Name: foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7
Uploaded: 2017-09-17T14:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Bu eser K-INBRE P20GM103418, Kansas soya Komisyonu (KSC/PSU 1663), ACS PRF 56629-UNI7 ve PSU polimer kimya başlangıç Fonu, tüm SS tarafından desteklenmektedir. Biz Üniversitesi videoları çekiyor, Bay Jacob Anselmi, video ile onun en iyi çalışma için teşekkür ederim. Biz de Bay Roger Heckert ve Bayan Katha Heckert cömert destekleri araştırma için teşekkür ederiz.

1. sentez ve çok parametrik Manyeto-floresan Nanosensors (MFnS) Functionalization.
<ol><li>Superparamagnetic demir oksit sentezi nano tanecikleri (IONPs) <ol><li>IONP sentezi için hazırlamak için aşağıdaki 3 çözümleri hazırlamak: çözüm 1: FeCl 3 (0.70 g) ve FeCl 2 H 2 O içinde (2 mL), çözüm 2: NH 4 OH (2.0 mL, 13.4 M) H 2 O (15 mL) ve çözüm 3: polyacrylic asit (0.855 g) H 2 O (5 mL).</li>
		<li>2 M hidroklorik asit (HCl) 90 µL çözüm 1'e ve daha sonra hemen vortexing 875 devirde iken çözüm 2 ile karıştırın. O zaman çözüm 3 ekleyin. Vortexing 60 dakika süreyle devam</li>
		<li>Santrifüj için 1,620 x g., 20 dk santrifüj kapasitesi süpernatant 2.880 x g., 20 dk</li>
		<li>Son süpernatant ile diyaliz arındırmak. Süpernatant bir Diyaliz Ekle (MWCO 6 &#8722; 8 K) ve fosfat tamponlu tuz (PBS) içeren bir ölçek yerleştirin (pH 7.4 =) ve spin bar. Her 2-3 h. ile taze tampon yerine 12 h için emmek izin</li>
	</ol></li> <li>IONPs yüzey için hedef özgü antikorların konjugasyon <ol><li>Aşağıdaki dört çözümleri hazırlamak: çözüm A: 5 mg 1-etil - 3-(3-dimethylaminopropyl) carbodiimide hidroklorid (EDC) 250 µL MES [2-(N- morpholino) ethanesulfonic asit] arabellek (0.1 M, pH 6.8 =), çözüm B: 3 mg, N-Hydroxysuccinimide (NHS) 250 µL MES arabelleği (0.1 M, pH 6.8 =), Igg1, E. coli mAb fosfat tamponlu tuz (PBS) (pH 7,4) 225 µL içinde çözüm C: 5 µg ve IONP çözüm D: 4 mL (5.0 mmol) PBS (pH 7,4) 1 ml.</li>
		<li>Ekleyin 2-(N-Morpholino) ethanesulfonic asit (MES) arabellek eriyik-e doğru A, o zaman hemen çözüm eklemek A çözüm D 15 içinde küçük artışlarla s, çözüm karıştırma için her eklenmesinden sonra ters çevirme.</li>
		<li>Hemen çözüm B çözüm D için küçük aralıklarla üç dakikalık bir süre içinde eklemek için başlayın. Her eklenmesinden sonra çözüm ters çevirme tarafından Mix.</li>
		<li>5 µL artırımlarıyla çözüm çözüm C eklemek çözüm karıştırma için her eklenmesinden sonra ters çevirme.</li>
		<li>Oda sıcaklığında 3 h için devam etmek ve o zaman kuluçka gecede 4 ° C'de devam etmek tepki izin.</li>
		<li>Arındırmak manyetik sütun PBS kullanma yolu ile elde edilen Ab Birleşik IONPs (pH 7.4, final [Fe] = = 3,5 mmol) herhangi bir çekimsiz antikorlar kaldırmak için.
		<ol><li>Yıkama manyetik sütun PBS (1 mL) ile. Sütun için manyetik plaka ekleyin ve IONP çözüm ekleyin.</li>
			<li>Manyetik sütun PBS (1 mL) ile tekrar yıkayın. Manyetik sütun Manyetik plaka kaldırın. PBS sütununu ekleyin ve çözüm toplamak. Mağaza 4 ° C'de</li></ol></li></ol></li> <li>Floresan 1,1 kapsülleme &#39; - Dioctadecyl - 3,3,3 &#39;, 3 &#39;-Tetramethylindocarbocyanine perklorat (dıı) boya kullanarak antikor Birleşik IONPs polyacrylic asit (PAA) kaplama içine bir Solvent Difüzyon yöntemi.
	<ol><li>IONPs 4 mL için dropwise 2.0 ekleyin dıı boya (2 mmol) 100 µL DMSO, sürekli 1100 devir / dakikada karıştırma ile µL.</li>
		<li>[Fe] son demir konsantrasyonu ile son bir MFnS çözüm oluşturma PBS karşı elde edilen çözüm (MWCO 6-8 KDa, 12 h) diyaliz 2 mmol =.</li>
	</ol></li> <li>MFnS karakterizasyonu <ol><li>spektrofotometrik analiz için bir plaka okuyucu ile floresan emisyon 595, kapsüllenmiş dıı boya algılamak için kullanın nM.</li>
		<li>Dinamik ışık saçılma <ol><li>ortalama boyutu ve MFnS yüzey ücret belirlemek için zetasizer MFnS çözüm örneği yerleştirin. 
			Not: Burada, ortalama boyutu ve fonksiyonel MFnS yüzey ücret 77.09 bulundu nm ve-22.3 mV, sırasıyla.</li>
		</ol></li></ol></li></ol> 2. Bakteriyel Culturing ve hisse senedi çözüm hazırlık <ol><li>bakteri kültürü <ol><li>besin suyu 1 ml dondurularak Pelet (E. coli O157: H7) hidrat ve bir ek 5 mL suyu ekleyin.</li>
		<li>Bu çözümün bir agar plaka ve çizgi uygulamak 100 µL kuluçka için 24 h 37 ° C'de ve ardından bir steril aşılama döngü kullanarak</li>
		<li>Sonra 24 h, izole bir koloni agar plaka seçin ve bir 15 mL kültür suyu ekleyin. Süre optik yoğunluk değeri izleme için 4-6 h, 37 ° C'de kuluçkaya (600 Absorbans nm).</li>
		<li>Bir optik yoğunluk değerini 0.1 alındıktan sonra kültür durdurmak ve seri dilutions gerçekleştirmek.</li>
	</ol></li> <li>Seri dilutions <ol><li>ekleyin 900 µL sekiz steril microcentrifuge tüpler için besin suyu.</li>
		<li>Ekle 100 µL bakteriyel süspansiyon ilk tüp (seyreltme 10 -1).</li>
		<li>Al 100 µL ilk tüp ve ikinci 10 -2 seyreltme elde etmek için ekleyin. Bu model 10 -8 son bir seyreltme ile biten kalan tüpler için devam.</li>
		<li>Transfer 100 µL Ağar kaplamalar ayrı ve 37 24 h için kuluçkaya her tüp ° C.</li>
		<li>Ertesi gün, her tabağa birimleri (CFUs) oluşturan colony saymak. ~ 100 CFUs ile kalıbı seçin. Karşılık gelen dilutions daha fazla deneyler için kullanın (100 µL ~ 100 CFUs =). 
		Not: Burada ~ 100 CFUs sonuçlanan 10 -6 seyreltme oldu.</li>
	</ol></li></ol> 3. MFnS kullanarak hızlı algılama E. coli O157: H7 <ol><li>1-100 arasında değişmektedir CFU sonuçlanan 10 -6 bakteriyel hisse senedi, miktarda artan çeşitli PBS Çözümleri (1 X, pH 7.4, 300 µL) Spike. MFnS tutarlı bir miktar eklemek (100 µL) Bu çözümler için.</li>
	<li>Sadece PBS (1 X, pH 7.4, 300 µL) içeren bir temel çözüm oluşturmak ve MFnS (100 µL).</li>
	<li>37 ° C'de 30 dk için çözümler kuluçkaya ve sonra oda sıcaklığında soğumaya izin.</li>
	<li>Manyetik relaxometer (0,47 Tesla) ve kayıt değişiklikleri gevşeme zamanlarda (T2) her çözümde CFUs göre transfer bireysel çözümler.
	<ol>T2 değerlerde değişiklikleri kaydetmek, sadece PBS ve MFnS içeren temel çözüm T2 değerini ölçerek başlamak için <li>.
		<ol><li>Yer manyetik çözümde relaxometer. İlgili yazılım açık, seçin &#34; T2 gevşeme &#34; ayarı ve basın &#34; ölçü birimi. &#34;</li></ol></li> <li>temel T2, topluluğu takip çeşitli ile çivili ek çözümler T2 değerini ölçmek bakteri konsantrasyonları. 
		Not: Taban çizgisi çivili T2 düşülen T2 T2 değişiklik eşdeğerdir.</li>
	</ol></li> <li>Manyetik relaxometer örnekleri kaldırmak ve 10 dakika süreyle 2880 x g de tüpler santrifüj kapasitesi</li>
	<li>Süpernatant dikkatle boşaltmak ve PBS (1 X, pH 7,4) 100 µL içinde bakteri granül resuspend.</li>
	<li>Eklemek 80 µL, her resuspension bir 96-şey plaka ve kayıt Floresans Koyulukları 595 nM. 
	Not: Test sonuçları bölümünde açıklandığı gibi göl su, süt ve diğerleri dahil olmak üzere farklı çözücüler kullanılarak tekrar edilebilir</li></ol>

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Bu nanoteknoloji gösterdiği uygulama henüz FDA tarafından onaylanmış değil.

Bu iletişim kuralı basit tamamen işlevsel MFnS üretmek için tasarlanmış mümkün olduğunca. Ancak, değişiklik Protokolü'nün kullanıcının nihai hedef bağlı olarak yararlı olabilecek çok önemli noktalar vardır. Örneğin, farklı antikor kullanımı birçok diğer patojenleri hedefleme için izin verecek. Ayrıca, bu iletişim kuralını molekülleri hedefleme olarak antikor kullanımı sınırlı değildir. Konak hücre reseptörleri gibi hedef patojenler için özel b...

Bakteriyel kontaminasyon hem de kalıcı geçtiği ticari gıda üretilen ve su kaynaklarının giderek hızlı ve spesifik tanı platformları için bir ihtiyaç yarattı. 1 , 2 , Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus ve Escherichia cins daha yaygın bakteriyel kirletici için yiyecek ve su kirlenmesi sorumlu bazılarıdır. 3 , 4 kez bakteriyel kontaminasyon Bu patojenler tarafından ateş, kolera, gastroenterit ve ishal gibi belirtiler olur. 4 su kaynaklarının kirlenme kez yeterince filtrelenmiş su erişimi olmayan topluluklar üzerinde ciddi ve olumsuz etkileri vardır ve gıda kontaminasyonu hastalıklar ve ürün geri çekme çabaları çok sayıda için açmıştır. 5 , 6
Bakteriyel kirlenme tarafından neden olduğu hastalıkların oluşumunu azaltmak için bir dizi hangi su ve yiyecek verimli satış veya tüketim önce taranabilir yöntemleri geliştirmek için çaba olmuştur. PCR,1,7,8,9,10 ELISA,11,12 döngü-aracılı izotermal amplifikasyon () gibi 3 teknikleri LAMBA),13,14 diğerleri arasında15,16,17,18,19,20,21, 22,23,24 son zamanlarda çeşitli patojenler tespiti için kullanılır. Geleneksel bakteriyel kültür yöntemleri ile karşılaştırıldığında, bu tekniklerin özgüllük ve zaman açısından çok daha verimli. Ancak, bu teknikler hala yanlış pozitif ve negatif, karmaşık prosedürleri ve maliyet ile mücadele. 1 , 3 , 25 multiparametric Manyeto-floresan nanosensors (MFnS) bakteriyel algılama için alternatif bir yöntem olarak önerilen bu nedenle içindir.
Bu nanosensors benzersiz olarak birlikte manyetik gevşeme ve floresan yöntemleri, hızlı ve doğru bir çift-algılama platformu için izin eşleyin. E. coli O157: H7 örnek kirletici kullanarak, MFnS dakika içinde olduğu kadar az 1 CFU algılama yeteneğini gösterilmiştir. Patojen özel antikorlar özgüllüğü artırmak için kullanılır ve algılama ve miktar her iki düşük ve yüksek kirlilik aralıklardaki bakteriyel kirletici için manyetik ve floresan modaliteleri kombinasyonu sağlar. 16 bakteriyel kontaminasyon durumunda patojen özgü antikorların hedefleme yetenekleri nedeniyle bakteri çevresinde nanosensors swarm. Manyetik nanosensors ile bakteri arasındaki manyetik demir çekirdekli ve çevresindeki su proton arasındaki etkileşimi sınırlar. Bu artış T2 gevşeme zamanlarda manyetik bir relaxometer tarafından kaydedildiği şekilde neden olur. Çözüm bakterilerde konsantrasyon yükselir gibi bakteri, alt T2 değerleri kaynaklanan artan sayıda nanosensors dağıtmak. Tersine, Floresans emisyon nanosensors patojen için doğrudan bağlı artan sayıda nedeniyle bakteri konsantrasyonu ile orantılı artar. Santrifüjü örnekleri ve bakteriyel Pelet yalıtım sadece doğrudan herhangi bir serbest yüzer nanosensors kaldırılması ve doğrudan Floresans emisyon sayısı ile korele bakteri iliştirilmiş nano tanecikleri tasarruf etmek bakteri içinde çözüm sunuyoruz. Bu mekanizma şematik gösterimi şekil 1' de temsil edilir.
Bu MFnS platform bakım noktası tarama unutmayın, düşük maliyetli ve taşınabilir özellikleri kaynaklanan ile dizayn edilmiştir. MFnS oda sıcaklığında kararlı olduğunu ve sadece doğru algılama bakteriyel kirletici için çok düşük konsantrasyonlarda gerekmektedir. Ayrıca, sentez sonra MFnS kullanımı basittir ve alanında eğitimli uzmanları kullanımı gerektirmez. Son olarak, bu tanı platform, bu tarafından bir platformda her türlü patojenler birçok farklı ortamlarda algılamak için kullanılan bir araç sağlayarak son derece özelleştirilebilir hedefleme için sağlar.

<table><tbody><tr><td>Ferrous Chloride Tetrahydrate</td><td>Fisher Scientific</td><td>I90-500</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Fisher Scientific</td><td>I88-500</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Polyacryllic Acid</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>EDC</td><td>Thermofisher Scientific</td><td>22980</td><td></td></tr><tr><td>NHS</td><td>Fisher Scientific</td><td>AC157270250</td><td></td></tr><tr><td>Anti-E. coli O111 antibody&amp;nbsp;</td><td>sera care</td><td>5310-0352</td><td></td></tr><tr><td>Anti-E. coli O157:H7 antibody [P3C6]&amp;nbsp;</td><td>Abcam</td><td>ab75244</td><td></td></tr><tr><td>DiI Stain</td><td>Fisher Scientific</td><td>D282</td><td></td></tr><tr><td>Nutrient Broth</td><td>Difco</td><td>233000</td><td></td></tr><tr><td>Freeze-dried E. coli O157:H7 pellet</td><td>ATCC</td><td>700728</td><td></td></tr><tr><td>Magnetic Relaxomteter&amp;nbsp;</td><td>Bruker</td><td>mq20</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern</td><td>NANO-ZS90</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Magnetic Column&amp;nbsp;</td><td>QuadroMACS</td><td>130-090-976</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 Series</td><td></td></tr><tr><td>Centrifuge (accuSpin Micro 17)</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>Floor Model Shaking Incubator</td><td>SHEL LAB</td><td>SSI5</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Open-Air Rocking Shaker</td><td>Fisher Scientific</td><td>02-217-765</td><td></td></tr></tbody></table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157: H7-var bağlı su bazlı hem de ve gıda kaynaklı hastalıklar ve bir tehdit rağmen gıda ve su filtreleme yöntemleri kullanılan şu anda kalır. Polimeraz zincir tepkimesi (PCR) ve enzim bağlı immunosorbent deneyleri (ELISA) özellikle patojenik kirleticiler, algılayabilir gibi konvansiyonel bakteri algılama yöntemleri, bunlar geniş numune hazırlama ve uzun bekleme süreleri gerektirir. Ayrıca, bu uygulamaları talep gelişmiş Laboratuvar Cihazları ve ayarları ve eğitimli profesyoneller tarafından yürütülmelidir. Burada, bir protokol manyetik ve floresan parametreleri nanoparçacık tabanlı bir platform, eşsiz kombinasyonu özellikleri basit bir tanı tekniği için önerilmiştir. Önerilen multiparametric Manyeto-floresan nanosensors (MFnS) E. coli O157: H7 kirlenme olduğu kadar az 1 adet az 1 h çözümünüzde mevcut koloni oluşturan ile algılayabilir. Ayrıca, MFnS gibi karmaşık medyada son derece işlevsel kalır yeteneği süt ve göl su doğrulanmadı. Ek özgüllük deneyleri de MFnS yalnızca belirli hedef bakteri, hatta huzurunda benzer bakteriyel tür algılama yeteneğini göstermek için kullanılmıştır. Manyetik ve floresan yöntemleri eşleştirme algılama ve patojen bulaşma her iki erken ve geç-aşamalı kirlenme algılama içinde yüksek performans sergileyen konsantrasyonları, geniş bir alanda miktar sağlar. Etkinliği, uygun fiyatta ve MFnS taşınabilirliği için bakım noktası tarama bakteriyel kirletici için ideal bir aday ayarları, geniş bir ticari olarak paketlenmiş gıda su depoları dan onları.

MFnS etki mekanizması şekil 1' de temsil edilir. MFnS bakteriyel kirletici yüzeyi kümeleme MFnS magnetic cores ve çevresindeki hidrojen çekirdekleri arasındaki etkileşimler etkilemektedir. Bu kümeleme, manyetik gevşeme sonucu olarak değerlerini artırmak. Bakteriyel kirletici konsantrasyonu arttıkça, kümeleme azaltır ve T2 değerleri değişikliği azalır. Bu nedenle, bir floresan modality eklenmesi önemlidir. Bakteri konsantrasyonu arttıkça, her iki düşük ve yüksek ko...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Hızlı algılama özellikle patojenik bakterilerin manyetik gevşeme ve floresan emisyon modaliteleri kombinasyonu ile hedef ve bu protokol genel amacı, düşük maliyetli, taşınabilir için fonksiyonel nanosensors sentez etmektir.

Gıda kaynaklı patojen tarama kullanarak Manyeto-floresan Nanosensor: E. Coli O157: H7 hızlı tespiti

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K Görüntüleme.  Pittsburg State University. Hızlı algılama özellikle patojenik bakterilerin manyetik gevşeme ve floresan emisyon modaliteleri kombinasyonu ile hedef ve bu protokol genel amacı, düşük maliyetli, taşınabilir için fonksiyonel nanosensors sentez etmektir.

Video: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Flourogenik DNAzymes kullanarak Bakterilerin Tespiti

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

İmmünoloji ve Enfeksiyon

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

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 model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Optik Algılama<em&gt; E. coli</emMezopor Silikon Biyosensörlerin tarafından&gt; Bakteri

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 ...

Biyomühendislik

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

İşlevselleştirilmiş Manyetik Nano partiküllerin sentezi, Siderofor Feroksamin ile Konjugasyonu ve Bakteri Tespiti Için Değerlendirilmesi

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Kimya

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Biyolojik Tahliller Manyetik Modülasyon Biyoalgılayıcı Sistemi kullanarak hızlı Homojen Algılama

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biyoloji

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Gıda kaynaklı patojen tespit etmek için bir elektrokimyasal DNA biyoalgılayıcı gelişimi

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors için noninvaziv tanı koyma proteaz aktivitesi Vivo tespit

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Escherichia coli'nin Sürekli Akış-PCR Mikroakışkan Çipinde Amplifikasyonu ve Kapiler Elektroforez Sistemi ile Tespiti

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Live Algılama<em&gt; Escherichia coli</em&gt; O157: PMA-qPCR H7 Hücreler

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

Turnusol Testi kullanarak Bakterilerin Kolorimetrik Tespiti

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Gıda kaynaklı patojen tarama kullanarak Manyeto-floresan Nanosensor: E. Coli O157: H7 hızlı tespiti

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