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

이 작품은 K-INBRE P20GM103418, 캔자스 콩 위원회 (KSC/PSU 1663), ACS PRF 56629-UNI7와 PSU 폴리머 화학 시작 기금, SS에 모두에 의해 지원 됩니다. 우리는 비디오와 그의 뛰어난 작품에 대 한 대학 동영상을, 씨 야 곱 Anselmi, 감사합니다. 우리는 또한 감사 로저 주었는지 씨와 부인 Katha 주었는지 연구에 대 한 그들의 관대 한 지원에 대 한.

1. 합성 및 다중 파라미터 자기 형광 Nanosensors (MFnS)의 기능화.
<ol><li>합성 superparamagnetic 산화 철 나노 입자 (IONPs) <ol>IONP 합성에 대 한 준비가 <li>준비 다음 3 솔루션: 솔루션 1: FeCl 3 (0.70 g) FeCl 2에서 H 2 O (2 mL), 솔루션 2: NH 4 오 (2.0 mL, 13.4 M) H 2 O (15 mL), 및 솔루션 3: polyacrylic 산 (0.855 g) H 2 O (5 mL).</li>
		<li>는 솔루션 1 2 M 염 산 (HCl)의 90 µ L을 추가 하 고 즉시 875 rpm에서 vortexing 동안 솔루션 2와 혼합. 그런 다음 추가 솔루션 3. 60 분에 대 한 vortexing 계속</li>
		<li>1,620 x g.에서 20 분 동안 원심 분리기 원심 2880 x g.에서 20 분 동안 상쾌한</li>
		<li>정화 투 통해 최종 상쾌한. 투 석의 가방에는 상쾌한 추가 (MWCO 6 &#8722; 8 K) 버퍼링 하는 인산 염 (PBS)를 포함 하는 비 커에 (pH = 7.4)와 스핀 바. 모든 2 3 h. 신선한 버퍼 교체 12 h 동안 담가 보자</li>
	</ol></li> <li>IONPs의 표면에 항 체 특정 대상의 활용 <ol><li>준비 하는 다음 4 개의 솔루션: 솔루션 a: 5 mg 1-에틸-3-(3-dimethylaminopropyl) carbodiimide 염 산 염 (EDC) 250 µ L MES에에서 [2-(N- morpholino) ethanesulfonic 산] 버퍼 (0.1 m M, pH 6.8 =), MES 버퍼의 250 µ L에서 솔루션 b: 3 mg N Hydroxysuccinimide (NHS)의 (0.1 m M, pH 6.8 =), 솔루션 c: 5 µ g IgG1, 대장균 mAb의 인산 염 버퍼 식 염 수 (PBS) (pH 7.4)의 225 µ L의 그리고 IONP의 솔루션 d: 4 mL (5.0 m m o l) 1 ml의 PBS (pH 7.4).</li>
		<li>추가 2-(N-Morpholino) ethanesulfonic 산 (MES) 솔루션, 버퍼 즉시 추가할 솔루션 솔루션 D 15에서 작은 단위로 혼합 각 추가 후에 해결책을 반전 하는 s.</li>
		<li>즉시 3 분 동안 조금씩에서 솔루션 d 솔루션 B를 추가 하기 시작 합니다. 각 추가 후에 해결책을 반전 하 여 믹스.</li>
		<li>솔루션 혼합 각 추가 후 반전 5 µ L의 단위로 솔루션 솔루션 C 추가.</li>
		<li>반응 실 온에서 3 h 동안 계속 계속는 외피 4 ° C에서 하룻밤 허용.</li>
		<li>정화 PBS를 사용 하 여 자기 열 통해 결과 Ab 활용 된 IONPs (pH 7.4, 최종 [Fe] = = 3.5 mmol) 어떤 결합형된 항 체를 제거 하.
		<li>PBS (1 mL)와 자기 열 세척 <ol>. 자기 접시에 열을 연결 하 고 IONP 해결책을 추가 하십시오.</li>
			<li>다시 PBS (1 mL)와 자기 열 세척. 자석 접시에서 자기 열을 제거 합니다. PBS는 열을 추가 하 고 솔루션을 수집 합니다. 4 ° C에서 저장소</li></ol></li></ol></li> <li>형광등 1, 1의 캡슐 &#39;-Dioctadecyl-3,3,3 &#39; 3, &#39;-Tetramethylindocarbocyanine 과염소산염 (DiI) 염료를 사용 하 여 항 체 활용 된 IONPs의 polyacrylic 산 (PAA) 코팅에는 용 매 확산 방법입니다.
	<ol><li>Dropwise 2.0 추가 IONPs의 4 ml, DiI 염료 (2 mmol) 1100 rpm에서 연속 혼합과 DMSO의 100 µ L의 µ L.</li>
		<li>PBS, [철]의 최종 철 농도와 최종 MFnS 솔루션을 만들기에 대 한 결과 솔루션 (MWCO 6-8 KDa, 12 h) dialyze = 2 mmol.</li>
	</ol></li> <li>특성의 MFnS <ol><li>spectrophotometric 분석를 사용 하 여 플레이트 리더 595에서 형광 방출으로 캡슐화 된 DiI 염료 검출 nM.</li>
		<li>동적 빛 산란 <ol><li>장소 샘플 MFnS 솔루션의 평균 크기는 MFnS의 표면 충전 확인 하 zetasizer. 
			참고: 여기, 평균 크기와 기능 MFnS의 표면 충전 했다 발견 77.09 및-22.3 mV, 각각.</li>
		</ol></li></ol></li></ol> 2. 세균성 Culturing 및 재고 솔루션 준비 <ol><li>세균 배양 <ol><li>영양 국물의 1 mL에 동결된 펠 릿 (대장균 O157:H7)를 수 화와 국물의 추가 5 mL를 추가 합니다.</li>
		<li>한 천 배지 및 행진을이 솔루션의 적용 100 µ L 24 h. 위해 37 ° C에 외피 다음 살 균 접종 루프를 사용 하 여</li>
		<li>후 24 h, 한 천 배지에서 고립 된 식민지 선택한 문화 국물의 15 mL를 추가 합니다. 광학 밀도 값을 모니터링 하는 동안 4-6 h 37 ° C에서 품 어 (흡 광도 600 nm).</li>
		<li>0.1의 광학 밀도 값을 얻은 배양, 중지 하 고 수행 하는 직렬 희석.</li>
	</ol></li> <li>직렬 희석 <ol><li>8 살 균 microcentrifuge 튜브 영양 국물의 추가 900 µ L.</li>
		<li>첫 번째 튜브 (희석 10 -1)에 세균 현 탁 액의 추가 100 µ L.</li>
		<li>걸릴 100 µ L 처음부터 튜브와 10 -2의 희석을 얻기 위해 두 번째 그것을 추가 합니다. 10 -8의 마지막 희석 끝나는 나머지 튜브에 대 한이 패턴을 계속.</li>
		<li>전송 100 µ L에서 각 튜브를 분리 한 천 배지, 그리고 37에서 24 h에 대 한 품 어 ° c.</li>
		<li>다음 날, 각 접시에 단위 (CFUs)를 형성 하는 식민지를 계산 합니다. ~ 100 CFUs와 플레이트를 선택 합니다. 해당 희석을 사용 하 여 추가 실험을 위한 (100 µ L = 100 ~ CFUs). 
		참고: 여기 그것은 귀착되 었 다 ~ 100 CFUs 10 -6 희석.</li>
	</ol></li></ol> 3. MFnS를 사용 하 여 급속 한 탐지의 E. 대장균 O157:H7 <ol><li>스파이크 1-100에서 CFU 범위 결과 10 -6 세균성 주식 양의 증가 함께 다양 한 PBS 솔루션 (1 X, pH 7.4, 300 µ L). MFnS의 일관 된 금액을 추가 (100 µ L) 이러한 솔루션을.</li>
	<li>PBS (1 X, pH 7.4, 300 µ L)만 포함 한 초기 솔루션 및 MFnS (100 µ L).</li>
	<li>37 ° C에서 30 분에 대 한 솔루션을 품 어 하 고 실내 온도에 냉각 하도록 허용 다음.</li>
	<li>자석 relaxometer (0.47 테슬라)와 휴식 시간 (T2) 각 솔루션에는 CFUs에 상대적인 레코드 변경 전송 개별 솔루션.
	<li>T2 값에 변화를 기록, PBS와 MFnS 포함 된 기준 솔루션의 T2 값을 측정 하 여 시작 <ol>.
		<li>장소는 자석에서 솔루션 <ol>relaxometer. 열고 해당 소프트웨어를 선택 합니다 &#34; T2 이완 &#34; 설정 및 프레스 &#34; 측정. &#34;</li></ol></li> <li>T2 값 추가 솔루션을 다양 한 아군은의 측정 다음 T2, 기준선의 컬렉션 박테리아의 농도. 
		참고: t 2에서 변화는 초기 아군 t2에서 감 t 2.</li>
	</ol></li> <li>자석 relaxometer에서 샘플을 제거 하 고 10 분을 위한 2880 x g에서 튜브 원심</li>
	<li>는 상쾌한 가만히 따르다와 100 µ L의 PBS (1 X, pH 7.4)에 세균성 펠 릿 resuspend.</li>
	<li>추가 80 µ L 96 잘 접시와 595에서 레코드 형광 강도를 각 물의 resuspension의 nM. 
	참고: 테스트 반복 될 수 있다 결과 섹션에 설명 된 대로 호수 물, 우유 등을 포함 한 다른 용 매를 사용 하 여</li></ol>

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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=Synergistic+Photothermal+and+Antibiotic+Killing+of+Biofilm-Associated+Staphylococcus+aureus+Using+Targeted+Antibiotic-Loaded+Gold+Nanoconstructs.">Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs.</a> ACS Infect Dis. 2 (4), 241-250 (2016).</li><li>Wang, Y., Ye, Z., Si, C., Ying, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtractive+inhibition+assay+for+the+detection+of+E.+coli+O157:H7+using+surface+plasmon+resonance.">Subtractive inhibition assay for the detection of E. coli O157:H7 using surface plasmon resonance.</a> Sensors (Basel). 11 (3), 2728-2739 (2011).</li><li>Zhao, X., 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=A+rapid+bioassay+for+single+bacterial+cell+quantitation+using+bioconjugated+nanoparticles.">A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles.</a> Proc Natl Acad Sci U S A. 101 (42), 15027-15032 (2004).</li></ol>

이 나노기술의 검증 된 응용 프로그램은 아직 FDA에 의해 승인 되지.

이 프로토콜 단순히 완전 한 기능 MFnS을 생산 하도록 설계 되었습니다 가능한. 그러나, 많은 핵심 포인트를 변경 하는 프로토콜의 사용자의 최종 목표에 따라 유용할 수 있습니다 있다. 예를 들어 다른 항 체를 사용 하 여 다른 많은 병원 체의 허용 것 이다. 또한,이 프로토콜 분자를 대상으로 항 체의 사용에 국한 되지 않습니다. 호스트 세포 수용 체, 등 대상 병원 체에 대 ?...

세균성 오염 둘 다의 지속적인 발생 상업적 식품 생산과 물 소스 점점 더 신속 하 고 구체적인 진단 플랫폼에 대 한 필요를 창조 했다. 1 , 2 일반적인 세균성 오염 물질 음식 및 물 오염에 대 한 책임의 일부는 살 모 넬 라, 황색, Listeria, 비 브리 오, 이질균, 균, 그리고 대장균 속에서 있습니다. 3 , 4 세균성 오염이 균에 의해 종종 열, 콜레라, 위장염, 설사 등 증상이 발생합니다. 4 오염 물 소스의 자주 충분히 필터링 된 물에 접근 없이 지역 사회에 크게 불리 한 효과 있으며 식품 오염 많은 질병 및 제품 회수 노력을 주도하 고 있다. 5 , 6
세균 오염으로 인 한 질병의 발생을 줄이기 위해 수는 물과 음식을 검색 될 수 있으며 효율적으로 이전 판매 또는 소비 하는 방법을 개발 하는 노력 되었습니다. 3 1,7,8,,910 ELISA,11,12 루프 중재 등온 증폭 (PCR 등 기법 램프),13,14 ,15,,1617,18,19,20,21, 22,,2324 최근 다양 한 병원 균의 검출에 대 한 사용 되었습니다. 전통적인 세균 배양 방법에 비해, 이러한 기술은 특이성 및 시간에 관하여 훨씬 더 효율적입니다. 그러나, 이러한 기술은 여전히 투쟁 가양성 및 원판, 복잡 한 절차 및 비용. 1 , 3 , 25 그것 세균 검출에 대 한 대체 방법으로 제안 하는 multiparametric 마그네틱 형광 nanosensors (MFnS)이 바로 그 이유입니다.
이러한 nanosensors 고유 쌍을 함께 자석 및 형광 modalities, 신속 하 고 정확한 이중 탐지 플랫폼에 대 한 허용. 샘플 오염으로 대장균 O157:H7를 사용 하 여, 분 이내 작은 1 CFU를 검색 하는 MFnS의 기능 설명 했다. 병원 체 특정 항 체 특이성, 증가 하는 데 사용 되 고 자석 및 형광 modalities의 조합 탐지 및 정량화 모두 낮은 높은 오염 범위에서 세균 오염 물질의 허용 합니다. 16 세균 오염에의 경우는 nanosensors 박테리아 병원 체 특정 항 체의 대상 능력 때문에 주위 메뚜기 것 이다. 마그네틱 nanosensors 및 박테리아 사이의 바인딩 자석 철 코어와 주변의 물 양성자 간의 상호 작용을 제한합니다. 그러면 증가 T2 이완 시간에 자석 relaxometer를 기준으로 기록 됩니다. 솔루션에는 박테리아의 농도 상승으로 nanosensors 박테리아, t 2 값의 결과로의 증가 수와 분산. 반대로, 형광 방출 박테리아, 병원 체에 직접 바인딩된 nanosensors의 증가 수의 농도와 비례하여에서 증가 합니다. 샘플, 원심 분리 및 절연 세균 펠 릿의 나노 박테리아, 어떤 부동성 nanosensors를 제거 하 고 직접 상호 연결의 수와 형광 방출에 직접 연결을 절약만 것입니다. 솔루션에 있는 박테리아. 이 메커니즘의 도식 대표는 그림 1에 표시 됩니다.
이 MFnS 플랫폼은 마음, 낮은-비용 및 휴대용 특성에 따른 포인트의 케어 상영과 함께 설계 되었습니다. MFnS, 실내 온도에 안정 하 고 세균 오염 물질의 정확한 탐지에 대 한 매우 낮은 농도에서 필요만. 또한, 합성, 후는 MFnS의 사용은 간단 하 고 분야에서 훈련 된 전문가의 사용을 필요로 하지 않습니다. 마지막으로,이 진단 플랫폼 고도로 사용자 정의 대상으로,이 의해 하나의 플랫폼 사용할 수 있습니다 많은 다른 설정에서 모든 종류의 병원 체를 검출 하는 수단을 제공 가능 합니다.

<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

둘 다 waterborne Enterohemorrhagic 대장균 O157:H7 연결 되었습니다 foodborne 병, 및 음식 및 물 심사 방법에도 불구 하 고 위협 사용 현재 남아 있다. 일반 세균 검출 방법, 동안과 같은 연쇄 반응 (PCR) 및 효소 연결 된 immunosorbent 분석 실험 (ELISA) 특히 병원 성 오염 물질을 감지할 수 있습니다 그들은 필요 광범위 한 샘플 준비 및 긴 대기 기간. 또한, 이러한 관행 정교한 실험실 계기 및 설정, 요구 하 고 훈련 된 전문가 의해 실행 되어야 합니다. 여기에, 프로토콜은 나노 기반 플랫폼에 자석 및 형광 매개 변수의 독특한 조합을 제공 하는 간단 하 게 진단 기술에 대 한 제안. 제안 된 multiparametric 마그네틱 형광 nanosensors (MFnS) 대장균 O157:H7 오염 조금 1 식민지 형성 장치 솔루션 미만 1 시간 내에 검색할 수 있습니다. 또한, MFnS의 능력을 유지와 같은 매우 복잡 한 미디어 기능을 우유와 호수 물 확인 되었습니다. 또한 추가 특이성 분석만 특정 대상 박테리아도 비슷한 세균성 종의 존재를 감지 하는 MFnS의 능력을 보여 주기 위해 사용 되었다. 자석 및 형광 modalities의 페어링 검색 및 병원 체 오염 농도, 모두와 늦은-초기 오염 탐지에 그것의 고성능 전시의 넓은 범위에서의 정량화에 대 한 수 있습니다. 효율성, 경제성, 그리고는 MFnS의 이동성 있도록 세균성 오염 물질에 대 한 포인트의 케어 심사를 위한 이상적인 후보자 설정의 넓은 범위에서 상업적으로 포장된 식품을 수생 저수지에서.

MFnS 행동의 메커니즘은 그림 1에 표시 됩니다. 세균 오염 물질의 표면 주위 MFnS의 클러스터링 방해는 MFnS의 magnetic cores와 주변 수소 핵 사이 상호 작용. 이 클러스터링, 자기 휴식의 결과로 값이 증가합니다. 세균 오염 물질의 농도 증가, 클러스터링, 줄이고 T2 값에 변화를 감소. 따라서, 형광 적임의 결정적 이다. 세균성 농도 증가, MFnS에 의해 생산 하는 형광 신호 증가, 모두 ?...

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

이 프로토콜의 전반적인 목표는 비용 효율적인, 노트북에 대 한 기능 nanosensors을 합성 하 고의 급속 한 탐지는 자석 및 형광 방출 modalities의 결합을 통해 병원 성 박테리아에 특히 표적으로.

E. 콜라이 O157:H7의 Foodborne 병원 체 검사를 사용 하 여 자기 형광 Nanosensor: 신속한 감지

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 조회수.  Pittsburg State University. 이 프로토콜의 전반적인 목표는 비용 효율적인, 노트북에 대 한 기능 nanosensors을 합성 하 고의 급속 한 탐지는 자석 및 형광 방출 modalities의 결합을 통해 병원 성 박테리아에 특히 표적으로.

비디오: 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).

형광 DNAzymes을 사용하여 박테리아의 탐지

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

연구

면역학 및 감염병학

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).

의 광학 검출<em&gt; E. 대장균</em메조 포러스 실리콘 바이오 센서에 의해&gt; 박테리아

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 ...

생체공학

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.

기능성 자기 나노 입자의 합성, 사이드로포레 페록사민과의 그들의 융합 및 박테리아 검출에 대한 평가

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

화학

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.

자기 변조 Biosensing 시스템을 사용하여 생물 학적 Assays의 급속한 동차 검색

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

생물학

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.

Foodborne 병원 체를 검출 하는 전기 화학 DNA 바이오 센서의 개발

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 프로 테아 제 활동에 감지 비보에 비 침 투 적인 진단에 대 한

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.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

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.

연속 유동 PCR 미세유체 칩에서 대장균 의 증폭 및 모세관 전기영동 시스템을 사용한 검출

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

라이브의 검출<em&gt; 대장균</em&gt; O157 : PMA-qPCR에 의한 H7 셀

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.

리트머스 테스트를 사용하여 박테리아의 비색 검출

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. ...

E. 콜라이 O157:H7의 Foodborne 병원 체 검사를 사용 하 여 자기 형광 Nanosensor: 신속한 감지

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