This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

מחקרים בבעלי חיים אושרו על ידי מועצת האתיקה בעלי חיים למחקר באוניברסיטת מקמאסטר בהתאם המועצה הקנדית על טיפול בבעלי חיים (CCAC) הנחיות. 1. Radiolabeling של הליגנדים TZ-tridentate עם 99m Tc זהירות: ההליכים הבאים מחייבים את השימוש של תרכובות רדיואקטיבי. העבודה צריכה להיעשות רק במעבדה מורשה עם דבקות תקנות הבטיחות וסילוק. תגובות מיקרוגל צריכות להתבצע במיקרוגל שתוכנן במיוחד עבור סינתזה כימית. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סינתזה של [99m Tc (CO) 3 (H 2 O) 3] + 19, 20 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בבקבוקון מיקרוגל, לשלב 8 מ&quot;ג K 2 [BH 3 CO 2], 15 מ&quot;ג Na 2 CO 3, 20 מ&quot;ג Na 2 B 4 O 7 · 10H 2 O, ו -25 מ&quot;ג קוקו [CH (OH)]2 COONa · 4H 2 O. טהר הבקבוקון במשך 10 דקות עם גז ארגון. </li><li style=";text-align:right;direction:rtl"> הוסף 4 מ&quot;ל של 99m TCO 4 - (~ 1,100 MBq, ~ 30 MCI) סליין 0.9% ל הבקבוקון. </li><li style=";text-align:right;direction:rtl"> מחמם את התגובה במיקרוגל במשך 3.5 דקות ב 110 מעלות צלזיוס לאחר 10 שניות של ערבוב כדי להבטיח ערבוב יסודי של חומרים כימיים. </li><li style=";text-align:right;direction:rtl"> התאם את ה- pH של הפתרון 3.5-4 באמצעות ~ 400 μL של 1 M HCl. אמת באמצעות נייר pH. </li></ol></li><li style=";text-align:right;direction:rtl"> Radiolabeling של Tetrazine הליגנדים 1-5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים 2 מ&quot;ג של כל ליגנד (תרכובות 1-5) ב 250 μL MeOH 21. </li><li style=";text-align:right;direction:rtl"> הוסף 250 μL של [99m Tc (CO) 3 (H 2 O) 3] + (~ 74 MBq, ~ 2 MCI) כל פתרון. </li><li style=";text-align:right;direction:rtl"> מחממים את תערובת התגובה באמצעות במיקרוגל למשך 20 דקות ב 60 מעלות צלזיוס. הערה: שלב זה היה זהה עבור כל 5 tetrazines. </li><li style=";text-align:right;direction:rtl"> עבור תרכובות 2- 5, להתאדות הממס מחדש dissolve המוצרים וכתוצאה מכך 1 מ&quot;ל של 1: 1 v / v DCM: TFA. </li><li style=";text-align:right;direction:rtl"> מחממים את תוצרי התגובה מומס (2-5) ב 60 ° C במיקרוגל במשך 6 דקות (2-4) או 10 דקות (5). </li><li style=";text-align:right;direction:rtl"> לאחר קירור לטמפרטורת החדר, להתאדות ממס באמצעות המאייד (36 מעלות צלזיוס, 8 mbar, 3 דקות, 6,000 סל&quot;ד) ו לפזר את המתחם מיובש 1: 1 ACN: H 2 O או 1: 1 MeOH: H 2 O, לפני טיהור HPLC. </li><li style=";text-align:right;direction:rtl"> לטהר את תרכובות Tc שכותרתו 99m (1-5), כולל הפרדת מוצר שכותרתו מ ליגנד tetrazine ללא תווית, באמצעות HPLC (C 18 התהפכה פאזי). בדרך כלל, להשתמש שיפוע elution של 30:70 ACN: H 2 O (שניהם עם 0.1% TFA) כדי 40:60 ACN: H 2 O מעל 20 דקות (18 דקות) ו טור 100 מ&quot;מ 4.6 x 18 C אנליטית. השתמש בשני UV (254 ננומטר) וזיהוי גמא. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לקחת דגימה קטנה של כל מוצר שהכותרת ולהשוות זמן שמירת HPLC שלה לזה של inj-שיתוףected, שאינם רדיואקטיביים, Re-שכותרתו סטנדרטי (0.125 מ&quot;ג ב 20% מתנול-H 2 O). התקן שכותרתו Re מזוהה עקבות UV HPLC, ויהיה elute באותו זמן כמו במתחם Tc שכותרתו 99m ב עקבות γ-HPLC. שיתוף הזרקה זה מראה פסגות בזמני שימור דומים, המאשרת את זהותו של 99m Tc שכותרתו מתחם. </li></ol></li><li style=";text-align:right;direction:rtl"> להתאדות הממס מתוך שברי HPLC באמצעות מאייד (36 מעלות צלזיוס, 8 mbar, 3 דקות, 6,000 סל&quot;ד). </li><li style=";text-align:right;direction:rtl"> נסח במתחם המטוהר בריכוז של 7.4 kBq / μL ב PBS, המכיל BSA 0.5% ו 0.01% Tween-80. </li><li style=";text-align:right;direction:rtl"> כדי להבטיח את התרכובות שכותרתו יציבות, לבצע מחקר יציבות חוץ גופייה. דגירה למתחם ניסח ב 37 מעלות צלזיוס למשך 1, 4 ו -6 שעות, הזרקת כמות קטנה (3.7 MBq) של התערובת על HPLC בכל נקודת זמן כדי להעריך את היציבות. </li></ol></li></ol> 2. מחקרים ביו-הפצה טרום ממוקד <ol style=";text-align:right;direction:rtl"><li&gt; הכנת חיות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש 7-9 בן שבוע, עכברים Balb / c נקבה (n = 3), לנהל TCO-BP מנוסחת מלוחים (20 מ&quot;ג / ק&quot;ג) (5 מיקרוגרם / μL), באמצעות הזרקת הזנב ורידים. </li><li style=";text-align:right;direction:rtl"> העבר את העכבר בהתקן ריסון פיזי, ולזהות את הוורידים ממוקמים על המשטחים לרוחב של הזנב ולנגב עם מטלית אלכוהול. בערך בשעה 2 ס&quot;מ מהקצה של הזנב, להכניס מחט 30-מד בזווית שטוחה, במקביל הווריד. לאט לוחץ על המתג כדי להזריק, להסיר מחט ולהחיל ספוג גזה נקי במקום ההזרקה עם לחץ קל עד עוצר דימום. </li><li style=";text-align:right;direction:rtl"> בשעה 1 הזרקת שעות שלאחר של TCO-BP, לנהל ~ 0.74 MBq (20 μCi) של 99m Tc-tetrazine מנוסחת 100 μL של 0.5% BSA, 0.01% Tween-80 ב PBS, באמצעות הזרקה-וריד הזנב. </li></ol></li><li style=";text-align:right;direction:rtl"> מחקרים ביו-הפצה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בנקודת הזמן הרצויה (t = 6 שעות), לטשטש את העכברים באמצעות 3% isoflurane תערובת גז חמצן 2%. להפגין כדידואר נסיגת קמצוץ על העכבר הרדים כדי לוודא שהם תחת מטוס של הרדמה כירורגית. </li><li style=";text-align:right;direction:rtl"> איסוף דם (1 מ&quot;ל) באמצעות לנקב לב באמצעות מזרק טרום שטופלו הפרין. מקום עכבר על גבית עם אף בחרוט אף הרדמה המשיכה ולאתר את תהליך xiphoid על החיה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנס מחט 25 G, מעט שמאלה של קו האמצע של החיה תחת תהליך xiphoid, בזווית 20 מעלות. הכנס את המחט, ולאט לאט למשוך בחזרה על הבוכנה לראות דם רכזת המחט אם הלב היה מנוקב. מעט מחדש את המחט תוך כדי לחיצה על הבוכנה במידת הצורך, כדי לנקב את הלב. לאט להקיז דם לתוך המזרק. </li></ol></li><li style=";text-align:right;direction:rtl"> להרדימו על ידי נקע בצוואר הרחם, בעוד בהרדמה. </li><li style=";text-align:right;direction:rtl"> מניח כל חיה בתוך שקית פלסטיק ולהשתמש כיל מנה (הגדרת Tc 99m) כדי למדוד את רמת פעילות הגוף כולו. </li><li style=";text-align:right;direction:rtl"> אסוף את רקמות ונוזלים הבאים טרום לשקולed לספור צינורות: דם, עצמות (ברך וכתף), כיס מרה, כליות, כבד, קיבה (עם תוכן), מעי דק (עם תוכן), מעי גס ואת מעי אטום (עם תוכן), בלוטת תריס ואת קנה נשימה, שלפוחית ​​שתן עם שתן , וזנב. </li><li style=";text-align:right;direction:rtl"> לשטוף רקמות מתאימות (למעט דם, כיס מרה, ואת שלפוחית ​​שתן) ב PBS להסיר דם כתם יבש לפני הנחת רקמות צינורות ספירה מתאימות. </li><li style=";text-align:right;direction:rtl"> מניחים פגר חיה בתוך שקית ניילון ולמדוד פעילות הגוף כולו שיורית באמצעות כיל מנה. </li><li style=";text-align:right;direction:rtl"> לשקול כל שפופרת המכילה דגימת רקמה. המשקל ההתחלתי הפחת של הצינור כדי לקבל מסה של רקמה. </li><li style=";text-align:right;direction:rtl"> השתמש כיל מנה (הגדרת Tc 99m) כדי למדוד את כמות פעילות מדגם בדיקה (100 μL) בעת ההזרקה עבור כל עכבר. הערה: זה מדגם בדיקה שווה את עוצמת הזריקה, ובכך נותן את ספירת הפעילות בזמן ההזרקה. </li><li style=";text-align:right;direction:rtl"> בעת מדידת רקמות,liquot 5 μL של מדגם הבדיקה השתמש בעבר. השתמש דלפק גמא רב-גלאי (הגדרת Tc 99m) ולספור להשיג הספירה לדקה (CPM) עבור מדגם בדיקת 5 μL. </li><li style=";text-align:right;direction:rtl"> השתמש בשני הערכים שהתקבלו ב 2.2.9 ו 2.2.10 לחשב את הפעילות ואת קשר CPM בעזרת משוואת 1 להשיג מקדם מרה (CPM μCi -1). (1) <img alt="משוואה 1" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li style=";text-align:right;direction:rtl"> השתמש הדלפק גמא למדוד את כמות רדיואקטיביות בכל רקמה או מדגם נוזל. </li><li style=";text-align:right;direction:rtl"> השתמש במשוואה 1 כדי לחשב את הסכום של הפעילות בכל רקמה או נוזל בזמן יחסית המדידה מהחומר המוזרק הכולל. ערך זה מנורמל אז לפי משקל איבר כפי שדווח מהחומר המוזרק אחוזים לגרם (כלומר,% מזהה / g) של רקמות. </li><li style=";text-align:right;direction:rtl"> בצע את השלבים 2.1.2 כדי 2.2.13 לנהל ניסוי ביקורת שלילית באמצעות הליגנדים tetrazine Tc שכותרתו 99m ב absence של TCO-BP. עכברי קורבן (n = 3) ברמה של 0.5, 1, 4 ו -6 הזרקת שעות שלאחר ולקבל רקמה או נוזל כמתואר לעיל. </li></ol></li></ol>

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The authors declare they have no competing financial interests.

אוסף של chelates tridentate צמוד tetrazine של קטבים שונים הוכן, ואת השירות של המתחמים שלהם 99m Tc תגובת IEDDA עם נגזרת TCO in vivo הוערך. שיטת תיוג יעילה לשחזור 99m Tc פותחה במשך חמש tetrazine-chelates, שבו הריכוז ליגנד היה 10 -3 M. שלב התיוג לווה deprotection של T- קבוצות בוטיל (עבור תרכ...

99m Tc נשאר רדיואיזוטופיים הדומיננטי בשימוש ברפואה גרעינית אבחון, עם למעלה מ -50 מיליון הליכי הדמיה שנערכו בשנת ברחבי עולם 1, 2, 3. רוב סוכני 99m Tc בשימוש קליני הם רדיואקטיביות סוג זלוף. ישנם מספר מצומצם של תרכובות ממוקדות פעיל אשר 99m Tc מופנה לאגד סמן ביולוגי ספציפי באמצעות קשירה לבנות מיקוד. יצירת רדיואקטיביות 99m Tc הממוקד מתעכבת לעתים קרובות על ידי השפעתו של 99m Tc-ליגנד מתחמים על היכולת של מולקולת המיקוד להיקשר הסמן הביולוגי של עניין, או איזוטופים מחצית החיים אינם ארוך מספיק לשימוש עם ביומולקולות משקל מולקולרי גבוה כמו נוגדנים. זה האחרון בדרך כלל דורש כמה ימים לפני תמונות נרכשות על מנת biomolecule כדי לנקות מן היעד הלא Tiss<html> UES. מיקוד טרום מציע גישה חלופית כדי להתגבר על האתגרים אלה. טרום מיקוד בשילוב עם כימית bioorthogonal הוכח להיות דרך יעילה לפתח בדיקות הדמיה מולקולריות חדשות לשני קרינת רדיו-הדמיה 4, 5, 6, 7, 8. הביקוש אלקטרונים הפוך- אלמון דילס (IEDDA) התגובה בין 1,2,4,5-tetrazine (טז) ו -cyclooctene טרנס (TCO) נגזרים, כפי שמוצג באיור 1, הוכח להיות יעיל במיוחד 6. התגובה IEDDA עם רכיבים אלה יכולים להפגין קינטיקה מהירה ב PBS (k 2 ≈ 6,000 M -1 s -1) סלקטיביות גבוהה, מה שהופך אותו אידיאלי עבור in vivo מראש מיקוד יישומים 9, 10. <html> e_content &quot;&gt; הגישה הנפוצה ביותר בשימוש כרוך בניהול וקטור מיקוד TCO הנגזר לאחר תקופת עיכוב מספיק, tetrazine radiolabeled מנוהל. tetrazines radiolabeled מבוסס על 11 C, 18 F, 64 Cu, 89 ZR, ו -111 ב כבר 11 דיווחו, 12, 13, 14, 15. לעומת זאת, יש רק אחד הדיווחים של 99m Tc שכותרתו טז, אשר הוכן באמצעות ליגנד סוג HYNIC המחייב שימוש-הליגנדים שיתוף למנוע התקשרות חלבונים והשפלה in vivo 16. כחלופה, אנו מדווחים כאן את הסינתזה של 99m Tc (I) שכותרתו tetrazines באמצעות משפחה של הליגנדים המהווים מתחמי tridentate יציבה עם [99m Tc (CO) 3] + הליבה. גיל = &quot;1&quot;&gt; <img alt="איור 1" src="/files/ftp_upload/55188/55188fig1.jpg" /> איור 1: תגובת IEDDA bioorthogonal בין tetrazine ו -cyclooctene טרנס. <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> בני משפחתו של הליגנדים מוכן לכלול chelates tridentate שמשתנות הקוטביות ואת לאופי הקבוצה מקשר בין אזור מתכת מחייב ואת טז (איור 2). המטרה היתה לזהות 99m Tc-Tetrazine לבנות שיכול למקם ביעילות ומגיבים עם אתרים שכותרתו TCO in vivo וברור במהירות כאשר לא מחויב, על מנת להניב גבוהה היעד ל-יעד שאינו יחסי. כדי לבדוק את הליגנדים, TCO-נגזרת של ביספוספונטים (TCO-BP) שימש 17. הראינו בעבר כי TCO-BP localizes לאזורים של מטבוליזם העצם פעיל ויכול להגיב עםtetrazines radiolabeled in vivo 18. זהו מגיב נוח לבדוק tetrazines חדש, כי זה יכול להיות מוכן בשלב אחד וניסויים יכול להתבצע בעכברים נורמליים שבו לוקליזציה מתרחשת בעיקר במפרקים (ברכיים וכתפיים).

<table border="0" cellpadding="0" cellspacing="0" width="1378">
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preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

הליגנדים היו מסונתזים באמצעות linkers ו chelators השונים באמצעות אסטרטגית amination רדוקטיבי פשוטה (איור 2), ואחריו צימוד של המוצר אל tetrazine הזמין מסחרי 22, 23. Radiolabeling בוצע תוך שימוש באותה השיטה לכל התרכובות והיה מאוד לשחזור...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

הכנה והערכה של<sup&gt; 99m</sup&gt; Tc שכותרתו Tridentate chelates לחינוך קדם-מיקוד שימוש Bioorthogonal לכימיה

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

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Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

8.1K Views.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

Video: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

הכנה ואפיון של SDF-1α-Chitosan-Dextran סולפט חלקיקים

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

הכנת מצעים נציץ והסיליקון לDNA אוריגמי ניתוח וניסויים

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

הכנת Aerogels biopolymer בעזרת ממיסים גרין

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

הכנת המאפיינים של Thermo-הפיך צולב הגומי Via דילס-אלדר לכימיה

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Simple Methods for the Preparation of Non-noble Metal Bulk-electrodes for Electrocatalytic Applications

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and composition of the material was characterized via powder X-ray diffraction (PXRD), M&#246;ssbauer spectroscopy (MB), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and energy dispersive X-ray spectroscopy (EDX). Two preparation methods of pentlandite bulk electrodes are presented. In the first approach a piece of synthetic pentlandite rock is directly contacted via a wire ferrule. The second approach utilizes pentlandite pellets, pressed from finely ground powder, which is immobilized in a Teflon casing. Both electrodes, whilst being prepared by an additive-free method, reveal high durability during electrocatalytic conversions in comparison to common drop-coating methods. We herein showcase the striking performance of such electrodes to accomplish the hydrogen evolution reaction (HER) and present a standardized method to evaluate the electrocatalytic performance by electrochemical and gas chromatographic methods. Furthermore, we report stability tests via potentiostatic methods at an overpotential of 0.6 V to explore the material limitations of the electrodes during electrolysis under industrial relevant conditions.

A facile preparation method of electrodes using the bulk material Fe4.5Ni4.5S8 is presented. This method provides an alternative technique to conventional electrode fabrication and describes prerequisites for unconventional electrode materials including a straightforward electrocatalytic testing method.

שיטות פשוטות להכנת הלא אצילי מתכת בתפזורת אלקטרודות עבור יישומים Electrocatalytic

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

אופטימיזציה של שיתוף גנטי של חומר כימי רגשים לתוך GPCRs עבור צילום-crosslinking מיפוי וכימיה Bioorthogonal בתאים בתרבית של חיה

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

הכנה ואפיון של C60/Graphene היברידית Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

הכנת יציב Aziridinium Bicyclic יונים, פתיחת הטבעת שלהם לסינתזה של Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

הכנת סיליקה פונקציונלי באמצעות שיטת Bioinspired

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

קידוד גנטי חומצת אמינו קאנונית לדור של נוגדן תרופתיים Conjugates דרך תגובה מהירה Bioorthogonal

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...