여기에보고 된 연구는 오번 대학교 AUDFS 및 USAF CRADA 07-277 - 60MDG-01에서 교부금에 의해 지원되었다. 이 문서에 표현 된 뷰는 저자의 사람이며, 공식적인 정책이나 미국 공군, 국방부, 또는 미국 정부의 입장을 반영하지 않습니다.

1. 스테이지 설정 <ol><li> 형 균주 S.를 얻을 구균 ATCC 12600, S. 구균 ATCC의 27690과 바실러스 서브 틸리 스 ATCC는 6051입니다. S.의 메티 실린 내성 구균 - MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis의 Sterne 다음, 살모넬라 티피 뮤 리움 LT2, 시겔 flexneri, 예 르시 니아 enterocolotica, 프로 테우스 mirabilis의, 폐렴 간균 13882, 용해성 파지 12600. </li><li> PBP 2a에 항체 결합 라텍스 구슬을 구하십시오. </li><li> 21 설명 된대로 NZY 매체를 준비합니다. </li><li> 인산를 얻기는 생리 식염수 (PBS)를 버퍼. </li><li> 랭 뮤어 - 블로 젯 (LB) 단층 증착 subphase로 증류수를 준비합니다. </li></ol> 2. 박테리오파지 전파 및 적정 <ol><li> S.의 하룻밤 문화 5 mL를 품다 파지의 500 μL (3 구균 ATCC 12600 (3.6 × 10 8 콜로니 형성 단위 (CFU) / ㎖) × 10 9 &lt;/ 37 ° C에서 하룻밤 쉐이커 인큐베이터에 2 L 플랙 500 ML의 NZY 매체에)&gt; PFU / ㎖을 먹다. </li><li> 4시 10 분 4,424 XG에 문화를 원심 분리기 ° C. </li><li> 4 10 분 11,325 XG에서 다시 원심 분리 상층 ° C. </li><li> 0.22 μm의 밀리 포아 필터를 통해 필터 상층 액. </li><li> 1.5 시간 동안 75,395 XG에서 여과 액을 원심 분리기. </li><li> 증류수 100 μL에 펠렛을 녹인다. </li><li> NZY 매체에 파지 현탁액의 10 배 희석을 준비합니다. </li><li> S.의 하룻밤 (ON) 문화 판 1 ML 모든 표면이 포함되어 있는지 확인하는 NZY 한천 2 플레이트의 각 상 구균 ATCC 12600을. 문화의 과잉을 제거하고 30 분 동안 표면을 건조 할 수 있습니다. </li><li> 판의 표면에 적절한 파지 희석 시료 (10 μL)을 발견하고 18-24 시간 동안 37 ℃에서 배양한다. </li><li> 플라크의 형성을 검토하고 파지 역가를 계산합니다. 살균 역가 (T)가 P의 수에 기초하여 추정laques (N)는 파지 서스펜션과 희석 계수 (F) 10 μL 부피 (V) 당 형성했다. . T는 = N / (VXF) 예를 들어, 희석 10-7에서 플라크의 수를 75 부피 10 μL (10 × 10 -3 ML)의 경우, 역가가 75 / 같습니다 : 역가가 공식으로 계산 하였다 (10 × 10 -3 × 10 -7) = 7.5 × 10 10 플라크 형성 단위 (PFU) 당 ML 정지. </li></ol> 3. 금 고정 파지 <ol><li> 다음 10 분 동안 아르곤 에칭 플라즈마에 의한 깨끗한 골드 코팅 된 석영 조각 (60mm 2) 살균 캐비닛에 UV 빛의 밑에 6 시간 동안 살균. </li><li> 멸균 페트리 접시에있는 각 조각의 금 표면에 1 × 10 9 PFU / ㎖ 파지 현탁액의 50 μl를 추가하고 실온에서 습기 챔버에서 하룻밤 배양한다. </li><li> 나머지 파지 현탁액을 제거하고 역가. </li><li> 언 바운드를 제거하는 PBS로 파지와 조각 5 번 씻으십시오파지. </li></ol> 4. 건조한 표면에 고정 파지 감염성 테스트 <ol><li> NZY 한천 배지에 S. 구균 ATCC 12600의 O / N 문화를 확산하고 건조 할 수 있습니다. </li><li> 플레이트 얼굴에 고정 파지와 금 조각을 아래로 놓습니다. </li><li> 37 12 시간 배양 한 후 용해의 영역을 관찰 ° C 그 감염을 나타냅니다. </li><li> 제어 실험없이 파지와 골드 커버 조각을 사용합니다. </li></ol> 5. 액체의 무료와 바인딩 파지의 용해성 활동의 테스트 <ol><li> S.의 ON 문화 추가 네 300 ML 사이드 암 플라스크의 모든 (6 × 10 6 CFU / 각 플라스크에 ml)에 NZY의 구균 ATCC 12600-10 ML. </li><li> 두 개의 플라스크 2 × 10 6 PFU / 무료 파지의 ML에 추가합니다. </li><li> 컨트롤과 같은 다른 두 플라스크를 사용합니다. </li><li> S.의 시간 경과를 모니터링 모든 플라스크 30 분 간격으로 광학 밀도 측정 (OD 600)의 570 분 구균 세포 용해.</li><li> 24 시간에서 최종 측정을 가져 가라. </li><li> 단계 5.1-5.5를 반복하지만, 제어 플라스크 없음 파지 골드 바운드 파지 대신 무료로 파지와 조각, 골드 조각을 사용합니다. </li></ol> 6. 부화하는 동안 바인딩 파지 손실 <ol><li> 실온에서 하룻밤 습도 챔버에 배치됩니다 페트리 접시에 무균 금 조각의 표면에 1 × 10 9 PFU / ㎖ 파지의 50 μl를 추가합니다. </li><li> 금 표면과 역가에서 언 바운드 파지와 서스펜션을 제거합니다. </li><li> 8 시간 동안 37 ° C에서 5 PBS로 시간과 장소 쉐이커 배양기에서 15 ML 튜브 1 ML의 NZY 솔루션 바운드 파지와 금 조각을 씻으십시오. </li><li> 2에 설명 된대로 분리 된 파지의 역가를 결정합니다. </li></ol> 7. 파지 상체 준비 <ol><li> 1 ML 유리 병의 분광 등급 클로로포름의 동일한 볼륨 스톡 파지 12600 서스펜션 (10 11 PFU / ㎖)의 400 μl를 결합실온에서. </li><li> 부드럽게 소용돌이 5-6 시간 (5 초 간격) 일분 기간 동안 파지 - 클로로포름 서스펜션. </li><li> 30 초 동안 안정하고 상체는 단층 준비를 위해 전원 피펫되었다가 포함 된 상위 단계의 피펫 현탁액을 허용했다. </li><li> 전송 및 주사 전자 현미경 상체 현탁액의 몇 마이크로 리터를 가져 가라. </li><li> 바이오 센서의 제조에 남아있는 회전 타원체 현탁액을 사용합니다. </li></ol> 8. 바이오 센서 제조 <ol><li> 10 분 PDC-32G Harrick 플라즈마 클리너 아르곤 플라즈마 에칭에 의한 청소 QCM-D 센서. </li><li> 모든 유기 불순물을 제거하는 헥산으로 센서를 씻어. </li><li> 준비하고 22에 설명 된 필름의 균형 구유를 청소하십시오. </li><li> subphase 솔루션 LB 트로프를 입력하고 여물통 장벽을 청소하고 20 통을 안정화 ± 0.1 ° C </li><li> 수성 suspensi에있는 파지 12600 300 μL 나누어지는 확산LB의 subphase 상 (10 11 PFU / ㎖)에. </li><li> 부분적으로 subphase 22,23으로 침수되는 경사 적실 유리 막대를 실행하는 파지 12600 현탁액 300 μL 나누어지는 (10 11 PFU / ㎖)함으로써 LB의 subphase 솔루션에 파지 단층을 준비합니다. </li><li> 10 분 동안 준비된 단일 층을 안정화하고 일정한 압력까지 19 N / m를 달성되어, 30mm / 분 (45cm 2 / 분)의 비율로 압축합니다. </li><li> 연속적으로 단층의 일곱 번 센서에서 찍기에 의해 밖으로 4.5 mm / min의 속도로 수직으로 QCM-D 센서에 수직 증착을 수행합니다. 박테리아 샘플에 노출 레이어 (데이터 표시되지 않음), 연속 균질하고 균일 한 것으로 나타났다 전에 전자 현미경으로는 파지 단일 층을 증착. </li><li> 반복 3 준비 파지 구형 서스펜션 4.5-4.8 단계를 반복합니다. </li><li> MRSA 검출, 전자에 대한 제작 파지 바이오 센서를 사용하여현미경, 및 타원. </li></ol> 9. 바이오 센서 테스트, 메티 실린 저항의 차별과 민감한 황색 포도상 구균 박테리아 <ol><li> 이 프로토콜에서는 수정되지 않은 수정 된 파지 및 파지 상체와 바이오 센서는 준비가되어 있습니다. 네 개의 센서 유량 용기를 가진 석영 크리스탈 마이크로는 QCM 센서의 표면에 고정 파지에 박테리아의 바인딩을 모니터링하는 데 사용됩니다. PBP 2a에 항체 결합 라텍스 구슬 메티 실린 내성 (MRSA) 및 민감한 황색 포도상 구균 박테리아를 구별하기 위하여 이용된다. 모든 측정은 5 MHz에서 유동 모드 (50 μL / 분)에서 실시하고 있습니다. </li><li> 물 (~ 30 분)에 QCM 센서의 기본 라인 공진 주파수를 설정합니다. </li><li> 테스트 셀을 통해 물에 살고있는 메티 실린 민감 황색 포도상 구균 박테리아 세포 (10 9 CFU / ㎖)의 현탁액을 그립니다. </li><li> 지속적으로 최초의 배음, USI에 대한 센서의 공진 주파수와 에너지 소모의 변화를 모니터링NG는 Q-소프트 소프트웨어입니다. </li><li> 센서의 공진 주파수와 손실의 변화가 포화 수준에 도달 할 때, 주파수와 소비의 지속적인 모니터링 중에 흐름 PBP 항체 결합 라텍스 구슬 (6.77 배 10 비즈 / ML)의 정지를 추가합니다. </li><li> 반복 메티 실린 내성 포도상 구균 박테리아 세포 (MRSA)에 대한 9.2-9.5 단계를 반복합니다. </li><li> C는 상수 (C = 17.7 NG X cm -2 X Hz에서 -1 5 MHz의 질량 감도 Sauerbrey 식 24,25 Δf에 =-ΔM XN / C에 의해 바인딩 세균에 의한 질량 변화를 통해 주파수 변화를 정의 ), M은 질량 n은 배음의 수 (N = 1)입니다. </li><li> 기록에서 에너지 소비의 변화 (Δ D)를 정의 진동의 지수 감쇠 (주파수와 진폭 진동의 한주기 동안 소비 및 저장 에너지의 정량화를 허용하는 완충,각각 E는 소비와 E가 저장 : Δ D = E 소산 / 2 πE가 저장됩니다. Δ D는 분산 단위 (DU), 하나의 DU = 10 -6 상대 단위)로 측정됩니다. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+shape+determination+by+the+Bacillus+subtilis+class+B+penicillin-binding+proteins+encoded+by+pbpA+and+pbpH.">Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH.</a> J. Bacteriol. 185, 4717-4726 (2003).</li><li>Grieco, S. H. H., Lee, S., Dunbar, W. S., MacGillivray, R. T. A., Curtis, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+filamentous+phage+yield+during+computer-controlled+fermentation.">Maximizing filamentous phage yield during computer-controlled fermentation.</a> Bioprocess and Biosystems Engineering. 32, 773-779 (2009).</li><li>Olsen, E. V., Pathirana, S. T., Samoylov, A. M., Barbaree, J. M., Chin, B. A., Neely, W. C., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+selective+biosensor+for+Salmonella+and+its+detection+in+the+environment.">Specific and selective biosensor for Salmonella and its detection in the environment.</a> J. Microbiol. Methods. 53, 273-285 (2003).</li><li>Pathirana, S. T., Barbaree, J., Chin, B. A., Hartell, M. G., Neely, W. C., Vodyanoy, V. <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+sensitive+biosensor+for+Salmonella.">Rapid and sensitive biosensor for Salmonella.</a> Biosens. Bioelectron. 15, 135-141 (2000).</li><li>Sauerbrey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+quartz+oscillators+for+weighing+thin+layers+and+for+microweighing.">The use of quartz oscillators for weighing thin layers and for microweighing.</a> Z. Phys. 155, 206-222 (1959).</li><li>Hook, F., Rodahl, M., Brzezinski, P., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+Dissipation+Kinetics+for+Protein+and+Antibody-Antigen+Adsorption+under+Shear+Oscillation+on+a+Quartz+Crystal+Microbalance.">Energy Dissipation Kinetics for Protein and Antibody-Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance.</a> Langmuir. 14, 729-734 (1998).</li><li>Griffith, J., Manning, M., Dunn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+bacteriophage+contract+into+hollow+spherical+particles+upon+exposure+to+a+chloroform-water+interface.">Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform-water interface.</a> Cell. 23, 747-753 (1981).</li><li>Hosseinidoust, Z., Van de Ven, T. 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V., Jonson, M., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+mass"+effect+in+biosensor's+QCM+applications.">Missing mass" effect in biosensor's QCM applications.</a> Biosens. Bioelectron. 17, 835-841 (2002).</li><li>Gervals, L., Gel, 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=Immobilization+of+biotinylated+bacteriophages+on+biosensor+surfaces.">Immobilization of biotinylated bacteriophages on biosensor surfaces.</a> Sensors and Actuators. 125, 615-621 (2007).</li><li>Nanduri, V., Sorokulova, I. B., Samoylov, A. M., Simonian, A. L., Petrenko, V. 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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

그것은 잘 파지는 세균 병원체 28 바이오 센서 프로브로 사용할 수있는 것으로 알려져 있습니다. 빠른 차별 항생제 내성 민감한 균주 : 그것은 PBP 2a에 항체과 함께 파지 이전 문제를 해결하기 위해 활용 될 수있는이 작품에서 보여줍니다. 그것은 그러나 이러한 일반적인 수정되지 않은 포도상 구균 파지들이 박테리아를 바인딩에도 불구하고, QCM 장치와 세균 검출에 ?...

황색 포도상 구균의 메티 실린 내성은 필수 감염과 원내 발생 4-8에 영향을 미치는 요인으로 제시되었다. 이러한 디스크를 확산 oxacillin 한천 스크린 테스트, 또는 국물 microdilution으로 메티 실린 저항의 인식의 일반적인 방법은, 저항의 표현을 향상시키기 위해 맞춤형 배양 조건에 의존한다. 변경은 oxacillin의 활용, ° C보다는 37 ° C 이상 30 35 배양 및 성장 매체의 NaCl의 포함이 (가) 있습니다. 또한, 기술의 이러한 유형으로 정확한 탐지를 위해, 24 시간의 긴 잠복기 대신에 16-18 시간의 필요합니다. 메티 실린 저항의 식별을위한 감도 적절한 (&gt; 96 %) 수준의 빠른 기술은 3-11 시간 후 결과를 같은 VITEK GPS-SA 카드, 신속한 ATB 포도상 구균 시스템 및 신속한 Microscan은 패널 시스템으로 자동 microdilution 기술을 포함 9-11. 크리스탈 MRSA ID 시스템은 S.의 성장 인식에 따라 빠른 방법입니다 2 % 염화나트륨과 산소에 민감한 형광 센서 리터 당 oxacillin 4 MG의 존재 구균. 주장 감도는 배양 12-14 4 시간 후에 100-91 % 사이가 다양합니다. 이러한 표현형 방법은 서로 다른 저항을 표현하는 유행 균주의 영향으로 자신의 정확도에 제한됩니다. 따라서 메티 실린 저항의 인식을위한 가장 널리 사용되는 방법은 메카 유전자 15의 PCR 또는 DNA 혼성화입니다. 그러나이 기술은 정제 된 DNA가 필요하며 세포 파편 16 등 각종 혼화제 (불순물)에 매우 민감합니다. 또한, 이러한 기술을 수행하는 데 오랜 시간이 필요합니다. 메카 유전자 제품, 단백질 PBP 2a에의 인식 전략은 저항을 결정하기 위해 활용 될 수 있으며 표준 시험 방법 17에 비해 더 신뢰할 수 있습니다. 1,2,18을 갖는 포함 황색 포도상 구균의 변종에 대한 인식 조사로 활용 될 수 있음을 표시했다. 이 작품에서 우리는 실시간으로 MRSA의 형태와 함께 박테리아의 인식과 같은 MRSA의 특정 인식 및 탐지에있는 새로운 기술을 제안 하였다. 이 특정한 목적 S.에 대한 단백질에 대한 단클론 항체와 결합 된 호스트의 다양한 스펙트럼 (MRSA 균주 포함) 구균 살균은 (PBP 2a에) 사용되었습니다. PBP 2a에는 세포벽 단백질이며 MRSA의 항생제 저항의 원인입니다. 그러나 PBP 2a에 항체는 S.에 대한 구체적인 없습니다 다른 박테리아 이후 구균 PBP 2a에 19,20에 서열의 유사성과 항생제 결합 단백질이 있습니다. 따라서 본 연구에서는 S. PBP 2a에 단백질에 대한 구균 살균 및 항체가 사용되었습니다. specifi에 바이오 센서를 개발 할 수 있으려면,으로 두 단계 동작을 활용 한로 장치를 인식하고 MRSA를 식별합니다. 초기 단계는 S.를 사용 센서 프로브와 같은 구균 살균의 단층, 두 번째 단계는 PBP 2a에 특정 항체를 고용하면서. 따라서 하나 S를 인식 단계 구균 박테리아, 다른 하나 항생제 결합 단백질에 민감 할 것입니다. 두 단계에서 수신 신호가 긍정적 인 경우는 MRSA의 특정 감지를 나타냅니다.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

황색 포도상 구균 균주의 광범위한 호스트 범위와 페니실린 결합 단백질 (PBP 2a에) 항체 결합 라텍스 구슬이 메티 실린 내성 (MRSA)에 민감 (MSSA) S의 차별을 위해 디자인 된 바이오 센서를 만드는 데 활용 된 데 구조적 변화 용해성 살균 . 구균 종 1,2. 용해성 파지는 물에 클로로포름 인터페이스와의 접촉에 의해 파지 상체로 변환되었습니다. 파지 회전 타원체 단층은 랭 뮤어 - 블로 젯 (LB) 기법에 의하여 바이오 센서의 표면에 이동되었습니다. 생성 된 바이오 센서는 박테리아 파지 상호 작용을 평가하기 위해 분산 추적을 가진 석영 크리스탈 마이크로 (QCM-D)로 조사되었다. 박테리아 회전 타원체 상호 작용은 감소 공진 주파수와 MRSA와 MSSA 균주 모두에 대한 분산 에너지 상승했다. 세균 바인딩 한 후,이 센서는 또한 페니실린 결합 단백질 항체 라텍스 비드에 노출 된의. MRSA로 분석 센서는 2A 항체 비즈 PBP에 응답, MSSA에 검사 센서는 응답을 준 없지만. 이 실험 구별은 메티 실린 저항하고 민감한 S. 사이에 명확한 차별을 결정 구균의 변종. 마찬가지로 바운드 및 언 바운드 살균은 표면에 물 현탁액에있는 박테리아의 성장을 억제합니다. 용해성 파지가 상체로 변경되면, 그들은 그들의 강한 용해성 활동을 유지하고 높은 세균 캡처 기능을 보여줍니다. 파지와 파지 상체는 항생제 내성 미생물의 테스트 및 살균을 위해 이용 될 수있다. 다른 응용 프로그램은 박테리오파지 요법과 항균 표면에서의 사용을 포함 할 수 있습니다.

파지는 S.의 시험 균주에 대한 용해성 활동을 보여 로 파지 현장 시험에 의해 표시 MRSA 균주를 포함 구균. 플라크의 크기는 일반적으로 5 ~ 15 mm까지였다. 활동은 다른 테스트 배양 (표 1)에 대해 찾을 수없는 경우. S.의 정상적인 성장 37 쉐이커 인큐베이터에 NZY 매체 구균 ATCC 12600는 ° C는 그림 1A (빈 원으로 표시된 곡선)에 표?...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

용해성 파지 바이오 센서 및 항체 비즈 메티 실린 내성 (MRSA) 및 민감한 황색 포도상 구균 박테리아를 구별 할 수 있습니다. 파지는 석영 크리스탈 마이크로 센서의 표면에 랭 뮤어 - 블로 젯 방식으로 고정 넓은 범위 포도상 구균 프로브로 근무 하였다. 항체 비즈 MRSA를 인식하고 있습니다.

항생제 내성 황색 포도상 구균 박테리아의 검출을위한 바이오 센서

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

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

24.0K 조회수.  Auburn University. 용해성 파지 바이오 센서 및 항체 비즈 메티 실린 내성 (MRSA) 및 민감한 황색 포도상 구균 박테리아를 구별 할 수 있습니다. 파지는 석영 크리스탈 마이크로 센서의 표면에 랭 뮤어 - 블로 젯 방식으로 고정 넓은 범위 포도상 구균 프로브로 근무 하였다. 항체 비즈 MRSA를 인식하고 있습니다.

비디오: Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

microfluidic 비말 플랫폼 형광 검출 방법

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

연구

생체공학

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

높은 이온 강도 솔루션에 감지하기위한 탄소 나노 튜브 고주파 나노 바이오 센서의 제작

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

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 ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

칩에 스트레스 유발 항생제 감수성 테스트

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRET 유동 세포 계측법과 Proteopathic의 시드 활동의 민감한 검출

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

의료 기기에 대한 박테리아의 높은 처리량 식별 구충제 폴리머

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

바이오 상호 작용을 검출 하는 β-lactamase 기반 Conductimetric 바이오 센서 분석 결과의 사용

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

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, ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

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

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

모 세관 배열에 여러 개의 핵 산의 비주얼 검색

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