이 프로토콜은 적게 10 μL의 혈청 샘플에서 세포 외 소체 (전기 자동차), 셀로부터 방출 멤브레인 작은 입자를 분리하는 방법을 자세히 설명. 이 접근법은, 초 원심 분리의 필요성을 회피 분석 시간의 몇 분을 필요로하고, 제한된 양의 시료로부터 전기차의 분리를 가능하게한다.
세포 외 소포 (전기 자동차), 각종 세포에서 방출 막의 입자는, 임상 적용을위한 할 수있는 잠재력을 가지고 있습니다. 그들은 핵산과 단백질화물을 포함 점점 진핵 생물과 원핵 세포 모두에 의해 사용되는 세포 간 의사 소통의 수단으로 인식하고 있습니다. 그러나, 작은 크기로, 전기 자동차의 분리를위한 현재의 프로토콜은 종종 시간이 소요, 복잡하고, 이러한 초 원심 분리기와 같은 큰 샘플 볼륨과 고가의 장비를 필요로합니다. 이러한 한계를 해결하기 위해, 우리는 쉽게 효율적이고, 10 μL로 낮은 샘플 볼륨을 필요 전기차의 서브 그룹들을 분리하기위한 종이 기반 면역 플랫폼을 개발했다. 생물학적 시료는 화학적으로 특정한 EV 표면 마커에 높은 친 화성을 가지고 포획 분자로 변성 된 종이 테스트 존에 직접 피펫 팅 될 수있다. 우리는 주사 전자 현미경 (SEM)을 사용하여 분석의 유효성을 확인, 종이 기반 효소 결합 immunosorbent 분석 (P-ELISA)과 전 사체 분석. 이 종이 기반의 디바이스는 건강과 질병에 EV 기능에 대한 우리의 이해를 발전하기 위해 병원과 연구 환경에서 전기 자동차의 연구를 가능하게 할 것이다.
Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met.
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.
세포 외 소포의 하위 그룹의 성공적인 분리를위한 가장 중요한 단계는 다음과 같습니다 용지 1) 좋은 선택; 종이 섬유의 표면 상에 포획 분자의 2)에 따르면 안정적이고 높은; 3) 시료의 적절한 처리; 4) 일반 실험실 위생 기준.
다공성 물질은 많은 저렴하고 장비가없는 분석에 사용되어왔다. 이들은 가변 공경, 다목적 기능, 저가 및 높은 표면 대 부피 비율 유체의 수동적 위킹?…
The authors have nothing to disclose.
이 작품은 대만 국립 과학위원회 grants- NSC 99-2320-B-007-005-MY2 (CC) 및 NSC 101-2628-E-007-011-MY3 (CMC) 및 재향 군인의 일반에 의해 부분적으로 지원 병원과 대만의 공동 연구 프로그램의 대학 시스템 (CC).
Chromatography Paper | GE Healthcare Life Sciences | 3001-861 | Whatman® Grade 1 cellulose paper |
(3-Mercaptopropyl) trimethoxysilane | Sigma Aldrich | 175617 | This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors. |
Ethanol | Fisher Scientific | BP2818 | Absolute, 200 Proof, molecular biology grade |
Bovine serum albumin (BSA) | BioShop Canada Inc. | ALB001 | Often referred to as Cohn fraction V. |
N-g-maleimidobutyryloxy succinimide ester (GMBS) | Pierce Biotechnology | 22309 | GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive. |
Avidin | Pierce Biotechnology | 31000 | NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin |
Biotinylated mouse anti-human anti-CD63 | Ancell | 215-030 | clone AHN16.1/46-4-5 |
biotinylated annexin V | BD Biosciences | 556418 | Annxin V has a high affinity for phosphotidylserine (PS) |
Primary anti-CD9 and secondary antibody | System Biosciences | EXOAB-CD9A-1 | The secondary antibody is horseradish peroxidise-conjugated |
Serum separation tubes | BD Biosciences | 367991 | Clot activator and gel for serum separation |
Annexin V binding buffer | BD Biosciences | 556454 | 10X; dilute to 1X prior to use. |
TMB substrate reagent set | BD Biosciences | 555214 | The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB) |
RNA isolation kit | Life Technologies | AM1560 | MirVana RNA isolation kit |
Polyvinylpyrrolidone-based RNA isolation aid | Life Technologies | AM9690 | Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides. |
RNA cleanup kit | Qiagen Inc. | 74004 | MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA. |
Plasma chamber | March Instruments | PX-250 | |
Scanning electron microscope | Hitachi Ltd. | S-4300 | |
Desktop scanner | Hewlett-Packard Company | Photosmart B110 | 8-bit color images were captured. Cameras and smart phones may be also used. |
Image-record system | J&H Technology Co | GeneSys G:BOX Chemi-XX8 | 16-bit fluroscence images were captured. Fluroscence microscopes may be also used. |