Riconosciamo l&#39;appoggio finanziario del premio NSF CMMI-1000724.

1A. Monostrato Formazione Primaria su Silicio <ol><li> Taglio wafer di silicio in 1 centimetro due substrati, polvere e risciacquare con acqua ed etanolo filtrata. </li><li> Eliminare la contaminazione organica immergendo i substrati di silicio in un piatto di vetro contenente Nano striscia a 75 º C. Dopo 15 minuti, risciacquare con acqua deionizzata ogni substrato, acqua filtrata. </li><li> Luogo ogni substrato in una soluzione al 5% HF (Attenzione: HF è un materiale estremamente pericoloso) per rimuovere lo strato di ossido nativo. Dopo 5 minuti a secco senza l&#39;ossido di silicio con l&#39;azoto </li><li> Per produrre un substrato clorurati, subito immergere ogni pezzo senza ossido di silicio in una fiala di scintillazione contenente 2 ml di satura PCl 5 in clorobenzene. Questa soluzione deve essere filtrata a 0,2 micron. </li><li> Montare un condensatore fiala sopra ogni flacone e metterli in una heatblock set a 112 ° C per un&#39;ora. </li><li> Dopo la reazione è completa, lasciate raffreddare e risciacquare fiale ogni surface con clorobenzene e secco sotto azoto filtrata. </li><li> Per formare un propenil-termina substrato, posto ogni superficie di silicio clorurati in un flaconcino di pressione contenente 4 ml di cloruro di magnesio propenil. Luogo ogni flacone pressione in un heatblock a 130 ° C per 24 ore. </li><li> Prendere ogni fiala di pressione fuori dal heatblock e lasciate raffreddare. </li><li> Sciacquare ogni superficie rapidamente con DCM ed etanolo e secco sotto azoto filtrata. </li></ol> 1B. Monostrato Formazione primaria sul Germanio <ol><li> Taglio wafer germanio nei supporti 1cm2, polvere e risciacquare con acqua ed etanolo filtrata. </li><li> Eliminare la contaminazione organica immergendo le superfici in un piatto di vetro contenente acetone per 20 minuti </li><li> Luogo ogni superficie in una soluzione al 10% di HCl per 15 minuti. Questo processo elimina contemporaneamente lo strato di ossido nativo e clorurati la superficie. Dopo 5 minuti asciugare i substrati con azoto. </li><li> Per formare un substrato ottil-terminated, place ogni clorurati superficie germanio in un flaconcino di pressione contenente 4 ml di cloruro di magnesio ottile (2 mm). Luogo ogni flacone pressione in un heatblock a 130 ° C per 48 ore. </li><li> Prendere ogni fiala di pressione fuori dal heatblock e lasciate raffreddare a temperatura ambiente. </li><li> Sciacquare ogni superficie rapidamente con DCM ed etanolo e secco sotto azoto filtrata. </li></ol> 2. NHS Funzionalizzazione substrato su silicio e germanio <ol><li> Preparare una filtrata 0,1 M NHS-diazirine soluzione in tetracloruro di carbonio. Attenzione: Mantenere l&#39;esposizione alla luce al minimo. </li><li> Pipetta alcune gocce della soluzione sulle superfici metile terminale. Permettere alla soluzione di diffondere su tutta la superficie. </li><li> Posizionare le superfici sotto una lampada UV (☐ = 254 nm, 4400/cm2 a 0,74 pollici). Lasciare le superfici di reagire ai raggi UV per 30 minuti, quindi aggiungere più NHS-diazirine alla superficie e lasciare che la reazione di proseguire per altri 30 minuti. </li><li> Sciacquare la modifica NHS surfaces con DCM ed etanolo e secco sotto azoto filtrata. </li></ol> 3. Molecola Funzionalizzazione piccoli <ol><li> Reagire NHS-modified substrati in una 20 mm ter-butil carbamoil (Boc-) etilendiammina soluzione in diclorometano (DCM) per due ore a temperatura ambiente. </li><li> Dopo la reazione, lavare i Boc-modified substrato con DCM e etanolo. </li><li> Deprotect il substrato Boc modificati utilizzando il 25% acido trifluoroacetico (TFA) in DCM per un&#39;ora a temperatura ambiente. </li><li> Sciacquare la superficie risultante con DCM, etanolo e 10% (w / v) di bicarbonato di potassio in acqua e asciugare sotto azoto filtrata. </li><li> Analizzare tutte le superfici con XPS per determinare la composizione elementare. </li></ol> 4. Acida poliuretano Acrilato Timbro (PUA) Preparazione <ol><li> Diluire acrilato A del 30% con B triacrylate trimetilolpropano etossilato per ridurre la viscosità. Aggiungi fotoiniziatori C e D per la miscela di reazione (Fig.URE 6). </li><li> Aggiungi sodio 2-mercaptoethanesulfonate (0,2 g, 1.22 mmol) ad una soluzione HCl 4N in diossano (10 ml) e mescolare a temperatura ambiente per 2 minuti. </li><li> Filtrare fuori il cloruro di sodio prima attraverso un filtro di vetro fine e poi attraverso una membrana di 0,2 μ m siringa in PTFE filtro per permettere una soluzione limpida di 2-mercaptoethanesulfonic acido diossano. </li><li> Diossano evaporare a pressione ridotta </li><li> Reagire l&#39;acido solfonico risultante con 2 ml di poliuretano acrilato miscela prepolymeric a temperatura ambiente e poi sotto vuoto a 50 ° C. Assicurati di liberare completamente la miscela di bolle d&#39;aria intrappolate. </li><li> Raffreddare la soluzione risultante a temperatura ambiente e polimerizzare tra due lastre di vetro microscopio o un vetrino e un maestro per esposizione a luce UV per 2 ore a temperatura ambiente. </li><li> Dopo la polimerizzazione, con attenzione buccia fuori il timbro del maestro e lavare il timbro con etanolo e l&#39;acqua e asciugare con nitroge filtratan. </li></ol> 5. Stampa catalitica e SEM / Analisi AFM <ol><li> Posizionare il corrispondente poliuretano acrilato timbro sulla parte superiore del NHS-modified substrato a temperatura ambiente per un minuto in assenza di carico esterno per tenerli insieme. </li><li> Dopo la reazione, a parte il timbro e il substrato. </li><li> Sciacquare il substrato con l&#39;etanolo, l&#39;acqua, etanolo e poi asciugare con azoto filtrato. </li><li> Sciacquare il timbro con l&#39;etanolo, l&#39;acqua, etanolo e poi asciugare con azoto filtrato. </li><li> Conservare i francobolli a temperatura ambiente prima della successiva applicazione. </li><li> Analizza la struttura prodotta utilizzando la modalità di contatto laterale microsopy forza atomica (AFM) e microscopia elettronica a scansione (SEM) </li></ol> 6. Proteine ​​Patterning e Microscopia a fluorescenza <ol><li> Immergere il SSN-fantasia substrato bifunzionale in lisina-N, N-diacetic acido (20 mm) e Et 3 N (100 mm) in DMF: H20 (1:1) a temperatura ambiente per 1 ora e poi sciacquato conacqua ed etanolo. </li><li> Incubare i substrati in 50 mM NiSO4 soluzione per 5 minuti a temperatura ambiente. </li><li> Sciacquare i substrati chelati con abbondante acqua e tampone vincolante (20 NaP mm, 250 mm NaCl, 10 mM imidazolo, pH 7,5) e immergere in una soluzione filtrata GFP (~ 40 μ M) per 1 ora a 0 ° C. </li><li> Sciacquare immediatamente i substrati con tampone vincolante seguito da PBS (pH 7,4). </li><li> Mantenere idratata substrati in PBS a 0 ° C fino a quando non erano pronti per l&#39;analisi microscopia a fluorescenza. </li></ol> 7. Proteine ​​Patterning e Microscopia a fluorescenza <ol><li> Immergere il SSN-fantasia substrato bifunzionale in lisina-N, N-diacetic acido (20 mm) e Et 3 N (100 mm) in DMF: H 2 0 (1:1) a temperatura ambiente per 1 ora e poi risciacquate con acqua ed etanolo. </li><li> Incubare i substrati in un 50 mm Niso 4 soluzione per 5 minuti a temperatura ambiente. </li><li> Sciacquare i substrati chelati eccessivamente wacqua-esima e binding buffer (20 mM NaP, 250 mM NaCl, 10 mM imidazolo, pH 7,5) e immergere in una soluzione filtrata GFP (~ 40 mM) per 1 ora a 0 ° C. </li><li> Sciacquare immediatamente i substrati con tampone vincolante seguito da PBS (pH 7,4). </li><li> Mantenere idratata substrati in PBS a 0 ° C fino a quando non erano pronti per l&#39;analisi microscopia a fluorescenza. </li></ol> 8. Rappresentante dei risultati: Un esempio di soft-patterning litografico nano catalitico è mostrata in Figura 7. L&#39;approccio crea pattern chemoselective sulla privo di ossidi di silicio e germanio, che può essere ortogonalmente funzionalizzati con prodotti chimici diversi e frazioni biologiche. La reazione tra il SSN-functioanlized substrato e il timbro catalitico fantasia porta alla idrolisi delle frazioni NHS nelle aree di contatto conformazionale, producendo una fantasia bifunzionale regioni substrato portante del NHS attivato e gli acidi carbossilici. A causa della diffusioni di natura gratuita del nostro metodo, ottenere una risoluzione vicina a quella della fotolitografia. Per esempio, la Figura 7 mostra caratteristiche di 125 nm, che sono stati riprodotti in modo uniforme su tutta la superficie del substrato di silicio. Sorprendentemente, il timbro catalitica può essere riutilizzato più volte senza perdere efficienza. Chemoselective funzionalizzazione di semiconduttori fantasia con biomolecole apre la prospettiva di integrare i tradizionali materiali elettronici con i substrati biologici altamente selettivi per applicazioni nel rilevamento, diagnostica, e le aree di analisi della ricerca. Un esempio di tale funzionalizzazione è mostrato in figura 8, dove NHS-fantasia silicio è stata selettivamente funzionalizzati con molecole proteiche. Sfruttando la reattività differenziale di acidi carbossilici attivati ​​e libero, per prima cosa apposta nitrilotriacetico acido-terminato (NTA) linker heterobifunctional al SSN-funzionalizzati regioni, e poi utilizzato la conseguenteNTA-superficie modellata come un modello per l&#39;attaccamento selettivo di esa-istidina-tagged GFP. Figura 8b mostra chiaramente l&#39;intensità di fluorescenza differenziale tra GFP-modificato e idrolizzato regioni indenni acido carbossilico. Le dimensioni e la forma delle caratteristiche replicato sono coerenti tra le due superfici NHS modellata (Fig. 8a) e GFP-modified superficie (Figura 8b), confermando la notevole stabilità del carbonio passivato superfici e la selettività del metodo di stampaggio. Il protocollo non si limita alla proteina His-tag, e può essere utilizzato per altre biomolecole modello compreso il DNA e gli anticorpi. <img src="/files/ftp_upload/3478/3478fig1.jpg" alt="Figura 1"/> Figura 1. Regime generale in rappresentanza di stampa catalitico microcontact <img src="/files/ftp_upload/3478/3478fig2.jpg" alt="Figura 2"/> Figura 2. Struttura del bi-strato msistema olecular su Ge e Si. Primaria monostrato alchilico forme stabili Ge-C o Si-C legami con il substrato e fornisce un sistema chimicamente inerte e chiudere imballato che protegge la superficie sottostante dal degrado. (B) overlayer secondaria forma legami stabili con primarie CC strato protettivo e fornisce terminali funzionali gruppi <img src="/files/ftp_upload/3478/3478fig3.jpg" alt="Figura 3"/> Figura 3. Schemi di reazione che rappresenta la formazione di monostrati primaria di protezione su Si (A) e Ge (B) <img src="/files/ftp_upload/3478/3478fig4.jpg" alt="Figura 4"/> Figura 4. Funzionalizzazione chimica del monostrato protettivo primario con un donatore carbene heterobifunctional <img src="/files/ftp_upload/3478/3478fig5.jpg" alt="Figura 5"/> Reazione Figura 5. Schema dimostrando modifiche piccola molecola di NHS-funzionalizzati subStrates e gli spettri corrispondenti XPS <img src="/files/ftp_upload/3478/3478fig6.jpg" alt="Figura 6"/> Figura 6. Composizione della catalitico pre-miscela polimerica, le condizioni di polimerizzazione, e le immagini SEM della fantasia solfonico acido-modificato il timbro e il corrispondente in PMMA-Si padrone <img src="/files/ftp_upload/3478/3478fig7.jpg" alt="Figura 7"/> Figura 7. SEM e le immagini attrito AFM di SAM modellata su Si e Ge con un timbro acido <img src="/files/ftp_upload/3478/3478fig8.jpg" alt="Figura 8"/> Figura 8 Soft-patterning litografico e funzionalizzazione del silicio passivato con molecole organiche e biologiche a:.. Immagine SEM della fantasia NHS-modified substrato b:. Micrografia di substrato fluorescente GFP modificate.

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Il protocollo presentato è una forma o di stampa microcontact senza inchiostro che può essere universalmente applicata a qualsiasi substrato in grado di supportare semplici ben ordinata monostrati. In questo metodo, un francobollo-catalizzatore immobilizzato trasferisce un modello per una superficie d&#39;appoggio corrispondente gruppi funzionali. Poiché il processo non si basa sul trasferimento di inchiostro da timbro alla superficie della limitazione diffusiva risoluzione di μCP tradizionale e reattiva è ovviato, permettendo di produzione di routine di oggetti su scala nanometrica. L&#39;incorporazione di un primario altamente ordinato sistema molecolare fornisce una protezione completa del semiconduttore sottostante dai danni dell&#39;ossidazione. Allo stesso tempo, il metodo supporta immobilizzazione di ingombranti gruppi reattivi utilizzando una overlayer secondaria reattiva; insieme il sistema raggiunge sia la protezione e la funzionalizzazione. La tecnica inizia con la formazione di stabili legami carbonio-superficie consentendo chimicamente inerte Primary monostrato che serve come una barriera efficace per la formazione di ossido. Formazione di una overlayer secondaria reattiva fornisce terminale gruppi NHS funzionali che fungono da punti di attacco per una serie di combinazione di sostanze chimiche e biologiche. Questo sistema stabile doppo strato molecolare è successivamente modellato utilizzando il nostro approccio catalitica μCP. L&#39;approccio presentato in questo studio offre un metodo generale per substrati semiconduttori patterning con una vasta gamma di materiali organici e biologici. La possibilità di creare disegni organici semiconduttori interfacce senza costoso, strumentazione complessa offre numerose opportunità in campi quali l&#39;elettronica, le nanotecnologie, la biochimica e la biofisica.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda / modello </th></tr><tr><td> XPS spettrometro </td><td> Kratos Axis Ultra </td></tr><tr><td> Microscopio a forza atomica </td><td> Veeco D3100 </td></tr><tr><td> SEM-FEG microscopio </td><td> FEI XL30 </td></tr><tr><td> Microscopio a fluorescenza </td><td> Zeiss Axio Imager </td></tr><tr><td> Heatblock </td><td> VWR </td></tr><tr><td> Pompa del vuoto </td><td> Boc Edwards </td></tr><tr><td> Acque dell&#39;impianto di depurazione </td><td> Millipore </td></tr><tr><td> TESP silicio sonde </td><td> Veeco </td></tr><tr><td> Silicio </td><td></td></tr><tr><td> Fiale di pressione </td><td> Chemglass </td></tr><tr><td> Pressione di aspirazione </td><td> Chemglass </td></tr><tr><td> Lampada UV </td><td> UVP </td></tr><tr><td> Timbro del materiale </td><td> Vedi riferimenti 20 e 18 </td></tr><tr><td> PFTE siringa filtri </td><td> VWR </td></tr><tr><td> Nano Striscia </td><td> Cyantek </td></tr><tr><td> HCl </td><td> Sigma </td></tr><tr><td> Etanolo </td><td> Sigma </td></tr><tr><td> Acetone </td><td> Sigma </td></tr><tr><td> HF </td><td> Sigma </td></tr><tr><td> Clorobenzene </td><td> Sigma </td></tr><tr><td> PCL5 </td><td> Sigma </td></tr><tr><td> Propenile cloruro di magnesio </td><td> Sigma </td></tr><tr><td> Cloruro di Magnesio ottile </td><td> Sigma </td></tr><tr><td> Tetracloruro di carbonio </td><td> Sigma </td></tr><tr><td> Boc protetto etilendiammina </td><td> Sigma </td></tr><tr><td> TFA </td><td> Sigma </td></tr><tr><td> Sodium 2-mercaptoethanesulfonate </td><td> Sigma </td></tr><tr><td> 4N HCl soluzione in diossano </td><td> Sigma </td></tr><tr><td> Lisina-N, N-diacetic acido </td><td> Sigma </td></tr><tr><td> Et 3 N </td><td> Sigma </td></tr><tr><td> DMF </td><td> Sigma </td></tr><tr><td> NISO 4 </td><td> Sigma </td></tr><tr><td> NaP </td><td> Sigma </td></tr><tr><td> NaCl </td><td> Sigma </td></tr><tr><td> imidazolo </td><td> Sigma </td></tr><tr><td> PBS </td&gt; <td> Sigma </td></tr></tbody></table>

soft lithographic functionalization and patterning oxide-free silicon and germanium

Lo sviluppo di dispositivi elettronici ibridi si basa in gran parte l&#39;integrazione di (bio) e materiali organici semiconduttori inorganici attraverso un interfaccia stabile che permette di trasporto efficienti elettroni e protegge i substrati di fondo dalla degradazione ossidativa. Gruppo di semiconduttori IV può essere efficacemente protetti con altamente ordinato monostrati auto-assemblati (SAM), composto di semplici catene alchiliche che agiscono da barriera impermeabile alle soluzioni sia organici e acquosi. Semplice SAM alchil, tuttavia, sono inerti e non riconducibili alle tecniche tradizionali di patterning. La motivazione per immobilizzare i sistemi organici molecolari su semiconduttori è quello di impartire una nuova funzionalità per la superficie in grado di fornire funzioni ottiche, elettroniche e meccaniche, così come l&#39;attività chimiche e biologiche. Stampa microcontact (CP μ) è un soft-tecnica litografica per SAM patterning su superfici miriade. 1-9 Nonostante la sua semplicity e versatilità, l&#39;approccio è stato in gran parte limitato a superfici di metallo nobile e non è stato ben sviluppato per il trasferimento di substrati modello tecnologicamente importanti, come l&#39;ossido senza silicio e germanio. Inoltre, poiché questa tecnica si basa sulla diffusione di inchiostro per trasferire il pattern da elastomero di substrato, la risoluzione di tali stampa tradizionale si limita essenzialmente a vicino a 1 μ m. 10-16 In contrasto con la stampa tradizionale, senza inchiostro patterning CP μ si basa su una specifica reazione tra una superficie immobilizzato sottofondo e un francobollo legato catalizzatore. Perché la tecnica non si basa su diffusive formazione SAM, si espande in modo significativo la diversità delle superfici patternable. Inoltre, la tecnica senza inchiostro evita le limitazioni delle funzionalità dimensioni imposte dalla diffusione molecolare, facilitando la replica di caratteristiche molto piccole (&lt;200 nm). 17-23 Tuttavia, fino ad ora, senza inchiostro μ CP è stato utilizzato principalmente per patterning sistemi relativamente disordinati molecolare, che non proteggono le superfici sottostanti dal degrado. Qui riportiamo un semplice, affidabile high-throughput metodo per patterning passivato silicio e germanio con reattivo monostrati organici e dimostrare funzionalizzazione selettiva dei substrati fantasia sia con piccole molecole e proteine. La tecnica utilizza una NHS-reattiva preformate sistema doppo strato di ossido senza silicio e germanio. La porzione NHS viene idrolizzato in un modello specifico modo con un acido-modificato timbro sulfonic acrilato di produrre modelli chimicamente distinti di NHS-attivato e gli acidi carbossilici. Un limite significativo alla risoluzione di molte tecniche CP μ è l&#39;uso di materiale PDMS che manca la rigidità meccanica necessaria per il trasferimento ad alta fedeltà. Per ovviare a questa limitazione abbiamo utilizzato un poliuretano acrilato polimeri, un materiale relativamente rigido che può esserefacilmente funzionalizzati con differenti frazioni organiche. Il nostro approccio patterning protegge completamente sia silicio e germanio da ossidazione chimica, fornisce un controllo preciso sulla forma e le dimensioni delle caratteristiche fantasia, e dà accesso immediato ai modelli chimicamente discriminati che può essere ulteriormente funzionalizzati con molecole sia organici e biologici. L&#39;approccio è generale ed applicabile ad altre superfici tecnologicamente rilevanti.

Watch this Scientific Journal Video about Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium at JoVE.com

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Qui si descrive un metodo semplice per patterning senza ossido di silicio e germanio funzionalizzazione con reattivo monostrati organici e dimostrare dei substrati fantasia con piccole molecole e proteine. L&#39;approccio protegge completamente le superfici da ossidazione chimica, fornisce un controllo preciso sulla morfologia caratteristica, e consente un accesso immediato ai modelli chimicamente discriminati.

Morbido Funzionalizzazione litografica e Patterning ossido senza silicio e germanio

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

soft-lithographic-functionalization-patterning-oxide-free-silicon

Research

JoVE Journal

Bioengineering

14.6K Views.  Duke University. Qui si descrive un metodo semplice per patterning senza ossido di silicio e germanio funzionalizzazione con reattivo monostrati organici e dimostrare dei substrati fantasia con piccole molecole e proteine. L'approccio protegge completamente le superfici da ossidazione chimica, fornisce un controllo preciso sulla morfologia caratteristica, e consente un accesso immediato ai modelli chimicamente discriminati.

Video: Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plasma Surface Patterning litografia per la creazione di reti di cella

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Ricerca

Bioingegneria

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

High-throughput sintesi dei carboidrati e funzionalizzazione di nanoparticelle polianidride

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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.

Un metodo versatile di patterning proteine ​​e cellule

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

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Procedura litografica morbido per la produzione di dispositivi microfluidici plastica con vista-porte trasparente alla luce visibile e infrarossa

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Progettazione di film di silicio poroso come elementi portanti del fattore di crescita del nervo

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

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.

Robot morbido bioispirato con microelettrodi incorporati

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

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Nanofili di silicio e stimolazione ottica per indagini sull'accoppiamento elettrico intra e intercellulare

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Modellazione semplice, economica e modulare delle cellule utilizzando il DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...