본 연구는 알레르기 및 전염병 국립 연구소의 협력 계약 상 AI075565 (DAH)로 의해 웬디와 소아 비뇨기과 연구의 우수 켄 루비 기금에 의해 지원되었다. BMC는 주디스와 소아 비뇨기과 로버트 윈스턴 의자입니다.

1. 전기 화학 센서의 작용 <ol><li> 300 μM에서 0.05 μM의 농도에서 thiolated 캡처 프로브를 준비하는 1,6 - hexanedithiol (HDT), 10 MM 트리스 - HCL, pH를 8.0, 0.3 M의 NaCl, 10 분 동안 상온에서 어둠 속에서 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산)과 부화 . HDT와 thiolated 캡처 프로브의 배양 더 일관성있는 결과의 결과로, 포획 프로브의 티올 그룹이 감소되도록합니다. </li><li> 수분 및 / 또는 미립자를 제거하기 위하여 5 초 동안 노출 된 금 16 센서 어레이 칩 (들)에 질소 스트림을 적용합니다. </li><li> 센서 배열의 모든 16 센서의 작동 전극에 HDT-thiolated 캡처 프로브 믹스의 6 μl를 적용하고 4 ° C 하룻밤에 덮여 페트리 접시에 센서 칩 (들)을 저장합니다. Thiolated 캡처 프로브는 벌거 벗은 금 전극에 직접 바인딩 및 HDT는 캡처 프로브의 내용물을 과도을 방지하고 목표 하이브리드을 촉진 확장 된 형태에서 그들을 유지하는 역할을합니다. </li><li>날 따라 5 초 동안 질소의 흐름에 따라 2 ~ 3 초 건조를위한 이온 H 2 O와 센서 칩을 씻는다. </li><li> 10 MM 트리스 - HCL, pH를 8.0, 0.3 M의 NaCl, 1 mM의 EDTA (에틸렌 다이아 민 테트라 초산), 1 mM의 16 개 센서와 50 분 동안 부화의 작업 전극 6 메르 캅토-1-헥산 (MCH)의 6 μl를 적용합니다. 이 이후의 모든 센서 칩에서 incubate은 상온에서 적용 페트리 접시에서 수행됩니다. MCH는 thiolated 캡처 프로브 또는 HDT가 전극 표면에 존재하지 않는 틈새를 채우는 차단제 역할을합니다. </li></ol> 2. 샘플 준비 <ol><li> 5 분 16,000 XG에 microcentrifuge 관 및 원심 분리기 성장의 로그 단계 (≈ 0.1 OD 600)에서 세균성 문화의 1 ML을 전송합니다. 배양 상청액을 제거합니다. 박테리아 펠렛은 즉시 처리 또는 -80 ° C 나중에 사용하기 위해 저장할 수 있습니다. </li><li> 철저 봇에 피펫 팁을 적용하여 1 M NaOH를 10 μL에서 박테리아 펠렛을 resuspend을microcentrifuge 관 및 여러 번 위아래로 피펫의 탐. 5 분 상온에서 현탁액을 품어. </li><li> 1 M 인산 버퍼, 산도 7.2의 50 μl를 추가 형광 - 수정 검출기 프로브의 2.5 % 소 혈청 알부민 (BSA)와 0.25 밀리미터를 포함하여 세균 해물을 중화. 실온에서 10 분 동안 중화 해물을 품어. 형광 - 수정 검출기 프로브는 세균성 rRNA의 대상 분자 잡종. </li></ol> 3. 전기 센서 분석 <ol><li> 5 초 질소의 흐름에 따라 2 ~ 3 초 건조를위한 이온 H 2 O와 센서 칩에서 MCH를 씻으십시오. </li><li> 14 각 센서의 작동 전극에 중화 세균 해물 4 μl를 적용하고 15 분 동안 품어. 대상 검출기 프로브 컴플렉스 고정 thiolated 캡처 프로브에 잡종. </li><li> 2.5 % BSA 및 0.25 &amp;를 포함, 1 M 인산 버퍼, 산도 7.2 올리고 뉴클레오티드를 브리징 1 nm의 4 μl를 적용MU, M 형광 - 수정 검출기 프로브 2 개의 긍정적 인 제어 센서 (신호 정상화를 위해 사용되는) 15 분 동안 부화. </li><li> 5 초 질소의 흐름에 따라 2 ~ 3 초 건조를위한 이온 H 2 O와 센서 칩을 씻으십시오. </li><li> 1 M 인산 버퍼, 산도 7.2에서 0.5 U / ㎖ 방지 형광-HRP 4 μl를 적용 16 개 센서의 작동 전극에 0.5 %의 카제인을 함유하고 15 분 동안 품어. 안티 - 형광-HRP가 고정화 된 형광 - 수정 검출기 프로브에 바인딩합니다. </li><li> 5 초 질소의 흐름에 따라 2 ~ 3 초 건조를위한 이온 H 2 O와 센서 칩을 씻으십시오. </li><li> 센서 칩 마운트에 센서 칩과 부하의 표면에 잘 필름 스티커를 적용합니다. 피펫 50 16 개 센서 위에 TMB 기질 ㎕의 센서와 칩 마운트를 닫습니다. </li><li> 헬리오스 칩 리더를 사용하는 모든 16 센서 전류 측정과 순환 전압 전류 법 측정을 얻습니다. 전류 측정 전류는 TMB R의 속도에 비례센서 표면에 배기관 (그림 1 참조). </li></ol>

<ol>
	<li>Wu, J., Campuzano, S., Halford, C., Haake, D. A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+Surface+Monolayers+for+Ultrasensitive+(Zeptomole)+Amperometric+Detection+of+Nucleic+Acid+Hybridization+without+Signal+Amplification.">Ternary Surface Monolayers for Ultrasensitive (Zeptomole) Amperometric Detection of Nucleic Acid Hybridization without Signal Amplification.</a> Anal. Chem. 82, 8830-8837 (2010).</li><li>Halford, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Antimicrobial+Susceptibility+Testing+by+Sensitive+Detection+of+Precursor+Ribosomal+RNA+Using+a+Novel+Electrochemical+Biosensing+Platform.">Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor Ribosomal RNA Using a Novel Electrochemical Biosensing Platform.</a> Antimicrob. Agents Chemother. 56, (2012).</li><li>Campuzano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ternary+monolayers+as+DNA+recognition+interfaces+for+direct+and+sensitive+electrochemical+detection+in+untreated+clinical+samples.">Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.</a> Biosens. 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Microbiol. 49, 4293-4296 (2011).</li><li>Mastali, 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=Optimal+probe+length+and+target+location+for+electrochemical+detection+of+selected+uropathogens+at+ambient+temperature.">Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature.</a> J. Clin. Microbiol. 46, 2707-2716 (2008).</li><li>Liao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrochemical+DNA+biosensors+for+rapid+molecular+identification+of+uropathogens+in+clinical+urine+specimens.">Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.</a> J. Clin. Microbiol. 44, 561-570 (2006).</li><li>Liao, J. 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A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+disposable+nucleic+acid+biosensors+for+direct+bioelectronic+detection+in+raw+biological+samples.">Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples.</a> Talanta. 85, 1330-1337 (2011).</li><li>Ecker, D. J., 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=Ibis+T5000:+a+universal+biosensor+approach+for+microbiology.">Ibis T5000: a universal biosensor approach for microbiology.</a> Nat. Rev. Microbiol. 6, 553-558 (2008).</li><li>Casalta, J. 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모든 저자는 설명 된 방법에 관련된 특허에 발명자이다. 이들 특허 중 하나가 Qvella 법인 허가되었습니다. BMC와 DAH는 Qvella 회사의 지분이 있습니다. VG는 전기 센서 어레이 칩의 제조 업체입니다 GeneFluidics 회장, 칩 마운트, 그리고이 문서에 설명 된 칩 리더입니다.

여기에 설명 된 전기 센서 분석은 핵산 표적을 신속하게 탐지 할 수 있습니다. 민감도와 특이도가 차례로 길이 캡처 및 검출기 프로브의 GC 내용에 따라 대상 프로브 하이브 리다이 제이션의 자유 에너지에 부분적으로 의존한다. 우리는 일반적으로 주위 온도 (~ 20 ° C) 5, 6에서 하이브리드 단계를 수행하십시오. 그러나 하이브리드 단계 (3.2, 3.3)는 칩이 포함 된 커버 챔버에 배치됩니다 경우...

세균 검출 및 식별을위한 표적 분자로 rRNA를 사용하면 장점을 가지고 있습니다. 박테리아 세포의 rRNA의의 풍부한 대상 증폭 1 필요없이 밀리리터 당 250 박테리아의 낮은 민감도 제한을 제공합니다. 세균성 rRNA의가 DNA 프로브와 하이브리드 액세스 할 수있는 고유 한 종의 특정 시퀀스를 포함하고 있습니다. 따라서, 전기 센서 배열은 각 센서가 다른 종의 특정 캡처 프로브 기능화되어 알 수없는 박테리아를 식별하는 데 사용할 수 있습니다. 양성 대조군 센서는 &quot;다리&quot;캡처 및 검출기 프로브 내부 교정 신호를 생성 할 수있는 합성 올리고 뉴클레오티드 대상에 대해 포함되어야한다. 전기 센서는 기본 및 translational 연구 애플 리케이션의 넓은 범위가 있습니다. 예를 들어, 분석은 정확하게 E.의 효과를 측정하기 위해 여기에 설명 된 사용되었습니다 RRN에서 대장균의 성장 단계세균 생리학 2에 관심있는 연구자들에게 큰 관심입니다 및 사전 rRNA의 사본 번호. 전기 센서 분석의 감도는 노이즈 비율로 신호에 의해 결정됩니다. 신호 증폭 및 소음 감소 방법의 다양한 탐구되었다. 우리는 센서 표면의 화학적 성질을 개선하는 검출기 프로브 및 / 또는 HRP 효소의 비특이적 결합을 줄이는 열쇠는 사실을 알게 될 것입니다. 특히, alkanedithiols 및 mercaptohexanol의 혼합 단층이 대상 하이브리드 3 캡처 프로브의 접근성을 유지하면서 더 완전 전극 표면을 커버하여 배경을 줄이기 위해 발견되었습니다. 이러한 표면 화학 처리는 복잡한 생물 학적 샘플을 포함하는 분석에 특히 중요하다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>6-mercapto-1-hexanol (MCH)</td>
 <td>Sigma</td>
 <td>451088</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>1,6-hexanedithiol (HDT)</td>
 <td>Sigma</td>
 <td>H-12005</td>
 <td>Store at room temperature</td>
 </tr>
 <tr>
 <td>Thiolated capture probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Fluorescein-modified detector probes</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Bridging Oligonucleotide</td>
 <td>Operon</td>
 <td>N/A</td>
 <td>Store at 100 &mu;M in 0.1x TE at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-Fluorescein-HRP, Fab fragments</td>
 <td>Roche</td>
 <td>11 426 346 910</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Helios Chip Reader</td>
 <td>GeneFluidics</td>
 <td>GFR-2009</td>
 <td></td>
 </tr>
 <tr>
 <td>Sensor Chip Mount</td>
 <td>GeneFluidics</td>
 <td>GFR-003</td>
 <td></td>
 </tr>
 <tr>
 <td>Film well sticker</td>
 <td>GeneFluidics</td>
 <td></td>
 <td>Shipped with sensor chips</td>
 </tr>
 <tr>
 <td>Bare gold 16-sensor array chips </td>
 <td>GeneFluidics</td>
 <td>SC1000-16X-B </td>
 <td>Store in 100% N2 at room temperature</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin </td>
 <td>Sigma</td>
 <td>A7906 </td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>1M Phosphate Buffer, pH 7.2 </td>
 <td></td>
 <td></td>
 <td>0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2</td>
 </tr>
 <tr>
 <td>Blocker Casein in PBS </td>
 <td>Pierce</td>
 <td>37528 </td>
 <td>Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 &deg;C</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

bacterial detection &amp; identification using electrochemical sensors

전기 센서가 널리 혈당의 신속하고 정확한 측정을 위해 사용되며, 분석의 다양한 검출에 적용 할 수 있습니다. 전기 센서는 유용한 전기 신호로 생물학적 인식 이벤트를 transducing에 의해 작동합니다. 신호 전달은 전류 측정 전극에 산화 환원 효소의 활동을 결합하여 발생합니다. 센서의 특이성은 고유의 효소의 특성, 포도당 센서의 경우 포도당 산화 효소 또는 효소 항체 또는 프로브 사이의 결합의 산물 중 하나입니다. 여기, 우리가 직접 감지하고 박테리아를 식별하는 전기 센서 분석 방법을 설명합니다. 모든 경우에, 여기에 설명 된 프로브 DNA 올리고 뉴클레오티드이다. 이 방법은 대상 리보솜 RNA (rRNA의)를 캡처 및 검출기 프로브 샌드위치 하이브리드를 기반으로합니다. 검출기 프로브가 시간에 연결되어있는 동안 포획 프로브는 센서 표면에 고정되어orseradish 퍼 옥시 데이즈 (HRP). 같은 3,3 &#39;, 5,5&#39;-tetramethylbenzidine (TMB)와 같은 기판 표면에 결합 캡처 표적 탐지기 단지와 전극에 추가되면, 기판은 HRP에 의해 산화 작업 전극에 의해 감소​​된다. 전극에 전류 흐름을 생산, 전극에서 HRP의 기질에 의해 전자의 왕복이 산화 환원 사이클 결과.

우리는 샌드위치 ELISA와 유사하게 구성되어 전기 화학적 분석을 설명합니다. 그림 1, 검출기 프로브에 3 &#39;형광 결합에 바인딩 방지 형광 항체 단편을 결합 HRP에 의해 촉매 산화 환원 반응에 의해 개발 캡처 및 검출기 프로브를 대상 리보솜 RNA (rRNA의) 하이브리드에 표시. 분석 감도의 중요한 구성 요소는 금 전극의 표면 화학입니다. 우리는 thiolated 캡처 프로브, mercaptohexanol 및 hexanedith...

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Bacterial Detection & Identification Using Electrochemical Sensors

우리는 빠른 세균 검출 및 식별을 위해 전기 센서 분석 방법을 설명합니다. 분석은 리보솜 RNA (rRNA의) 종의 특정 시퀀스 DNA 올리고 뉴클레오타이드 캡처 프로브 작용 센서 어레이를 포함한다. 캡처 프로브와 고추 냉이 과산화 효소 연결된 DNA 올리고 뉴클레오타이드 검출기 프로브 대상 rRNA의의 샌드위치 하이브리드는 측정 전류 측정 전류를 생성합니다.

전기 화학 센서를 사용하여 세균의 검출 및 식별

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

bacterial-detection-identification-using-electrochemical-sensors

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

28.1K 조회수.  Veterans Affairs Greater Los Angeles Healthcare System. 우리는 빠른 세균 검출 및 식별을 위해 전기 센서 분석 방법을 설명합니다. 분석은 리보솜 RNA (rRNA의) 종의 특정 시퀀스 DNA 올리고 뉴클레오타이드 캡처 프로브 작용 센서 어레이를 포함한다. 캡처 프로브와 고추 냉이 과산화 효소 연결된 DNA 올리고 뉴클레오타이드 검출기 프로브 대상 rRNA의의 샌드위치 하이브리드는 측정 전류 측정 전류를 생성합니다.

비디오: Bacterial Detection & Identification Using Electrochemical Sensors

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

보조 전기 분무 이온화 질량 분석법 (SESI - MS)를 사용하여 특성화 세균 휘발성 물질

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

연구

생체공학

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

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

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

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Photoacoustic Flowmetry를 사용하여 순환 흑색증 전지의 탐지 및 격리

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

다중 Transdermal 전기 감지를위한 빈 Microneedle 기반 센서

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

유기 전기 트랜지스터와 장벽 조직 혼란의 감지

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

유전자 인코딩 된 센서를 사용 글루타민 유출 영상

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

라벨없는 DNA의 혼성화 분석 용 미세 유체 기반 전기 바이오칩

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

저렴한 센서와 곤충 검출 및 분류 플라잉

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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

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

Microtoroid 광 공진기를 사용하여 라벨이없는 단일 분자 검출

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

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

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

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

물방울 미세 유체에 대한 여러 가지 빛깔의 형광 검출 광학 섬유를 사용하여

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