생물 액체 배지에서 확장 성, 금속 높은 가로 세로 비율 나노 복합 재료의 생성
Summary
여기서 우리는 생물학적 조건하에 액체 배지에서 신규 한 고 종횡비 biocomposites를 합성 프로토콜을 제시한다. biocomposites 각각의 직경 및 길이에서 마이크로 나노 미터 스케일. 시스틴 결합 구리 나노 입자 (CNPS) 및 황산구리는 합성 용 핵심 부품이다.
Abstract
이 프로토콜의 목적은 높은 종횡비 구조를 갖는 두 개의 신규 biocomposites의 합성을 설명하는 것이다. biocomposites 구리 나노 입자 (CNPS) 또는 금속 성분을 기여 황산구리 중 하나와, 구리 및 시스틴 이루어져있다. 합성 생물학적 조건 (37 ° C)와 24 시간 후의 자기 조립 복합 형태 하에서 액상에서 수행된다. 형성되면, 이러한 복합 재료는 모두 액체 매체와 건조 폼에서 매우 안정적이다. 복합 길이 범위를 마이크로 -하는 나노에서 확장, 몇 미크론에서 직경 25 nm의. 에너지 분산 형 X 선 분광법 (EDX)을 전계 방출 주사 전자 현미경은 따라서 최종 나노 복합 재료에서의 황 원으로서 시스테인을 확인, 출발 CNP 재료 결석 동안 황, NP 유래의 선형 구조 내에 존재하는 것을 입증 . 이러한 선형 나노 및 마이크로 복합 재료의 합성, STR의 길이의 다양한 중uctures는 합성 용기 내에서 형성된다. 합성 후 액상 혼합물의 초음파 처리는 초음파 처리 시간 증가로 평균 길이를 감소하여 구조물의 평균 크기를 제어하는데 도움이 입증되었다. 형성된 구조는 고도로 응집하지 않는 안정한 액상이 형성되어 있기 때문에, 원심 분리는 또한 집중 형성된 복합체 편석을 돕기 위해 사용될 수있다.
Introduction
Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 °C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called “urchin” and “nanoflower” type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 °C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described.
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells.
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described “coffee ring effect” 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.
Protocol
Representative Results
Discussion
CNPS 포함 된 나노 물질의 잠재적 인 독성을 평가하는 동안, 이것은 장기적, CNPS는 큰 응집 형태로 초기에 더 분산 미립자 분포 (도 2)로 형질 전환시키고 있음을 관찰 하였다. 일부의 경우, 생물학적 조건 하에서 세포 배양 접시에서 생산 된 이러한 고도로 응집 된 구조물은 "게"를 포함하는 전술 구리 연상시키는 중앙 집합체에서 높은 선형 돌기를 형성 6. 그것은 여기에 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).
Materials
| Mini Vortexer | VWR (https://us.vwr.com) | 58816-121 | |
| CO2 Incubator Model # 2425-2 | VWR (https://us.vwr.com) | Contact vendor | Current model calalog # 98000-360 |
| Eppendorf Centrifuge (Refrigerated Microcentrifuge) | Labnet (http://labnetinternational.com/) | C2500-R | Model Prism R |
| Cell Culture Centrifuge Model Z323K | Labnet (http://labnetinternational.com/) | Contact vendor | Current model Z206A catalog # C0206-A |
| Sonicator (Ultrasonic Cleaner) | Branson Ultrasonics Corporation (http://www.bransonic.com/) | 1510R-MTH | |
| Balance | Sartorius (http://dataweigh.com) | Model CP225D similar model CPA225D | |
| Olympus IX51 Inverted Light Microscope | Olympus (http://olympusamerica.com | Contact vendor | |
| Olympus DP71 microscope digital camera | Olympus (http://olympusamerica.com | Contact vendor | |
| external power supply unit- white light for Olympus microscope | Olympus (http://olympusamerica.com | TH4-100 | |
| 10x, 20, and 40x microscope objectives | Olympus (http://olympusamerica.com | Contact vendor | |
| Scanning Electron Microscope | Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html) | model S-4800 | |
| Transmission Electron Microscope | Zeiss (http://zeiss.com/microscopy/en_de/products.html) | model Libra 120 | |
| Table Top Work Station Unidirectional Flow Clean Bench | Envirco (http://envirco-hvac.com) | model PNG62675 | Used for sterile cell culture technique |
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