עבודה זו נתמכה על ידי הקרן הלאומית למדע (מענק מס 1118-1108 ומענק מס 1146-1112) ומינה קרול זיכרון קרן המחקר. ES מכיר בהכרת תודה על התמיכה הכספית של ראסל ברי למכון.

1. הכנת החמצון הנקבובי SiO 2 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוופלים לחרוט סי (צד אחד מלוטש ב&lt; 100&gt; פנים ומסוממים בכבדות, מסוג p, 0.0008 Ω · ס&quot;מ) בתמיסה 03:01 (V / V) של HF מימיים ואתנול מוחלט ל30 שניות בזרם קבוע צפיפות של 385 mA / 2 סנטימטר. שים לב שHF הוא נוזל מאכל מאוד, וזה צריך להיות מטופל בזהירות רבה. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את פני השטח של סרט סי (PSI) הנקבובי וכתוצאה מכך עם אתנול מוחלט מספר פעמים; לייבש את הסרטים תחת גז חנקן יבש. </li><li style=";text-align:right;direction:rtl"> חמצן דגימות PSI הטרי החרוטות בתנור צינור ב800 מעלות צלזיוס במשך שעה 1 באוויר הסביבה (למקם את המדגם בתנור בטמפרטורת חדר, לחמם את התנור ל800 מעלות צלזיוס, להשאיר בתנור במשך שעה 1, כבה את תנור ולהסיר את הדגימות מהתנור רק בטמפרטורת חדר). </li></ol> 2. Biofunctionalization של 2 פיגומים PSiO <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה PSI חמצון (PSIO 2 מדגם) במשך שעה 1 בmercaptopropyl (פתרון trimethoxysilane-3) 95% (MPTS) (108 מ&quot;מ בטולואן). </li><li style=";text-align:right;direction:rtl"> יש לשטוף את מדגם PSiO 2 שטופלו silane עם טולואן, מתנול, אצטון ו; יבש מתחת לגז חנקן יבש. </li><li style=";text-align:right;direction:rtl"> דגירה מדגם PSiO 2-הותאם silane ל30 דקות ב 1 מיליליטר של 100 פתרון ביוטין PEO-iodoacetyl מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את מדגם PSiO 2 שטופלו ביוטין עם 0.1 פוספט M שנאגרו פתרון (PBS) מספר פעמים. </li><li style=";text-align:right;direction:rtl"> דגירה מדגם PSiO 2-הותאם ביוטין ל30 דקות ב 1 מיליליטר של מיקרוגרם / מיליליטר streptavidin פתרון 100 (SA). </li><li style=";text-align:right;direction:rtl"> יש לשטוף את המדגם שטופל ב-SA PSiO 2 עם 0.1 פתרון M PBS מספר פעמים. </li><li style=";text-align:right;direction:rtl"> דגירה PSiO 2 מדגם הותאם-SA וכתוצאה מכך ל30 דקות ב 1 מיליליטר של 100 מיקרוגרם / ה biotinylated מיליליטר פתרון נוגדן חד שבטי coli (אימונוגלובולין G, IgG) או עם 100 מיקרוגרם / IgG biotinylated ארנב מיליליטר (כמודל לנוגדנים חד שבטיים). </li></ol> 3. תיוג פלורסנט ומיקרוסקופ פלואורסצנטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה המשטחים שונה-IgG עם 15 מיקרוגרם / מיליליטר והעמסת (FITC) מתויג ארנב IgG נגד ל40 דקות ועם 15 מיקרוגרם / מיליליטר והעמסת (FITC) מתויג IgG עכבר אנטי כביקורת. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את הדגימות שונה עם 0.1 פתרון M PBS מספר פעמים. </li><li style=";text-align:right;direction:rtl"> לבחון את הדגימות תחת מיקרוסקופ פלואורסצנטי. </li></ol> 4. תרבות חיידקים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לטפח E. חיידקי coli K12 בצינור 10 מיליליטר עם 5 מיליליטר של לוריא Bertani בינוני (LB) (הרכב בינוני ב1 ליטר של מים deionized: 5 גרם של NaCl, 5 גרם של תמצית שמרים, ו10 גרם של tryptone). דגירה החיידקים רועדים לילה בשעה 37 ° C. </li><li style=";text-align:right;direction:rtl"> לנטר את ריכוז חיידקים על ידי קריאת הצפיפות האופטית (OD) באורך גל של 600 ננומטר. לאחר צמיחת הלילה במדיום LB, קרא את OD 600 באמצעות ספקטרופוטומטר כדי לקבוע את ריכוז החיידקים. מספר התאים הוא קשהctly פרופורציונאלי לOD 600 מדידות (OD 1 600 = 10 8 תאים / מיליליטר). </li></ol> 5. חיידקי חישה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח את PSiO הותאם-IgG 2 ומסודרים PSiO 2 (כמו השליטה) דגימות בתא זרימת פרספקס בהזמנה אישית. תקן את תא הזרימה כדי להבטיח שרפלקטיביות המדגם נמדדה באותה הנקודה במשך כל המדידות. </li><li style=";text-align:right;direction:rtl"> דגירה הדגימות עם א &#39; ההשעיה coli K12 (10 4 תאים / מיליליטר) ל30 דקות בטמפרטורת חדר. ואז להסיר את ההשעיה חיידקים על ידי שטיפת התא עם 0.85% w / תמיסת מלח v ל30 דקות. </li><li style=";text-align:right;direction:rtl"> לעקוב אחר השינויים בנתונים רפלקטיביות לאורך כל הניסוי. כל המדידות אופטיות צריכה להתבצע במימי שמסביב. ספקטרום צריך להיות שנאסף באמצעות ספקטרומטר CCD ונותח על ידי יישום Fast Fourier Transform (FFT), כפי שתואר 25,26 בעבר. </li><li style=";text-align:right;direction:rtl"> לאשר את קיומו של החיידקים עלמשטח biosensor, על ידי תצפית של הדגימות תחת מיקרוסקופ אור זקופה מייד לאחר ניסוי biosensing. </li></ol>

<ol>
	<li>Velusamy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+foodborne+pathogen+detection:+In+the+perspective+of+biosensors.">An overview of foodborne pathogen detection: In the perspective of biosensors.</a> Biotechnol. Adv. 28 (2), 232-23 (2010).</li><li>Doyle, M. P., Beuchat, L. R., Montville, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food Microbiol.: Fundamentals and Front. 2, (2001).</li><li>Radke, S. M., Alocilja, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+biosensor+for+detecting+foodborne+bioterrorism+agents.">A microfabricated biosensor for detecting foodborne bioterrorism agents.</a> IEEE Sens. J. 5 (4), 744 (2005).</li><li>Glynn, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+emerging+molecular+diagnostic+technologies+applicable+to+bacterial+food+safety.">Current and emerging molecular diagnostic technologies applicable to bacterial food safety.</a> Int. J. of Dairy Technol. 59 (2), 126 (2006).</li><li>Leonard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+biosensors+for+detection+of+pathogens+in+food+and+water.">Advances in biosensors for detection of pathogens in food and water.</a> Enzyme Microb. Technol. 32 (1), 3 (2003).</li><li>Alvarez, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+porous+silicon+photonic+crystal+for+bacterial+cell-based+biosensing.">Using a porous silicon photonic crystal for bacterial cell-based biosensing.</a> Physica Status Solidi a-Applications and Materials Science. 204 (5), 1439 (2007).</li><li>Archer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+porous+silicon+microarray+for+DNA+hybridization+detection.">Electrical porous silicon microarray for DNA hybridization detection.</a> Micro- and Nanosystems. 782, 385 (2004).</li><li>Chan, S., Horner, S. R., Fauchet, P. M., Miller, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Gram+Negative+Bacteria+Using+Nanoscale+Silicon+Microcavities.">Identification of Gram Negative Bacteria Using Nanoscale Silicon Microcavities.</a> J. Am. Chem. Soc. 123, 11797 (2001).</li><li>Dancil, K. -. P. S., Greiner, D. P., Sailor, M. J., Canham, L. T., Sailor, M. J., Tanaka, K., Tsai, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of a Porous Silicon Based Biosensor. 536, 557-562 (1999).</li><li>D'Auria, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+silicon-based+biosensors+for+the+selective+identification+of+analytes+of+social+interest.">Nanostructured silicon-based biosensors for the selective identification of analytes of social interest.</a> J Phys - Condens Matter. 18 (33), S2019 (2006).</li><li>de Leon, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+culturing+and+biophotonic+sensing+using+porous+silicon.">Neurons culturing and biophotonic sensing using porous silicon.</a> Appl Phys Lett. 84 (22), 4361 (2004).</li><li>Janshoff, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporous+p-type+silicon+Fabry-Perot+layers.+Fabrication,+characterization,+and+applications+in+biosensing.">Macroporous p-type silicon Fabry-Perot layers. Fabrication, characterization, and applications in biosensing.</a> J. Am. Chem. Soc. 120 (46), 12108 (1998).</li><li>Orosco, M. M., Pacholski, C., Miskelly, G. M., Sailor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-coated+porous+silicon+photonic+crystals+for+amplified+optical+detection+of+protease+activity.">Protein-coated porous silicon photonic crystals for amplified optical detection of protease activity.</a> Adv. Mater. 18, 1393 (2006).</li><li>Pacholski, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+using+porous+silicon+double-layer+interferometers:+reflective+interferometric+Fourier+transform+spectroscopy.">Biosensing using porous silicon double-layer interferometers: reflective interferometric Fourier transform spectroscopy.</a> J. Am. Chem. Soc. 127 (33), 11636 (2005).</li><li>Pacholski, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflective+Interferometric+Fourier+Transform+Spectroscopy:+A+Self-Compensating+Label-Free+Immunosensor+Using+Double-layers+of+Porous+SiO2.">Reflective Interferometric Fourier Transform Spectroscopy: A Self-Compensating Label-Free Immunosensor Using Double-layers of Porous SiO2.</a> J. Am. Chem. Soc. 128, 4250 (2006).</li><li>Sailor, M. J., Link, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart+Dust:+nanostructured+devices+in+a+grain+of+sand.">Smart Dust: nanostructured devices in a grain of sand.</a> Chem. Comm. , 1375 (2005).</li><li>Schwartz, M. P., Alvarez, S. D., Sailor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+SiO2+interferometric+biosensor+for+quantitative+determination+of+protein+interactions:+Binding+of+protein+a+to+immunoglobulins+derived+from+different+species.">Porous SiO2 interferometric biosensor for quantitative determination of protein interactions: Binding of protein a to immunoglobulins derived from different species.</a> Anal. Chem. 79 (1), 327 (2007).</li><li>Schwartz, M. i. c. h. a. e. l. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+smart+petri+dish:+A+nanostructured+photonic+crystal+for+real-time+monitoring+of+living+cells.">The smart petri dish: A nanostructured photonic crystal for real-time monitoring of living cells.</a> Langmuir. 22, 7084 (2006).</li><li>Stewart, M. P., Buriak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+biological+applications+of+porous+silicon+technology.">Chemical and biological applications of porous silicon technology.</a> Adv. Mater. 12 (12), 859 (2000).</li><li>Zhang, D., Alocilja, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+nanoporous+silicon-based+DNA+biosensor+for+the+detection+of+Salmonella+enteritidis.">Characterization of nanoporous silicon-based DNA biosensor for the detection of Salmonella enteritidis.</a> IEEE Sens J. 8 (5-6), 775 (2008).</li><li>Bonanno, L. M., Segal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+porous+silicon-polymer-based+hybrids:+from+biosensing+to+drug+delivery.">Nanostructured porous silicon-polymer-based hybrids: from biosensing to drug delivery.</a> Nanomedicine. 6 (10), 1755 (2011).</li><li>Jane, A., Dronov, R., Hodges, A., Voelcker, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+silicon+biosensors+on+the+advance.">Porous silicon biosensors on the advance.</a> Trends Biotechnol. 27 (4), 230 (2009).</li><li>Li, S., Huang, J., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porous+silicon+optical+microcavity+for+sensitive+bacteria+detection.">A porous silicon optical microcavity for sensitive bacteria detection.</a> Nanotechnology. 22 (42), 425502 (2011).</li><li>Massad-Ivanir, N., Shtenberg, G., Segal, E., Zahavy, E., Ordentlich, A., Yitzhaki, S., Shafferman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nano Bio-Technology for Biomedical and Diagnostics Research. 733, (2012).</li><li>Massad-Ivanir, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Nanostructured+Porous+SiO2+Surfaces+for+Bacteria+Detection+via+"Direct+Cell+Capture".">Engineering Nanostructured Porous SiO2 Surfaces for Bacteria Detection via "Direct Cell Capture".</a> Anal. Chem. 83 (9), 3282-32 (2011).</li><li>Massad-Ivanir, N., Shtenberg, G., Zeidman, T., Segal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+porous+SiO2/hydrogel+hybrids+as+optical+biosensors+for+rapid+detection+of+bacteria.">Construction and characterization of porous SiO2/hydrogel hybrids as optical biosensors for rapid detection of bacteria.</a> Adv Funct Mater. 20 (14), 2269-22 (2010).</li><li>Mathew, F. P., Alocilja, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+silicon-based+biosensor+for+pathogen+detection.">Porous silicon-based biosensor for pathogen detection.</a> Biosens. Bioelectron. 20 (8), 1656 (2005).</li><li>Ouyang, H., Archer, M., Fauchet, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Frontiers in Surface Nanophotonics. 133, 49 (2007).</li><li>Ouyang, H., DeLouise, L. A., Miller, B. L., Fauchet, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+quantitative+detection+of+protein+using+macroporous+silicon+photonic+bandgap+biosensors.">Label-free quantitative detection of protein using macroporous silicon photonic bandgap biosensors.</a> Anal. Chem. 79 (4), 1502-15 (2007).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , (1996).</li><li>Piervincenzi, R. T., Reichert, W. M., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+a+single-chain+antibody+fragment+for+surface+immobilization+in+an+optical+biosensor.">Genetic engineering of a single-chain antibody fragment for surface immobilization in an optical biosensor.</a> Biosensors and Bioelectronics. 13 (3-4), 305 (1998).</li><li>Saerens, D., Huang, L., Bonroy, K., Muyldermans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+Fragments+as+Probe+in+Biosensor+Development.">Antibody Fragments as Probe in Biosensor Development.</a> Sensors. 8 (8), 4669 (2008).</li><li>Shtenberg, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picking+up+the+Pieces:+A+Generic+Porous+Si+Biosensor+for+Probing+the+Proteolytic+Products+of+Enzymes.">Picking up the Pieces: A Generic Porous Si Biosensor for Probing the Proteolytic Products of Enzymes.</a> Anal. Chem. 85 (3), 1951 (2013).</li><li>Bonanno, L. M., DeLouise, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steric+Crowding+Effects+on+Target+Detection+in+an+Affinity+Biosensor.">Steric Crowding Effects on Target Detection in an Affinity Biosensor.</a> Langmuir. 23 (10), 5817 (2007).</li><li>Banada, P. P., Bhunia, A. K., Mohammed, E., Zourob, S., Turner, A. P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. , 567 (2008).</li><li>Poma, A., Whitcombe, M., Piletsky, S., Whitcombe, M. J., Piletsky, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Designing receptores for the next generation of biosensors. , 105 (2013).</li><li>Dudak, F. C., Boyaci, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+immunosensor+based+on+surface+plasmon+resonance+for+enumeration+of+Escherichia+coli+in+water+samples.">Development of an immunosensor based on surface plasmon resonance for enumeration of Escherichia coli in water samples.</a> Food Res. Int. 40 (7), 803 (2007).</li><li>Dudak, F. C., Boyaci, I. H. <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+label-free+bacteria+detection+by+surface+plasmon+resonance+(SPR)+biosensors.">Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors.</a> Biotechn J. 4 (7), 1003 (2009).</li><li>Skottrup, P. D., Nicolaisen, M., Justesen, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+on-site+pathogen+detection+using+antibody-based+sensors.">Towards on-site pathogen detection using antibody-based sensors.</a> Biosens. Bioelectron. 24 (3), 339 (2008).</li><li>Taylor, A. D., Ladd, J., Homola, J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. , 83 (2008).</li></ol>

המחברים מצהירים שום אינטרסים כלכליים מתחרים.

Immunosensor אופטי ללא תווית, המבוסס על nanostructure PSiO 2 (סרט דק פברי פרו-) הוא מפוברק, ותחולתה הפוטנציאלית כbiosensor לגילוי חיידקים הוא אישרה. שינויים ופתרון בעיות אחד החששות הגדולים בעת תכנון immunosensor ה?...

מוקדם ומדויק זיהוי של חיידקים פתוגניים הוא חשוב ביותר עבור מזון ובטיחות מים, ניטור סביבתי, ואבחון נקודה של טיפול 1. כמו טכניקות מיקרוביולוגיה מסורתיות הן זמן רב, מייגע, ואין להם את היכולת לזהות מיקרואורגניזמים ב&quot; בזמן אמת &quot;, או מחוץ לסביבת המעבדה, biosensors מתפתח כדי לעמוד באתגרים אלה 2-5. בשנים האחרונות, סי נקבובית (PSI) התפתחה כפלטפורמה מבטיחה לעיצוב של חיישנים וbiosensors 6-20. בעשור האחרון מחקרים רבים בנוגע לחיישנים אופטיים המבוסס על PSI וbiosensors פורסמו 21,22. שכבת PSI nanostructured היא מפוברק בדרך כלל על ידי תחריט anodic אלקטרוכימיים מSi רקיק יחיד גביש. ננו PSI כתוצאה תערוכה רבים מאפייני יתרון, כגון פנים גדול ונפח חופשי, להתעמק בגדלים שניתן לשלוט וopti מתכונןמאפייני קאל 10,16. התכונות אופטיות של שכבת PSI, כגון photoluminescence 8,11 ואינטרפרומטריה מבוססת החזרה אור הלבן 7,19, מושפעות במידה רבה על ידי תנאים סביבתיים. לכידתו של analytes מולקולות / יעד אורחים בתוך תוצאות השכבה נקבוביות בשינוי במקדם השבירה הממוצעת של הסרט, כפי שנצפה אפנון בספקטרום photoluminescence או כשינוי באורך גל בספקטרום רפלקטיביות 10. למרות החדשנות העצומה בתחום טכנולוגית biosensor האופטי PSI, יש רק כמה דיווחים על פלטפורמות מבוססות PSI לגילוי חיידקים 6,8,20,23-29. בנוסף, רוב מחקרי הוכחה של קונספט אלה הוכיחו זיהוי &quot;עקיף&quot; חיידקים. לכן, בדרך כלל תמוגה המוקדמת של התאים נדרשת כדי לחלץ את שברי החלבון / DNA הממוקד, אופייניים לחיידקים למדו 29. הגישה שלנו היא ללכוד ישירות חיידקי היעדתאים על גבי biosensor PSI. לפיכך, נוגדנים חד שבטיים, שהם ספציפיים כדי למקד חיידקים, הם משותקים על פני השטח הנקבובי. כריכה של תאי חיידקים, דרך אינטראקציות נוגדן אנטיגן, על פני השטח של biosensor לגרום לשינויים באמפליטודה (עוצמה) של ספקטרום רפלקטיביות 24-26. בעבודה זו, אנו מדווחים על הבנייה של biosensor מבוסס PSI אופטי ולהדגים היישום שלה כפלטפורמת biosensing ללא תווית לזיהוי של Escherichia coli (E. coli) חיידקי K12 (המשמשים כמודל מיקרואורגניזם). פיקוח אות אופטי הוא האור מוחזר מnanostructure psi עקב ההפרעה פברי פרו-סרט דק (איור 1 א). שינויים במשרעת / עוצמת האור מתואמים לקיבוע מסוים של תאי חיידקי היעד על גבי משטח biosensor, המאפשרים איתור וכימות של החיידקים מהירים.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

<html> Biosensor אופטי ללא תווית המבוסס על Si הנקבובי nanostructured מיועד ללכידה וזיהוי מהירים של חיידקי Escherichia coli K12, כמיקרואורגניזם מודל. Biosensor מסתמך על כריכה ישירה של תאי חיידקי היעד על פני השטח שלו, בעוד שאף טיפול מקדים (למשל על ידי תמוגה תא) של המדגם למד נדרשת. סרט דק Si mesoporous משמש כאלמנט מתמר האופטי של biosensor. תחת תאורת אור לבנה, השכבה נקבובית מציגה דפוסי שוליים פברי פרו-נפתרו היטב בספקטרום רפלקטיביות שלה. החלת Fast Fourier Transform (FFT) לתוצאות נתוני רפלקטיביות בשיא אחד. שינויים בעוצמת שיא FFT נמצאים תחת פיקוח. לפיכך, חיידקי היעד ללכוד על גבי משטח biosensor, דרך אינטראקציות נוגדן אנטיגן, גורם לשינויים מדידים באינטנסיביות של פסגות FFT, המאפשרים תצפית &quot;בזמן אמת&quot; של קובץ מצורף חיידקים. NT &quot;&gt; סרט Si mesoporous, מיוצר על ידי תהליך anodization אלקטרוכימיים, הוא מצומדת עם נוגדנים חד שבטיים, ספציפיים לחיידקי היעד. חוסר התנועה, immunoactivity והספציפיות של הנוגדנים אושר על ידי ניסויי תיוג ניאון. רגע biosensor חשוף ל חיידקי יעד, התאים שנתפסו ישירות על גבי משטח Si הנקבובי-הותאם נוגדן. אירועי לכידה ספציפיים אלה לגרום לשינויים בעוצמה בספקטרום ההפרעות אופטיות סרט דק של biosensor. אנו מדגימים כי biosensors אלה יכולים לזהות ריכוזי חיידקים נמוכים יחסית (זיהוי מגבלה של 10 4 תאים / מיליליטר) בפחות משעה.

סרטי PSI חמצון (PSiO 2) מוכנים כפי שתוארו בסעיף טקסט הפרוטוקול. 1B איור מראה מיקרוסקופ אלקטרונים סורק ברזולוציה גבוהה של סרט PSI וכתוצאה מכך לאחר חמצון תרמי. PSiO 2 השכבה מאופיינת בנקבוביות גליליות מוגדרות היטב בקוטר בטווח של 30-80 ננומטר. <p class="jove_content" style=...

Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Biosensor אופטי ללא תווית לזיהוי חיידקים מהירים הוא הציג. Biosensor מבוסס על Si הנקבובי nanostructured, שנועד ללכוד באופן ישיר את תאי חיידקי היעד על פני השטח שלו. אנו משתמשים בנוגדנים חד שבטיים, משותקים על המתמר הנקבובי, כמו הבדיקות ללכוד. המחקרים שלנו מראים את הישימות של biosensors אלה לצורך זיהוי של ריכוזי חיידקים נמוכים תוך דקות ללא עיבוד לפני מדגם (כגון תמוגה תא).

זיהוי אופטי של<em&gt; א coli</em&gt; חיידקים על ידי Biosensors הסיליקון Mesoporous

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

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JoVE Journal

Bioengineering

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

16.8K Views.  Technion - Israel Institute of Technology.  Biosensor אופטי ללא תווית לזיהוי חיידקים מהירים הוא הציג. Biosensor מבוסס על Si הנקבובי nanostructured, שנועד ללכוד באופן ישיר את תאי חיידקי היעד על פני השטח שלו. אנו משתמשים בנוגדנים חד שבטיים, משותקים על המתמר הנקבובי, כמו הבדיקות ללכוד. המחקרים שלנו מראים...

Video: Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Cre-P לקס גישה רקומבינציה לצורך זיהוי של Fusion Cell In vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

הוספת בדיקות ביולוגיות Biosensors סיליקה אופטי באמצעות סוכני צימוד silane

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

ניתוח הפנמה נייד של חלקיקים וחיידקים על ידי Cytometry Multi-Flow דימות ספקטרלי

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensor לאיתור של חיידקי סטפילוקוקוס עמידים לאנטיביוטיקה

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

שימוש בBiosensors האופטי ללא תווית לזיהוי מודולציה של תעלות אשלגן על ידי קולטנים מצמידים G-חלבון

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

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 ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

ללא תווית מולקולה בודדת איתור באמצעות תהודה אופטית Microtoroid

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

ססגוניות קרינת איתור עבור מיקרופלואידיקה אגל באמצעות סיבים אופטיים

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.