We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

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The authors have nothing to disclose.

Salvi הוא ממשק microfluidic המאפשר פני הנוזל דינמי וניתוח ממשק נוזל-מוצק על ידי מכשירים מבוססי ואקום, כגון TOF-SIMS במיקרוסקופ אלקטרונים סורק (SEM). בשל השימוש של פתחים קטנים לחשוף נוזל ישירות בריק, Salvi מתאים טכניקות ספקטרוסקופיה ודימות רבות ממוקדות דק ללא כל שינוי; 22 את הטלטל...

הידרציה חיוני למבנה, 1 קונפורמציה, 2 והפעילות הביולוגית 3 של חלבונים. חלבונים ללא מולקולות מים המקיף אותם לא יצטרכו פעילויות ביולוגיות קיימא. באופן ספציפי, מולקולות מי אינטראקציה עם פני שטח ואת המבנה הפנימי של חלבונים, ומדינות הידרציה שונות של חלבונים לעשות אינטראקציות כאלה ברורים. 4 האינטראקציה של חלבונים עם משטחים מוצקים היא תופעה בסיסית עם השלכות בננוטכנולוגיה, בביו-חומרים ותהליכי הנדסת רקמות. מחקרים ארוכים הראו כי שינויים מרחביים עלולים להתרחש כחלבון מפגשי משטח. TOF-SIMS כבר שנחזה הטכניקה שיש לו פוטנציאל ללמוד ממשק החלבון-מוצק. 5-7 חשוב להבין את הידרציה של חלבונים על משטחים מוצקים, אשר באופן פוטנציאלי מספק הבנה בסיסית של המנגנון של מבנה שלהם, קונפורמציה, ו ביולוגיפעילות אל. עם זאת, שיטות אנליטיות פני השטח העיקריים הם בעיקר מבוססי ואקום יישומים ישיר ללימודי נוזל נדיף קשה עקב אידוי מהיר של נוזל נדיף תחת סביבת ואקום. פיתחנו ואקום תואם microfluidic ממשק, מערכת לניתוח בבית ממשק אבק נוזלי (Salvi), כדי לאפשר תצפיות ישירות של משטחים נוזלי אינטראקציות מוצק נוזלי באמצעות ספקטרומטריית מסה זמן של הטיסה משני יון (TOF-SIMS). 8- 11 ההיבטים הייחודיים כוללים את הפעולות הבאות: 1) בחלון הזיהוי צמצם של 2-3 מיקרומטר קוטר המאפשר הדמיה ישירה של פני הנוזל, 2) מתח הפנים משמש להחזיק את הנוזל בתוך הצמצם, ו -3) Salvi הוא נייד בקרב פלטפורמות מרובות אנליטי. 11,12 Salvi מורכב סיליקון ניטריד (חטא) קרום כאזור זיהוי וכן microchannel עשויים polydimethylsiloxane (PDMS). זה fabricated בחדר הנקי, וגורמי הייצור ועיצוב מפתח כבר מפורט ניירות ופטנטים קודמים. 8-12 היישומים של TOF-SIMS ככלי אנליטי הודגמו באמצעות מגוון של תמיסות מימיות ותערובות נוזל מורכבות, חלק שהכיל חלקיקים. 13-17 באופן ספציפי, נוזל Salvi TOF-SIMS מאפשר דינמי חיטוט של הממשק הנוזל-מוצקה של מערכות ביולוגיות חיות (כלומר, biofilms), תאים בודדים, וממשק מוצקים אלקטרוליטים, פתיחת הזדמנויות חדשות עבור בשלב באתרו מרוכז מחקרים כוללים נוזלים באמצעות TOF-SIMS. עם זאת, העיצוב הנוכחי אינו מאפשר אינטראקציות נוזלי גז עדיין. זהו כיוון לפיתוח עתידי. Salvi נעשה שימוש כדי ללמוד את סרט חלבון hydrated בעבודה זו בפעם הראשונה. פיברונקטין הוא דימר חלבון נפוץ, מורכב משני מונומרים כמעט זהה מקושרים על ידי זוג אג&quot;ח דיסולפיד, 18 אשר אניS-ידוע ביכולתה לאגד תאים. 19,20 נבחר כמערכת מודל כדי להמחיש כי סרט חלבון hydrated יכול להיות נחקר באופן דינמי באמצעות נוזל Salvi TOF-SIMS גישה. פתרון החלבון הוכנס microchannel. לאחר דוגר במשך 12 שעות, סרט חלבון hydrated עטף את הצד האחורי של הממברנה SiN. מים ללא יונים (DI) שימש כדי לשטוף לערוץ לאחר ההקדמה חלבון. מידע נאסף מולקולות חלבון פיברונקטין hydrated ב microchannel Salvi באמצעות TOF-SIMS דינמי. מים די נחקרו גם כביקורת להשוות עם תוצאות שהתקבלו סרט דק hydrated פיברונקטין. הבדלים ברורים נצפו בין סרט חלבון hydrated ומי DI. עבודה זו ממחישה כי חלבון ספיחה על פני שטח בסביבת הנוזלי ניתן ללמוד באמצעות Salvi רומן גישת TOF-SIMS נוזלי. פרוטוקול סרטון נועד לספק הדרכה טכנית עבור אנשים המעונייניםניצול כלי אנליטי חדש ליישומים מגוונים של Salvi עם TOF-SIMS ולהפחית טעויות מיותרות בטיפול נוזלי, כמו גם רכישת וניתוח נתונים TOF-SIMS.

<table border="0" cellpadding="0" cellspacing="0" width="1170">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">ToF-SIMS</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">TOF.SIMS 5</td>
			<td style="width:400px;">Resolution: &#62; 10,000 m/&#916;m for mass resolution; &#62; 4,000 m/&#916;m for high spatial resolution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">System for Analysis at the Liquid Vacuum Interface (SALVI)</td>
			<td style="width:260px;">Pacific Northwest National Laboratory</td>
			<td style="width:121px;">N/A</td>
			<td style="width:400px;">SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">PEEK Union</td>
			<td style="width:260px;">Valco</td>
			<td style="width:121px;">ZU1TPK</td>
			<td>for connecting the inlet and outlet of SALVI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">5 Axes Sample Stage</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">N/A</td>
			<td>Stage is self-made for mounting SALVI in ToF-SIMS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Barnstead Nanopure Water Purification System</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">D11921</td>
			<td>ROpure LP Reverse Osmosis filtration module (D2716)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:121px;">309659</td>
			<td style="width:400px;">1 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">21-377-821</td>
			<td>Range: 100 to 1000 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette Tip</td>
			<td style="width:260px;">Neptune</td>
			<td style="width:121px;">2112.96.BS</td>
			<td>1000 &#181;L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Centrifuge Tube</td>
			<td style="width:260px;">Corning</td>
			<td style="width:121px;">430791</td>
			<td>15 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Fibronectin</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:121px;">F1141</td>
			<td>1 mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Ethanol</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">S25310A</td>
			<td>95% Denatured</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Gibco PBS</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">10010-023</td>
			<td>pH 7.4</td>
		</tr>
	</tbody>
</table>

in situ characterization of hydrated proteins in water by salvi and tof-sims

עבודה זו ממחישה באפיון באתרו של ביומולקולות חלבון בתמיסה המימית באמצעות מערכת לניתוח בבית ממשק האבק הנוזלי (Salvi) וזמן של הטיסה ספקטרומטריית משני יון המוני (TOF-SIMS). סרט חלבון פיברונקטין מאובן על סיליקון ניטריד (חטא) קרום הנוצר באזור זיהוי Salvi. במהלך ניתוח TOF-SIMS, שלושה מצבים של ניתוח נערכו כולל ספקטרומטריית מסה ברזולוציה מרחבית גבוהה, דו מימדי (2D) הדמיה, ו פרופיל עומק. ספקטרה המונית נרכשה בשני מצבים חיוביים ושליליים. מים ללא יונים נותחו גם כמדגם התייחסות. התוצאות שלנו מראה כי סרט פיברונקטין במים יש יותר פסגות אשכול מים ברורות וחזקות נמשלו למים לבד. פסגות מאפיין של שברי חומצת אמינו ניתנים לצפייה גם בחלבון hydrated ספקטרה TOF-SIMS. תוצאות אלו ממחישות כי ספיחת מולקולת חלבון על משטח ניתן ללמוד דינמיקהlly באמצעות Salvi ו TOF-SIMS בסביבת הנוזל בפעם הראשונה.

כמה תוצאות נציג מוצגים כדי להדגים את היתרונות של הפרוטוקול המוצע. באמצעות ממשק microfluidic Salvi, אלומת יונים העיקרי (Bi 3 +) יכול להפציץ ישירות על הסרט פיברונקטין hydrated במים DI. לפיכך המיפוי הכימי המולקולרי של פני הנוזל ניתן לרכוש בהצלחה. <p class="jove_...

Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

עבודה זו מציג פרוטוקול טיפול נוזל וההכנסה מדגם על microchannel עבור באתרו זמן של טיסת ניתוח ספקטרומטריית משני יון המוני של ביומולקולות חלבון בתמיסה מימית.

באפיון באתרו של חלבונים מימה במים על ידי Salvi ו TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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JoVE Journal

Chemistry

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

8.2K Views.  Pacific Northwest National Laboratory.  עבודה זו מציג פרוטוקול טיפול נוזל וההכנסה מדגם על microchannel עבור באתרו זמן של טיסת ניתוח ספקטרומטריית משני יון המוני של ביומולקולות חלבון בתמיסה מימית. 

Video: In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

שינויי קונפורמציה Submillisecond בחלבונים נפתר על ידי photothermal Beam הסטה

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

בSIMS אתרו וIR ספקטרוסקופיה של משטחים מוגדרים היטב הוכן על ידי רכה נחיתה של יונים המוניים שנבחרו

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

המים אמולסיות שמן: מערכת חדשה להרכבת חלבונים מסיסים במים כלורופיל מחייב עם פיגמנטים הידרופובי

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

הערכה של בורון מסוממים יהלומי אלקטרודה איכות ובקשה ל<em&gt; באתר</em&gt; שינוי של pH המקומי על ידי אלקטרוליזה המים

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiolabeling וכימות של רמות סלולריות של Phosphoinositides ידי נצנץ זרימה מצמיד כרומטוגרפיה נוזלית ביצועים גבוהים

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

בחיי עיר אפיון Biofilms MR1 Shewanella oneidensis על ידי SALVI וסימס תוף

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

בחיי עיר איפיון חלקיקים Boehmite במים באמצעות נוזלי SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

חלקיקים מגנטיים-DNA מחייב ניתוח לפי פיזור אור דינאמי, Electrophoretic

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

שיפור האות Nanoprobe מהירה על ידי באתרו בתוך Nanoparticle זהב סינתזה

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Photoelectron הדמיה של אניונים מאויר על ידי ניתוק 310 ננומטר F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

באפיון באתרו של חלבונים מימה במים על ידי Salvi ו TOF-SIMS

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