이 작품은 건강의 국립 연구소 부여 번호 NIH / NCRR P41 - RR 016,292 (ACERT에 대한)에 의해 지원되었다.

1. 자료 이 작품에 사용된 모든 용매는 시약 등급 있었다. 스핀 N - tert - 부틸 - α - phenylnitrone (PBN)와 표준 스핀 라벨을 포함하여 트랩, 2,2,6,6 - tetramethyl - 1 - piperinyoxyl이 (템포) 시그마에서 얻은되었고 공급으로 사용되었습니다. 공장 산화 억제제는 Swanson, 주식 회사 미국에서 상업적으로 획득했다. 2. 자유 래디 칼의 담배 연기 및 분석의 준비 <ol><li> lycopene은 아세톤에 녹아있는 동안 필터에 산화 억제제를 소개하려면, 산화 억제제의 pycnogenol과 포도 종자 추출물 먼저, 95 % 에탄올에 용해되었다. 솔벤트 볼륨 항산화 용해도에 따라 달랐다. </li><li> 0.4의 산화 억제제의 양은 MG / 필터가 사용되었습니다. 그들은 다음 활성탄의 10 MG로 코팅했다. 이런 목적을 위해 활성 탄소 필터와 진공 하에서 건조 무산소 조건에서 항산화 솔루션 ~ 12 시간을 위해 흔들되었다. </lI&gt; <li> 산화 억제제는 다음 종래의 아세테이트 필터 (CA 필터)에 도입되었습니다. 이를 위해 필터를 두 조각으로 절단되었다. 항산화 필터 샌드위치 (그림 1A) - 코팅 공장 산화 억제제는 필터를 형성하기 위해 필터와 테이프의 조각으로 싼 두 조각 사이에 삽입되었습니다. 이 결합된 필터는 이후에 담배를 포함하는 담배 막대에 붙어 있 었지. 필터가없이 항산화 제외 항산화 필터와 같은 방식으로 만들어진 컨트롤이 추가되었습니다. </li><li> 전에 흡연 시뮬레이션하는 연구 담배는 압축을 푼 2 일 최소 포화 NaBr 솔루션을 사용하여 일정한 습도 환경 (20 ° C, 60 % 상대 습도)에 보관되었다. </li><li> 일상적인 분석을 위해 흡연 시뮬레이션이로 Fig.1과 같이 단일 포트 흡연 장치를 사용하여 주위 온도에서 공연되었다, 물 흡인기 또는 GAST DOA - P104 - BN의 진공 펌프 / 공기 압축기 (벤톤 하버, Mich) 연결로 구성되어 있습니다 스핀 - 트래핑하기오픈 한 쪽 끝에있는 T - 교차점을 통해 조립. 퍼프는 퍼프 사이에 열어 두었던 오픈 엔드를 연결해에 의해 수행되었다. 가스 흐름이 유량계에 의해 체크 ~ 2.2에 설정된 SCFH = 17.5 펌프와 통풍구 사이에 위치 밸브를 조정하여 ML / s의. </li><li> 연구 담배는 1과 비슷한 60 초마다 반복 이초의 기간 동안 35 ML의 퍼프 볼륨의 상태에 따라 훈제했다. 가스 상 자유 래디 칼의 양적 추정을위한 흡연 시뮬레이션으로 그림에 표시된 스핀 트래핑 시스템과 연계하여 수행되었다. 1. </li><li> 열 강한 퍼프 (35 ML / 퐁퐁)은 각 담배에 대한 촬영했다. 가스 상 자유 래디 칼은 캠브리지 필터 패드를 통해 MCS를 전달하여 수집된 후 스핀 트래핑 솔루션 (벤젠, 2.0 ML에서 0.05 M PBN)에 도입되었습니다. </li><li> 마지막 퍼프 후, 부풀어 오른 트래핑 솔루션은 동일한 벤젠과 초기 볼륨 (2.0 ML)로 재조정했다. 나누어지는는 ~ 25 안으로 옮겨졌 다가길이 0mm, 한쪽 끝에 봉인 3mm의 ID 유리관. </li><li> 트래핑 솔루션은 냉동 펌프 - 해동 절차를 사용 deoxygenated되었습니다. 그것은 액체 질소에 따라 냉동었고, 진공이 적용되었습니다. 그렇다면 그것은 함정에 가스 거품이 탈출 수 있도록, 아르곤 분위기 하에서 해동되었고, 다시 냉동. 이 사이클은 세 번 반복 후, 튜브했습니다 불꽃 - 밀봉 진공하에 추가적인 ESR 측정에 사용. 벤젠에 잘 용해됩니다 산소, 유기 래디 칼의 ESR 라인을 넓혀 때문에이 단계가 필요합니다. 탈산소 반응은 극적으로 신호 대 잡음 비율을 향상시킵니다. </li><li> X - 밴드 ESR 스펙트럼은 표준 조건에서 9.34 GHz의 주파수에서 Bruker EMX 분광계에 기록되었다. 대부분의 실험에 사용되는 분광계 설정되었습니다 : 센터 현장 3312.5G, 스캔 폭 80G, 변조 진폭 0.5G, 시간 상수 82 마이크로초, 검사 시간 40 초. </li><li> 스핀 트랩 부가물 어떤 경우에는 req 실험 조건에서 비교적 안정일반적으로 ~ 20 분 데려 25 쌓일, uired. 그러나, 12 시간 이후 벤젠 용액의 ESR 신호의 강도가 ~ 5 요소에 의해 감소​​. </li><li> 트래핑 adduct의 농도를 정량하기 위해 초기 처음 유도체 ESR 스펙트럼이 통합되었습니다. 그 결과 흡수 스펙트럼의 연기에 포함된 검댕 / 타르 제품에 따라 가능성이 가장 광범위한 일중 배경을 보여줍니다. 뺄셈 후에이 배경의 트래핑 adduct의 분리 삼인승는 한 번 더 (그림 2) 통합되었습니다. </li></ol> 3. 대표 결과 담배 생산 연기 (기상)를 굽기에있는 자유 래디 칼의 대부분은 즉시하고 불안정하고 있습니다. 이러한 래디 칼을 관찰하기 위해 스핀 트랩 기술을 채용합니다. 그것은보다 안정되고 ESR (Fig.1)에 의해 감지 수있는 스핀 adduct로 그들을 변환하여 가스 상 자유 래디 칼을 캡처합니다. 0.05의 현재 연구에서 스핀 - 트랩 솔루션M PBN 15을 구분하기가 어렵습니다 산소와 탄소 중심의 래디 칼의 혼합 아르 자유 래디 칼, 위상 연기 가스를 수집하기 위해 사용되었다. 우리의 경우에는, 그러나, 관찰 초미세 분리 상수 N = 13.7G와 H = 1.95G들이 주요 제품에 것을 제안, alkoxyl 자유 래디 칼 (RO.) 2 부가물를 트래핑에 해당하는 값이 매우 유사합니다. 우리는 연기의 흐름에 습기로 인해 있다고 약한 ESR 신호 (그림 2) 초기 측정에서 관찰 낮은 재현성을 보여주었다. 이 문제를 해결하기 위해, 우리는 캠브리지 필터와 스핀 트랩 솔루션 사이에 액체 질소 (LN2) 트랩을 추가했습니다. 에선이 함정 급속 냉동하여 MCS 흐름에서 물을 제거하고 유리관의 내벽에 캡처. 이것은 크게 ESR 신호를 개선하고 높은 재현성 결과를 (그림 3) 허용. 함정 자유 래디 칼의 금액을 사용하여 결정되었다참고 샘플. 컨트롤 샘플 산화 억제제없이, 템포 알려진 농도의 이중 적분 스펙트럼과의 이중 적분 스펙트럼의 비교에 의해 예상 벤젠의 전형적인 adduct 농도는 1.24 μm의 (Fig.2)되었습니다. 공기의 양이 흡연하는 동안 각각의 담배 통과 이후 ~ 350 ML, 이것은 ~ 7.1X10 -9 M의 MCS의 가스 단계에서 자유 라디칼 농도에 대한 견적을 제공하고, 래디 칼의 총 숫자는 가스 단계에서 덫에 걸렸다 ~ 1.5X10 15 전체 담배. 가스 및 미립자 단계 모두 포함한 전체 담배의 연기에 자유 래디 칼의 총 금액에 대한 견적은 16 ~ 10 자유 래디 칼 9했습니다. 메인 스트림 담배 연기의 가스 단계 자유 래디 칼에 식물 산화 억제제의 청소 효과의 다른 수준이 관찰되었다. 그들의 청소 요금은 그림에서 소개되었습니다. 4. Lycopene과 포도 씨앗 추출물은 가장 높은 속도를 보여주 낮은 반면어 속도는 pycnogenol (Fig.4)에 대한 관찰되었다. <img alt="그림 1" src="/files/ftp_upload/3406/3406fig1.jpg" /> 그림 1. 스핀 트랩을 사용하여 주류 담배 연기 (MCS)에서 가스 위상 자유 래디 칼을 수집에 대한 개선 흡연 시뮬레이션 설계의 도표. MCS는 다음 CA 필터, 캠브리지 필터 (노란색 필터)를 통해 물 흡인기에 의해 그려진 및 H 2 O.을 제거하는 액체 질소 트랩 통과되었다 가스 상 제품은 마침내 함정을 스핀에 가서 스핀 트랩 솔루션을 통해 부풀어 오른 모양이었다. 식물 항산화는 MCS의 자유 래디 칼을 청소하기 위해 담배에 부착 (동그라미로 확대) 종래의 아세테이트 필터의 두 가지 사이에 위치했다. <img alt="그림 2" src="/files/ftp_upload/3406/3406fig2.jpg" /> 그림 2. 벤젠의 스핀 - 트래핑의 adduct 농도에 대한 양적 평가는 첫 번째 integra에서 광범위한 배경 신호를 빼서 필요내 스펙트럼. 스핀 - 트래핑의 adduct에 대한 초미세 분리 매개 변수 H = 1.95 G, N = 13.7G입니다. <img alt="그림 3" src="/files/ftp_upload/3406/3406fig3.jpg" /> 그림 3. LN2 트랩을 통해 전달이 상당히 MCS의 스핀 - 트래핑 얻은 ESR 신호의 품질을 향상시킵니다. <img alt="그림 4" src="/files/ftp_upload/3406/3406fig4.jpg" /> 그림 4. MCS의 자유 래디 칼의 농도에 자연 산화 억제제의 효과. 상대 신호 농도는 다음과 같습니다 제어 - pycnogenol 100 % - 55 %, 포도 종자 추출물 - 12 %, lycopene - 10 %.

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Rep. 6, 161-171 (2001).</li><li>Valavanidis, A., Vlachogianni, T., Fiotakis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke:+involvement+of+reactive+oxygen+species+and+stable+free+radicals+in+mechanisms+of+oxidative+damage,+carcinogenesis+and+synergistic+effects+with+other+respirable+particles.">Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.</a> International Journal of Environmental Research and Public Health. 6, 445-462 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

담배 연기에있는 다른 자유 라디칼 청소부의 효과의 신뢰성 추정은 자유 래디 칼의 양적 검출을위한 기술을 재현할 수 있어야합니다. 이전 1, 그것은 비 극성 용매에서 높은 농도 스핀 트랩 솔루션의 작은 볼륨이 담배 연기에서 자유 래디 칼을 트래핑에 가장 효과적인 것으로 나타났습니다. 담배 연기는 항상 유기 화합물의 연소 및 트래핑 용매에 끝날 수있는 담배의 잔류 수분에서 수증기가 포함되어 있습니다. PBN의 스핀 트랩 용액에 물이 혼합물은 크게 스핀 트래핑 부가물의 수명과 ESR 신호의 강도를 감소시킵니다. 가스 상 래디 칼의 일부 분율도 얼어붙은 표면에 갇혀있을 경우에도 액체 질소로 냉각 U 자형 튜브를 통하여 간단하게 통과 MCS하여 수분을 제거하면 크게, 우리의 실험에서 ESR 스펙트럼의 품질을 개선. t를 사용그의 기술은 우리가 담배 필터에 도입된 천연 화합물을 다른 급진 -​​ 소기의 상대적 효율성을 비교. 우리는 lycopene과 포도 씨앗 담배 필터로 통합 후 기상 MSC에서 자유 래디 칼의 90 %까지 청소하실 수 있습니다 즉시 추출 것으로 나타났습니다. 이러한 높은 청소 능력은 헤모글로빈과 shikonine 8,14 같은 가장 효율적인보고 자유 라디칼 프로텍터 중에서이 저렴한 즉시 사용 가능한 천연 화합물을 차지한다. 그러나, 공부 천연 산화 억제제와 함께 로드된 실험의 담배 필터에 실온에서 스토리지 일주일 후 그들의 소기 용량의 눈에 띄는 부분을 잃었습니다. 이 문제를 해결하는 것은 lycopene과 상업 담배 필터의 포도 종자 추출물 미래의 애플 리케이션을 자극 수 있습니다.

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

흡연은 인간의 암와 관련된 것입니다. 이것은 폐암 사망자의 대부분은 흡연 5,6,7,12로 인해 발생하는 것이보고되었습니다. 담배 연기의 입자 단계에서 담배 tars 및 관련 제품이 발암성 및 mutagenic 관련 질병의 주요 원인이지만, 담배 연기도 9,10 물질들의 중요한 그룹으로 간주됩니다 자유 래디 칼의 상당 금액을 포함하고 있습니다. 자유 래디 칼은 손상 단백질 구조, lipids과 DNA 시퀀스에 의해 세포 성분을 공격하고 암 다양한 형태의 개발의 위험을 증가시킵니다. 흡입 급진파는 폐 3 담배 연기의 부정적인 건강 효과를 많은 기여 부가물을 생산합니다. 연구는 흡연 유발 손상의 위험을 감소하기 위해 담배 연기에 자유 래디 칼을 줄이기 위해 실시되었습니다. 이것은 헤모글로빈과 헴이 함유 화합물 것은 부분적으로 질산 산화, 반응을 청소 수있는 것으로보고되었습니다oxidants와 담배 연기의 4 발암성 휘발성 nitrosocompounds. 헤모글로빈과 활성탄은 자유 래디 칼을 청소하고 담배 연기 14에서 자유 래디 칼의 90 %까지 제거하는 데 사용된의 &#39;바이오 필터&#39;는 구성된. 그러나, 비용 비효율로 인해, 그것은 성공적으로 상용화되지 않았습니다. 또 다른 연구는 shikonin, 중국 한방 의학 8 구성 요소의 좋은 청소 효율을 보여주었다. 현재 연구에서는, 우리는 스핀 - 트래핑을 사용하여 주류 담배 연기에 가스상 자유 래디 칼의 청소 효과 (MCS)의 담배 연기 및 측정의 가스상 자유 래디 칼을 청소에 대한 담배 필터에 일반 자연 항산화 추출물을 도입을위한 프로토콜을보고 전자 스핀 공명 (ESR) 분광학 1,2,14. 우리는 lycopene과 담배 필터에 자신의 미래의 응용 프로그램을 가리킨 수있는 포도 종자 추출물의 높은 청소 능력을 보여주었다. 이 프로의 중요한 이점pective 청소부들은 각각 토마토 또는 와인 산업의 부산물 11,13에서 대량으로 얻을 수있다는 것입니다

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

스핀 - 트래핑 ESR 분광법은 공장 산화 억제제 lycopene, pycnogenol와 담배 연기의 가스 상에게 자유 래디 칼을 청소에 포도 종자 추출물의 효과를 연구하는 데 사용되었다.

메인 스트림 담배 연기에 자유 래디 칼을 감지하고 가스 단계 청소를위한 프로토콜

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

a-protocol-for-detecting-scavenging-gas-phase-free-radicals

Nanyang Technological University

Research

JoVE Journal

Bioengineering

19.3K 조회수.  CDCF-AOX Lab. 스핀 - 트래핑 ESR 분광법은 공장 산화 억제제 lycopene, pycnogenol와 담배 연기의 가스 상에게 자유 래디 칼을 청소에 포도 종자 추출물의 효과를 연구하는 데 사용되었다.

비디오: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

연구

생체공학

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

마이크로 기둥 미세 유체 장치에서 바이오 필름 보리 형성을위한 프로토콜

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

낮은 풍부 단백질을 검출하기위한 플라즈마 또는 소변의 하이드로 겔 나노 입자 수확

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

전체 담배 연기에의 Organotypic 3D 기관지와 코 조직 문화 모델의 반복 노출의 영향 평가

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

시토크롬을 캡슐화<em&gt; C</em&gt; 금속 나노 입자없이 실리카 에어로젤 Nanoarchitectures에 가스 상 생체 활성을 유지하면서

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

교차 reactivity 무료 및 정찰 병 무료 스냅 칩 샌드위치 Immunoassays 다중화

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

전통적인 담배 연기와 전자 담배 연 무질에서 액체 추출의 세대에 대 한 장치를 운영 하는 마이크로컨트롤러

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

엄밀한 포함의 고정 및 스테인드, 전체, 마이크로 단층에 의해 섹션-무료 3D 조직학에 대 한 밀리미터 단위 표본

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

식물 반응 산소 종에서 Vivo에서 음이온 세 륨 산화물 나노 입자의 촉매 청소

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

개별 리포좀 간의 조성 불균질성을 검출하기 위한 정량적 형광 현미경 검사법 기반 단일 리포좀 분석

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...