Gli autori riconoscono (1) sostegno finanziario del Dipartimento di Scienza e Nanobiomedical BK21 PLUS NBM Globale Centro di Ricerca di Medicina Rigenerativa in Dankook University e dal Priority Centri di Ricerca del programma (n ° 2009-0093829) finanziato dalla NRF, Repubblica di Corea, ( 2) le strutture e l&#39;assistenza scientifica e tecnica, del Centro australiano per la microscopia e microanalisi presso l&#39;Università di Sydney. HKC è grato al Australian Research Council per i supporti finanziari attraverso una sovvenzione Discovery progetto (DP0985367 e DP120102778). WCH è grato al Australian Research Council per i supporti finanziari attraverso una borsa di linkage progetto (LP120200489, LP110200316).

1. Preparazione della sonda colloidale e AFM substrato <ol><li> Per preparare sonde colloidali, utilizzare un metodo sviluppato precedentemente dagli autori. 19 <ol><li> In breve, utilizzare un supporto angolo di 45 ° di apporre un cantilever tipless allo specifico angolo di 45 ° (Figura 1A). </li><li> Preparare un vetrino epossidica da spalmare uno strato sottile di resina epossidica su un vetrino da microscopio. Utilizzare una spatola pulita o un lento flusso di azoto per assicurare che lo strato di resina epossidica aggiunto al vetrino è di altezza minima. </li><li> Fissare il vetrino resina epossidica ad un ottico di lente del microscopio zoom 40X con un supporto progettato su misura (Figura 1B). Quindi utilizzare il cantilever avvicinarsi al vetrino epossidica e acquisire una piccola quantità di resina epossidica sulla cantilever. </li><li> Ripetere questi passaggi per allegare anche una singola particella di interesse all&#39;apice del cantilever (Figura 1C). </li></ol></li><li> Preparare il substrato AFM apponendo par colloidaleticelle su un vetrino AFM utilizza un adesivo di montaggio termoplastico. <ol><li> Scaldare una copertura antiscivolo rotondo 35 mm a 120 ° C, e applicare una piccola quantità di adesivo per il coprioggetto. L&#39;alta temperatura è necessaria per fondere l&#39;adesivo termoplastico per applicazione. </li><li> Poi raffreddare, il coprioggetto a 40 ° C prima di spolverare le particelle colloidali sulla colla. NOTA: A 40 ° C la colla è sufficientemente impostato che le particelle non si sono inseriti nella colla, ma la colla è abbastanza appiccicosa per assicurare che le particelle aderiscono al substrato. </li><li> Ulteriori raffreddare il vetrino di RT e utilizzare una leggera corrente di azoto per soffiare via eventuali particelle libere in eccesso. </li><li> Lavare le substrato più volte con il mezzo liquido che verrà utilizzato per misurazioni sonda colloidali per garantire che tutti particelle libere vengono rimossi dal substrato. NOTA: Questo è importante per ridurre gli effetti di particelle a flusso libero nel corso della misura, che può tra l&#39;agire con la mensola e introdurre errori nei risultati. </li></ol></li></ol> 2. Montaggio della sonda colloidale, Allineamento Laser, e riequilibranti del sistema <ol><li> Montare il vetrino coprioggetto con le particelle colloidali nella metà inferiore di una cella di liquido, facendo in modo che l&#39;O-ring sia posizionato correttamente per evitare perdite. </li><li> Posizionare un foglio trasparente idrofobo sul palco microscopio per premunirsi contro qualsiasi liquido che può fuoriuscire durante l&#39;esperimento, soprattutto se solo con la metà inferiore di una cella liquido per la misura, e posizionare la cella liquido sul palco microscopio. NOTA: per semplicità si può utilizzare solo la metà inferiore di una cella liquido, dato che il sistema può essere equilibrato adeguatamente; punta - l&#39;evaporazione cambia lo stato della misurazione e impatti / lettura dei risultati. </li><li> Fissare la sonda colloidale alla testa di scansione AFM e assemblare sul AFM. Con il software dello strumento AFM, utilizzare le manopole sulscansione testa per portare la punta a sbalzo a fuoco. NOTA: Tutte le fasi procedurali e le misure sono state completate utilizzando un MFP-3D-Bio AFM con il software Asylum Research. </li><li> Per massimizzare l&#39;intensità, allineare il laser sulla punta del cantilever usando le manopole di regolazione appropriati sulla testa di scansione. </li><li> Lasciare che il sistema si riequilibri per 5-10 minuti o fino a quando il valore di deflessione stabilizza. Utilizzare la manopola di regolazione deflessione per portare la deformazione a zero o leggermente negativa. </li><li> Dopo che il sistema è equilibrata in aria, utilizzare il software AFM (Pannello termica nella finestra Master Panel) per calcolare le termicamente InvOLS (sensibilità) e la costante elastica della sonda colloidale. NOTA: Questa sensibilità sarà utilizzato temporaneamente fino alla vera sensibilità è misurata al termine della misura (vedi punto 4). <ol><li> Selezionare &quot;Cal Costante molla&quot; o &quot;Cal InvOLS&quot; e quindi fare clic su &quot;Capture dati termici&quot;. </li><li> Una voltaun picco significativo è evidente, interrompere l&#39;acquisizione dei dati e fare clic per ingrandire il picco principale. </li><li> Clicca su &quot;Inizializza Fit&quot; seguito da &quot;Fit dati termici,&quot; per avere la costante elastica calcolati automaticamente o valori InvOLS. </li></ol></li><li> Aggiungere lentamente 2 ml di terreno liquido alla cella liquido con una siringa e accertarsi che non siano presenti bolle intorno cantilever. Riallineare il laser, poiché l&#39;indice di rifrazione del mezzo è cambiata, e ancora una volta equilibrare il sistema che consente il valore della freccia di stabilizzarsi prima di regolare la deformazione a zero. NOTA: Se una grande differenza di temperatura esistente tra l&#39;ambiente e il liquido, in equilibrio richiederà più tempo. </li></ol> 3. Imaging and Data Acquisition <ol><li> Impostare il formato di scansione iniziale a 20 micron, velocità di scansione a 1 Hz, angolo di scansione di 90 °, set point a 0,2 V e di ottenere una scansione del campione. Regolare il guadagno come necessario per ottenere la traccia sovrappostae ripercorrere le curve. </li><li> Una volta trovata una particella di interesse, zoom subito su quella particella di limitare le interazioni sonda estesi con il substrato prima di ottenere misurazioni del volume forza. </li><li> Una volta ingrandita, acquisire un&#39;immagine sufficiente di una singola particella o porzione di una singola particella. Poi passare al Pannello di forza nel software. Portare la barra di posizione rossa nella posizione più alta, impostare la distanza di forza per 5 micron, velocità di scansione di 0,1 Hz, il canale di trigger a nessuno e condurre una singola misura della forza. Assicurarsi che la sonda non contatta il substrato. </li><li> Dal grafico singola misura ottenuta, calcolare la linea di deflessione virtuale facendo clic destro sulla finestra del grafico, e selezionando l&#39;opzione &quot;Calcola virtuale Def Line&quot;. Questo calcolerà automaticamente la deformazione virtuale e aggiornare il valore come necessario all&#39;interno del software. </li><li> Cambiare il canale di trigger a flessioni e impostare il punto di trigger a 20 nm. Impostare il dist forzazione a 1 micron e regolare la velocità di scansione come desiderato in base alle forze misurate di interesse. </li><li> Regolare manualmente il valore per la deflessione Inverse Optical Leva Sensibilità (InvOLS) nel pannello di rassegna Forza dopo aver condotto 2-3 consecutivi preliminari misure di forza unico. <ol><li> Condurre una misura della forza sola, quindi fare clic sul pulsante &quot;Review&quot; sul pannello di Forza che apre un Master Panel Forza. </li><li> Evidenziare la misurazione della forza più recentemente completato. Sotto l&#39;intestazione &quot;Asse&quot; garantire che solo &quot;DeflV&quot; sia selezionata. Modificare il campo di immissione &quot;X-Axis&quot; a &quot;Settembre&quot; utilizzando il menu a tendina e cliccare su &quot;make grafico.&quot; </li><li> Fare clic sulla scheda &quot;Parm&quot; nel Pannello di Forza Maestri e regolare il valore di &quot;InvOLS&quot; fino alla regione di contatto del grafico è completamente verticale. Poi popolare questo valore nel campo &quot;DEFL InvOLS&quot; che si trova sotto il Cal60; sub-tab nella scheda Forza situato sulla finestra Pannello Maestro principale. </li><li> Ripetere questo 2-3 volte per assicurare che il valore InvOLS non cambia significativamente. </li></ol></li><li> Ora che tutti i parametri sono stati istituiti, assicurarsi che il livello del liquido è ancora sufficiente e che la flessione è ancora stabile. NOTA: In questo momento, forza unica curve o le mappe di forza possono essere ottenuti. Se si desiderano misure di forza di sosta, le opzioni di sosta è possibile accedere nel pannello di Forza. </li></ol> 4. Post-tuning di sensibilità per l&#39;analisi <ol><li> Dopo il completamento dell&#39;acquisizione misurazione, misurare la vera sensibilità della sonda colloidale. A tale scopo, effettuare una misurazione della forza utilizzando una relativamente grande deflessione / forza con la sonda colloidale nello stesso mezzo liquido contro un &quot;infinitamente&quot; superficie dura come mica. NOTA: La sensibilità è stata ottenuta dopo il completamento degli esperimenti, perché il grande deflessione / force potrebbe danneggiare collosonde Idal preparati con colloidi porose o fragili. </li><li> La pendenza della regione di contatto è utilizzato dal software per calcolare automaticamente la sensibilità (Figura 2). Utilizzare questo vero valore di sensibilità durante l&#39;analisi dei dati di tutte le curve ottenute utilizzando quel particolare sonda colloidale. </li></ol>

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The authors declare that they have no competing financial interests.

Diverse fonti di instabilità presenti durante liquido sonda nanoscopia colloidale sistema può essere facilmente mitigati attraverso procedure di equilibrazione corrette. Instabilità come discusso in precedenza producono risultati errati e le curve di forza che sono più difficili da analizzare obiettivamente. Se tutte le fonti di instabilità sono stati teso e grafici simili a quello mostrato in Figura 4 sono ancora presenti, un altro parametro di misura può essere la ragione. Altri parametri di mis...

Microscopia a forza atomica (AFM) è una tecnica che permette l&#39;imaging qualitativa e quantitativa e preciso di una superficie del materiale. 4-6 Tradizionalmente, AFM è utilizzato per la valutazione della topografia della superficie, morfologia e struttura dei materiali multi-fasiche. AFM ha la capacità di valutare quantitativamente le interazioni nano-scala, come carica, attrazione, repulsione e di adesione forze tra una sonda specifica e del substrato in aria e mezzi liquidi. 7,8 L&#39;AFM originariamente sviluppato da Binning, Quate e Gerber 9 usi una sonda di nota sensibilità / determinato e costante elastica di avvicinarsi e / o la scansione di un campione. A causa delle interazioni fisiche tra la sonda e il campione, il cantilever viene deflesso durante il contatto o la vicinanza e seconda della modalità di funzionamento, tali deviazioni può essere tradotto per acquisire la topografia delle forze presenti tra la sonda e il campione o campione di misura. Modifiche al tecnico AFMque, come sonda colloidale nanoscopia, 10 hanno permesso scienziato per valutare direttamente le interazioni nano-forza tra due materiali presenti in un sistema colloidale di interesse. In colloidale sonda nanoscopia, una particella sferica di scelta è fissato al vertice di un cantilever, in sostituzione delle punte coniche e piramidali tradizionali. Una particella sferica è l&#39;ideale per permettere il confronto con i modelli teorici, come la Johnson, Kendal, Roberts (JKR) 11 e Derjaguin, Landau, Vervwey, Overbeek (DLVO) 12-14 teorie e ridurre al minimo l&#39;influenza della rugosità superficiale sulla misura. 15 Queste teorie vengono utilizzati per definire i meccanismi di contatto e le forze fra le particelle previsti all&#39;interno di un sistema colloidale. La teoria DLVO combina le attraente forze di van der Waal e forze elettrostatiche repulsive (a causa di doppi strati elettrici) per spiegare quantitativamente il comportamento di aggregazione dei sistemi colloidali acquose, mentre la JTeoria KR incorpora l&#39;effetto della pressione di contatto e l&#39;adesione al modello di contatto elastico tra due componenti. Una volta che una sonda appropriata viene prodotto, viene usato per rivolgersi a qualsiasi altro materiale / particella per valutare le forze tra le due componenti. Utilizzando uno standard fabbricato punta sarà in grado di misurare le forze di interazione tra punta e che un materiale di scelta, ma il vantaggio di utilizzare una sonda colloidale su misura permette la misurazione delle forze presenti tra materiali presenti all&#39;interno del sistema studiato. Interazioni misurabili includono:.. Adesivo, attraente, repellenti, carica, e anche forze elettrostatiche presenti tra le particelle 16 Inoltre, la tecnica sonda colloidale può essere utilizzato per esplorare forze tangenziali presenti tra le particelle e l&#39;elasticità del materiale 17,18 La capacità di effettuare misurazioni in vari media è uno dei principali vantaggi di sonda colloidale nanoscopia. Condizioni ambientali, liquido media, o condizioni di umidità controllata possono essere utilizzati per simulare condizioni ambientali del sistema studiato. La capacità di effettuare misurazioni in un ambiente liquido consente lo studio di sistemi colloidali in un ambiente che si trova in natura; pertanto, essere in grado di acquisire dati quantitativamente che è direttamente traducibile al sistema nel suo stato naturale. Ad esempio, le interazioni delle particelle presenti all&#39;interno inalatori (MDI) possono essere studiate usando un modello di propellente liquido con proprietà simili al propellente utilizzato in MDI. Le stesse interazioni misurati in aria, non siano rappresentativi del sistema esistente all&#39;interno dell&#39;inalatore. Inoltre, il mezzo liquido può essere modificato per valutare l&#39;effetto di infiltrazioni di umidità, un tensioattivo secondario, o temperatura sulle interazioni delle particelle in un MDI. La capacità di controllare la temperatura può essere utilizzato per simulare alcuni passi nella produzione di sistemi colloidali di valutare come temperatura sia nella produzione di omemorizzazione dei sistemi colloidali può avere un impatto sulle interazioni delle particelle. Misurazioni che possono essere ottenuti utilizzando sonde colloidali includono; Scansione Topografia, singole curve forza-distanza, le mappe di adesione forza-distanza, e dimorare misure di forza-distanza. I parametri fondamentali che vengono misurati con il metodo della sonda nanoscopia colloidale presentata in questo documento includono i valori di energia separazione snap-in, carico massimo, e. Snap-in è una misura delle forze di attrazione, max caricare il valore della forza massima adesione, e l&#39;energia di separazione trasmette l&#39;energia necessaria per ritirare la particella da contatto. Questi valori possono essere misurati mediante misure istantanee o forza di sosta. Due diversi tipi di misurazioni dwell includono deflessione e rientro. La lunghezza e il tipo di misura di permanenza può essere specificamente scelti per imitare interazioni specifiche che sono presenti all&#39;interno di un sistema di interesse. Un esempio sta usando deflessione di sosta - che detienei campioni in contatto ad un valore desiderato deflessione - valutare i legami adesivi che si sviluppano in aggregati formati in dispersioni. I legami adesivo formato può essere misurata come una funzione del tempo e possono fornire una conoscenza delle forze necessarie amalgamare aggregati dopo stoccaggio prolungato. La pletora di dati che possono essere ottenuti con questo metodo è un testamento alla versatilità del metodo.

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Double-Bubble Epoxy</td>
			<td style="width:179px;">Hardman</td>
			<td style="width:119px;">4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Veeco Tipless Probes</td>
			<td style="width:179px;">Veeco</td>
			<td style="width:119px;">NP-O10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Porous Particles</td>
			<td style="width:179px;">Pearl Therapeutics</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Atomic Force Microscope (MFP)</td>
			<td style="width:179px;">Asylum&#160;</td>
			<td style="width:119px;">MFP-3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SPIP Scanning Probe Image Processor Software</td>
			<td style="width:179px;">NanoScience&#160; Instruments</td>
			<td style="width:119px;">N/A</td>
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			<td height="21" style="height:21px;width:319px;">35 mm Coverslips</td>
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quantitative and qualitative examination of particle-particle interactions using colloidal probe nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Sistemi colloidali liquidi sono utilizzati per diversi sistemi di drug delivery farmaceutiche. Per la somministrazione di farmaci per inalazione, un sistema colloidale comune è la sospensione pressurizzato (pMDI). Interazioni tra le particelle presenti nel pMDI svolgono un ruolo fondamentale nella formulazione stabilità fisica, lo stoccaggio e l&#39;uniformità consegna della droga. In questo manoscritto, forze fra le particelle tra particelle a base di lipidi porosi (~ 2 micron ottica diametro particellare medio) in ...

Watch this Scientific Journal Video about Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy at JoVE.com

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Esame quantitativa e qualitativa delle interazioni particella-particella colloidale Uso Probe Nanoscopia

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

quantitative-qualitative-examination-particle-particle-interactions

Research

JoVE Journal

Chemistry

10.9K Views.  University of Sydney. Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems. 

Video: Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Identificazione qualitativa di acidi carbossilici, acidi bo e ammine Utilizzando Fluorofori Cruciform

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

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Chimica

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

La proteomica quantitativa, utilizzando riduttiva dimethylation per Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

Sintesi e purificazione di Iodoaziridines Coinvolgere quantitativa Selezione del Ottimale fase stazionaria per cromatografia

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Sintesi ed esfoliazione della discotici zirconio Fosfati di ottenere cristalli liquidi colloidale

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

Un quantitativo Glycomics e proteomica combinata strategia Purificazione

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Norme per quantitativa Metalloproteomic analisi utilizzando Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

Intuizioni le interazioni di aminoacidi e peptidi con materiali inorganici Uso singola molecola Forza Spettroscopia

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Faccetta--sfaccettatura che collega di cadmio colloidale forma-anisotropo Chalcogenide nanostrutture

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Photodeposition of Pd onto Colloidal Au Nanorods by Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot electrons upon SPR irradiation drive reductive deposition of Pd on colloidal AuNR in the presence of [PdCl4]2-. Plasmon-driven reduction of secondary metals potentiates covalent, sub-wavelength deposition at targeted locations coinciding with electric field &#8220;hot-spots&#8221; of the plasmonic substrate using an external field (e.g., laser). The process described herein details a solution-phase deposition of a catalytically-active noble metal (Pd) from a transition metal halide salt (H2PdCl4) onto aqueously-suspended, anisotropic plasmonic structures (AuNR). The solution-phase process is amenable to making other bimetallic architectures. Transmission UV-vis monitoring of the photochemical reaction, coupled with ex situ XPS and statistical TEM analysis, provide immediate experimental feedback to evaluate properties of the bimetallic structures as they evolve during the photocatalytic reaction. Resonant plasmon irradiation of AuNR in the presence of [PdCl4]2- creates a thin, covalently-bound Pd0 shell without any significant dampening effect on its plasmonic behavior in this representative experiment/batch. Overall, plasmonic photodeposition offers an alternative route for high-volume, economical synthesis of optoelectronic materials with sub-5 nm features (e.g., heterometallic photocatalysts or optoelectronic interconnects).

A protocol for anisotropic photodeposition of Pd onto aqueously-suspended Au nanorods via localized surface plasmon excitation is presented.

Fotodeposizione di Pd su Colloidal Au Nanorods di Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot ...

Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation

In this work, we demonstrate a facile method for colloidal lead halide perovskite nanoplatelet synthesis (Chemical formula: L2[ABX3]n-1BX4, L: butylammonium and octylammonium, A: methylammonium or formamidinium, B: lead, X: bromide and iodide, n: number of [BX6]4- octahedral layers in the direction of nanoplatelet thickness) via ligand-assisted reprecipitation. Individual perovskite precursor solutions are prepared by dissolving each nanoplatelet constituent salt in N,N-dimethylformamide (DMF), which is a polar organic solvent, and then mixing in specific ratios for targeted nanoplatelet thickness and composition. Once the mixed precursor solution is dropped into nonpolar toluene, the abrupt change in the solubility induces the instantaneous crystallization of nanoplatelets with surface-bound alkylammonium halide ligands providing colloidal stability. Photoluminescence and absorption spectra reveal emissive and strongly quantum-confined features. X-ray diffraction and transmission electron microscopy confirm the two-dimensional structure of the nanoplatelets. Furthermore, we demonstrate that the band gap of perovskite nanoplatelets can be continuously tuned in the visible range by varying the stoichiometry of the halide ion(s). Lastly, we demonstrate the flexibility of the ligand-assisted reprecipitation method by introducing multiple species as surface-capping ligands. This methodology represents a simple procedure for preparing dispersions of emissive 2D colloidal semiconductors.

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.