עבודה זו נתמכה על ידי TU Delft, ארגון הולנד למחקר מדעי (NWO), הקרן למחקר בסיסי בעניין, ועל ידי קרן המדע האירופית.

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torque+measurement+at+the+single-molecule+level.">Torque measurement at the single-molecule level.</a> Annu Rev Biophys. 42, 583-604 (2013).</li></ol>

פטנט הקשור לעבודה זו כבר הגיש תחת PCT/NL2011/050446 התייחסות.

בשנים האחרונות, טכניקות מולקולה בודדת הוכיחו התחולה רחבה שלהם במחקר של חלבוני מנוע התהליכית ואנזימים אחרים, מניב תובנה קינטיקה שלהם וmechanochemistry הבסיסי. בהקשר של כוח ספקטרוסקופיה, תרומות חשובות שנעשו על ידי זרימת מיקרוסקופ כוח אטומי מתיחה, ופינצטה אופטית והמגנטיות. פינצטה אופטית והמגנטי (MT) הצליחה בעיקר בשילוב של גמישות רבה במונחים של מניפולציה מולקולרית גבוה במרחב וברזולוציה של זמן. כאן, אנו מתמקדים בMT, אשר יכול להחיל את שני הכוחות והמומנטים המשתרעים למולקולות ביולוגיות קשורות בין פני השטח וחרוזים פאראמגנטי 1-3. פינצטה המגנטית (MT, איור 1 א) הן טכניקת מולקולה בודדת מאוד תכליתית, כי נעשתה שימוש כדי לעקוב אחר שני התכונות מכאניות של חומצות גרעין, כמו גם יחסי הגומלין שלהם עם חלבונים. יש לי MT רבים כוחים, ובם פשטות כוללת וחוסנו של היישום הניסיוני, יישום קליל של מומנט, פעולה טבעית וכיול פשוט במצב כוח קבוע 4, הרחבה במקביל מדידות 5, 6, והעדר חימום מדגם וניזקים. בהשוואה למולקולה בודדת גישות אחרות, MT מספק דרך לבצע מדידות כוח תלות בכוחות נמוכים כמו 10 ≈ Fn ויש לי את היכולת לשלוט בצורה ישירה את מידת supercoiling. בעוד MTs יש בעיקר שימש ככלי ניסיוני כדי לחקור תהליכים ביולוגיים מעורבים חומצות גרעין 7, 8, הם מצאו גם יישום במחקרים של התכונות מכאניות של חלבונים 9-13 או תאים 10, 14-17. אזכור שימושי רב זמין המתארים כיצד לבנות ולהפעיל את 4 MT, 18-20. Howevאה, MT הקונבנציונלי לא לעקוב אחר תנועה סיבובית באופן ישיר, ותוך שהם חלים מומנט, הם לא מודדים מומנט באופן ישיר. בנוסף, הם להגביל את הסיבוב החופשי של לקשור חומצות הגרעין. כאן, אנו מציגים שתי שלוחות של פינצטה מגנט. פינצטה הראשונה, המכונה באופן חופשי, במסלול המגנטית (FOMT, איור 1b) 21, מאפשר המדידות של תנודות זווית שיווי משקל ושינויים בטוויסט של מולקולות חומצות גרעין קשורות, ללא הגבלת התנועה הסיבובית סביב הציר לקשור. השני, המכונה פינצטה מומנט המגנטית (MTT, איור 1 ג), שבו יש לו את היכולת ליישם ולמדוד ישירות את שני הכוחות ומומנטים לביומולקולות אחת 22-27. בפרוטוקול הבא, אנו מניחים שהקורא יש באופי שלו / שלה מכשיר MT &quot;קונבנציונלי&quot;. אנו מפנים את הקורא לדיון לאזכור על איך לבנות ולהפעיל MT להגדיר, כמו גם consideמנות שחייבות להילקח בחשבון בבחירה של חרוזים מגנטיים, מגנטים, ואת שגרת מעקב. בנוסף, סעיפי 1 ו -2 של טקסט הפרוטוקול לתאר איך אנחנו בדרך כלל להכין ודגירת דגימת DNA לשימוש בMT, כמו גם מדידות הראשוניות שניתן לבצע על ה-DNA בודד בMT הקונבנציונלי. סעיפים 3 ו -4 לפרוטוקול הטקסט להמחיש עד כמה מכשיר MT ניתן להתאים בקלות ומשמש למדידות FOMT וMTT.

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			<td height="42" style="height:42px;width:252px;">Sandblaster</td>
			<td style="width:123px;">Great Lake Orthodontics</td>
			<td style="width:119px;">190-070 Microetcher II</td>
			<td style="width:209px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Nitrocellulose</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">LC2001</td>
			<td></td>
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			<td height="42" style="height:42px;">Magnetic particle concentrator</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">12002D</td>
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			<td height="42" style="height:42px;width:252px;">Non-magnetic latex beads (0.5 &#956;m radius)</td>
			<td style="width:123px;">Polysciences</td>
			<td style="width:119px;">17010</td>
			<td></td>
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			<td height="42" style="height:42px;width:252px;">Non-magnetic latex beads (1.5 &#956;m radius)</td>
			<td style="width:123px;">Sanbio</td>
			<td style="width:119px;">PV05N/2179&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Antidigoxigenin</td>
			<td style="width:123px;">Roche</td>
			<td style="width:119px;">11 214 667 001</td>
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			<td height="63" style="height:63px;width:252px;">Streptavidin-coated superparamagnetic beads (0.25 &#956;m radius)</td>
			<td style="width:123px;">Ademtech</td>
			<td style="width:119px;">3150</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Streptavidin-coated superparamagnetic beads (0.5 &#956;m radius, &#8220;MyOne&#8221;)</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">650.01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Streptavidin-coated superparamagnetic beads (1.4 &#956;m radius, &#8220;M270&#8221;)</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">653.05</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Biotin-coated latex beads (0.5 &#956;m radius)</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:119px;">F-8768</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Cubic magnets for conventional tweezers</td>
			<td style="width:123px;">Supermagnete</td>
			<td style="width:119px;">W-05-N50-G</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Cylindrical magnet for MTT and FOMT</td>
			<td style="width:123px;">Supermagnete</td>
			<td style="width:119px;">R-06-02-02G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Side magnet for MTT</td>
			<td style="width:123px;">Supermagnete</td>
			<td style="width:119px;">S-04-07-N</td>
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			<td height="42" style="height:42px;width:252px;">Linear stage</td>
			<td style="width:123px;">Physik Instrumente</td>
			<td style="width:119px;">M-126.PD</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Rotary stage</td>
			<td style="width:123px;">Physik Instrumente</td>
			<td style="width:119px;">C-150</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">High-resolution automated sample stage</td>
			<td style="width:123px;">Physik Instrumente</td>
			<td style="width:119px;">P-733.2D</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Software for coding analysis routines</td>
			<td style="width:123px;">&#160;The Mathworks</td>
			<td style="width:119px;">Matlab</td>
			<td style="width:209px;">custom-written routines are available from the authors</td>
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magnetic tweezers for the measurement of twist and torque

טכניקות מולקולה בודדת מאפשרות לחקור את ההתנהגות של מולקולות ביולוגיות בודדות בפתרון בזמן אמת. טכניקות אלו כוללות מה שנקרא גישות הכוח ספקטרוסקופיה כגון מיקרוסקופ כוח אטומי, פינצטה אופטית, לזרום מתיחה, ופינצטה המגנטית. בין גישות אלה, פינצטה מגנטית הצטיינו ביכולתם ליישם את המומנט תוך שמירה על כוח מתיחות מתמיד. הנה, זה בא לידי ביטוי איך כגון תצורה ניסיונית פינצטה המגנטית &quot;קונבנציונלית&quot; יכולה, באמצעות שינוי פשוט של תצורת השדה שלה כדי למזער את עוצמת השדה הרוחבי, להיות מותאמת על מנת למדוד את מידת הפיתול במולקולה ביולוגית. התצורה וכתוצאה מכך שמכונה פינצטה המגנטית באופן חופשי, במסלול. בנוסף, הוא הראה כיצד שינוי נוסף של תצורת השדה יכול להניב שדה רוחבי עם גודל ביניים בין זו של &amp; #8220; &quot;פינצטה המגנטית הקונבנציונלית ופינצטה המגנטית באופן חופשי, במסלול, המאפשרת למדוד את המומנט מאוחסן במולקולה ביולוגית באופן ישיר. תצורה זו מכונה פינצטה המומנט המגנטית. הווידאו הנלווה מסביר בפירוט כיצד ההמרה של פינצטה המגנטית הקונבנציונלית לפינצטה מגנטית באופן חופשי-מקיפה ופינצטה מומנט מגנטי יכולה להיות מושלמת, ומדגים את השימוש בטכניקות אלה. עיבודים אלה לשמור על כל נקודות החוזק של פינצטה המגנטית הקונבנציונלית תוך הרחבת צדדיות של המכשיר הזה חזק מאוד.

Watch this Scientific Journal Video about Magnetic Tweezers for the Measurement of Twist and Torque at JoVE.com

Magnetic Tweezers for the Measurement of Twist and Torque

פינצטה המגנטית, טכניקת מניפולציה מולקולה בודדת חזקה, יכולה להיות מותאמת למדידות הישירות של טוויסט (שימוש בתצורה הנקראת באופן חופשי, המקיף פינצטה המגנטית) ומומנט (באמצעות תצורה כינתה פינצטה מומנט מגנטי) במקרומולקולות ביולוגיות. הנחיות לביצוע מדידות כאלה מקבלות, כוללים יישומים למחקר של ה-DNA וחוטי Nucleo-חלבון קשורים.

מלקחיים מגנטיים למדידה של טוויסט ומומנט

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

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23.0K Views.  Delft University of Technology.  פינצטה המגנטית, טכניקת מניפולציה מולקולה בודדת חזקה, יכולה להיות מותאמת למדידות הישירות של טוויסט (שימוש בתצורה הנקראת באופן חופשי, המקיף פינצטה המגנטית) ומומנט (באמצעות תצורה כינתה פינצטה מומנט מגנטי) במקרומולקולות ביולוגיות. הנחיות לבי?...

Video: Magnetic Tweezers for the Measurement of Twist and Torque

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

חלקיקים ניאון למדידה של ריכוז יון במערכות ביולוגיות

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

מתיחת רצפים קצרים של DNA עם פינצטה חיל הצירי האופטית קבועה

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

מרובב יחיד מולקולה proteolysis חיל מדידות באמצעות מלקטת מגנטיים

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

תהודה מגנטית elastography מתודולוגיה להערכת של רקמות מהונדסות צמיחה Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipulation של יחיד RNA מולקולות על ידי מלקחיים אופטיים

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

המצאה של Microchip הפקת דיפול בסיוע שלב מוצק עבור ניתוח מתכת עקבות בדגימות מים

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

פרוטוקול עבור הדמיה מגנטית תחום עם בחיי עיר היסטרזיס מדידת וההתקנה ממוטבת

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

מדידת רשת ספין-סריג הרפיה השדה המגנטי של היפרקוטב [1-13ג] פיבט

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

מיקרו-גיבלים מגנטיים, אקוסטיים ואופטיים-משולשים-רספונסיביים לטיפול בסרטן משולב של פוטוטותרמיה

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

ביו-פונקציונליזציה של ננו-חומרים מגנטיים

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...