlenmemiş ve hemen elde etmek için parlak MTS etiketsiz için etiketli 1:2 oranında kombine. Birkaç kez aşağı yukarı pipetleme karıştırın. Isıtılmış su banyosunda 20-40 dakika tubulin polimerize 37 ° C. MTS uzunluğu uzun polimerizasyon kez daha büyük olacaktır. Tubulin polimerize olmasına rağmen, 20 mM için taksol BORU tampon içeren bir mikrosantrifüj tüpe ekleyerek BORU taksol tampon hazırlamak. Yukarı ve aşağı pipetleme birkaç kez ve vorteks taksol iyi karıştırın ve daha sonra 37 ısıtılmış banyoya tüp yeri ° C. Polimerizasyon sonra, sıcak BORU taksol tampon ve kalkan 1 / 100 MTS sulandırmakışıktan. Bu şekilde hazırlanan MTS zaman ve paket, daha fazla seyreltme azaltılabilir uzayacaktır. </li><li> 30 ml soğuk BORU tampon (0-4 ° C), 2-mercaptoethanol% 50, 5 ml katalaz glukoz oksidaz, 5 ml ve 5 ml glikoz 5 ml birleştirerek 10x oksijen süpürücü karışımı (OS) hazırlayın. 2 Glikoz son ilave edilmelidir. Her bileşen ekledikten sonra 3-5 kez pipetleme ve aşağı çevirerek karıştırın. OS karışımı buz koyun, yaklaşık 2 saat boyunca etkili olmaya devam edecektir. </li><li> MT polimerizasyon başarılı oldu ve MTS görüntüleme 1x OS karışımı ile hazırlanan MTS floresans uyarma birkaç dakika altında stabil olduğunu doğrulayın. Görüntüleme için bu şekilde hazırlanan MTS, rodamin floresan için uygun bir uyarma / emisyon filtre kombinasyonu kullandığınızdan emin olun. BORU 1x OS karışımı ile tampon ve görüntüleme kullanarak 1 / 10 ila 1 / 100 MTS seyreltilmesi görüntüleme bireysel filamentler için en iyi çalışma seyreltme belirleyin. </li><li> Bir akış odası, iki adet çift taraflı bant dışında 2-3 mm yerleştirilir büyük (22 x 50 mm) ve küçük (22 x 22 mm) cam lamel sandviç hazırlayın. Bant cam slayt üzerine yerleştirerek ve bir jilet kesim tarafından kolayca şeritler halinde kesilir. Mikropipet kullanarak akış hücre içine MTS bir kaç mikrolitre tanıtın. Yüksek büyütme görüntüleme için, uzun çalışma mesafesi hedefleri üst yüzey akış odasının görüntüleme için gerekli olabilir. Lamel / numune yüzey akış odasının üst yüzeyi arasındaki mesafe bant kalınlığına göre belirlenir ve 60-100 mm olmalıdır. </li></ol> Desen MTS elektroforez kullanarak <ol><li> Elektrot bant (Şekil 2) dik çalışan bağlantı yolu ile şeritler arasında olduğu gibi, desenli, SAM-kaplı numune üzerinde çift taraflı bant iki ince şeritler koyarak bir elektroforez odasının hazırlayın. Yaklaşık 3 mm (Şekil 2) tarafından örnek çıkıntılar counterelectrode kenarında çift taraflı bant, teyp gibi dokunmadan metal yüzü counterelectrode yerleştirin. Birkaç 15 cm uzunlukta, yalıtılmış bakır mıknatıs tel kesin ve her iki ucunda 1 cm yalıtım kapalı şerit. Counterelectrode (bu bir multimetre ile kontrol edilebilir) ile elektrik kontağı yapmaktan kaçınmaya dikkat çekerken, küçük bir damla gümüş boya kullanarak substrat maruz Au alan bir tel takın. Counterelectrode gümüş boya kullanarak ikinci bir tel takın. Teller gümüş boya ile bağlı bant ile örnekler daha güvenli olabilir. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="Şekil 2" /> Şekil 2 ters bir floresan mikroskop Flow hücre montajı. Lütfen Şekil 2&#39;de büyük halini görmek için <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1390/1390fig2.jpg">buraya tıklayın</a> . </li><li> 1x final konsantrasyon OS ile birlikte BORU tampon kullanarak istenilen seyreltme MTS tanıtın. 20X büyütme Görüntü ve bir voltaj uygulamadan önce silikon gofret desen bulmak. İsteğe bağlı olarak, akış odasının, akışkan akışı ve akış odasının düzlemde MT göçü önlemek için oje kullanılarak kapatılmalıdır. </li><li> 1 V kadar bir gerilim uygulayın ve MTS göç gözlemlemek. MTS, geri dönüşümlü hapsi istenen ise daha düşük voltaj (yaklaşık 0.5 V) kullanılıyor olabilir, ancak bu yanal kayma önlemek için akış odasının sızdırmazlık gerektirir. Tırnak cilası akış odasının açık biter sızdırmazlık için iyi çalışır. V 0.7 hakkında yukarıda gerilimler, bazı MT yapışma zayıf olsa, desen olabilir. Yaklaşık 0,9 V Yukarıdaki gerilimler, MT göçü daha hızlı olacak ve yapışma güçlü olacak. Mikroelektronlar yok etmek için de indükleyici elektroliz (gaz kabarcığı oluşumu) ve yaklaşık 1,2 V üstünde olasılığını önemli ölçüde artırır. Göç mikroskop odak dikey düzlemde ayarlayarak takip edilebilir. Doğru şekilde yapılmazsa, MTS elektrot yüzeylerinde lokalize. </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

MTS, göç nedeniyle parlaklık ve düşük eşiğe kolayca floresan mikroskopi kullanılarak görüntülenmiştir. Sadece biyomoleküllerin tampon pH uygun ayarı ile net ücret taşımak için ikna olmasını gerektirir Bu yöntem, genellikle biyomolekülün desenlendirme için geçerli. Kapiler elektroforez kullanılan silika duvarlar gibi hiçbir ücret yüzeyleri vardır çünkü Electroosmosis Bu kurulum kaçınılmalıdır. Bu yöntem çeşitli değişiklikler yapılabilir. Sızdırma...

No: 1 borosilikat cam lamelleri (22 X 50 mm) VWR satın alınmıştır. Cilalı elektronik sınıf p-tipi &lt;111&gt; silikon gofret Yüksek saflıkta gümüş boya Yapısı Sonda, Inc (Katalog # 5001 AB) elde edilmiştir Addison Mühendisliği, Inc satın alındı. Yalıtımlı ince göstergesi Bakır Bobin Teli (~ 0.08 mm (0.003) çap), elektronik tedarik şirketlerinden elde edilebilir.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Biz, altın Mikroelektronlar, bir silikon taban poli (etilen glikol) (PEG) trimethoxysilane kendinden montajlı tek tabaka (SAM) oluşturmak için bir prosedür mevcut. PEG-SAM tek bir montaj adımda oluşturulur ve silikon ve altın yüzeyler üzerinde biyolojik kirlenmeye engeller. SAM standart, pozitif ton litografi desenli ceket Mikroelektronlar kullanılır. Mikrotübül bir örnek olarak kullanarak, geri dönüşümlü bir şekilde SAM-örtülü elektrot elektroforetik göç ikna etmek için bir DC gerilim uygulanır. Bir akış odası, görüntüleme için elektroforetik göç ve mikrotübül desenlendirme kullanılan<em&gt; In situ</em&gt; Epifloresans mikroskopi kullanılarak. Hedef moleküller hareketsiz ve böylece spesifik moleküler etkileşimler gerektirmez elektroforez çalışanı olarak bu yöntem, genellikle biyomolekülün desenlendirme uygulanabilir. Ayrıca, desen SAM moleküllerin doğrudan litografik teknikleri kullanarak çalışırken karşılaşılan sorunları önler. Elektron demeti litografi ile uyumluluk, bu yöntem nano ölçekli desen biyomoleküllerin kullanılmasına izin verir.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Biz altın Mikroelektronlar bir silikon taban üzerinde bir poli (etilen glikol), kendinden montajlı tek tabaka (PEG-SAM) oluşturmak için bir prosedür mevcut. PEG-SAM, tek bir adımda oluşan ve silikon ve altın yüzeyler biofouling engeller. Elektroforez sonra nano ölçekli desenlendirme biyomoleküllerin için kullanılır.

Desenlendirme Biyomoleküller Mikroelektronlar Kendinden monte mono tabakaları Zehirli

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.4K Görüntüleme.  Texas A&M University (TAMU). Biz altın Mikroelektronlar bir silikon taban üzerinde bir poli (etilen glikol), kendinden montajlı tek tabaka (PEG-SAM) oluşturmak için bir prosedür mevcut. PEG-SAM, tek bir adımda oluşan ve silikon ve altın yüzeyler biofouling engeller. Elektroforez sonra nano ölçekli desenlendirme biyomoleküllerin için kullanılır.

Video: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Biyomühendislik

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Optik Şeffaf indiyum kalay oksit Elektrotlar Desenlendirme Hücreler

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Biyoloji

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomik Kuvvet Spektroskopi Tek Molekül Yapışma incelenmesi

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Mühendislik

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Photolithographically Tanımlı Parylene-C hücre Desenlendirme: SiO<sub&gt; 2</sub&gt; Yüzeyleri

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Bir Çekçek tabanlı Meclisi Yöntemi ile Biomembrane Mikroarray&#39;ler oluşumu

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Tek Hücre Hassas Nevronik Co-kültürler hazırlanması

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Nörobilim

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Sıralı Capillarity destekli Montaj yoluyla Mikroorganizmaların ve Mikropartiküllerin Deseni

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

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

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

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

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

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Desenlendirme Proteinlerin ve Hücreleri Çok Yönlü Yöntemi

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Anonim Mikroelektrotlu Bioinspired Yumuşak Robot

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Desenlendirme Biyomoleküller Mikroelektronlar Kendinden monte mono tabakaları Zehirli

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