אנו בתודה להכיר במימון DARPA פוקוס בחסות מיקרו / ננו יסודות fluidics (MF3) מרכז תחת חוזה מספר N66001-10-1-4003, ומן DARPA מענק N660001-09-C-2082.

<ol>
	<li>Jung, B., Bharadwaj, R., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+millionfold+sample+stacking+using+transient+isotachophoresis.">On-chip millionfold sample stacking using transient isotachophoresis.</a> Anal. Chem. 78 (7), 2319-2319 (2006).</li><li>Jung, B., Zhu, Y., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+100+am+fluorophores+using+a+high-sensitivity+on-chip+CE+system+and+transient+isotachophoresis.">Detection of 100 am fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis.</a> Anal. Chem. 79 (1), 345-345 (2007).</li><li>Everaerts, F. M., Beckers, J. L., Verheggen, T. P. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotachophoresis:+Theory,+instrumentation,+and+applications.">Isotachophoresis: Theory, instrumentation, and applications.</a> Elsevier Science &#38; Technology. , (1976).</li><li>Khurana, T. K., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconcentration,+separation,+and+indirect+detection+of+nonfluorescent+analytes+using+fluorescent+mobility+markers.">Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers.</a> Anal. Chem. 80 (1), 279-279 (2008).</li><li>Bercovici, M., Kaigala, G. V., Backhouse, C. J., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carrier+ampholytes+assay+for+portable,+label-free+detection+of+chemical+toxins+in+tap+water.">Fluorescent carrier ampholytes assay for portable, label-free detection of chemical toxins in tap water.</a> Anal. Chem. 82 (5), 1858-1858 (2010).</li><li>Bercovici, M., Kaigala, G. V., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+analyte+identification+using+isotachophoresis+and+a+fluorescent+carrier+ampholyte+assay.">Method for analyte identification using isotachophoresis and a fluorescent carrier ampholyte assay.</a> Anal. Chem. 82 (5), 2134-2134 (2010).</li><li>Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+system+for+isotachophoresis+assays.">Miniaturized system for isotachophoresis assays.</a> Lab Chip. 10 (17), 2242-2242 (2010).</li><li>Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantification+of+isotachophoresis+zones+using+nonfocusing+fluorescent+tracers.">Imaging and quantification of isotachophoresis zones using nonfocusing fluorescent tracers.</a> Anal. Chem. 81 (8), 3022-3022 (2009).</li><li>Schoch, R. B., Ronaghi, M., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+selective+extraction,+isolation,+preconcentration,+and+quantitation+of+small+RNAs+from+cell+lysate+using+on-chip+isotachophoresis.">Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.</a> Lab Chip. 9 (15), 2145-2145 (2009).</li><li>Persat, A., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+from+whole+blood+using+isotachophoresis.">Purification of nucleic acids from whole blood using isotachophoresis.</a> Anal. Chem. 81 (22), 9507-9507 (2009).</li><li>Bercovici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+urinary+tract+infections+using+isotachophoresis+and+molecular+beacons.">Rapid detection of urinary tract infections using isotachophoresis and molecular beacons.</a> Anal. Chem. 83 (11), 4110-4110 (2011).</li><li>Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+malaria-infected+erythrocytes+using+isotachophoresis.">Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.</a> Anal. Chem. 83 (24), 9715-9715 (2011).</li><li>Persat, A., Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+electrolyte+chemistry+for+microfluidic+electrokinetics.+Part+I:+Acid-base+equilibria+and+pH+buffers.">Basic principles of electrolyte chemistry for microfluidic electrokinetics. Part I: Acid-base equilibria and pH buffers.</a> Lab Chip. 9 (17), 2437-2437 (2009).</li><li>Garcia-Schwarz, G., Bercovici, M., Marshall, L. A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+dispersion+in+isotachophoresis.">Sample dispersion in isotachophoresis.</a> J. Fluid Mech. 1 (1), 1-1 (2011).</li><li>Kohlrausch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+concentrations-verschiebungen+durch+electrolyse+im+inneren+von+l&#214;sungen+und+l&#214;sungsgemischen.">&#220;ber concentrations-verschiebungen durch electrolyse im inneren von l&#214;sungen und l&#214;sungsgemischen.</a> Ann. Phys. 298 (10), 209-209 (1897).</li><li>Alberty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+boundary+systems+formed+by+weak+electrolytes.+Theory+of+simple+systems+formed+by+weak+acids+and+bases.">Moving boundary systems formed by weak electrolytes. Theory of simple systems formed by weak acids and bases.</a> J. Am. Chem. Soc. 72 (6), 2361-2361 (1950).</li><li>Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+zone+electrophoresis.+I.+Steady-state+moving-boundary+systems+formed+by+different+electrolyte+combinations.">Multiphasic zone electrophoresis. I. Steady-state moving-boundary systems formed by different electrolyte combinations.</a> Biochem. 12 (5), 871-871 (1973).</li><li>Persat, A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling+by+simultaneous+selective+isotachophoresis+and+hybridization+with+molecular+beacons.">MicroRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons.</a> Anal. Chem. , (2011).</li></ol>

אין לנו מה למסור.

השיטות ITP שהוצג לאפשר זיהוי מהיר, רגיש, ויציב וטיפול מולקולות יוניים. האתגר העיקרי בשימוש ITP היא בחירה נכונה של מאגרים לה טמפ ו TE. ב ITP anionic, אנחנו בדרך כלל לבחור כלוריד כמו אניון מובילה כי היא חומצה חזקה עם הניידות המוחלטת גבוהה מאוד, ולכן יש מאפיינים מאוד לחיזוי. לכן, הבחירה חיץ anionic ITP הוא מופחת בדרך כלל בחירה נכונה TE counterion. עבור ניסויים התמקדות סלקטיבית, בחירה TE הוא קריטי כדי הרחקה של זיהום יונים. ביישומים שבהם מזהמים אינם נוכחים, נמוך ניידות TE יעיל מוביל לשיעור מהיר יותר של התמקדות. אנו ממליצים להשתמש הבאות, יחסית מחונך anionic יונים טה: MES, מגבים, HEPES, tricine. הבחירה של counterion משפיע הן pH מערכת וניידות TE יעיל. Counterions נפוצים הם טריס (pKa 8.1) ו-BIS טריס (pKa 6.4). מבחני הדורשים pH נמוך או גבוה, מומלץ פירידין (pKa 5.25) וethanolamine (pKa 9.5), בהתאמה. בלוחות 1 ו -2, עבור ITP anionic קטיוני, בהתאמה, אנו מסכמים כמה דוגמאות חיץ שימושיים. אנחנו לוקחים HCl כמו יון נתרן LE anionic וכפי קטיוני LE יון, ואנחנו מניחים 100 כוח יונית מ&quot;מ למאגר LE. הקורא יהיה לציין כי יונית מאגר כוח הפרוטוקולים שלנו (וגם לוחות 1-2) תמיד גדול או שווה ל 10 מ&quot;מ. אמנם באופן תיאורטי את הפיסיקה של ITP חל על כוח יונית נמוך 10 מ&quot;מ, מזהמים טבעיים (חומצה פחמתית למשל מתגובה בין מים דו תחמוצת הפחמן באטמוספירה) ליד רמה 1 mM לעתים קרובות להגביל את השימוש המעשי של מאגרים נמוכים כוח יוניים. נציין, כי מנגנון התמרה האות אינו מוגבל מבחני שהוצגו בפרוטוקול זה. כפי שנאמר בקצרה חלק 5, במצב הרמה מטוהרים אזורי ניתן לאתר באמצעות שינוי המוליכות המקומי, absor UV, טמפרטורה bance, או אינדקס השבירה. בקבוצה שלנו, פיתחנו שיטה אשר מנצל ampholytes הספק ניאון לאיתור זיהוי רגיש של analytes ידוע. 5 בניסויים מצב שיא, בדיקות ספציפיות ניתן לתייג analytes ממוקדים. בדוגמה אחת, אנו משתמשים באלומות מולקולריות - בדיקות oligonucleotide כי לזרוח על ההכלאה רצף היעד שלהם - כדי לזהות DNA היעד או מולקולות רנ&quot;א עם סגוליות גבוהה 11,18. בעוד ITP היא איתנה יחסית פיזור הנגרמת על ידי זרימת הלחץ מונע EOF, EOF מוגזמת עלולה לגרום לקיפאון של ממשק ITP, שם מהירות EOF אומר הופך שווה למהירות ITP. 14 EOF חזקה כזו מתרחשת בעיקר בתנאים של pH גבוה וחוזק יוני נמוך. מסיבה זו, אנו ממליצים על שימוש מאגרים עם pH 8 או נמוך יותר ועם כוח יונית בסדר גודל של 100 מ&quot;מ, אם נוח. מניסיוננו, PVP הואביותר ציפוי יעיל לדיכוי EOF שבבי בורוסיליקט. עם זאת, בגלל המאפיינים שלה סנון, PVP לא יכול להיות מתאים עבור יישומים מסוימים, כגון התמקדות הדנ&quot;א הגנומי. בניסויים, אנו צופים כי התוספת של PVP יכול לצמצם במידה ניכרת את ניידות הדנ&quot;א הגנומי המוחלט (עד כדי כך שהוא לא מתמקד). במקרים אלה ציפוי silanol כגון Sigmacote ב conjuction עם פעילי שטח (למשל טריטון X-100) יכול להיות גם יעיל בהפחתת EOF. 10 <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td colspan="4"> TE אניון (pKa -1) </td></tr><tr><td> Counterion חציצה (pKa 1) </td><td> MES (6.10) </td><td> מגבים (7.20) </td><td> HEPES (7.50) </td><td> tricine (8.15) </td></tr><tr><td> ethanolamine (9.50) </td><td> 21.41 </td><td> 20.40 </td><td> 17.38 </td><td> 22.85 </td></tr><tr><td> טריס (8.08) </td><td> 21.00 </td><td> 19.23 &lt;/ Td&gt; <td> 15.84 </td><td> 17.56 </td></tr><tr><td> BIS-טריס (6.40) </td><td> 18.22 </td><td> 10.41 </td><td> 7.30 </td><td> 5.23 </td></tr><tr><td> פירידין (5.18) </td><td> 1.01 </td><td> 3.72 </td><td> 2.42 </td><td> 1.48 </td></tr></tbody></table> טבלה 1. ניידות גודל אפקטיבי (× 10 -9 מ &#39;2 / V / s) של אזור מותאם אנליטי טהור ITP anionic שם LE הוא 100 מ&quot;מ HCl ו 200 חציצה counterion מ&quot;מ. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td colspan="4"> TE קטיון (pKa 1) </td></tr><tr><td> Counterion חציצה (pKa -1) </td><td> ethanolamine (9.50) </td><td> טריס (8.08) </td><td> BIS-טריס (6.40) </td><td> פירידין (5.18) </td></tr><tr><td> MES (6.10) </td><td> 35.57 </td><td> 21.96 </td><td> 16.39 </td><td> 10.45 </td></tr><tr><td> MOPS (7.20) </td><td> 35.47 </td><td> 20.92 </td><td> 9.76 </td><td> 3.93 </td></tr><tr><td> HEPES (7.50) </td><td> 35.35 </td><td> 19.97 </td><td> 7.77 </td><td> 2.88 </td></tr><tr><td> Tricine (8.15) </td><td> 34.77 </td><td> 16.72 </td><td> 4.50 </td><td> 1.44 </td></tr></tbody></table> טבלה 2. גודל ניידות אפקטיבית (× 10 -9 מ &#39;2 / V / s) של אזור מותאם אנליטי טהור ITP קטיוני שבו LE הוא 100 מ&quot;מ ו 200 נתרן חציצה counterion מ&quot;מ.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="4"> ריאגנטים </td></tr><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> מים מזוקקים </td><td> GIBCO </td><td> 10977 </td><td> RNase / DNase חינם </td></tr><tr><td> Clorox Ultra </td><td> Clorox </td><td> 02489CT </td><td></td></tr><tr><td> סודיום הידרוקסיד (NaOH) </td><td> Mallinckrodt </td><td> 7708 </td><td></td></tr><tr><td> חומצה הידרוכלורית (HCL) </td><td> EMD כימיקלים </td><td> HX0603-4 </td><td></td></tr><tr><td> Trizma הבסיס (טריס) </td><td> Sigma-Aldrich </td><td> T6066 </td><td></td></tr><tr><td> polyvinylpyrrolidone (PVP) </td><td> Polysciences בע&quot;מ </td><td> 06067 </td><td> MW 1000000 </td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> H-4034 </td><td></td></tr><tr><td> Alexa פלואוריד 488 חומצה carboxylic </td><td> Invitrogen </td><td> A20000 </td><td></td></tr><tr><td> tricine </td><td> Sigma-Aldrich </td><td> T-9784 </td><td></td></tr><tr><td> BIS-טריס </td><td> Sigma-Aldrich </td><td> B4429 </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> AM9260G </td><td></td></tr><tr><td> טריטון X-100 </td><td> Sigma-Aldrich </td><td> 100 x </td><td></td></tr><tr><td> ליזוזים </td><td> Sigma-Aldrich </td><td> L6876 </td><td></td></tr><tr><td> SYBR ירוק II </td><td> Invitrogen </td><td> S7564 </td><td></td></tr><tr><td> ethanolamine </td><td> Sigma-Aldrich </td><td> 411000 </td><td></td></tr><tr><td> Rhodamine 6G </td><td> Acros Organics (Geel, בלגיה) </td><td> CAS 989-38-8 </td><td></td></tr><tr><td> L-חומצות אמינו </td><td> Sigma-Aldrich </td><td> LAA21 </td><td></td></tr><tr><td> כוח SYBR גרין RNA ל-C T 1-Step קיט </td><td> יישומי Biosystems </td><td> 4389986 </td><td></td></tr><tr> &lt;td&gt; PCR primers </td><td> Integrated DNA Technologies </td><td></td><td></td></tr><tr><td colspan="4"> ציוד </td></tr><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> שבב בורוסיליקט microfluidic </td><td> Caliper מדעי החיים בע&quot;מ </td><td> NS12A </td><td> מסופק עם או בלי נושא כלים מפלסטיק </td></tr><tr><td> משאבת ואקום </td><td> גאסט </td><td> DOA-P104-AA </td><td></td></tr><tr><td> Sourcemeter </td><td> Keithley </td><td> 2410 </td><td> קבועים שוטפים וקבועים פעולה מצבי מתח </td></tr><tr><td> מיקרוסקופ epifluorescent הפוכה </td><td> אולימפוס </td><td> IX70 </td><td> השתמש כספית מנורה (אולימפוס) או LED (Thorlabs) לתאורה </td></tr><tr><td> מסנן הקוביה </td><td> אומגה </td><td> XF115-2 </td><td> עירור / פליטה: כחול / ירוק </td></tr><tr><td> בטוח, לנעול צינורות microcentrifuge </td><td> Eppendorf </td><td> 022363204 </td><td> 1.5 מ&quot;ל קיבולת </td></tr><tr><td> סרכזת </td><td> Eppendorf </td><td> 5417C </td><td></td></tr></tbody></table> לוח 3. חומרים כימיים ספציפיים וציוד.

on-chip isotachophoresis for separation of ions and purification of nucleic acids

טכניקות Electrokinetic הם מרכיב עיקרי של יישומים microscale בגלל היכולת הייחודית לבצע מגוון של תהליכים fluidic ו electrophoretic ב, מערכות קומפקטיות פשוטות ללא חלקים נעים. Isotachophoresis (ITP) היא טכניקה electrokinetic פשוט מאוד חזקים שיכולים להשיג הפרדה של פי 1,2 מיליון preconcentration ויעיל והפקת המבוסס על ניידות יוניים. 3 לדוגמה, אנו הוכיחו את היישום של ITP להפרדה וזיהוי רגיש ללא תווית מולקולות יוניים (רעלים כגון DNA, rRNA, מירנה) עם מעט או ללא הכנה מדגם 4-8 ו מיצוי וטיהור של חומצות גרעין של מטריצות מורכבות כולל תרבית תאים, שתן ודם. 9-12 ITP משיגה מיקוד ההפרדה באמצעות שדה חשמלי היישומי שני מאגרים במערכת ערוץ fluidic. עבור analytes anionic, מובילה את האלקטרוליט (LE) חיץ נבחר שלuch כי אניונים שלה יש יותר ניידות electrophoretic יעיל יותר אניונים של אלקטרוליט נגרר (TE) חוצץ (ניידות אפקטיבית מתאר את מהירות סחיפה הנצפה של יון ולוקח בחשבון את המדינה יינון של יון, כפי שתואר על ידי Persat ואח&#39; בפירוט . 13). לאחר כינון הממשק בין TE ולה, שדה חשמלי מוחל כך יונים לה טמפ להתרחק מהאזור הכבוש על ידי יוני te. יונים לדוגמה גזע ביניים ניידות יעילה לקראת יוני טה אבל אני לא מצליח לעקוף יונים לה טמפ, ולכן הם מתמקדים בממשק LE-TE (להלן &quot;ממשק ITP&quot;). יתר על כן, TE ואזורים לה טמפ טופס של מוליכות נמוכה בהתאמה גבוהה, אשר להקים שיפוע תלול שדה חשמלי על הממשק ITP. זה שיפוע בתחום preconcentrates מינים המדגם כפי שהן להתמקד. בחירה נכונה של תוצאות טה LE להתמקד וטיהור של מינים שאינם יעד ממוקד מינים אחרים, ובסופו של דבר ההפרדההפרדה ד מינים לדוגמה. אנחנו כאן לסקור ITP עקרונות פיסיקליים בבסיס ולדון בשני מצבים סטנדרטיים של המבצע: &quot;השיא&quot; מצבי &quot;מישור&quot;. במצב שיא, יחסית לדלל יונים מדגם להתמקד יחד בתוך חופפים פסגות צרים על הממשק ITP. במצב הרמה, יונים מדגם שופע יותר להגיע לריכוז המצב היציב ואת לבודד את הרמה, כמו אזורים סמוכים שהוזמנו על ידי ניידות יעילה שלהם. מצבי שיא הרמה נובעים מתוך פיזיקה בסיסית אחת, אלא לייצג משטרים שונים הנבדלות ריכוז אנליטי הראשונית ו / או את משך הזמן המוקצב הצטברות המדגם. אנחנו הראשונים לתאר בפירוט השיא מודל הניסוי מצב ואז להראות assay מצב שיא החילוץ של חומצות גרעין מ - א &#39; תא קולי התרבות. אנו מסיקים על ידי הצגת מצב assay הרמה, שבה אנו משתמשים שאינם מתמקדים נותבים (NFT אזור) מינים לדמיין את ההפרדה ליוצרים quantitation של חומצות אמינו.

Watch this Scientific Journal Video about On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids at JoVE.com

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Isotachophoresis (ITP) היא הפרדה electrokinetic חזקים בטכניקה preconcentration עם יישומים החל איתור הרעלן הכנת המדגם. אנו בודקים את עקרונות פיסיקליים של ITP ו המתודולוגיה של יישום טכניקה זו שני יישומים דוגמה ספציפית: הפרדה וזיהוי של מולקולות קטנות וטיהור של חומצות גרעין מ lysate תרבית תאים.

על שבב Isotachophoresis להפרדה של יוני וטיהור של חומצות גרעין

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

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24.3K Views.  Stanford University. Isotachophoresis (ITP) היא הפרדה electrokinetic חזקים בטכניקה preconcentration עם יישומים החל איתור הרעלן הכנת המדגם. אנו בודקים את עקרונות פיסיקליים של ITP ו המתודולוגיה של יישום טכניקה זו שני יישומים דוגמה ספציפית: הפרדה וזיהוי של מולקולות קטנות וטיהור של חומצות גרעין מ lysate תרבית תאים.

Video: On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

המצאה של אלקטרוכימי-DNA Biosensors לגילוי Reagentless של חומצות, חלבונים ומולקולות קטנות גרעין

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Lensfree על שבב טומוגרפית מיקרוסקופית העסקת רב זווית תאורה פיקסל סופר רזולוציה

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

על שבב phenotyping דלקתיות האנדותל

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

פלטפורמה מבוססת Photoresist ביוסנסור אלקטרוכימי Microfluidic הסרט יבש: ייצור המכשיר, הכנה על שבב Assay, ותפעול המערכת

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

ייצור ואימות מערכת איברים על שבב עם אלקטרודות משולבת ישירות לכמת התנגדות חשמלית Transendothelial

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

זיהוי ויזואלי של מספר חומצות גרעין ב מערך נימי

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

הכנה, טיהור, השימוש ליפוזומים המכילים חומצת שומן

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

כימות בו זמני של קינורנינים נבחרים שנותחו על ידי ספקטרומטריית כרומטוגרפיה-מסה נוזלית במדיום שנאסף מתרביות תאים סרטניים

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

הרכבה עצמית של חומצות פפטידיות פפטיד שעברו שינוי גמא לתוך ננו-מבנים מורכבים בתערובות ממס אורגניות

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.